CN101020719B - Composite angelica polysaccharide and its preparation process and use - Google Patents
Composite angelica polysaccharide and its preparation process and use Download PDFInfo
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Abstract
The present invention relates to composite angelica polysaccharide, which is extracted from dry root of angelica as one kind of Chinese medicine material, has weight average molecular weight of 20,000-80,000 dalton, and consists of polygalacturonic acids, AGPs, Rhamnogalacturonans and alpha-polyglucans mainly. The composite angelica polysaccharide with or without pharmaceutically acceptable supplementary material may be prepared into injection for treating neutrophilic granulocytopenia, anemia, thrombocytopenia, etc and for the auxiliary treatment of cancer.
Description
The present invention relates to field of traditional Chinese medicine pharmacy.Be specifically related to weight-average molecular weight that a kind of extraction from the dry root of Chinese medicinal materials Radix Angelicae Sinensis (AngelicaSinensis (Oliv) Diels), separation, purifying obtain 20,000-80, the compound polysaccharide of Radix Angelicae Sinensis between 000 dalton.Content＞80%.It is characterized in that this complex polysaccharide is mainly by gala aldehydic acid glycan (Polygalacturonic acids), sandlwood gala aldehydic acid glycan (Rhamnogalacturonans), Arabinogalactan-Protein (AGPs) and alpha-glucan (α-Polyglucans) form.Its characteristics are: 1. polysaccharide percentage composition: albumen percentage composition=95-99%: 1-5%.2. sugar is formed: mainly by pectinose (35-45%), and galacturonic acid (20-28%), semi-lactosi (18-25%), glucose (5-15%), rhamnosyl (3-8%), 4-methyl glucoside aldehydic acid (1-5%) is formed.3. glycosidic link link: pectinose is mainly with α-3, and α-5, a α end and α-3,5 glycosidic link are connected.Galacturonic acid mainly is connected with α-4 glycosidic link.Semi-lactosi is mainly with α-3, and α-6 and α-3,6 glycosidic link are connected.Glucose is mainly with α-4, and α-6 and α-4,6 glycosidic link are connected.Rhamnosyl mainly is connected with α-2 and α-2,4 glycosidic link.4-methyl glucoside aldehydic acid mainly is connected with end with α-4.4. amino acid is formed: form by 19 kinds of different amino acid, wherein, and arginine, Serine, L-glutamic acid, glycine, L-Ala, Methionin and hydroxyproline content are higher, account for the 65-75% of total amino acid content.5. weight-average molecular weight: 20,000-80,000 dalton.Other: soluble in water for white or off-white powder, be insoluble to ethanol, acetone and other organic solvent.Aqueous solution pH is 4-6, is slightly acidic.The invention still further relates to preparation technology, preparation of compound polysaccharide of Radix Angelicae Sinensis and uses thereof.
When the dry root that is classified as umbelliferae (umbuiferae) angelica per nnial herb Radix Angelicae Sinensis (Angelica Sinensis (Oliv) Diels).
The traditional Chinese medical science is thought: the Radix Angelicae Sinensis nature and flavor are sweet, hot, warm.Effect is invigorated blood circulation for enriching blood, and menstruction regulating and pain relieving relaxes bowel.Cure mainly: the deficiency of blood is sallow, dizzy palpitaition, menoxenia, asthenia cold abdominalgia, the dry constipation of intestines, cards such as injury from falling down.Radix Angelicae Sinensis mainly contains volatile oil and water soluble component, and early stage research mainly concentrates on volatile oil component, soluble small molecular composition and pharmacological action aspect thereof.In recent years, along with modern medicine, pharmacy and development of molecular biology, the water-soluble macromolecule part-Radix Angelicae Sinensis Crude polysaccharides (AP) and the pharmacological action thereof of Radix Angelicae Sinensis also there has been more deep understanding.(the Chinese Pharmacological circular, 2003,19 (4): 448-451) find: Radix Angelicae Sinensis Crude polysaccharides (AP) can strengthen the non-specific immune function of normal and immunosuppressed mice to Yang Tiehong etc.Wang Ya equality (Chinese Journal of Hematology, 1993,14 (12): 650-651) find: Radix Angelicae Sinensis Crude polysaccharides (AP) can promote anaemia mouse peripheral red blood cells, oxyphorase to go up, and stimulating anaemia mouse marrow red is quick-fried formula colony forming cell (BFU-E) and CFU-E in late period (CFU-E) propagation of increasing.(Medical University Of Chongqing's journal, 2002,27 (4): 373-375) find: Radix Angelicae Sinensis Crude polysaccharides (AP) can be induced fetal thymus cell and splenocyte expression of GM-CSF and IL-3 to Zheng Min etc.Therefore, discussion is used for green blood with Radix Angelicae Sinensis polysaccharide as efficient part, improves immunologic function etc., and prospect is arranged very much.
The invention provides weight-average molecular weight that a kind of extraction from the dry root of Chinese medicinal materials Radix Angelicae Sinensis (Angelica Sinensis (Oliv) Diels), separation, purifying obtain 20,000-80, the compound polysaccharide of Radix Angelicae Sinensis between 000 dalton.Content＞80%.
The main technique flow process is as follows: after the clean system of angelica sinensis, add water or with 0.2-0.6M potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC 50-105 ℃ of extraction (amount of water is 3-4 times, extracts 1-3 hour, extracts 2-3 time altogether) at every turn.Extracting solution 60-80 ℃ is evaporated to rare thick paste shape, carries out the 20-40% alcohol precipitation after the cooling, and 2-8 ℃ left standstill 8-24 hour.Collect supernatant liquor, spraying drying promptly gets the Radix Angelicae Sinensis polysaccharide crude.With Radix Angelicae Sinensis polysaccharide crude product water dissolution, concentration is 2-6%, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration (MWCO=5000-10 of 5-10K, 000), keep SP or CM Sepharose cationic exchange coloum on the liquid, elutriant is pH5-6, the damping fluid of 20-100mM (is got analytically pure acid and salt thereof, water is prepared the solution of desired concn respectively, pressing different ratios mixes promptly), damping fluid can be selected from citric acid-sodium citrate damping fluid, Sodium phosphate dibasic-citrate buffer solution, acetate-sodium acetate buffer etc.Collect 2-5 column volume of effluent liquid, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration (MWCO=5 of 5-10K, 000-10,000), keep Q or DEAE Sepharose anion-exchange column on the liquid, elutriant is pH5-6, the damping fluid of 20-100mM (is got analytically pure acid and salt thereof, water is prepared the solution of desired concn respectively, presses different ratios and mixes promptly), damping fluid can be selected from citric acid-sodium citrate damping fluid, Sodium phosphate dibasic-citrate buffer solution, acetate-sodium acetate buffer etc.Collecting 2-5 column volume of effluent liquid, is the ultra-filtration membrane ultrafiltration (MWCO=5,000-10,000) of 5-10K with molecular weight cut-off, keeps liquid through 65-85% ethanol sedimentation twice, centrifugal, collecting precipitation.It is anti-molten that precipitation adds water, and concentration is 20-30%, and spraying or lyophilize promptly get content at the compound polysaccharide of Radix Angelicae Sinensis more than 80%.
Or the supernatant liquor behind the collection 20-40% alcohol precipitation, be the ultra-filtration membrane ultrafiltration of 5-10K and continue later step with molecular weight cut-off behind the recovery ethanol, obtain compound polysaccharide of Radix Angelicae Sinensis equally.
The present invention also provides the preparation method of compound polysaccharide of Radix Angelicae Sinensis injection: get an amount of compound polysaccharide of Radix Angelicae Sinensis, a kind of extremely multiple pharmaceutically acceptable solubility promoter, dispersion agent, carrier or vehicle do not added or added, suitable injection such as powder pin (freeze-dried powder or powder direct packaging), liquid drugs injection made after the mixing.Each injected dose unit contains 50mg thing of the present invention at least.
Compound polysaccharide of Radix Angelicae Sinensis provided by the invention is used for the treatment of neutrophilic granulocytopenia, anaemia, thrombocytopenia, disease of viral infection, hypoimmunity and as anticancer adjuvant.
Compound polysaccharide of Radix Angelicae Sinensis provided by the invention shows in the result of the experiment in vitro of green blood and Immune Function, use compound polysaccharide of Radix Angelicae Sinensis, its concentration can stimulate the peripheral blood lymphocytes with phytohaemagglutinin (PHA) activatory people to produce interleukin 6 (IL-6), grain/macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interferon-(IFN-γ) when 25-400 μ g/ml effectively.Use compound polysaccharide of Radix Angelicae Sinensis, its concentration can stimulate the splenocyte propagation of Balb/C mouse effectively when 31.25-250 μ g/ml.
Experimental result shows in the body of green blood aspect, the Balb/C mouse of accepting radiotherapy is used compound polysaccharide of Radix Angelicae Sinensis, when dosage is 50-200mg/kg/d, the subcutaneous injection administration, used 7 days continuously, compare with the physiological saline group, white corpuscle, red corpuscle, oxyphorase, pcv and hematoblastic recovery have all shifted to an earlier date.Studies show that further administration group mouse bone marrow cells CFU-E, grain, monosystem progenitor cell and macronucleus hemopoietic forebody cell number increase.The Balb/C mouse of accepting chemotherapy is used compound polysaccharide of Radix Angelicae Sinensis, when dosage is 50-200mg/kg/d, the subcutaneous injection administration is when using 7 days continuously, compare with the physiological saline group, white corpuscle, red corpuscle, oxyphorase, pcv and hematoblastic recovery have also all shifted to an earlier date.
Experimental result shows in the body of immunology: the consumption of compound polysaccharide of Radix Angelicae Sinensis can stimulate the Balb/C mouse boosting cell of accepting chemotherapy to produce interferon-gamma (IFN-γ) and interleukin II (IL-2) at 25-100mg/kg/d effectively.Compound polysaccharide of Radix Angelicae Sinensis can also stimulate Balb/C mouse normal or that accept chemotherapy bovine serum to be induced the reactivity of tardy property super quick (DTH) effectively.
Experimental result shows in the antiviral body: attack malicious chick to bursal disease virus of chickens and use compound polysaccharide of Radix Angelicae Sinensis, consumption used 5 days when oral 50mg/d or intramuscular injection 50mg/d continuously, continued to observe 28 days.Compare 100% survival of medication treated animal with control animals dead 67%.Suffer from dog for Canine parvovirus infection and hundstaupe disease and use compound polysaccharide of Radix Angelicae Sinensis, consumption used 3-7 days when 100-200mg/d continuously, continued to observe 28 days.Compare with the conventional treatment that adopts clinically at present, curative ratio obviously raises, and does not have the situation of recurrence.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but the present invention is not done any restriction.
Embodiment 1
The preparation of compound polysaccharide of Radix Angelicae Sinensis
Clean system Radix Angelicae Sinensis 40kg, extracting in water 3 times adds water 160kg at every turn, extracts 3.0 hours, and extracting temperature is 100 ℃.United extraction liquid is evaporated to rare thick paste shape for 65 ℃, adjusts volume to 20L, and standing over night adds 95% ethanol to final concentration 35%, stir, placed the centrifugal 10min of 5000 * g 12 hours for 4 ℃, collect supernatant liquor, spraying drying gets Radix Angelicae Sinensis polysaccharide crude 504g.With Radix Angelicae Sinensis polysaccharide crude product 504g water dissolution, concentration is 4%, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 10K, retaining liquid is long-pending to be 8.6L, last 200 * 300mm SPSepharose cationic exchange coloum, elutriant is acetate-sodium acetate buffer of pH 5.250mM, collects effluent liquid 30L altogether, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 5K, must keep liquid 9.0L, keep DEAE Sepharose anion-exchange column on the liquid, elutriant is pH5.2, acetate-sodium acetate buffer of 50mM, collecting effluent liquid 28L altogether, is ultra-filtration membrane ultrafiltration (MWCO=5, the 000-8 of 5K with molecular weight cut-off, 000), must keep liquid 5.3L.Add 95% ethanol to final concentration and placed the centrifugal 10min of 5000 * g, collecting precipitation 12 hours for 70%, 4 ℃.It is anti-molten to 5.5L that precipitation adds water,, add 95% ethanol to final concentration and placed the centrifugal 5min of 5000 * g, collecting precipitation 12 hours for 70%, 4 ℃.It is anti-molten to 6L that precipitation adds water, spraying drying, and proper reversion closes polysaccharide products 124g.Compound polysaccharide of Radix Angelicae Sinensis content is 97%.
Detected result is as follows:
1. sugar is formed (Mol%)
| Pectinose (Ara) | Rhamnosyl (Rha) | Glucose (Glc) |
| 40.86? | 5.03? | 7.99? |
| Semi-lactosi (Gal) | Galacturonic acid (GalA) | 4-methyl glucoside aldehydic acid (4-Me-GluA) |
| 20.29? | 23.88? | 1.94? |
2. amino acid is formed (mol%)
| Oxyproline (Hyp) | Serine (Ser) | Threonine (Thr) | L-glutamic acid (Glu) | Glycine (Gly) | Aspartic acid (Asp) |
| 8.14? | 6.96? | 5.36? | 8.06? | 8.63? | 5.04? |
| L-Ala (Ala) | Proline(Pro) (Pro) | Gelucystine (Cys) | Tyrosine (Tyr) | Xie Ansuan (Val) | Methionine(Met) (Met) |
| 6.10? | 3.09? | 0.06? | 1.11? | 2.62? | 0.19? |
| Leucine (Leu) | Phenylalanine (Phe) | Histidine (His) | Methionin (Lys) | Arginine (Arg) | Isoleucine (Ile) |
| 1.90? | 1.24? | 2.56? | 7.51? | 27.92? | 1.30? |
| γ-An Jidingsuan (γ-ABA) | ? | ? | ? | ? | ? |
| 2.21? | ? | ? | ? | ? | ? |
3. protein content and weight-average molecular weight
Protein content: 2.56%.
Weight-average molecular weight: 56,703 dalton
Embodiment 2
The preparation of compound polysaccharide of Radix Angelicae Sinensis lyophilized injectable powder
Get embodiment 1 gained 50g compound polysaccharide of Radix Angelicae Sinensis, with containing 1.0% sodium-chlor, 0.4 ‰ tween-80, pH5.2, the acetate of 50mM-sodium acetate buffer dissolving is adjusted final volume to 2000ml.0.22um the filter membrane Sterile Filtration divides to install in 1000 10ml cillin bottles, freeze-drying promptly.
Embodiment 3
Compound polysaccharide of Radix Angelicae Sinensis is induced PHA activatory people's peripheral blood lymphocytes
Generate G-CSF, IL-6 and IFN-γ
Separation from healthy people's whole blood, washing obtain the human peripheral blood mononuclear cell, adjust cell with the RPMI-1640 substratum and count to 4 * 10
6/ ml.Embodiment 1, Radix Angelicae Sinensis Crude polysaccharides (AP) (the Radix Angelicae Sinensis Crude polysaccharides (AP) of using existing laboratory method extraction, purifying to obtain) (Shang Peng etc. with RPMI-1640 substratum preparation different concns, the journal .2001 of The Fourth Military Medical University, 22 (14): 1311-1314), with positive control drug polyporusum bellatus (Lianyun Harbour east wind pharmaceutical factory) solution, Department of Pharmacy classifies 400,200 as, 100,50,25 μ g/ml, each concentration is done multiple hole.Add PHA liquid, final concentration 4 μ g/ml.Add the monocyte suspension, final concentration of cells is 2 * 10
6/ ml.Cell control well only adds monocyte suspension and PHA liquid, and the nutrient solution control wells only adds the RPMI-1640 substratum.37 ℃, 5% CO2gas incubator was cultivated 24 hours, and is centrifugal, collects supernatant.Detect the growing amount of granulocyte colony-stimulating factor (G-CSF), interleukin 6 (IL-6) and interferon-(IFN-γ) respectively with corresponding ELISA test kit, cytokine amount (S/C) expression that the cytokine amount/cell control group that produces with the dosing group produces is active.
The compound polysaccharide of Radix Angelicae Sinensis of following table demonstration different concns is induced generation
The quantity of G-CSF, IL-6 and IFN-γ
As can be seen from the above results: Radix Angelicae Sinensis polysaccharide can induce PHA activatory people's peripheral blood lymphocytes to generate G-CSF, IL-6 and IFN-γ, and growing amount and drug level are proportionate.The effect of inducing of compound polysaccharide of Radix Angelicae Sinensis obviously is better than the Radix Angelicae Sinensis Crude polysaccharides (AP) of Isodose.
Embodiment 4
Compound polysaccharide of Radix Angelicae Sinensis stimulates Balb/C mouse boosting cell propagation
Preparation Balb/C mouse boosting cell suspension is adjusted cell and is counted to 2.5 * 10
6/ ml.Embodiment 1, Radix Angelicae Sinensis Crude polysaccharides (AP) and the positive control drug polyporusum bellatus solution of preparation different concns, Department of Pharmacy classifies 500,250 as, 125,62.5 and 31.25 μ g/ml, each concentration is done multiple hole.Add splenocyte suspension, final concentration of cells is 1.25 * 10
6/ ml.Cell control well only adds splenocyte suspension.37 ℃, 5% CO2gas incubator was cultivated 48 hours, and every hole adds 50 μ l, 0.5 μ Ci's
3HTdR continues to cultivate 18 hours.Use the cell harvestor collecting cell, on liquid scintillation instrument, read the cpm value, active with administration group cpm value/cell control group cpm value (S/C) expression.
Following table shows that the compound polysaccharide of Radix Angelicae Sinensis of different concns stimulates
The situation of mouse boosting cell propagation
As can be seen from the above results: compound polysaccharide of Radix Angelicae Sinensis can stimulate Balb/C mouse boosting cell propagation, and growing amount and drug level are proportionate.And the effect of compound polysaccharide of Radix Angelicae Sinensis obviously is better than the Radix Angelicae Sinensis Crude polysaccharides (AP) of equivalent.
Embodiment 5
Compound polysaccharide of Radix Angelicae Sinensis promotes to accept radiation murine marrow
The recovery of hemopoietic forebody cell
With female Balb/c mouse (body weight 18-22g), be divided into 5 groups at random, 10 every group: the 1st group: the normal control group.The 2nd group: irradiation+physiological saline group.The 3rd group: irradiation+embodiment 1, consumption 100mg/kg/d.The 4th group:. irradiation+Radix Angelicae Sinensis Crude polysaccharides (AP), consumption 100mg/kg/d.The 5th group.: irradiation+polyporusum bellatus, consumption 200mg/kg/d.
0 day except that the normal control group, with whole body sublethal dose (total amount 600cGy) roentgen radiation x mouse.Since 0 day, each administration group mouse subcutaneous injection administration, once a day, successive administration 7 days.Normal control group 0 day, 0-6 days isopyknic physiological saline of subcutaneous injection of irradiation+physiological saline group.Put to death mouse in 8,11,16 days that test in batches, the aseptic femur of getting, the preparation bone marrow cell suspension, adjusting cell count is 1 * 10
5/ ml adds substratum, puts CO
2Incubator was cultivated 6 days for 37 ℃, counting marrow grain, monosystem progenitor cell (CFU-GM).Cultivated 7 days for 37 ℃, counting marrow red is the quick-fried formula colony forming cell (BFU-E) that increases.Cultivated 8 days counting marrow macronucleus hemopoietic forebody cell (CFU-MEG) for 37 ℃.
Following table shows that compound polysaccharide of Radix Angelicae Sinensis promotes to accept radiation murine marrow
The recovery of hemopoietic forebody cell
Remarks: each group compares with the physiological saline group,
*Expression P＜0.05,
*Expression P＜0.01,
* *Expression P＜0.001.
As can be seen from the above results: compound polysaccharide of Radix Angelicae Sinensis can promote to accept marrow grain-monosystem progenitor cell, the CFU-E of radiation murine, the recovery of macronucleus hemopoietic forebody cell, Radix Angelicae Sinensis Crude polysaccharides (AP) can only promote the recovery of preceding two kinds of medullary cells, and effect is weaker than compound polysaccharide of Radix Angelicae Sinensis.
Embodiment 6
Compound polysaccharide of Radix Angelicae Sinensis promotes to accept the recovery of radiotherapy mouse peripheral hemogram
The Balb/c female mice, body weight 18-22g, random packet, 15 every group.Reagent is divided into 4 groups: the 1st group is embodiment 1, and consumption is 50mg/kg/d.The 2nd group is embodiment 1, and consumption is 200mg/kg/d.The 3rd group is Radix Angelicae Sinensis Crude polysaccharides (AP), and consumption is 200mg/kg/d.The 4th group is polyporusum bellatus, and consumption is 200mg/kg/d.The 5th group is the physiological saline group.The 6th group is the normal control group.
1-5 group mouse was accepted x-ray bombardment at 0 day, the irradiation total amount is 550cGy.Began the subcutaneous injection administration same day from shining, once a day, one week of continuous use.7th, got tail vein respectively on the 10th, 14,17,21,24, counting peripheral blood leucocyte (WBC), red corpuscle (RBC), oxyphorase (Hb), pcv (HCT) and thrombocyte (PLT).
Peripheral blood WBC (* 10
9/ L) variation
Peripheral blood RBC (* 10
12/ L) variation
The variation of peripheral blood Hb (g/L)
The variation of peripheral blood HCT (%)
Peripheral blood PLT (* 10
9/ L) variation
Remarks: each group compares with the physiological saline group,
*Expression P＜0.05,
*Expression P＜0.01,
* *Expression P＜0.001.
As can be seen from the above results: the consumption of compound polysaccharide of Radix Angelicae Sinensis can promote to accept radiotherapy mouse peripheral blood RBC when 50-200mg/kg/d, Hb, and HCT, the recovery of PLT and WBC, and situation and the dosage recovered are proportionate.Radix Angelicae Sinensis Crude polysaccharides (AP) can not promote the recovery of PLT, and effect is worse than the compound polysaccharide of Radix Angelicae Sinensis of Isodose.
Embodiment 7
Compound polysaccharide of Radix Angelicae Sinensis promotes the recovery of chemotherapy mouse peripheral hemogram
The Balb/c female mice, body weight 18-22g, random packet, 15 every group.Reagent is divided into 4 groups: the 1st group is embodiment 1, and consumption is 50mg/kg/d.The 2nd group is embodiment 1, and consumption is 200mg/kg/d.The 3rd group is Radix Angelicae Sinensis Crude polysaccharides (AP), and consumption is 200mg/kg/d.The 4th group is polyporusum bellatus, and consumption is 200mg/kg/d.The 5th group is the physiological saline group.The 6th group is the normal control group.
1-5 group mouse was accepted mitomycin in 0 day-1, and tail intravenously administrable, dosage are 3mg/kg/d.Chemotherapy began the subcutaneous injection administration same day, once a day, and continuous use 7 days.2nd, tail vein blood 1 time respectively on the 5th, 7,9,12,14,16,19,22, counting peripheral blood leucocyte (WBC), red corpuscle (RBC), oxyphorase (Hb), pcv (HCT) and thrombocyte (PLT).
Peripheral blood WBC (* 10
9/ L) variation
Peripheral blood RBC (* 10
12/ L) variation
The variation of peripheral blood Hb (g/L)
The variation of peripheral blood HCT (%)
Peripheral blood PLT (* 10
9/ L) variation
Remarks: each group compares with the physiological saline group,
*Expression P＜0.05,
*Expression P＜0.01,
* *Expression P＜0.001.
As can be seen from the above results: the consumption of compound polysaccharide of Radix Angelicae Sinensis can promote to accept radiotherapy mouse peripheral blood RBC when 50-200mg/kg/d, Hb, and HCT, the recovery of PLT and WBC, and situation and the dosage recovered are proportionate.Radix Angelicae Sinensis Crude polysaccharides (AP) can not promote the effect of the recovery of PLT, and effect is worse than the compound polysaccharide of Radix Angelicae Sinensis of Isodose.
Embodiment 8
The Balb/c of compound polysaccharide of Radix Angelicae Sinensis immune stimulatory hypofunction
The mouse boosting cell secretion of gamma-IFN
The Balb/c female mice, body weight 18-22g, random packet, 10 every group.Reagent is divided into 4 groups: the 1st group is embodiment 1, and consumption is 25mg/kg/d.The 2nd group is embodiment 1, and consumption is 100mg/kg/d.The 3rd group is Radix Angelicae Sinensis Crude polysaccharides (AP), and consumption is 100mg/kg/d.The 4th group is Zadaxin, and consumption is 5mg/kg/d.The 5th group is the physiological saline group.The 6th group is the normal control group.
From the 1st day, 1-5 group subcutaneous injection administration was given 7 days continuously.The 4th day, except that the normal control group, each organized intraperitoneal injection of cyclophosphamide 1 time, and dosage is 200mg/kg.Put to death mouse on the 11st day, the preparation splenocyte suspension, adjusting cell count is 2 * 10
6/ ml, every hole adds 1ml, does multiple hole.Add ConA to final concentration 2.5 μ g/ml, no ConA person is contrast.At 5% carbonic acid gas incubator, hatched 48 hours for 37 ℃.Centrifugal, collect supernatant liquor, detect the content of cytokine interferon-(IFN-γ) with the ELISA method.
Following table shows compound polysaccharide of Radix Angelicae Sinensis immune stimulatory hypofunction
Balb/c mouse boosting cell secretion of gamma-IFN
Remarks: each group compares with the physiological saline group.
As can be seen from the above results: the consumption of compound polysaccharide of Radix Angelicae Sinensis can immune stimulatory hypofunction mouse boosting cell secretion of gamma-IFN when 25-100mg/kg/d.And growing amount and dosage are proportionate.The effect of Radix Angelicae Sinensis Crude polysaccharides (AP) is weaker than the compound polysaccharide of Radix Angelicae Sinensis of Isodose.
Embodiment 9
Compound polysaccharide of Radix Angelicae Sinensis is to the interior resisting virus effect of bursal disease virus of chickens
4 the week age chick, body weight 200-250g is divided into 3 groups at random after bursal disease virus of chickens is attacked poison, 25 every group, male and female half and half.The 1st group: control group, not administration.The 2nd group: the oral administration group, embodiment 1 adds in the drinking-water, 50mg/ days.Successive administration 5 days.The 3rd group, the administered intramuscular group, embodiment 1 consumption is 50mg/ days, the injection cumulative volume is 1ml.Successive administration 5 days.Continuous 2 weeks are observed the death condition of medication group and control animals.Experimental session, dead animal need take out the fabricius bursa, weighs.The 14th day, put to death all surviving animals, take out the fabricius bursa, weigh.
Following table shows general state, mortality ratio and the fabricius bursa weight of the 14th day medication group and control animals
| Group | General state | Mortality ratio | Fabricius bursa weight |
| Control group | Become thin poor growth | 67％? | 4.5-6.4g? |
| The oral administration group | Well, weight increase | 0? | 1-2g? |
| The drug administration by injection group | Well, weight increase | 0? | 1-2g? |
As can be seen from the above results: oral or intramuscular injection gives compound polysaccharide of Radix Angelicae Sinensis, and consumption can obviously alleviate the infection and the pathology of chicken bursa in the time of 50mg/ days, prevents that bursal disease virus of chickens from attacking the death of malicious animal.
Embodiment 10
Compound polysaccharide of Radix Angelicae Sinensis clinically to canine distemper and
The curative effect of Canine parvovirus infection
Experiment is divided into 4 groups.The 1st group: clinical definite is 41 of the small-sized dogs of Canine parvovirus infection, body weight 1500-4000g, and vein is given embodiment 1.The animal 100mg/d of body weight 1000-2000g, the animal 200mg/d of body weight＞2000g, every day 1 time, continuous use 3-7 days.The 2nd group: clinical definite is 20 of the small-sized dogs of Canine parvovirus infection, body weight 1000-4600g, and therapy for treating routinely, in contrast.The 3rd group: clinical definite is 47 of the small-sized dogs of canine distemper, body weight 1000-5000g, and vein is given embodiment 1, and usage is the same.Continuous use 3-7 days.The 4th group: clinical definite is 28 of the small-sized dogs of canine distemper, body weight 1000-5000g, and therapy for treating routinely, in contrast.Observe healing situation and the death condition of animal, follow up a case by regular visits to 4 weeks, understanding has or not recurrence.
Following table shows the death condition and the palindromia situation of each treated animal of different time sections
| Group | 3 days mortality ratio/recurrence rates | 7 days mortality ratio/recurrence rates | 28 days mortality ratio/recurrence rates |
| 1? | 10％/0? | 15％/0? | 15％/0? |
| 2? | 30％/0? | 55％/0? | 55％/15％? |
| 3? | 20％/0? | 26％/0? | 26％/0? |
| 4? | 34％/0? | 48％/0? | 52％/18％? |
Canine distemper and Canine parvovirus infection are two kinds of serious animal infectious diseases, without treatment mortality ratio＞95%.The compound polysaccharide of Radix Angelicae Sinensis consumption is when 100-200mg/d, and with the treatment plan comparison of present clinical employing, the mortality ratio of infected animal is low, cures animal not recurrence in 28 days.
Compound polysaccharide of Radix Angelicae Sinensis can be used for the treatment of neutrophilic granulocytopenia, anaemia, thrombocytopenia, hypoimmunity, disease of viral infection and as anticancer adjuvant according to above result.
Claims (10)
1. compound polysaccharide of Radix Angelicae Sinensis that extraction from the dry root of Chinese medicinal materials Radix Angelicae Sinensis (Angelica Sinensis (Oliv) Diels), separation, purifying obtain; It is characterized in that this complex polysaccharide mainly by gala aldehydic acid glycan, sandlwood gala aldehydic acid glycan, Arabinogalactan-Protein and alpha-glucan are formed; Its characteristics are: 1. polysaccharide percentage composition: albumen percentage composition=95-99%: 1-5%; 2. sugar is formed: main pectinose by 35-45%, and the galacturonic acid of 20-28%, the semi-lactosi of 18-25%, the glucose of 5-15%, the rhamnosyl of 3-8%, the 4-methyl glucoside aldehydic acid of 1-5% is formed; 3. glycosidic link link: pectinose is mainly with α-3, and α-5, a α end and α-3,5 glycosidic link are connected; Galacturonic acid mainly is connected with α-4 glycosidic link; Semi-lactosi is mainly with α-3, and α-6 and α-3,6 glycosidic link are connected; Glucose is mainly with α-4, and α-6 and α-4,6 glycosidic link are connected; Rhamnosyl mainly is connected with α-2 and α-2,4 glycosidic link; 4-methyl glucoside aldehydic acid mainly is connected with end with α-4; 4. amino acid is formed: form by 19 kinds of different amino acid, wherein, and arginine, Serine, L-glutamic acid, glycine, L-Ala, Methionin and hydroxyproline content are higher, account for the 65-75% of total amino acid content; 5. weight-average molecular weight: 20,000-80,000 dalton; Other: soluble in water for white or off-white powder, be insoluble to ethanol, acetone and other organic solvent; Aqueous solution pH is 4-6, is slightly acidic.
2. method for preparing compound polysaccharide of Radix Angelicae Sinensis as claimed in claim 1 is characterized in that may further comprise the steps: Radix Angelicae Sinensis dry root water is carried or with 0.2-0.6M potassium primary phosphate or sodium dihydrogen phosphate extraction, extracting temperature is 50-105 ℃; The 20-40% ethanol sedimentation, collect supernatant liquor, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 5-10K, keep liquid through cationic exchange coloum and anion-exchange column, with the buffer solution elution of pH5-6,20-100mM, collect 2-5 column volume of effluent liquid, effluent liquid is the ultra-filtration membrane ultrafiltration of 5-10K through molecular weight cut-off, gained keeps the ethanol sedimentation twice of liquid through 65-85% after the ultrafiltration, and the gained throw out is described compound polysaccharide of Radix Angelicae Sinensis.
3. the described method of claim 2 is characterized in that ion exchange resin used in preparation process is SP Sepharose positively charged ion and DEAE Sepharose anionite-exchange resin.
4. according to claim 2 or 3 described methods, it is characterized in that damping fluid used in the preparation process is citric acid-sodium citrate damping fluid, Sodium phosphate dibasic-citrate buffer solution, acetate-sodium acetate buffer.
5. according to claim 2 or 3 described methods, it is characterized in that: preparing the complex polysaccharide post precipitation, add water, obtain the purified complex polysaccharide through spraying or lyophilize with resolution of precipitate through 2 65-80% alcohol precipitation processes.
6. with any complex polysaccharide that obtains of claim 2-5, its weight-average molecular weight is 20,000-80,000 dalton.
7. with any complex polysaccharide that obtains of claim 2-5, polysaccharide content＞80%.
8. contain the described compound polysaccharide of Radix Angelicae Sinensis injection of arbitrary claim among the with good grounds claim 1-7, comprise liquid drugs injection, the powder pin; Be not add or add a kind of extremely multiple pharmacy acceptable auxiliary material by the compound polysaccharide of Radix Angelicae Sinensis that described method obtains to make, contain compound polysaccharide of Radix Angelicae Sinensis 50mg in each injected dose unit at least.
9. according to the purposes of the described compound polysaccharide of Radix Angelicae Sinensis of arbitrary claim among the claim 1-7 in preparation treatment neutrophilic granulocytopenia, anaemia, thrombocytopenia, hypoimmunity, disease of viral infection and anticancer ancillary drug.
10. purposes according to claim 9, it is characterized in that described anticancer ancillary drug for put, the chemotherapy adjuvant drug; Described disease of viral infection is disease and the hundstaupe disease that bursal disease virus of chickens, Canine parvovirus infection cause; Described hypoimmunity comprises that the human or animal suffers from many reasons such as various infection or noninfectious disease, pharmacological agent, environmental pollution, radiation, overtired, improper diet, stress and causes.
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| CN103864947A (en) * | 2014-03-17 | 2014-06-18 | 重庆医科大学 | Process for separating and extracting angelica polysaccharide and application thereof |
| CN104522683A (en) * | 2014-12-19 | 2015-04-22 | 无限极（中国）有限公司 | Application of Chinese angelica polypeptide with effects of resisting oxidization and delaying ageing in preparation of food |
| CN105617356A (en) * | 2016-01-29 | 2016-06-01 | 程潜 | Medicine for treating asthma |
| CN105617354A (en) * | 2016-01-29 | 2016-06-01 | 程龙 | Medicine for treating rheumatism and rheumatoid |
| CN105597081A (en) * | 2016-01-29 | 2016-05-25 | 程龙 | Medicine for reducing hypertension |
| CN106376544B (en) * | 2016-08-31 | 2020-05-26 | 天津海友佳音生物科技股份有限公司 | Protective solution for preserving artemia nauplii and preserving method |
| CN108607092B (en) * | 2018-05-04 | 2021-06-01 | 福州大学 | Application of angelica sinensis protein in preparation of medicine for assisting tumor treatment |
| CN112076208A (en) * | 2019-06-13 | 2020-12-15 | 青岛海洋生物医药研究院股份有限公司 | Application of glucan in preparation of medicine |
| CN111560082B (en) * | 2020-07-16 | 2020-10-27 | 鲁东大学 | Phosphonic acid angelica polysaccharide compound and preparation method thereof |
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