CN105037447A - Achyranthes bidentata glycopolymer as well as preparation method and application thereof - Google Patents
Achyranthes bidentata glycopolymer as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a glycopolymer in achyranthes bidentata as well as a preparation method and application thereof. In the method, the achyranthes bidentata is used as a raw material, and is subjected to water-extraction alcohol precipitation and alkaline-extraction alcohol precipitation, so that polymers at all the parts of the achyranthes bidentata are obtained. The extraction method is implemented under a relatively gentle condition, and components of the glycopolymer are intactly preserved. The achyranthes bidentata glycopolymer has a remarkable effect at the aspect of anti-osteoporosis symptom; in addition, a glycopolymer pure product is obtained through separation and purification processes including Sevag method deproteinization, DEAE cellulose column chromatography, Sephacryl column chromatography and the like, and structural analysis shows that the achyranthes bidentata glycopolymer obtained through the method comprises polyose and oligose. Through the adoption of the achyranthes bidentata glycopolymer as well as the preparation method and application thereof, a basis is provided for the application of the achyranthes bidentata glycopolymer in the fields of health care products, medicines and the like.
Description
Technical field
The invention belongs to medicine and technical field of health care food, be specifically related to the preparation method of glycopolymers in plant, relate to the preparation method of Radix Achyranthis Bidentatae raw sugar polymkeric substance, refined sugar polymkeric substance in particular, also relate to this root of bidentate achyranthes raw sugar polymkeric substance and preventing and/or treating the application in osteoporosis.Glycopolymers in the present invention comprises polysaccharide and oligosaccharides.
Background technology
Osteoporosis (osteoporosis, OP) is that one reduces with bone mineral quantity, bone micro-structure destroys, bone strength reduces, thus causes the fragility of bone to increase and be easy to the disease that generation fracture is feature.Osteoporosis is a kind of multiple geriatric disease, and along with the aging of population is on the rise, osteoporosis is serious harm senior health and fitness.Postmenopausal women, because being subject to double influence that the is old and feeble and decline of hormone in vivo level, becoming the crowd that is injured that this disease is maximum, accounts for total number of patients more than 70%.The shortcomings such as if the drug main bone resorption inhibitor of current protect against osteoporosis, bone formation-promoter and mineralising medicine, these medicines all belong to replacement therapy, and in life-time service process, have toxic side effect large, targeting is low, and compliance is poor, and expense is high.Therefore, a kind of medicine of efficient, low toxicity is necessary to look for treat osteoporosis.Chinese medicine has long history in treatment osteoporosis, has the advantages such as whole body is nursed one's health, toxic side effect is little, cheap, more and more receives the concern of people.And in numerous Chinese medicine, nourishing disney and strengthening bone class Chinese medicine is subject to the favor of people due to good curative effect always.
The root of bidentate achyranthes (
achyranthesbidentatab.l) be the dry root of the amaranthaceous plant root of bidentate achyranthes, be one of " four-Huai medicine ".There is invigorating the liver and kidney, strengthening the bones and muscles, the merit of stimulating the menstrual flow by the stasis of blood, ensurining proper downward flow of the blood, be mainly used in soreness of waist and knee joint, muscles and bones unable, through closing the disease of abdominal mass, dizziness due to hyperactivity of liver-YANG.History tree clearly records the prescription totally 21 for the treatment of " heumatism ", " atrophic debility of bones ", and relate to medicine 77 kinds, wherein the root of bidentate achyranthes occurs 11 times.Root of bidentate achyranthes glycopolymers is as one of the main active ingredient of the root of bidentate achyranthes, and be in the news multiple pharmacologically actives such as having immunomodulatory, antitumor, hypoglycemic, protection liver kidney.
At present, the research of root of bidentate achyranthes glycopolymers is mainly concentrated on that it is antitumor, anti-oxidant both at home and abroad, immunomodulatory, hypoglycemic, anti-ageing aspect of waiting for a long time activity research, to its research in protect against osteoporosis, there is not been reported.
Summary of the invention
The object of the present invention is to provide a kind of root of bidentate achyranthes glycopolymers ABW3-1.
Another object of the present invention is to the extracting method that glycopolymers in a kind of root of bidentate achyranthes is provided.
Another object of the present invention is to provide the application of above-mentioned root of bidentate achyranthes glycopolymers in preparation control medicine for treating osteoporosis or protective foods 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 this glycopolymers is made up of glucose and fructose, its structure is:
.
An extracting method for glycopolymers in the root of bidentate achyranthes, the method comprises following operation steps:
1) shear: root of bidentate achyranthes dry root is cut into segment, cleans up with water, dry;
2) water extraction: the root of bidentate achyranthes sheared is carried out water extraction, collects extracting solution and residue respectively;
3) classification alcohol precipitation: after extracting solution is concentrated, add ethanol and make ethanol contend concentration be a%, leave standstill, collecting precipitation, obtains raw sugar polymer A B1; Again concentrated by supernatant liquor, add ethanol and make ethanol contend concentration be b%, leave standstill, collecting precipitation, obtains raw sugar polymer A B2; Again concentrated by supernatant liquor, add ethanol and make ethanol contend concentration be c%, leave standstill, collecting precipitation, obtains raw sugar polymer A B3; Wherein 10≤a < b < c < 100;
4) alkali is carried: be soaked in by the residue after water extraction in 0.1 ~ 1MNaOH solution, leave standstill 1 ~ 4h, supernatant liquor 0.1 ~ 1MHCl neutralizes, the pH of supernatant liquor is made to be 6 ~ 8, centrifuging and taking supernatant, concentrated supernatant, adding ethanol makes ethanol contend concentration be 50 ~ 90%, leave standstill, collecting precipitation, obtains alkali and carries raw sugar polymer A BB;
5) purifying: purifying is carried out to above-mentioned raw sugar polymer A B1, AB2, AB3, ABB, obtains root of bidentate achyranthes glycopolymers.
Further, step 2) described in the concrete operations of water extraction be with 60 ~ 100 DEG C of hot water of 5 ~ 15 times of volumes, the root of bidentate achyranthes cut is extracted, extraction time 1 ~ 4h.
Further, all described simmer down tos 40 ~ 70 DEG C of concentrating under reduced pressure in step 3), all described standing time is 10 ~ 28h.
Further, 10≤a < 60,60 < b < 80,80 < c < 100 in step 3).
Further, the glycopolymers AB3 after purifying in step 5) is carried out ion-exchange chromatography again, carries out wash-out with distilled water, use phend-sulphuric acid to follow the tracks of elution curve, collect sugar moieties according to elution curve, concentrated, lyophilize; Then water dissolution is used, centrifugal, get supernatant liquor and carry out molecular sieve gel column chromatography, carry out wash-out with water, use phend-sulphuric acid to follow the tracks of elution curve, collect sugar moieties according to elution curve, concentrated, obtain root of bidentate achyranthes glycopolymers ABW3-1 after lyophilize.
Root of bidentate achyranthes glycopolymers, the extracting method of this glycopolymers is extracting method described above.
The application of root of bidentate achyranthes glycopolymers ABW3-1 in the preparation osteoporotic medicine of control or protective foods or functional food.
The root of bidentate achyranthes glycopolymers AB1, AB2, AB3, ABB application in the preparation osteoporotic medicine of control or protective foods or functional food.
The application of root of bidentate achyranthes glycopolymers in the preparation osteoporotic medicine of control or protective foods or functional food.
The invention has the beneficial effects as follows:
1. the present invention utilizes ion exchange chromatography and molecular sieve gel column chromatography method purifying root of bidentate achyranthes glycopolymers, prepare a kind of root of bidentate achyranthes essence oligosaccharides first, and the Analysis and Identification of system is carried out to the physico-chemical property, molecular weight, monose composition etc. of this smart oligosaccharides, and has successfully drawn the feature structure of this oligosaccharides.The invention provides raw sugar polymkeric substance in the root of bidentate achyranthes, the preparation method of refined sugar polymkeric substance and the activity research of root of bidentate achyranthes glycopolymers in osteoporosis, for root of bidentate achyranthes glycopolymers provides foundation in the application in the fields such as medicine, healthcare products.
2. compared with extracting glycopolymers with traditional water-boiling method, the present invention adopts the combination of water extraction and alcohol precipitation method and alkaline extraction, alcohol concn carries out classification alcohol precipitation from low to high, initial gross separation is carried out to root of bidentate achyranthes glycopolymers, high concentration ethanol can be separated by glycopolymers glycopolymers that is little with polarity, poorly water-soluble that is polarity is large, good water solubility simultaneously, makes extracted glycopolymers composition contain a greater variety of glycopolymers composition.And this preparation technology is simple, easy to operate, and can scale operation.
3. by column chromatography, separation and purification is carried out, Be very effective to root of bidentate achyranthes raw sugar polymkeric substance in the present invention, prepare a kind of root of bidentate achyranthes oligosaccharides sterling first.
4. the present invention identifies a kind of root of bidentate achyranthes oligosaccharide structure prepared, and specify that physico-chemical property and the structure of this oligosaccharides, provides structure foundation for probing into its pharmacologically active mechanism.
5. the invention provides in the root of bidentate achyranthes preparation method of the composition with anti-osteoporosis activity, and filter out high, the active strong glycopolymers position of content.
Accompanying drawing explanation
Fig. 1 is the HPGPC color atlas of ABW3-1;
Fig. 2 is the infared spectrum of ABW3-1;
Fig. 3 is ABW3-1's
13cNMR collection of illustrative plates;
Fig. 4 is ABW3-1's
1hNMR collection of illustrative plates;
Fig. 5 is the HSQC collection of illustrative plates of ABW3-1;
Fig. 6 is the HMBC collection of illustrative plates of ABW3-1;
Fig. 7 is the impact of root of bidentate achyranthes glycopolymers on ovariectomized female rats body weight;
Fig. 8 is the impact of root of bidentate achyranthes glycopolymers on ovariectomized female rats Uterine coefficient;
Fig. 9 is the impact of root of bidentate achyranthes glycopolymers on femur of mature ovariectomized rats bone density;
Figure 10 is the impact of root of bidentate achyranthes glycopolymers on ovariectomized female rats lumbar vertebra bone density;
Figure 11 is the impact of root of bidentate achyranthes glycopolymers on femur of mature ovariectomized rats bone mineral quantity;
Figure 12 is the impact of root of bidentate achyranthes glycopolymers on ovariectomized female rats lumbar vertebra bone mineral quantity.
Embodiment
1. the extracting method of glycopolymers in the root of bidentate achyranthes, the method comprises following operation steps:
1) shear: root of bidentate achyranthes dry root is cut into segment, by cold water Rapid Cleaning, dries;
2) water extraction: the root of bidentate achyranthes sheared is carried out water extraction, collects extracting solution and residue respectively;
3) classification alcohol precipitation: after extracting solution is concentrated, add ethanol and make ethanol contend concentration be a%, leave standstill, collecting precipitation, obtains raw sugar polymer A B1; Again concentrated by supernatant liquor, add ethanol and make ethanol contend concentration be b%, leave standstill, collecting precipitation, obtains raw sugar polymer A B2; Again concentrated by supernatant liquor, add ethanol and make ethanol contend concentration be c%, leave standstill, collecting precipitation, obtains raw sugar polymer A B3; Wherein 10≤a < b < c < 100; Preferred 10≤a < b < c < 100, and 10≤a < 60,60 < b < 80,80 < c < 100;
4) alkali is carried: be soaked in by the residue after water extraction in 0.1 ~ 1MNaOH solution, leave standstill 1 ~ 4h, supernatant liquor 0.1 ~ 1MHCl neutralizes, the pH of supernatant liquor is made to be 6 ~ 8, centrifuging and taking supernatant, concentrated supernatant, adding ethanol makes ethanol contend concentration be 50 ~ 90%, leave standstill, collecting precipitation, obtains alkali and carries raw sugar polymer A BB;
5) purifying: purifying is carried out to above-mentioned raw sugar polymer A B1, AB2, AB3, ABB, obtains root of bidentate achyranthes glycopolymers.
Preferably, segment described in step 1) is the segment of 1 ~ 2cm.
Preferably, step 2) described in the concrete operations of water extraction be with 60 ~ 100 DEG C of hot water of 5 ~ 15 times of volumes, the root of bidentate achyranthes is extracted, extraction time 1 ~ 4h.
Preferably, all described simmer down tos 40 ~ 70 DEG C of concentrating under reduced pressure in step 3), the time that all alcohol precipitations leave standstill is 10 ~ 28h.
Preferably, in step 4), NaOH solution consumption volume is 10 ~ 20 times of residue volume.
Preferably, the concrete operations of purifying described in step 5), for utilizing Sevag method respectively to raw sugar polymer A B1, AB2, AB3, ABB removing protein, are carried out dialysing through dialysis tubing after removing protein, freeze-drying again.
Preferably, the molecular weight cut-off of above-mentioned dialysis tubing is 3000Da.
Preferably, following chromatography purification is carried out to the glycopolymers AB3 after purifying in step 5):
1) ion-exchange chromatography: the glycopolymers AB3 after purifying carries out ion-exchange chromatography, carries out gradient elution with distilled water, uses phend-sulphuric acid to follow the tracks of elution curve, collects sugar moieties respectively according to elution curve, then concentrated, lyophilize; Dissolve with water respectively again, centrifugal, get supernatant liquor;
2) molecular sieve gel column chromatography: the supernatant liquor of previous step gained is carried out molecular sieve gel column chromatography again, carry out wash-out with water, use phend-sulphuric acid to follow the tracks of elution curve, collect sugar moieties according to elution curve, glycopolymers sterling is obtained, by its called after ABW3-1 after concentrated, lyophilize.
Preferably, above-mentioned ion exchange column is DEAE ion exchange column.
Preferably, above-mentioned molecular sieve gel chromatography selects Sephacryl molecular sieve chromatography post.
Preferably, the oligosaccharides that above-mentioned root of bidentate achyranthes glycopolymers ABW3-1 is made up of glucose and fructose, primarily of → 2)-β-D-Fru-(1 → and → 2)-α-D-Fru-(6 → composition, terminal sugar residues is made up of α-D-Glc-(1 → and → 2)-β-D-Fru, and the structure of ABW3-1 is:
。
Preferably, the molecular weight of ABW3-1 is 1074Da.
Root of bidentate achyranthes glycopolymers, the preparation method of this glycopolymers is the extracting method of glycopolymers in the root of bidentate achyranthes described above.
The application of root of bidentate achyranthes glycopolymers ABW3-1 in the preparation osteoporotic medicine of control or protective foods or functional food.
The root of bidentate achyranthes glycopolymers AB1, AB2, AB3, ABB application in the preparation osteoporotic medicine of control or protective foods or functional food.
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
the extracting method of glycopolymers in embodiment 1 root of bidentate achyranthes
1) shear: the root of bidentate achyranthes dry root scissors of 10kg is cut to 1 ~ 2cm, uses cold water Rapid Cleaning, dry;
2) water extraction: by the root of bidentate achyranthes sheared, extracts with 10 times of volume hot water (60 ~ 100 DEG C), extraction time 2h, and collect extracting solution, residue dries.
3) classification alcohol precipitation: extracting solution, after 60 DEG C of concentrating under reduced pressure, adds ethanol and makes ethanol contend concentration be 50%, after room temperature leaves standstill 24h, centrifugal, collecting precipitation, and weigh 323g, is raw sugar polymer A B1; Collect supernatant liquor after 60 DEG C of concentrating under reduced pressure, adding ethanol makes ethanol contend concentration be 70%, after room temperature leaves standstill 24h, centrifugal, collecting precipitation, weigh 893g, for raw sugar polymer A B2, repeat, by above-mentioned operation (except ethanol contend concentration is except 90%, other operations are all the same) equally, to obtain 671g raw sugar polymer A B3.
4) alkali is carried: be immersed in by the dregs of a decoction after water extraction in the 0.3MNaOH solution of 15 times of volumes, and room temperature places 2h, and supernatant liquor 0.5MHCl neutralizes, its pH is made to be 6 ~ 8, get supernatant liquor to carry out centrifugal (5000r/min, 10min), get supernatant liquor, 60 DEG C of concentrating under reduced pressure, then adding ethanol makes ethanol contend concentration be 75%, centrifugal after leaving standstill 24h, collecting precipitation, be weighed as 410g, this carries raw sugar polymer A BB for alkali.
5) purifying: utilize Sevag method to carry out removing protein to each component raw sugar polymkeric substance respectively, after removing protein raw sugar polymkeric substance dialysis tubing (molecular weight cut-off is 3000Da) carry out dialysing, freeze-drying, obtain root of bidentate achyranthes glycopolymers.
the extracting method of embodiment 2 root of bidentate achyranthes glycopolymers ABW3-1
1) shear: the root of bidentate achyranthes dry root scissors of 10kg is cut to 1 ~ 2cm, uses cold water Rapid Cleaning, dry;
2) water extraction: by the root of bidentate achyranthes sheared, extracts with 10 times of volume hot water (60 ~ 100 DEG C), extraction time 2h, and collect extracting solution, residue dries.
3) alcohol precipitation: extracting solution, after 60 DEG C of concentrating under reduced pressure, adds ethanol and makes ethanol contend concentration be 50%, leaves standstill 24h, collect supernatant liquor after 60 DEG C of concentrating under reduced pressure, adding ethanol makes ethanol contend concentration be 70%, leaves standstill 24h, repeats by above-mentioned operation equally (except ethanol contend concentration is 90%, other operations are all the same), after room temperature leaves standstill 24h, centrifugal, collecting precipitation, weigh 671g, is raw sugar polymer A B3;
4) purifying: utilize Sevag method to carry out removing protein to raw sugar polymer A B3, after removing protein raw sugar polymkeric substance dialysis tubing (molecular weight cut-off is 3000Da) carry out dialysing, freeze-drying, obtain root of bidentate achyranthes glycopolymers AB3.
5) ion-exchange chromatography: get the glycopolymers AB3 after the above-mentioned purifying of 100mg, be dissolved in the deionized water of 5mL, be splined on DEAE-Cellulose52 post, there are 2 peaks under the elutriant condition of different salt concn, wherein peak one is water (0MNaCl) elution fraction, peak two is that 0.1MNaCl elution fraction (uses phend-sulphuric acid to follow the tracks of elution curve in elution process, sugar moieties is collected respectively) according to elution curve, respectively by after concentrated for gained elutriant, lyophilize, obtain two kinds of glycopolymers (peak one glycopolymers, peak two glycopolymers).
6) molecular sieve gel chromatography: by the peak one glycopolymers sample after above-mentioned freeze-drying, dissolve with water, centrifugal, get supernatant liquor, upper SephacrylS-100HR post, wash-out is carried out with water, use phend-sulphuric acid to follow the tracks of elution curve, occur a single symmetrical peak, collect main peak, concentrated, obtain root of bidentate achyranthes glycopolymers ABW3-1 after lyophilize.
Purity and molecular weight detection: the aqueous solution glycopolymers ABW3-1 of above-mentioned acquisition being made into 2% concentration (W/V), HPGPC method records retention time, calculates molecular weight according to typical curve.
As shown in Figure 1, the component ABW3-1 obtained after ion-exchange and gel filtration method separation and purification is single symmetrical peak to result, illustrates that ABW3-1 is homogeneous glycopolymers, and recording molecular weight is 1074Da.
Below to state above extraction and isolation in embodiment to ABW3-1 do further structural analysis.
root of bidentate achyranthes oligosaccharidesaBW3-1
structural analysis
(1) monosaccharide composition analysis
From complete acid hydrolysis products chromatography of ions HPAEC-PAD collection of illustrative plates, ABW3-1 monose consists of glucose and fructose.
(2) infrared spectra detects
The infrared spectra detected result of ABW3-1 as shown in Figure 2, therefrom can be found out, the charateristic avsorption band that ABW3-1 contains oligosaccharides is: 3368.98cm
-1for O-H stretching vibration, 2935.72cm
-1for C-H stretching vibration, 1643.45cm
-1for carbonyl absorption peak.927.69cm
-1and 813.60cm
-1for the charateristic avsorption band of furan nucleus; 873.72cm
-1there is absorption peak at place, illustrates that ABW3-1 is for form primarily of β type saccharide residue.
(3) methylation analysis
Sample through methylating, hydrolysis, reduction, after acetylize GC-MS analyze, result show ABW3-1 contain → 1)-β-D-Fru
f-(2 → and → 2)-β-D-Fru
f-(6 → saccharide residue.
(4) nuclear magnetic resonance spectroscopy of glycopolymers
Homogeneous glycopolymers ABW3-1 sample is placed in nuclear magnetic tube, uses D
2o surveys spectrum after dissolving, and acquired results as illustrated in figures 3-6.
According to the ownership of the nuclear magnetic spectrum of above-mentioned Fig. 3 ~ 6 each carbon known and hydrogen, as shown in table 1 below.
Table 1ABW3-1 nuclear magnetic resonance spectroscopy result
aUnresolvedfromothersignals.
Through above-mentioned complete acid hydrolysis, methylation analysis, infrared spectra detect and nmr analysis, result display ABW3-1 be a kind of oligosaccharides be made up of with 1:5 ratio glucose and fructose, from methylation analysis illustrate its contain → 1)-β-D-Fru
f-(2 → and → 2)-β-D-Fru
f-(6 → saccharide residue, the order of connection between different sugar residue is drawn by two-dimentional nuclear-magnetism HMBC spectrum analysis, show that the structure of ABW3-1 is by above analysis:
, its molecular weight is 1074Da.
In addition, we, to be isolated to Achyranthan ABW2-1, ABP2-2 structure as follows in early stage.
The structure of Achyranthan ABW2-1:
Wherein m is 0 ~ 19, n is 0 ~ 19, and m+n is 2 ~ 19.
The structure of Achyranthan ABP2-2:
Its molecular-weight average is 3406Da.
Above homogeneous polysaccharide, oligosaccharides may be the activeconstituentss of root of bidentate achyranthes glycopolymers osteoporosis, and thus, the Structural Identification of homogeneous glycopolymers provides strong foundation by for follow-up mechanism of probing into root of bidentate achyranthes glycopolymers osteoporosis.
Below the activity of the root of bidentate achyranthes glycopolymers that the present invention extracts is detected.
the function of resisting osteoporosis research of embodiment 3 root of bidentate achyranthes glycopolymers
1 experimental technique
1.1 experimental designs and grouping: 3 monthly age non-mating female sd inbred rats 70, SPF level (being provided by Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center), 7 groups are divided at random by body weight principle of reciprocity, each treated animal number and medication are in table 1, wherein positive controls E2 is 17 beta estradiols, and model group OVX and sham operated rats SHAM is distilled water.Post operation gastric infusion every day, weighs weekly once, adjusts corresponding dosage by body weight change, continuous gavage 90d.
Table 1 experimental design and grouping
1.2 ovariectomized rats operation methods:
By rat with the Nembutal sodium solution of 45mg/kg dosage (4%) intraperitoneal injection of anesthesia, abdomen position is fixed.Under its most end rib, midaxillary line exposes surgical field of view with the infall apart from about 1cm outside backbone, unhairing.First appoint and get side and perform the operation, with betagen solution sterilization, cut skin, muscle of back and sarolemma, with pincet gently by outside white shiny liparitosis pull-out otch, separating out fat group, just can see ovary.By the uterine tube operating sutures ligation of ovary lower end, then extract ovary.After myometrial suture, outside deposited with antiinfectious powder (benzylpenicillin sodium), extract opposite side ovary with method.The treatment process of sham operated rats (SHAM) is the same, and just excision fritter fatty tissue, does not extract ovary.Postoperatively be placed in warm environment, nurse is observed.
1.3 draw materials and preservation
Concrete operations are as follows:
1) weigh, anaesthetize;
2) dissect rat from belly and expose internal organ, press close to muscle of back place tweezers and clamp artery and get 2ml blood in heparin sodium pipe, change common blood vessel of getting immediately and get whole blood (after leaving standstill lh, get serum after the centrifugal 10min of 3000rmp ,-80 DEG C of stored frozen are for subsequent use).
3) get right side shin bone, femur and fifth lumbar vertebra, put in the urine cup that 4%PBS paraformaldehyde solution is housed, after 24h, change the ethanolic soln of 70%.-20 DEG C of freezen protective.
4) get left femur, shin bone and third and fourth lumbar vertebrae, physiological saline gauze wraps up and uses masking foil external parcel, and in sealing bag, packing is placed in-80 DEG C of stored frozen.
1.4 rat bone densities measure
Dual-energy X-rays absorptionmetry HologicDiscoveryWI85003DXA is adopted to detect the full bone density (bonemineraldensity of fourth lumbar vertebra bone (L4) and fl, BMD), front end femoral bmd (1cm), distal femoral bone density (2cm) and bone mineral content (bonemineralcontent, BMC).
1.5 bone biomechanical tests
During test ,-80 DEG C of femurs preserved and vertebra are placed in-20 DEG C and thaw in advance, then normal temperature unfreezing, wets again with physiological saline.Mini858Bionix material testing system is adopted to analyze the biomechanical property of fl (three-point bending test).During test on MTS trier, with the pressure head of 1mm diameter, loading velocity is 6mm/min, and span (L) is 15mm, and sensor is 500N.Instrument records load and the radial degree changing value of each moment point automatically, draws load-radial degree curve, and obtains corresponding parameter.
experimental result
2.1 root of bidentate achyranthes glycopolymers ring removal ovary female rats ghost image
Root of bidentate achyranthes glycopolymers rings removal ovary female rats ghost image sees Fig. 7, and compare with before administration after each treated animal administration, body weight all obviously increases.Compare with SHAM group, OVX ovariectomized rats is after ten weeks, and body weight obviously increases (P<0.01), and prompting removal ovary makes rat body weight obviously increase.Compared with OVX group, the root of bidentate achyranthes glycopolymers AB1, AB2, AB3 and ABB administration group is after the 7th week, and body weight increase starts to alleviate (P<0.05), and obvious suppression also appears in the positive controls trend that after the tenth week, body weight increases.Administration group and the positive increase organized E2 and all can suppress ovariectomized female rats body weight are described.
2.2 root of bidentate achyranthes glycopolymers are on the impact of removal ovary female rats Uterine coefficient
Fig. 8 is shown in the impact of root of bidentate achyranthes glycopolymers on removal ovary female rats Uterine coefficient, compares with SHAM group, and after OVX ovariectomized rats, Uterine coefficient obviously reduces (P<0.05), and prompting removal ovary makes rat uterus atrophy, weight saving.Compared with OVX group, AB1, AB2, AB3 administration group organizes E2 with the positive can obviously increase ovariectomized female rats uterus weight.Illustrate that these three administration groups and positive group can make ovariectomized female rats metratrophia improve.
2.3 root of bidentate achyranthes glycopolymers are on the impact of removal ovary female rats femur and lumbar vertebra bone density
Root of bidentate achyranthes glycopolymers is to removal ovary female rats femur and lumbar vertebra bone density (BMD, bonemineraldensity) Fig. 9,10 is shown in impact, compare with SHAM group, obviously reducing (P<0.01) appears in femur (Fig. 9) and lumbar vertebra (Figure 10) bone density of OVX ovariectomized female rats, and prompting ovariectomized female rats successfully copies primary osteoporosis.Compared with OVX group, AB1, AB2, AB3 administration group organizes with positive the bone density (P<0.05) that E2 obviously can increase femur of mature ovariectomized rats (Fig. 9) and lumbar vertebra (Figure 10).Illustrate that corresponding administration group can the osteoporosis symptoms of effectively preventing rat with the positive E2 that organizes.
2.4 root of bidentate achyranthes glycopolymers are on the impact of removal ovary female rats femur and lumbar vertebra bone mineral quantity
Figure 11,12 is shown in the impact of root of bidentate achyranthes glycopolymers on removal ovary female rats femur and lumbar vertebra mineral quantity, compare with SHAM group, obviously reducing (P<0.01) appears in OVX rat femur and lumbar vertebra mineral quantity, and prompting ovariectomized female rats successfully copies primary osteoporosis.Compared with OVX group, AB1, AB2, AB3 administration group organizes E2 with the positive all obviously can increase femur of mature ovariectomized rats mineral quantity (see Figure 11); AB1, AB2 administration group obviously increases ovariectomized female rats lumbar vertebra mineral quantity (P<0.05) (see Figure 12), and the above results illustrates that each root of bidentate achyranthes glycopolymers administration group all has the effect of certain protect against osteoporosis.
2.5 root of bidentate achyranthes glycopolymers are on the impact of biomechanical properties
Table 2 root of bidentate achyranthes glycopolymers is on the impact of biomechanical properties
In table, all numerical value is mean value ± SD.*P<0.05, * * P<0.01vs.OVX; #P<0.05, ##P<0.01vs.SHAM.
Root of bidentate achyranthes glycopolymers, compares with SHAM group in table 2 impact of biomechanical properties, and reducing all appears in OVX rat femur biomechanics correlation parameter, and prompting ovariectomized rats biomechanical property declines; Compared with OVX group, administration group all can effectively improve ovariectomized female rats biomechanical property, and the above results illustrates that administration group all has the effect of certain protect against osteoporosis.
The root of bidentate achyranthes oligosaccharides ABW3-1 that the present invention extracts may be one of effective constituent playing above-mentioned function of resisting osteoporosis, also have the effect significantly increasing femur of mature ovariectomized rats and lumbar spine bmd and bone mineral quantity, its mechanism of action obviously improving bone biomechanical property needs to be studied further.
conclusion
After the oophorectomize of female rats, estrogen level reduces, bone metabolism is active, bone conversion strengthens, bone mineral quantity is lost, the model of height conversion hysteria osteoporosis when being classical imitation postmenopausal women.This experimental study shows, the root of bidentate achyranthes glycopolymers at each position of being extracted by aforesaid method all significantly can increase femur of mature ovariectomized rats and lumbar spine bmd and bone mineral quantity, obviously improve bone biomechanical property, thus play the effect of its treatment rat model osteoporosis.
Follow-uply need to study the osteoporosis mechanism of root of bidentate achyranthes glycopolymers, and the mechanism for probing into root of bidentate achyranthes glycopolymers osteoporosis is provided strong foundation by the Structural Identification of homogeneous glycopolymers.
Above-described is only some embodiments of the present invention, but embodiments of the present invention are not restricted to the described embodiments.For the person of ordinary skill of the art, the change done under other any does not deviate from spirit of the present invention and principle, modification, replacement, simplification, all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a root of bidentate achyranthes glycopolymers ABW3-1, is characterized in that: the oligosaccharides that this glycopolymers is made up of glucose and fructose, and its structure is:
.
2. the extracting method of glycopolymers in the root of bidentate achyranthes, is characterized in that: the method comprises following operation steps:
1) shear: root of bidentate achyranthes dry root is cut into segment, cleans up with water, dry;
2) water extraction: the root of bidentate achyranthes sheared is carried out water extraction, collects extracting solution and residue respectively;
3) classification alcohol precipitation: after extracting solution is concentrated, add ethanol and make ethanol contend concentration be a%, leave standstill, collecting precipitation, obtains raw sugar polymer A B1; Again concentrated by supernatant liquor, add ethanol and make ethanol contend concentration be b%, leave standstill, collecting precipitation, obtains raw sugar polymer A B2; Again concentrated by supernatant liquor, add ethanol and make ethanol contend concentration be c%, leave standstill, collecting precipitation, obtains raw sugar polymer A B3; Wherein 10≤a < b < c < 100;
4) alkali is carried: be soaked in by the residue after water extraction in 0.1 ~ 1MNaOH solution, leave standstill 1 ~ 4h, supernatant liquor 0.1 ~ 1MHCl neutralizes, the pH of supernatant liquor is made to be 6 ~ 8, centrifuging and taking supernatant, concentrated supernatant, adding ethanol makes ethanol contend concentration be 50 ~ 90%, leave standstill, collecting precipitation, obtains alkali and carries raw sugar polymer A BB;
5) purifying: purifying is carried out to above-mentioned raw sugar polymer A B1, AB2, AB3, ABB, obtains root of bidentate achyranthes glycopolymers.
3. method according to claim 2, is characterized in that: step 2) described in the concrete operations of water extraction be with 60 ~ 100 DEG C of hot water of 5 ~ 15 times of volumes, the root of bidentate achyranthes cut is extracted, extraction time 1 ~ 4h.
4. method according to claim 2, is characterized in that: all described simmer down tos 40 ~ 70 DEG C of concentrating under reduced pressure in step 3), all described standing time is 10 ~ 28h.
5. method according to claim 2, is characterized in that: 10≤a < 60,60 < b < 80,80 < c < 100 in step 3).
6. method according to claim 2, is characterized in that: the glycopolymers AB3 after purifying in step 5) is carried out ion-exchange chromatography again, carries out wash-out with distilled water, phend-sulphuric acid is used to follow the tracks of elution curve, sugar moieties is collected according to elution curve, concentrated, lyophilize; Then water dissolution is used, centrifugal, get supernatant liquor and carry out molecular sieve gel column chromatography, carry out wash-out with water, use phend-sulphuric acid to follow the tracks of elution curve, collect sugar moieties according to elution curve, concentrated, obtain root of bidentate achyranthes glycopolymers ABW3-1 after lyophilize.
7. root of bidentate achyranthes glycopolymers, is characterized in that: the extracting method of this glycopolymers is arbitrary described method in claim 2 ~ 6.
8. the application of root of bidentate achyranthes glycopolymers ABW3-1 according to claim 1 in the preparation osteoporotic medicine of control or protective foods or functional food.
9. the root of bidentate achyranthes glycopolymers AB1, AB2, AB3, ABB according to claim 2 application in the preparation osteoporotic medicine of control or protective foods or functional food.
10. the application of root of bidentate achyranthes glycopolymers according to claim 7 in the preparation osteoporotic medicine of control or protective foods or functional food.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107226870A (en) * | 2017-05-31 | 2017-10-03 | 广东药科大学 | Root of bidentate achyranthes glycopolymers and its preparation method and application |
| CN109796539A (en) * | 2019-01-24 | 2019-05-24 | 广东药科大学 | Intelligence development glycopolymers and its preparation method and application |
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| CN101508720A (en) * | 2003-06-27 | 2009-08-19 | 南京大学 | Triterpene extracts of achyranthes bidentata and uses in osteosporosis resistant medicament |
| WO2009137534A2 (en) * | 2008-05-06 | 2009-11-12 | Bionovo, Inc. | Estrogenic extracts for use in treating 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101508720A (en) * | 2003-06-27 | 2009-08-19 | 南京大学 | Triterpene extracts of achyranthes bidentata and uses in osteosporosis resistant medicament |
| WO2009137534A2 (en) * | 2008-05-06 | 2009-11-12 | Bionovo, Inc. | Estrogenic extracts for use in treating 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107226870A (en) * | 2017-05-31 | 2017-10-03 | 广东药科大学 | Root of bidentate achyranthes glycopolymers and its preparation method and application |
| CN109796539A (en) * | 2019-01-24 | 2019-05-24 | 广东药科大学 | Intelligence development glycopolymers and its preparation method and application |
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