CN107698688A - A kind of Radix Codonopsis homogeneous polysaccharide and preparation method and application - Google Patents
A kind of Radix Codonopsis homogeneous polysaccharide and preparation method and application Download PDFInfo
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Abstract
The present patent application discloses a kind of Radix Codonopsis homogeneous polysaccharide, and its molecular weight is 1.14 × 105, and be made up of rhamnose, arabinose, glucose, galactolipin and galacturonic acid, its construction unit is：The preparation method of Radix Codonopsis homogeneous polysaccharide, Radix Codonopsis medicine materical crude slice is taken, with alcohol degreasing, the dregs of a decoction after degreasing extract Thick many candies with water extraction and alcohol precipitation method, then plus pepsin Sevage method removing proteins, add ethanol alcohol precipitation and obtain Codonopsis pilosula polysaccharide powder, through the gel post separations of Sephadex G 75, eluted with NaCl solution, it is corresponding to collect the 34th 40 pipe, after concentration, then separated again with Sephacryl S 200HR posts, NaCl solution elutes, and collects the main peak part in elution curve；With bag filter dialysis desalting, the solution in collecting bag carries out concentrated frozen drying, obtains Radix Codonopsis homogeneous polysaccharide.Codonopsis pilosula polysaccharide made from the inventive method is homogeneous polysaccharide, and yield is high, has anti-oxidant and enhancing immunocompetence, can remove DPPH free radicals, available for preparing antioxidant and enhancing immune formulation.
Description
Technical field
The present invention relates to pharmaceutical technology, and in particular to a kind of Radix Codonopsis homogeneous polysaccharide and preparation method and application.
Background technology
Radix Codonopsis (Codonopsis Radix) is campanulaceae of the genus codonopsis, there is the effect of strengthening spleen and tonifying lung, nourishes blood and promotes the production of body fluid, main
Control in spleen-lung Qi deficiency, eat less and tired, false asthma cough, insufficiency of vital energy and blood, sallow complexion, palpitation, injury thirst, Heat Diabetes etc.
Disease, it is distributed mainly on the provinces such as Shanxi, Shaanxi, Sichuan, Gansu.Research in recent years finds that the main component in Radix Codonopsis includes steroid
Alcohols, triterpenes, Radix Codonopsis glycoside, lactone, alkaloids and polysaccharide.Constantly there is experiment to study Codonopsis pilosula polysaccharide in recent years
Analysis, it has been found that it has extensive pharmacological action, including antitumor, regulation immunity of organism, anti anoxia, resisting stress etc..
Polysaccharide is the macromolecule carbon aqueous polymer formed by the monose of more than 10 by glycosidic bond links, can use formula
(C6H10O5)nRepresent.Polysaccharide has extremely complex structure, while also has the ability for loading abundant biological information.Because polysaccharide
Structures shape its bioactivity and function, and the elaboration of structure is the basis for understanding its activity and preferably being developed.
Codonopsis pilosula polysaccharide also has antioxidation activity, and by establishing D- galactolipin inducing mouse aging models, research Radix Codonopsis is thick
The antioxidation of polysaccharide (CPPS), the results showed that CPPS has the function that certain resisting oxidation and delaying senility (Guo Xiaonong, Qi Huan
Sun, Wang Bing, wait Codonopsis pilosula polysaccharides antioxidation and its influence [J] Food Sciences to life span of drosophila melanogaster, 2013,34 (15):
285-288.)；The external antioxidation removed free radical experiment and compare Codonopsis pilosula polysaccharide (CP) and sulphation CP (sCP), is adopted
Compare liver-protective function in CP and sCP bodies with mouse liver injury model, wherein CP does not have clear and definite structure, and as a result the two is equal
With anti-oxidant and liver protection function, and sCP activity significantly (Liu C, Chen J, Li E, et al.The
comparison of anti oxidatIve and hepatoprotective activities of Codonopsis
pilosula polysaccharide(CP)and sulfated CP[J]. International
immunopharmacology,2015,24(2):299-305.)。
Codonopsis pilosula polysaccharide have enhancing immunocompetence, Yang Guang etc. (Yang Guang, Li Fasheng, Liu Hui, etc..Codonopsis pilosula polysaccharide is exempted to mouse
Influence [J] Pharmacology and Clinics of Chinese Materia Medicas of epidemic disease function, 2005,21 (4):39.) find that Codonopsis pilosula polysaccharide is piercing to mouse under study for action
Generated after swashing on antibody level apparently higher than the not mouse to Codonopsis pilosula polysaccharide, thus it is speculated that be that macromolecular substances stimulate siberian crabapple in Radix Codonopsis
Unite so as to produce immunological enhancement.Zhang Xiaojun etc. (Zhang Xiaojun, Zhu Chen Gall, Hu Li, etc..Codonopsis pilosula polysaccharide is to mouse immune and makes
Influence [J] the new Chinese medicines and clinical pharmacology of blood function, 2003,14 (3):174-176.) to giving the mouse after Codonopsis pilosula polysaccharide
Find in the contrast of immune and hematopoiesis function, after giving Codonopsis pilosula polysaccharide, spleen caused by ray can be resisted, atrophy of thymus gland phenomenon, can delay
Anemia is solved, shows that Radix Codonopsis has certain promotion spleen hematopoiesis, improves the function of immunity.(Yu Lan, the Wang Yi such as Yu Lan.Road
Zhen Luolong Codonopsis pilosula polysaccharides are to influence [J] Zunyi Medical College journal of mouse immune activity, 2016,39 (1):10-13.) imperial Radix Codonopsis
Polysaccharide can mitigate the suppression of caused by cyclophosphamide immune function of mice, the mouse that different molecular weight Codonopsis pilosula polysaccharide is induced endoxan
Hereditary thing, which changes, has antagonism.
The content of the invention
The technical problems to be solved by the invention are by being classified by molecular weight to Codonopsis pilosula polysaccharide, then further dividing
From purifying, structural characterization is carried out, there is provided a kind of polysaccharide formed by a kind of condensation of monosaccharide molecule, have the Radix Codonopsis of medical value homogeneous
Polysaccharide, and the Radix Codonopsis homogeneous polysaccharide to obtaining carries out antioxidation activity and the immunocompetent Primary Study of enhancing.
It is an object of the invention to provide a kind of Radix Codonopsis homogeneous polysaccharide, its molecular weight is 1.14 × 105, and by rhamnose, Ah
Uncle's sugar, glucose, galactolipin and galacturonic acid is drawn to form, its construction unit is：
It is a further object to provide the preparation method of the Radix Codonopsis homogeneous polysaccharide, comprise the following steps：
1) Radix Codonopsis medicine materical crude slice will be dried, degreasing is carried out 2~3 times with 5~8 times of amount ethanol heating condensing refluxes；
2) dregs of a decoction after degreasing in step 1) are measured into water extraction 3 times with 5~8 times, temperature is 80~100 DEG C, each 2h, by water
After extract is concentrated under reduced pressure, absolute ethyl alcohol is added to alcohol content up to 80~85%, 24h is stood, abandoning supernatant, collects precipitation, obtain
To Radix Codonopsis Thick many candies；
3) after Radix Codonopsis Thick many candies are redissolved plus pepsin to its content is 0.1~0.4%, the water bath with thermostatic control at 37 DEG C
12h, precipitation is removed, is repeated 3 times, collected upper solution, concentration, add Sevage reagents, acutely vibrate 30min, stand 12h,
Collect upper strata polysaccharide solution, repeat to UV scanning without protein specificity absworption peak when stop；
4) to adding 4~6 times of 95%~100% ethanol of amount after step 3) at the middle and upper levels polysaccharide solution concentration, 24h is stood, is collected
Precipitation, is freeze-dried after being repeated 3 times, obtains Codonopsis pilosula polysaccharide powder；
5) through Sephadex G-75 gel columns after the Codonopsis pilosula polysaccharide powder obtained in step 4) is completely dissolved with distilled water
Separation, with 0.9% NaCl solution isocratic elution, flow velocity 0.5mL/min collects a test tube per 3min, then with phenol sulfuric acid
Method detects, and collects the 34th~40 pipe；
6) by after the eluent concentration collected in step 5), separated again with Sephacryl S-200HR posts, 0.9%
NaCl solution elutes, flow velocity 0.5mL/min, after phend-sulphuric acid detection, collects the main peak part in elution curve；
7) it is saturating with 3500Da bag filter after the solution concentration of the main peak part in the elution curve that will be collected in step 6)
2d desalinations are analysed, the solution in bag filter is finally subjected to concentrated frozen drying, obtains Radix Codonopsis homogeneous polysaccharide.
Further, in the Sevage reagents, chloroform：N-butanol=5:1.
Present invention finds being extracted by water extraction and alcohol precipitation method, Sephadex G-75 and Sephacryl S-200HR gel columns
The scheme such as isolate and purify, it is 1.14 × 105Da to have obtained molecular weight, by rhamnose, arabinose, glucose, galactolipin and half
The Radix Codonopsis homogeneous polysaccharide of lactobionic acid composition, yield is high, and obtained Radix Codonopsis homogeneous polysaccharide has antioxidation activity and enhancing immune
Activity, DPPH free radicals can be removed, available for anti-oxidant and enhancing immunocompetence product is prepared, there is certain application prospect.
It is a still further object of the present invention to provide Radix Codonopsis homogeneous polysaccharide to prepare oxidation resistant product or immune preparing enhancing
The application of biologically active prod.
Further, hydroxypropyl methylcellulose, carboxyrnethyl starch sodium, microcrystalline cellulose, lactose, medical starch and magnesium stearate are taken
Mixed with Radix Codonopsis homogeneous polysaccharide and tablet is made, the tablet is oxidation resistant product or enhancing immunocompetence product.
Further, take microcrystalline cellulose and medical starch to be mixed with Radix Codonopsis homogeneous polysaccharide and tablet is made, the tablet
For oxidation resistant product or enhancing immunocompetence product.
Further, take microcrystalline cellulose, medical starch and Radix Codonopsis homogeneous polysaccharide to mix, insert in hard shell capsules, make
Into capsule, the capsule is oxidation resistant product or enhancing immunocompetence product.
Brief description of the drawings
Fig. 1 is the elution curve that Radix Codonopsis Thick many candies component crosses Sephadex G-75 gel columns.
Fig. 2 is the HPGPC gel chromatography figures of Radix Codonopsis homogeneous polysaccharide.
Fig. 3 is Dextran series polysaccharide molecular weight canonical plottings.
Fig. 4 is five kinds of standard monose GC-MS total ion chromatograms, note：(left → right) 1 (Rha 9.381min), 2 (Ara
9.613min), 3 (Man 14.496min), 4 (Glc 14.653min) and 5 (Gal 14.793min).
Fig. 5 is Radix Codonopsis homogeneous polysaccharide GC-MS total ion chromatograms, note：(left → right) 1 (Rha 9.354min), 2 (Ara
9.608min), 3 (Glc 14.621min) and 4 (Gal 14.777min).
Fig. 6 is GC-MS total ion chromatograms after the reduction of Radix Codonopsis homogeneous polysaccharide uronic acid, is noted：(left → right) 1 (Rha
9.368 min), 2 (Ara 9.605min), 3 (Glc 14.641min) and 4 (Gal 14.794min).
Fig. 7 is the IR spectrograms of Radix Codonopsis homogeneous polysaccharide.
Fig. 8 is the total ion chromatogram after Radix Codonopsis homogeneous polysaccharide methylates, and is noted：(left → right) 1 (2,3-Me2-Rhap),
2 (2,3-Me2-Araf)、3(2,3,6-Me3-Galp)、4(2,3,4,6-Me4-Glcp)、5(2,6-Me2-Galp)、6 (2,
3,4-Me3-Galp), 7 (2,3,5-Me3-Araf) and 8 (2,3,4-Me3-Rhap).
Fig. 9 is the total ion chromatogram to be methylated after Radix Codonopsis homogeneous polysaccharide uronic acid reduces, is noted：(left → right) 1 (2,
3-Me2-Rhap)、2(2,3-Me2-Araf)、3(2,3,6-Me3-Galp)、4(2,3,4,6-Me4-GalAp)、5 (2,3,4,
6-Me4-Glcp), 6 (2,6-Me2-Galp), 7 (2,3,4-Me3-Galp), 8 (2,3,5-Me3-Araf) and 9 (2,3,4-
Me3-Rhap)。
Figure 10 is the 1H spectrograms of Radix Codonopsis homogeneous polysaccharide, is noted：(left → right) B (→ 5)-α-L-Araf- (1 →), C (→ 4)-β
- D-Galp- (1 →), A (→ 4)-α-L-Rhap- (1 →), J (α-L-Rhap- (1 →) and D (α-D-GalAp- (1 →).
Figure 11 is the 13C spectrograms of Radix Codonopsis homogeneous polysaccharide, is noted：(left → right) I (α-L-Araf- (1 →), C (→ 4)-β-D-
Galp-(1 →)、E(β-D-Glcp-(1→)、G(→3,4)-β-D-GalAp-(1→)、F(→3,4)-β-D-Galp-(1→)
With H (→ 6)-α-D-Galp- (1 →).
Figure 12 is clearance rate comparison diagrams of the Vc to DPPH free radicals.
Figure 13 is the clearance rate comparison diagram of Radix Codonopsis homogeneous polysaccharide and Vc to DPPH free radicals.
Embodiment
Below by embodiment, the present invention is further detailed explanation：
The preparation of Radix Codonopsis homogeneous polysaccharide
1. the extraction of Radix Codonopsis Thick many candies：
Codonopsis pilosula is taken in round-bottomed flask, by 1:8(g:ML solid-liquid ratio) adds ethanol, heats condensing reflux 2h, enters
Solid-liquid ratio is 1 during second of degreasing of row:7(g:ML), flow back 2h.The dregs of a decoction after degreasing are dried, the ethanol of residual is vapored away, presses
1:8(g:ML solid-liquid ratio) adds distilled water into the Radix Codonopsis dregs of a decoction, extracts 3 times, 2h/ times, temperature is 90 DEG C, and extraction is taken out after terminating
Filter, merge the Aqueous extracts of 3 times.Extract solution is concentrated under reduced pressure, adds the ethanol of 4 times of amounts, it is stirring while adding, it is quiet at 4 DEG C
Filtered after putting 24h, abandoning supernatant, collect precipitation, obtain Thick many candies.
2. the purifying of Radix Codonopsis homogeneous polysaccharide：
Weigh Radix Codonopsis Thick many candies to be dissolved with distilled water, be added to Sephadex G-75 ((2.6 × 140cm, Whatman))
In gel column, the NaCl solution for preparing 0.9% carries out isocratic elution as eluent, flow velocity 0.5mL/min, and every 3min is received
Collect a test tube, detected using phend-sulphuric acid, collect the concentration of 34-40 pipes, then with Sephacryl S-200HR post separations,
0.9% NaCl solution elution, flow velocity 0.5mL/min, collects the main peak part in elution curve, the solution concentration being collected into
The bag filter for being afterwards 3500Da with molecular cut off dialyses 2d come desalination, and solution is concentrated in collecting bag, is freeze-dried afterwards,
Obtain Radix Codonopsis homogeneous polysaccharide.
3. the Purity of Radix Codonopsis homogeneous polysaccharide：
High-efficient liquid phase chromatogram condition：The high performance liquid chromatographs of Agilent 1260, the chromatographic columns of Ultrahydrogel 250,
Mobile phase is 0.2mol/L NaNO3Solution, flow velocity 0.6mL/min, 35 DEG C of column temperature, detector are that RID (examine by differential refraction
Survey device).The Codonopsis pilosula polysaccharide that step 3 is obtained is dissolved with suitable quantity of water, and sample size is 20 μ L, as a result in single symmetrical in chromatogram
Peak, it is homogeneous polysaccharide to illustrate the component.
4. the determination of Radix Codonopsis homogeneous polysaccharide molecular weight：
The pulullan polysaccharide for taking 1mg molecular weight to be 50K, 80K, 150K, 270K, 410K and 670K Da is dissolved in 1mL respectively
In water, chromatographic condition is same as above, and is analyzed with the high performance liquid chromatographs of Agilent 1260.Retention time is recorded, with pair of molecular weight
Number (logM) is ordinate, and retention time (t) is abscissa, obtains standard curve y=10.00-0.384x, by Radix Codonopsis homogeneous polysaccharide
Appearance time substitutes into curvilinear equation, and the molecular weight for obtaining Radix Codonopsis homogeneous polysaccharide is 1.14 × 105Da.
The structural characterization of Radix Codonopsis homogeneous polysaccharide
1. polysaccharide component is analyzed：
The Radix Codonopsis homogeneous polysaccharide of 2mg dryings is weighed in 25mL round-bottomed flasks, addition 4mL 2mol/L TFA (trifluoro second
Acid), 6h is hydrolyzed at 110 DEG C, afterwards plus 4mL methanol is spin-dried for, and is repeated 3 times, and with the complete TFA for removing residual, adds 0.5mL
Water dissolves, then weighs 1mg Ara (arabinose), Man (mannose), Rha (rhamnose), Gal (galactolipin) and Glc respectively
(glucose) standard items are dissolved in 1mL water, and the sodium borohydride (NaBH that 20mg is dried is added in each sample4), at room temperature instead
3h is answered, after terminating, 25% glacial acetic acid to bubble-free is added dropwise and produces, is spin-dried at 60 DEG C.2mL methanol is added into each reduzate
It is spin-dried for, is repeated 3 times.There is white product to be dropped from bottle wall, add 1.5mL acetic anhydrides, react 1h at 110 DEG C.Reaction terminates
Repeat afterwards plus 2mL dimethylbenzene is spin-dried for (80 DEG C), 3 times, to remove unnecessary acetic anhydride.Respectively add 1mL chlorine into acetylate again
Imitative and water, is repeated 3 times, and collects lower floor's organic phase, adds a small amount of anhydrous sodium sulfate to remove water, with 0.45 μm of membrane filtration, GC- to be entered
MS is analyzed.
GC-MS chromatographic conditions：Agilent 6890-5973N type gas chromatograph-mass spectrometer (GC-MS)s, chromatographic column：Agilent
HP-5MS capillary columns (30m × 0.25 μm of 250 μ m)；Carrier gas：Helium (He)；Heter temperature：250℃；Temperature programming：
140 DEG C of initial temperature, 200 DEG C are risen to 10 DEG C/min, keeps 5min, then 240 DEG C are risen to 8 DEG C/min；Split ratio：50:1；
Sample size：5μL.
Uronic acid reduction reaction：5mg Radix Codonopsis homogeneous polysaccharide is weighed, is dissolved in 5mL water, adds 100mg 1- hexamethylenes
Base -2- (morpholine ethyl) Carbodiimide metho tosilate (CMC), after stirring and dissolving, adjusted with 0.01mol/L HCl
The pH value of solution remains at 4.7 or so, after reacting 2h, then 5mL 2mol/L NaBH is added dropwise4Solution, with 2mol/L's
The pH value of HCl regulation solution is maintained at 7.0 or so, reacts 1h, then the bag filter dialysis 24h with 3500Da, is evaporated, obtains Radix Codonopsis
Homogeneous polysaccharide uronic acid reduzate.After the series reactions such as complete sour water solution, reduction and acetylation are carried out to reduzate,
Enter GC-MS analyses again.The front and rear sample total ion chromatogram of uronic acid reduction is mutually compareed with the chromatogram of standard monose,
The monosaccharide component that Radix Codonopsis homogeneous polysaccharide can be determined is rhamnose, arabinose, glucose, galactolipin and galacturonic acid.
2. Radix Codonopsis homogeneous polysaccharide IR is analyzed：
Sample carries out IR scannings using KBr pressed disc methods, and it is special that typical polysaccharide absorption is presented in Radix Codonopsis homogeneous polysaccharide from IR figures
Sign, wherein 3439.48cm-1For the stretching vibration peak of O-H keys, 2926.91cm-1For CH2The stretching vibration peak of middle c h bond；
1742.82cm-1There is the characteristic absorption peak of uronic acid in place, and it is a kind of acid sugar to illustrate the sugar；1630.74cm-1For CO2Or altogether
Stretching vibration peak caused by unboiled water, 1434.25cm-1For the angle vibration peak of c h bond, 1244.54cm-1It is to be drawn by primary alconol β-OH
The stretching vibration peak risen；1107.47 and 1018.88cm-1The absworption peak at place illustrates to contain pyranoid ring in Radix Codonopsis homogeneous polysaccharide, and
910.40cm-1The absworption peak that place occurs adds the structure that furan nucleus in Radix changii homogeneous polysaccharide be present；756.09cm-1For pyrans
The symmetrical stretching vibration peak of ring.
3. Radix Codonopsis homogeneous polysaccharide methylation analysis：
Methylation reaction：The Radix Codonopsis homogeneous polysaccharide and Radix Codonopsis homogeneous polysaccharide uronic acid reduzate for taking 10mg dryings respectively add
Enter into 25mL round-bottomed flask, add the anhydrous DMSO solution ultrasound 30min dissolvings of 2mL, the NaOH powder ultrasonics for adding 30mg to dry
3h is reacted, 1mL CH are slowly added dropwise along wall under ice bath3I, lucifuge ultrasonic reaction 2h, adds 3mL water to terminate to react, by 1:1 plus CHCl3
Extraction, organic phase is collected, is repeated 3 times, repeat to methylate after drying does not have hydroxyl peak into IR scanning spectras.Whole course of reaction
At 20 DEG C, N2Protection is lower to be carried out.
Subsequent reactions：Add 4mL 2mol/L TFA into the sample after methylating, 5h is hydrolyzed at 110 DEG C, add after hydrolysis
4mL methanol is spin-dried for removing unnecessary TFA, is repeated 3 times, then adds the dissolving of 0.5mL water；0.2mL hydrolyzates are taken, add 30mg NaBH4,
3h is reacted at room temperature, is intermittently shaken in course of reaction, and 25% glacial acetic acid to bubble-free is added dropwise after terminating and produces, adds 2mL methanol to revolve
It is dry, it is repeated 3 times；Add 1.5mL acetic anhydrides into reduzate, after reacting 1h at 100 DEG C, add 2mL dimethylbenzene to be spin-dried for, repeat 3
It is secondary to remove unnecessary acetic anhydride；Respectively add 2mL chloroforms and water into acetylate, vibrate, stand, collect lower floor's organic phase,
With 1mL water washings 3 times, then add a small amount of anhydrous sodium sulfate water removal, treat that GC-MS is analyzed.
GC-MS chromatographic conditions：Agilent 6890-5973N type gas chromatograph-mass spectrometer (GC-MS)s, chromatographic column：HP-5MS hairs
Capillary column (30m × 0.25 μm of 250 μ m)；Carrier gas：He；Heter temperature：250℃；Temperature programming：140 DEG C of initial temperature, with
10 DEG C/min rises to 200 DEG C, keeps 5min, then rise to 240 DEG C with 8 DEG C/min；Split ratio：50:1；Sample size： 5μL.
The Radix Codonopsis homogeneous polysaccharide methylation analysis data of table 1
The Radix Codonopsis homogeneous polysaccharide uronic acid reduzate methylation analysis data of table 2
The result to methylate shows that the saccharide residue of Radix Codonopsis homogeneous polysaccharide sugar chain shares 8 kinds of connected modes, and Radix Codonopsis is homogeneous more
The saccharide residue of sugared uronic acid reduzate shares 9 kinds of connected modes, and the end group Galp for illustrating to have more is GalA existing way,
1,3,4-linked Galp ratio significantly increases, and illustrates GalA existing way and also includes 1,3,4-linked GalAp；
Non-reducing end residue (end group GalAp, end group Glcp, end group Arap and end group Rhap) after COP-W1 ' methylation reactions with
The mol ratio of branch saccharide residue (1,3,4-linked Galp) is 5.2:5.4, show that it methylates completely.Therefore, it is equal in Radix Codonopsis
In one polysaccharide, Rha is with 1 → and 1 → 4 connection, and Ara is with 1 → and 1 → 5 connection, and Glc is mainly with 1 → connection, and Gal is with 1 → 4,1
→ 3,4 and 1 → 6 connection, and GalA is mainly with 1 → and 1 → 3,4 connections.
4. Radix Codonopsis homogeneous polysaccharide NMR is analyzed：
The Radix Codonopsis homogeneous polysaccharide for taking 30mg to dry, is dissolved in 0.5mL D2In O, NMR analyses are carried out.
From1H spectrograms and13The anomeric proton signal that C spectrograms can be seen that Radix Codonopsis homogeneous polysaccharide includes BH1(δ5.98)、CH1
(δ 5.09)、AH1(δ5.00)、JH1(δ 4.97) and DH1(δ 4.94), and anomeric carbon signals have IC1(δ107.3)、CC1(δ
106.2)、EC1(δ 103.8)、GC1(δ102.1)、FC1(δ100.0)、HC1(δ 98.2), show two kinds of structures of α and β in the sugar be present
Type；1There are methyl signals in Rha residues at the high field region δ 1.17 of H spectrograms, the presence with composition analysis result Rha is phase
It is corresponding；13In C spectrograms, there is the carbon signal δ 170.7 of uronic acid, illustrate to contain uronic acid in Radix Codonopsis homogeneous polysaccharide, be
A kind of acid sugar is identical with component analysis and IR result.The anomeric carbon hydrogenation degree of each saccharide residue in Radix Codonopsis homogeneous polysaccharide
Devolve category and be shown in Table 3.
The hydrocarbon chemical shift ownership of the Radix Codonopsis homogeneous polysaccharide of table 3
To sum up analyze, the possible construction unit of the homogeneous polysaccharide predicted sees below formula：
5. the antioxidation activity experiment of Radix Codonopsis homogeneous polysaccharide
Precision weighs DPPH 3.9mg, is dissolved in 100mL ethanol solutions, lucifuge shaking 30min, makes it fully molten
Solution, it is configured to the DPPH ethanol solutions that concentration is 0.1mmol/L, matching while using, the spectral scan under UV, its maximum absorption wavelength
For 516nm.COP-W1 is dissolved in pure water, is made into 0.15,0.3,0.6,1.2,2.4 and 4.8mg/mL initial concentration gradient,
Ascorbic acid (Vc) is used as positive reference substance, be made into 0.01875,0.0375,0.075,0.15,0.3,0.6,1.2,2.4,
4.8mg/mL initial concentration gradient.
1mL sample solutions and DPPH solution are measured respectively, is mixed, and react 30min under the conditions of lucifuge, it is molten with 50% ethanol
Liquid is reference, and absorbance A is determined at 516nms, each concentration samples replication 3 times；Measure respectively 1mL pure water and
DPPH solution, the absorbance A of DPPH blank solutions is determined after mixingo；Measure 1mL sample solutions and absolute ethyl alcohol respectively again,
The absorbance A of blank sample is determined after mixingb；According to same method, measure reference substance Vc absorbance.Polysaccharide is to DPPH
The clearance rate of free radical calculates according to the following equation：
It is calculated according to the formula of free radical scavenging activity, changes of the COP-W1 to the clearance rates of DPPH free radicals with concentration
It has been fluctuated that, it is small during to the clearance rates of DPPH free radicals than 0.60mg/mL during final concentration of 1.20mg/mL, and 2.40
During mg/mL, clearance rate 46.6%, illustrate the ability that COP-W1 has certain removing free radical, and Radix Codonopsis homogeneous polysaccharide pair
Half clearance rate IC of DPPH free radicals50For 2.607mg/mL.
The clearance rate (%) of the Radix Codonopsis homogeneous polysaccharide of table 4 and Vc to DPPH free radicals
6. the enhancing immunocompetence experiment of Radix Codonopsis homogeneous polysaccharide
Extraction, ethanol precipitation and Sevage method deproteinizeds are decocted using aqueous solvent and prepare Radix Codonopsis homogeneous polysaccharide COP-W2, ring
Phosphamide is injected intraperitoneally as immunodepressant, establishes immunologic hypofunction animal model, gavage Radix Codonopsis homogeneous polysaccharide
COP-W2, measure mouse Carbon clearance rate, thymus gland, index and spleen index.Kunming mouse (20 ± 2) g of 72 male and female half and half, with
Machine is divided into Normal group, model group, Radix Codonopsis homogeneous polysaccharide COP-W2 low dose groups, middle dose group, high dose group, positive drug group
Totally 6 groups, every group 12, in addition to Normal group, mouse uses intraperitoneal injection of cyclophosphamide modeling, and wherein Normal group is injected
Physiological saline, other group of polysaccharide gavage, successive administration 7d, 1 time/d.Positive administration group gives radix astragali particle gavage, is determined after 7 d
Mouse thymus and index and spleen index.Mouse Thymus and spleen index reduces (P ＜ 0.01) compared with Normal group in results model group,
Positive drug group, the mouse thymus of Radix Codonopsis homogeneous polysaccharide COP-W2 groups and index and spleen index are higher than model group (P ＜ 0.01).Conclusion
Radix Codonopsis homogeneous polysaccharide COP-W2 can mitigate the suppression of caused by cyclophosphamide immune function of mice, and Codonopsis pilosula polysaccharide is induced endoxan
Mouse genetic thing, which changes, has antagonism.
Application example：Radix Codonopsis homogeneous polysaccharide is preparing anti-oxidant or enhancing immunocompetence function medicine application
The preparation of Radix Codonopsis homogeneous polysaccharide tablet
Radix Codonopsis homogeneous polysaccharide 2g, hydroxypropyl methylcellulose 4g, carboxyrnethyl starch sodium 10g, the microcrystalline cellulose obtained in Example 1
Plain 10g, lactose 115g, medical starch 50g, magnesium stearate 1g, put into after main ingredient and auxiliary material are fully mixed in homogenizer,
Spraying plus appropriate amount of water, whole grain, moisture control then tabletting, are made 1000, film coating 3~4%.Every containing main ingredient into
Divide 0.2mg.The tablet is oxidation resistant product or enhancing immunocompetence product.
The preparation of the another tablet of Radix Codonopsis homogeneous polysaccharide
Radix Codonopsis homogeneous polysaccharide 3g, carboxyrnethyl starch sodium 15g, microcrystalline cellulose 15g, the medical starch obtained in Example 1
50 g, magnesium stearate 2g, put into after main ingredient and auxiliary material are fully mixed in homogenizer, spraying plus appropriate amount of water, whole grain, moisture
Control then tabletting, is made 1000 3~4%.The every 0.3mg of composition containing main ingredient.The tablet is oxidation resistant product or increasing
Strong immunocompetence product.
The preparation of Radix Codonopsis homogeneous polysaccharide capsule
Radix Codonopsis homogeneous polysaccharide 2g, microcrystalline cellulose 25g, the medical starch 70g obtained in Example 1, by medical starch
First dry, cross 120 mesh sieves, then mixed with Radix Codonopsis homogeneous polysaccharide, microcrystalline cellulose, after 120 mesh sieve twice, inserted in hard shell capsules,
1000 capsules of the present invention are made.Every hard shell capsules composition containing main ingredient 0.2mg, the capsule are that oxidation resistant product or enhancing are immune
Biologically active prod.
Finally be necessary described herein be：Above example is served only for further detailed to technical scheme work
Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to the above of the invention
Some the nonessential modifications and adaptations made belong to protection scope of the present invention.
Claims (7)
1. a kind of Radix Codonopsis homogeneous polysaccharide, it is characterised in that its molecular weight is 1.14 × 105, and by rhamnose, arabinose, grape
Sugar, galactolipin and galacturonic acid composition, its construction unit are：
2. the preparation method of Radix Codonopsis homogeneous polysaccharide according to claim 1, it is characterised in that：Comprise the following steps：
1) Radix Codonopsis medicine materical crude slice will be dried, degreasing is carried out 2~3 times with 5~8 times of amount ethanol heating condensing refluxes；
2) dregs of a decoction after degreasing in step 1) are measured into water extraction 3 times with 5~8 times, temperature is 80~100 DEG C, each 2h, by Aqueous extracts
After being concentrated under reduced pressure, absolute ethyl alcohol is added to alcohol content up to 80~85%, 24h is stood, abandoning supernatant, collects precipitation, obtain party
Join Thick many candies；
3) after Radix Codonopsis Thick many candies are redissolved plus pepsin to its content is 0.1~0.4%, and water bath with thermostatic control 12h, goes at 37 DEG C
Except precipitation, it is repeated 3 times, collects upper solution, concentration, add Sevage reagents, acutely vibrate 30min, stand 12h, in collection
Layer polysaccharide solution, repeat to UV scanning without protein specificity absworption peak when stop；
4) 95%~100% ethanol are measured to adding 4~6 times after step 3) at the middle and upper levels polysaccharide solution concentration, standing 24h, collect precipitation,
It is freeze-dried after being repeated 3 times, obtains Codonopsis pilosula polysaccharide powder；
5) through Sephadex G-75 gel columns point after the Codonopsis pilosula polysaccharide powder obtained in step 4) is completely dissolved with distilled water
From with 0.9% NaCl solution isocratic elution, flow velocity 0.5mL/min, every 3min collects a test tube, then uses Phenol sulfuric acid procedure
Detection, collect the 34th~40 pipe；
6) by after the eluent concentration collected in step 5), separated again with Sephacryl S-200HR posts, 0.9% NaCl is molten
Liquid elutes, flow velocity 0.5mL/min, after phend-sulphuric acid detection, collects the main peak part in elution curve；
7) after the solution concentration of the main peak part in the elution curve that will be collected in step 6), dialysed 2d with 3500Da bag filter
Desalination, the solution in bag filter is finally subjected to concentrated frozen drying, obtains Radix Codonopsis homogeneous polysaccharide.
3. the preparation method of Radix Codonopsis homogeneous polysaccharide according to claim 2, it is characterised in that：In the Sevage reagents,
Chloroform：N-butanol=5:1.
4. Radix Codonopsis homogeneous polysaccharide according to claim 1 is preparing oxidation resistant product or is strengthening immunocompetence product preparing
Application.
5. Radix Codonopsis homogeneous polysaccharide according to claim 4 is preparing oxidation resistant product or is strengthening immunocompetence product preparing
Application, it is characterised in that：Take hydroxypropyl methylcellulose, carboxyrnethyl starch sodium, microcrystalline cellulose, lactose, medical starch and stearic acid
Magnesium mixes with Radix Codonopsis homogeneous polysaccharide and tablet is made.
6. Radix Codonopsis homogeneous polysaccharide according to claim 4 is preparing oxidation resistant product or is strengthening immunocompetence product preparing
Application, it is characterised in that：Take microcrystalline cellulose and medical starch to be mixed with Radix Codonopsis homogeneous polysaccharide and tablet is made.
7. Radix Codonopsis homogeneous polysaccharide according to claim 4 is preparing oxidation resistant product or is strengthening immunocompetence product preparing
Application, it is characterised in that：Take microcrystalline cellulose, medical starch and Radix Codonopsis homogeneous polysaccharide to mix, insert in hard shell capsules, make
Into capsule.
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| CN110025622A (en) * | 2019-04-25 | 2019-07-19 | 兰州大学 | A kind of isolation and purification method of Radix Codonopsis oligosaccharides and application |
| CN110151813A (en) * | 2019-05-24 | 2019-08-23 | 重庆市科学技术研究院 | A kind of Radix Codonopsis extract and its preparation method and application |
| CN110243972A (en) * | 2019-07-04 | 2019-09-17 | 无限极（中国）有限公司 | Radix Codonopsis quality determining method based on spectrum effect relationship |
| CN112961258A (en) * | 2021-02-26 | 2021-06-15 | 重庆医科大学附属第二医院 | Red ginseng homogeneous polysaccharide and extraction method and application thereof |
| CN114057907A (en) * | 2021-12-21 | 2022-02-18 | 中国药科大学 | Method for extracting, separating and purifying red ginseng polysaccharide |
| CN116655820A (en) * | 2023-06-07 | 2023-08-29 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics |
| CN116731217A (en) * | 2023-06-07 | 2023-09-12 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics |
| CN116731217B (en) * | 2023-06-07 | 2024-06-04 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics |
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| CN110025622A (en) * | 2019-04-25 | 2019-07-19 | 兰州大学 | A kind of isolation and purification method of Radix Codonopsis oligosaccharides and application |
| CN110025622B (en) * | 2019-04-25 | 2022-01-28 | 兰州大学 | Method for separating and purifying codonopsis pilosula oligosaccharide and application |
| CN110151813A (en) * | 2019-05-24 | 2019-08-23 | 重庆市科学技术研究院 | A kind of Radix Codonopsis extract and its preparation method and application |
| CN110151813B (en) * | 2019-05-24 | 2021-06-18 | 重庆市科学技术研究院 | Codonopsis pilosula extract and preparation method and application thereof |
| CN110243972A (en) * | 2019-07-04 | 2019-09-17 | 无限极（中国）有限公司 | Radix Codonopsis quality determining method based on spectrum effect relationship |
| CN112961258A (en) * | 2021-02-26 | 2021-06-15 | 重庆医科大学附属第二医院 | Red ginseng homogeneous polysaccharide and extraction method and application thereof |
| CN114057907A (en) * | 2021-12-21 | 2022-02-18 | 中国药科大学 | Method for extracting, separating and purifying red ginseng polysaccharide |
| CN116655820A (en) * | 2023-06-07 | 2023-08-29 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics |
| CN116731217A (en) * | 2023-06-07 | 2023-09-12 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics |
| CN116655820B (en) * | 2023-06-07 | 2024-05-14 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics |
| CN116731217B (en) * | 2023-06-07 | 2024-06-04 | 科乐美(广州)生物科技有限公司 | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics |
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