CN1676530A - Matrimony vine arabinogalactan protein, and its preparing process, use and composition - Google Patents
Matrimony vine arabinogalactan protein, and its preparing process, use and composition Download PDFInfo
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Abstract
This invention relates to Arabinogalactan proteins AGPs distilled, separated and purified from Lycium barbarum L and its heavy molecular weight from 30.000-100.000 Dolton, and its content is more than 80%. Add one or more adjuvant into proper AGPs in to make injection agent and every unit dosage of this injection agent contains at least 30mg this invention. This invention cures neutral granutocyte decrease symptom, anemia, haematoblast decrease symptom and is used as anticancer adjuvant. It can be used singly and also be used with G-CSF.
Description
The present invention relates to field of traditional Chinese medicine pharmacy.Be specifically related to a kind of weight-average molecular weight that extraction, purifying obtain from lycium barbarum (Lyciumbarbarum L.) 30,000-100, Arabinogalactan-Protein between 000 dalton (Arabinogalactan proteins.AGPs) is called for short matrimony vine AGPs.Content＞80%.The chemical characteristic that possesses higher plant AGPs: 1. polysaccharide: albumen=80-95%: 5-20%.2. sugared compositing characteristic: mainly contain pectinose (Ara) and semi-lactosi (Gal), both molar percentage sum＞50%.3. sugar chain: Ara is mainly 5-, 2,5-and 3, and 5-connects.Gal is mainly 6-and 3, and 6-connects.4. amino acid (AA) compositing characteristic: oxyproline (Hyp): 15-20%, Serine (Ser): 10-20%, Threonine (Thr): 5-10%, L-glutamic acid (Glu): 5-10%, glycine (Gly): 5-10%, aspartic acid (Asp): 5-10%, L-Ala (Ala): 5-10%.5. weight-average molecular weight: 30,000-100,000 dalton.6. solvability: water-soluble.The invention still further relates to preparation technology, preparation of matrimony vine AGPs and uses thereof, the application also provides the composition of described matrimony vine AGPs and Radix Astragali extract, Radix Angelicae Sinensis extract or G-CSF simultaneously, can make each component synergy by making up.
Wolfberry fruit is the dry mature fruit of Solanaceae Lycium plant lycium barbarum.Matrimony vine is a machaka, the florescence 5-6 month, the fruit phase 6-11 month.
The traditional Chinese medical science is thought: the wolfberry fruit nature and flavor are sweet, flat.Effect for nourish the liver to improve visual acuity, tonifying kidney and benefiting sperm, moistening lung.Be used for the treatment of hepatic and renal YIN deficiency, asthenia of essence and blood, soreness of the waist and knees, have a dizzy spell.Along with modern medicine, pharmacy and development of molecular biology, people have also had further understanding to the pharmacological action of wolfberry fruit.(the periodical .1987 (11) of institute of Military Medical Science Institute: 476) such as Geng Changshan, (herbal medicine, 1988.19. (7): 25) pharmacological research to lycium barbarum polysaccharide (LBP) shows: lycium barbarum polysaccharide (LBP) can significantly increase the heavy and raising periphery blood T lymphocyte percentage ratio of spleen of normal mouse.(Chinese J Pharmacol Toxicol .1990 (4): 1) find: lycium barbarum polysaccharide (LBP) can resist the immunosuppressive action of endoxan to mouse T cell, CTL cell and NK cell to Wang Baikun etc.Zhou Zhiwen etc. (Chinese J Pharmacol Toxicol .1991 (5): 1) find lycium barbarum polysaccharide (LBP) can promote mouse bone marrow cells hemopoietic stem cell (CFU-S), grain-monosystem progenitor cell (CFU-GM) and red be the quick-fried propagation that increases formula colony forming cell (BFU-E), therefore, discussion is used for green blood with lycium barbarum polysaccharide as efficient part, improve immunologic function, prospect is arranged very much.
The invention provides a kind of weight-average molecular weight that extraction, purifying obtain from wolfberry fruit 30,000-100, the Arabinogalactan-Protein between 000 dalton (Arabinogalactanproteins.AGPs) is called for short matrimony vine AGPs.Content＞80%.
The main technique flow process is as follows: after wolfberry fruit is cleaned, add water 100 ℃ of extractions (amount of water is 3-4 times, extracts 1.0 hours, extracts altogether 2 times) at every turn.Extracting solution 60-65 ℃ is evaporated to rare thick paste shape, carries out the 30-45% alcohol precipitation after the cooling, and 4 ℃ left standstill 8-12 hour.Collect supernatant liquor, spraying drying promptly gets matrimony vine AGPs crude product.With matrimony vine AGPs crude product water dissolution, concentration is 4-6%, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration (MWCO=10 of 10K, 000), keep SP Sepharose ion exchange column on the liquid, elutriant is pH5-6, the damping fluid of 30-50mM (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.Collect effluent liquid totally 3 column volumes, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration (MWCO=5 of 5K, 000-8,000), keep DEAE Sepharose ion exchange column on the liquid, elutriant is pH5-6, the damping fluid of 30-50mM (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 effluent liquid totally 3 column volumes, is the ultra-filtration membrane ultrafiltration (MWCO=5,000-8,000) of 5K with molecular weight cut-off, keeps liquid through 70% 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 matrimony vine AGPs more than 80%.
Or collect supernatant liquor behind the 30-45% alcohol precipitation, reclaiming behind the ethanol with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 10K and later step, obtains matrimony vine AGPs equally.
The present invention also provides the preparation method of matrimony vine AGPs injection: get an amount of matrimony vine AGPs, add one or more pharmaceutically acceptable solubility promoter, dispersion agent, carrier or vehicle, make suitable injection such as powder pin, liquid drugs injection or freeze-dried powder after the mixing.Each injected dose unit contains 30mg thing of the present invention at least.
Matrimony vine AGPs provided by the invention is used for the treatment of neutrophilic granulocytopenia, anaemia, thrombocytopenia and as anticancer adjuvant.Can be independent medication, also can with medication combined medications such as other polysaccharide or G-CSF.
The pharmacological action of matrimony vine AGPs provided by the invention aspect green blood is: the result of experiment in vitro shows, use matrimony vine AGPs separately, 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) and granulocyte colony-stimulating factor (G-CSF) when 50-400 μ g/ml effectively.
After matrimony vine AGPs and astragalus polysaccharides, Radix Angelicae Sinensis polysaccharide or the G-CSF combination, can bring into play synergistic function.When matrimony vine AGPs and astragalus polysaccharides (with reference to envelope Silan etc. the astragalus polysaccharides process study. the technology among Chinese patent medicine .1997.19 (12) .5-6. and improve after extraction, purifying and getting, polysaccharide content is more than 95%) drug combination, matrimony vine AGPs concentration is 100 μ g/ml, when astragalus polysaccharides concentration is 100 μ g/ml, with independent use matrimony vine AGPs or astragalus polysaccharides, concentration is that 200 μ g/ml compare, and stimulates the ability enhancing that produces IL-6, GM-CSF and G-CSF with PHA activatory people's peripheral blood lymphocytes.When matrimony vine AGPs and G-CSF drug combination, matrimony vine AGPs consumption is 50mg/kg/d, the G-CSF consumption is 100 μ g/kg/d, with independent use matrimony vine AGPs, consumption is 50mg/kg/d, or to use the G-CSF consumption separately be that 100 μ g/kg/d compare, stimulate the Balb/c mouse produce grain/macrophage colony formation cell (GM-CFC) and red be that the quick-fried ability that increases formula colony forming cell (BFU-E) obviously strengthens.
Result of experiment shows in the body, and the Balb/C mouse of accepting radiotherapy is used matrimony vine AGPs separately, and when dosage is 25-100mg/kg/d, the subcutaneous injection administration was used 7 days continuously, with placebo relatively, white corpuscle and hematoblastic recovery have shifted to an earlier date 4-7 days.Studies show that further administration group mouse bone marrow cells hemopoietic stem cell and grain, monosystem progenitor cell number increase, and induce spleen cell to generate IL-3, IL-6, GM-CSF and G-CSF.When matrimony vine AGPs and astragalus polysaccharides drug combination, dosage is matrimony vine AGPs 50mg/kg/d, astragalus polysaccharides 50mg/kg/d, when usage is the same, with independent use matrimony vine AGPs or astragalus polysaccharides, dosage is that the 100mg/kg/d group is compared, and leukocytic recovery has shifted to an earlier date 2-4 days.When matrimony vine AGPs and Radix Angelicae Sinensis polysaccharide (with reference to Zhang Linwei etc. the research of the separation and purification of Radix Angelicae Sinensis polysaccharide and part character thereof. biology magazine .1998.15 (3) .12-14. and improve after extraction, purifying and getting.Polysaccharide content is more than 95%) during drug combination, dosage is matrimony vine AGPs 50mg/kg/d, Radix Angelicae Sinensis polysaccharide 50mg/kg/d, when usage is the same, with independent use matrimony vine AGPs or Radix Angelicae Sinensis polysaccharide, dosage is that the 100mg/kg/d group compares, and white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date.When matrimony vine AGPs and G-CSF drug combination, dosage is matrimony vine AGPs100mg/kg/d, G-CSF 1-3 μ g/kg/d, when usage is the same, with independent use G-CSF, dosage is 1-3 μ g/kg/d or uses matrimony vine AGPs separately, dosage be the 100mg/kg/d group relatively, white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date 3-4 days.When the Balb/C mouse of accepting chemotherapy is used matrimony vine AGPs separately, when dosage is 25-100mg/kg/d, the subcutaneous injection administration, when using 7 days continuously, with placebo relatively, white corpuscle and hematoblastic recovery have shifted to an earlier date 4-7 days.When matrimony vine AGPs and astragalus polysaccharides drug combination, dosage is matrimony vine AGPs 50mg/kg/d, astragalus polysaccharides 50mg/kg/d, when usage is the same, with independent use matrimony vine AGPs or astragalus polysaccharides, dosage is that the 100mg/kg/d group is compared, and leukocytic recovery has shifted to an earlier date 2-4 days.When matrimony vine AGPs and Radix Angelicae Sinensis polysaccharide drug combination, dosage is matrimony vine AGPs 50mg/kg/d, Radix Angelicae Sinensis polysaccharide 50mg/kg/d, when usage was the same, with independent use matrimony vine AGPs or Radix Angelicae Sinensis polysaccharide, dosage was that the 100mg/kg/d group compares, white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date.When matrimony vine AGPs and G-CSF drug combination, dosage is matrimony vine AGPs 100mg/kg/d, G-CSF 1-3 μ g/kg/d, when usage is the same, with independent use G-CSF, when dosage is 1-3 μ g/kg/d or use separately matrimony vine AGPs, dosage be 100mg/kg/d group relatively, white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date 3-4 days.
The pharmacological action of matrimony vine AGPs provided by the invention aspect enhancing immunity is: result of experiment shows in the body; when the consumption of matrimony vine AGPs at 25-100mg/kg/d, can stimulate Balb/C mouse boosting cell normal or that accept chemotherapy to produce interferon-gamma (IFN-γ), interleukin II (IL-2) and interleukin-4 (IL-4) effectively.Matrimony vine AGPs also can improve S-180 tumor-bearing mice spleen nk cell activity, stimulates S-180 tumor-bearing mice splenocyte to produce interleukin II cytokines such as (IL-2).
Embodiment
Embodiment 1
The preparation of matrimony vine AGPs
Wolfberry fruit 40kg, extracting in water 2 times adds water 160kg at every turn, extracts 1.0 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 38%, stir, placed the centrifugal 10min of 5000 * g 10 hours for 4 ℃, collect supernatant liquor, spraying drying gets matrimony vine AGPs crude product 598g.With matrimony vine AGPs crude product 598g 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 9.2L, and last 200 * 300mm SPSepharose ion exchange column, elutriant are pH5.2, acetate-sodium acetate buffer of 50mM, collecting effluent liquid 30L altogether, is the ultra-filtration membrane ultrafiltration of 5K with molecular weight cut-off, must keep liquid 9.1L, keep DEAE Sepharose ion exchange column on the liquid, elutriant is pH5.2, and acetate-sodium acetate buffer of 50mM is collected effluent liquid 32L altogether, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration (MWCO=5 of 5K, 000-8,000), must keep liquid 6L.Add 95% ethanol to final concentration and placed the centrifugal 5min of 5000 * g, collecting precipitation 10 hours for 70%, 4 ℃.It is anti-molten to 6L that precipitation adds water,, add 95% ethanol to final concentration and placed the centrifugal 5min of 5000 * g, collecting precipitation 10 hours for 70%, 4 ℃.It is anti-molten to 7L that precipitation adds water, and spraying drying gets matrimony vine AGPs138g.AGPs content is 83%.
Detected result is as follows:
1. sugar is formed (Mol%)
| Pectinose (Ara) | Rhamnosyl (Rha) | Wood sugar (Xyl) | Seminose (Man) |
| ????35.2 | ???8.9 | ????5.1 | ??3.9 |
| Semi-lactosi (Gal) | Galacturonic acid (GalA) | Glucose (Glu) | Glucuronic acid (GluA) |
| ????20.7 | ???18.5 | ????2.9 | ??4.8 |
2. amino acid is formed (%)
| Oxyproline (Hyp) | Serine (Ser) | Threonine (Thr) | L-glutamic acid (Glu) | Glycine (Gly) | Aspartic acid (Asp) |
| ????16.60 | ??19.50 | ????5.57 | ???7.59 | ??9.40 | ??5.63 |
| L-Ala (Ala) | Proline(Pro) (Pro) | Halfcystine (Cys) | Tyrosine (Tyr) | Xie Ansuan (Val) | Methionine(Met) (Met) |
| ????7.11 | ??6.48 | ????0.018 | ???3.72 | ??3.55 | ??0.20 |
| Leucine (Leu) | Phenylalanine (Phe) | Histidine (His) | Methionin (Lys) | Arginine (Arg) | Isoleucine (Ile) |
| ????3.62 | ??1.872 | ????1.13 | ???2.23 | ??2.28 | ??3.50 |
3. protein content and weight-average molecular weight
Protein content: 10.9%.
Weight-average molecular weight: 87,600 dalton
Embodiment 2
The preparation of matrimony vine AGPs lyophilized injectable powder
Get embodiment 1 gained 30g matrimony vine AGPs, with containing 0.5% sodium-chlor, 0.4 ‰ tween-80, pH5.0, 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
Matrimony vine AGPs induces PHA activatory people's peripheral blood lymphocytes
Generate GM-CSF, G-CSF and IL-6
It is 6 groups that experiment is divided into: the 1st group of reagent is embodiment 1, and concentration is 50 μ g/ml.The 2nd group of reagent is embodiment 1, and concentration is 100 μ g/ml.The 3rd group of reagent is embodiment 1, and concentration is 200 μ g/ml.The 4th group of reagent is embodiment 1, and concentration is 400 μ g/ml.The lycium barbarum polysaccharide (LBP) (Wang Ling etc., unming Medical College journal .1995.16 (2)) of the 5th group of reagent for obtaining with existing laboratory method extraction, purifying, concentration is 400 μ g/ml.The 6th group of reagent is polyporusum bellatus injection (Lianyun Harbour east wind pharmaceutical factory), and concentration is 400 μ g/ml.As positive control.The 7th group is blank.
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 1 * 10
6/ ml adds each pipe, every pipe 1.0ml successively.Add soup 0.5ml (except that blank) again, PHA liquid 0.5ml (to final concentration 4 μ g/ml).37 ℃, 5% CO2gas incubator was cultivated 24 hours, and is centrifugal, collects supernatant.Detect GM-CSF respectively with corresponding ELISA test kit, the growing amount of G-CSF and IL-6, the cytokine amount that produces with the dosing group is the active unit of expression than the cytokine amount (S/C) that blank group produces.
Following table shows the matrimony vine AGPs of different concns
Induce and generate GM-CSF, the quantity of G-CSF and IL-6
| Grouping | Reagent concentration (μ g/ml) | ???G-CSF ???(S/C) | ??GM-CSF ???(S/C) | ????IL-6 ????(S/C) |
| ????1 | ????50 | ????5.8 | ????1.8 | ????76.4 |
| ????2 | ????100 | ????18.9 | ????7.0 | ????130.6 |
| ????3 | ????200 | ????24.5 | ????9.7 | ????386.6 |
| ????4 | ????400 | ????30.7 | ????12.7 | ????551.1 |
| ????5 | ????400 | ????7.2 | ????3.9 | ????58.1 |
| ????6 | ????400 | ????11.2 | ????5.9 | ????9.0 |
As can be seen from the above results: matrimony vine AGPs can induce PHA activatory people's peripheral blood lymphocytes to generate GM-CSF, G-CSF and IL-6, and growing amount and drug level are proportionate.The effect of inducing of matrimony vine AGPs obviously is better than the LBP of Isodose.
Embodiment 4
Matrimony vine AGPs and astragalus polysaccharides share the peripheral blood of strengthening PHA activatory people
Monocyte is induced and is generated GM-CSF, G-CSF and IL-6
It is 4 groups that reagent is divided into: the 1st group of reagent is embodiment 1, and concentration is 200 μ g/ml.The 2nd group of reagent is astragalus polysaccharides, and concentration is 200 μ g/ml.The 3rd group of reagent is embodiment 1+ astragalus polysaccharides, and concentration respectively is 100 μ g/ml.The 4th group of reagent is polyporusum bellatus injection (Lianyun Harbour east wind pharmaceutical factory), and concentration is 400 μ g/ml.As positive control.Not dosing group is as blank.
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 1 * 10
6/ ml.Add each pipe, every pipe 1.0ml.Each pipe adds soup 0.5ml (except the blank), PHA liquid 0.5ml (to final concentration 4 μ g/ml) again.37 ℃, 5% CO2gas incubator was cultivated 24 hours, and is centrifugal, collects supernatant.Detect GM-CSF with corresponding ELISA test kit, the growing amount of G-CSF and IL-2, cytokine amount (S/C) expression that the cytokine amount/blank group that produces with the dosing group produces is active.
Following table shows that matrimony vine AGPs and astragalus polysaccharides share reinforcement and induce generation
G-CSF, GM-CSF and IL-6
| The reagent title | Reagent concentration (μ g/ml) | ???G-CSF ???(S/C) | ???GM-CSF ????(S/C) | ????IL-6 ????(S/C) |
| Embodiment 1 | ????200 | ????29.2 | ????8.7 | ????430.6 |
| Astragalus polysaccharides | ????200 | ????24.0 | ????9.5 | ????280.2 |
| Embodiment 1+ astragalus polysaccharides | ????100+ ????100 | ????35.1 | ????14.8 | ????650.8 |
| Polyporusum bellatus | ????400 | ????6.4 | ????5.4 | ????33.9 |
As can be seen from the above results: when matrimony vine AGPs and astragalus polysaccharides share, stimulate the peripheral blood lymphocytes with PHA activatory people to produce IL-6, the ability of GM-CSF and G-CSF strengthens.
Embodiment 5
Matrimony vine AGPs and G-CSF share stimulates the Balb/c mouse to produce
The ability of GM-CFC and BFU-E obviously strengthens.
Reagent is divided into 4 groups: the 1st group. and embodiment 1, consumption 50mg/kg/d.The 2nd group of .G-CSF (recombinant methionyl human G-CSF. trade(brand)name: Hui Er blood. kylin roc (China) Bioceuticals Inc.) consumption 100 μ g/kg/d.The 3rd group. embodiment 1+G-CSF, consumption are embodiment 150mg/kg/d, G-CSF100 μ g/kg/d.The 4th group. placebo.
Female Balb/c mouse, body weight 18-22g, random packet, 5 every group.The subcutaneous injection administration, once a day, successive administration 7 days.Get peripheral blood, separating monocytic cell, it is 1 * 10 that cell count is adjusted with the substratum that contains EPO (erythropoietin), IL-3, IL-6 and STEM CELL FACTOR etc. in the washing back
5Individual/ml, add the culture dish of 35mm successively, every ware adds 1.0ml.5%CO
2Incubator, 37 ℃ of saturated humidities were cultivated 7 days, contained the colony (BFU-E) of 50 cells at the microscopically counting.Each ware adds staining agent 1ml, 5%CO
2Incubator, 37 ℃ of saturated humidities were cultivated 3.5 hours, at the brown cell colony (GM-CFC) of microscopically counting.
Following table shows that matrimony vine AGPs and G-CSF share stimulation Balb/c mouse and produce
The ability of GM-CFC and BFU-E obviously strengthens.
As can be seen from the above results: obviously strengthen when matrimony vine AGPs and G-CSF share the ability that stimulates the Balb/c mouse to produce GM-CFC and BFU-E.
Embodiment 6
Matrimony vine AGPs 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 25mg/kg/d.The 2nd group is embodiment 1, and consumption is 50mg/kg/d.The 3rd group is embodiment 1, and consumption is 100mg/kg/d.The 4th group is LBP, and consumption is 100mg/kg/d.The 5th group is placebo.The 6th group is the normal control group.
1-5 group mouse was accepted x-ray bombardment at 0 day, irradiation dose is 500cGy.Began the subcutaneous injection administration same day from shining, once a day, one week of continuous use.Each organizes per 3 days tail vein bloods of mouse 1 time, counting peripheral blood leucocyte (WBC), and red corpuscle (RBC) and thrombocyte (PLT) observed for 4 weeks continuously.
Peripheral blood WBC (* 10
9/ L) variation
Peripheral blood RBC (* 10
12/ L) variation
Peripheral blood PLT (* 10
9/ L) variation
As can be seen from the above results: the recovery of the 1st group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 4 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 3 days than control group, P＜0.05.The recovery of the 2nd group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of the 3rd group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of the 4th group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 4 days than control group, P＜0.01.The recovery of PLT is similar to control group, P＞0.05.
As can be seen from the above results: when the consumption of matrimony vine AGPs during, can promote to accept the recovery of radiotherapy mouse peripheral hemogram, particularly promote the recovery of PLT and WBC, and situation and the dosage recovered be proportionate at 25-100mg/kg/d.LBP has only the effect of the recovery that promotes WBC, and effect is worse than the matrimony vine AGPs of Isodose.
Embodiment 7
Matrimony vine AGPs and Radix Angelicae Sinensis polysaccharide share promotion
Radiotherapy mouse peripheral hemogram recovers comprehensively
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 100mg/kg/d.The 2nd group is Radix Angelicae Sinensis polysaccharide, and consumption is 100mg/kg/d.The 3rd group is embodiment 1+ Radix Angelicae Sinensis polysaccharide, and consumption is each 50mg/kg/d.The 4th group is placebo.The 5th group is the normal control group.
1-4 group mouse was accepted x-ray bombardment at 0 day, irradiation dose is 500cGy.Began the subcutaneous injection administration same day from shining, once a day, one week of continuous use.Each organizes per 3 days tail vein bloods of mouse 1 time, counting peripheral blood WBC, RBC and PLT.Observed for 4 weeks continuously.
Peripheral blood WBC (* 10
9/ L) variation
Peripheral blood RBC (* 10
12/ L) variation
Peripheral blood PLT (* 10
9/ L) variation
Found that: the recovery of the 1st group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of the 2nd group of RBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of WBC and PLT and control group are approximate, P＞0.05.The recovery of the 3rd group of RBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of WBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 7 days than control group, P＜0.01.
As can be seen from the above results: unite and use matrimony vine AGPs and Radix Angelicae Sinensis polysaccharide, has complementary action, both shift to an earlier date time of recovery, use shift to an earlier date the time of recovery of WBC, the PLT of Radix Angelicae Sinensis polysaccharide than independent again, can make the peripheral hemogram recovery comprehensively as early as possible of irradiation back mouse than independent RBC with matrimony vine AGPs.
Embodiment 8
Matrimony vine AGPs and astragalus polysaccharides share further promotion and accept the chemotherapy mouse
The recovery in advance of peripheral blood WBC
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 100mg/kg/d.The 2nd group is astragalus polysaccharides, and consumption is 100mg/kg/d.The 3rd group is embodiment 1+ astragalus polysaccharides, and consumption is each 50mg/kg/d.The 4th group is placebo.The 5th group is the normal control group.
1-4 group mouse was accepted ametycin, tail intravenously administrable, dosage 3.5mg/kg/d at 0 day.Continuous use 3 days.Chemotherapy began the subcutaneous injection administration same day, once a day, and continuous use 14 days.Each organizes per 3 days tail vein bloods of mouse 1 time, counting peripheral blood WBC, RBC and PLT.Observed for 4 weeks continuously.
Peripheral blood WBC (* 10
9/ L) variation
Peripheral blood RBC (* 10
12/ L) variation
Peripheral blood PLT (* 10
9/ L) variation
Found that: the recovery of the 1st group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of the 2nd group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 7 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 3 days than control group, P＜0.05.The recovery of the 3rd group of RBC is similar to control group, P＞0.05.The recovery of WBC has shifted to an earlier date 11 days than control group, P＜0.01.The recovery of PLT has shifted to an earlier date 7 days than control group, P＜0.01.
As can be seen from the above results: unite and use matrimony vine AGPs and astragalus polysaccharides, compare with the two independent use group, both had complementary action, make the WBC that accepts mouse after the chemotherapy, PLT all recovers in advance.And the recovery to WBC has synergism.
Embodiment 9
Matrimony vine AGPs and G-CSF share promotion
Chemotherapy mouse peripheral hemogram recovers comprehensively
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 100mg/kg/d.The 2nd group is G-CSF, (recombinant methionyl human G-CSF. trade(brand)name: Hui Er blood. kylin roc (China) Bioceuticals Inc.) consumption is 3 μ g/kg/d.The 3rd group is embodiment 1+G-CSF, and consumption is embodiment 1 100mg/kg/d, G-CSF 3 μ g/kg/d.The 4th group is placebo.The 5th group is the normal control group.
1-4 group mouse was accepted ametycin, tail intravenously administrable, dosage 3.5mg/kg/d at 0 day.Continuous use 3 days.Chemotherapy began the subcutaneous injection administration same day, once a day, and continuous use 14 days.Each organizes per 3 days tail vein bloods of mouse 1 time, counting peripheral blood WBC, RBC and PLT.Observed for 4 weeks continuously.
Peripheral blood WBC (* 10
9/ L) variation
Peripheral blood RBC (* 10
12/ L) variation
Peripheral blood PLT (* 10
9/ L) variation
As can be seen from the above results: when matrimony vine AGPs and G-CSF drug combination, compare with independent use G-CSF or matrimony vine AGPs, have synergism, white corpuscle, hematoblastic recovery have all shifted to an earlier date 3-4 days, and erythrocytic recovery has also shifted to an earlier date.
Embodiment 10
Matrimony vine AGPs induces normal mouse and endoxan chemotherapy mouse boosting cell to produce
IL-2, IL-4, IL-3, IL-6 and IFN-γ
The Balb/c male mice, body weight 18-22g, random packet, 8 every group.1st, 2 groups since the 1st day subcutaneous injection embodiment 1, and consumption is 100mg/kg/d, successive administration 7 days.The 3rd group since the 1st day subcutaneous injection polyporusum bellatus, and consumption is 100mg/kg/d, and successive administration 7 days is as positive control.The 4th group since the 1st day subcutaneous injection LBP, and consumption is 100mg/kg/d, successive administration 7 days.5th, 6 groups since the 1st day subcutaneous injection equal-volume physiological saline, successive administration 7 days.1,3,4,5 groups of the 4th day intraperitoneal injection of cyclophosphamide 200mg/kg, all mouse are put to death, the preparation splenocyte suspension in injection the 5th day behind the endoxan.With 24 porocyte culture plates, add each 1ml of splenocyte suspension (final concentration of cells: 5 * 10 respectively
6/ ml), ConA 1ml (final concentration is 2.5 μ g/ml) or OVA 1ml ((final concentration is 200 μ g/ml), 5%CO
2, cultivated 48 hours for 37 ℃, centrifugal, get supernatant liquor, detect the content of cytokine with the ELASA method.The results are shown in following table:
Table 1
| Developing medium | ????????????????OVA | ???????????????ConA | ||||
| Grouping | IL-2 (pg/ml) (mean) | IL-2 (standard deviation) | IL-2 (P value) | IL-3 (pg/ml) (mean) | IL-3 (standard deviation) | IL-3 (P value) |
| ????6 | ???11.26 | ???0.43 | ??100.33 | ????2.49 | ||
| ????2 | ???9.06 | ???0.30 | ??1061.22 | ????19.24 | ??? *＜0.01 | |
| ????5 | ???7.48 | ???0.13 | ??186.7 | ????4.56 | ||
| ????1 | ???12.17 | ???0.32 | ?? **＜0.01 | ??2487.09 | ????1.3 | ??? **＜0.01 |
| ????3 | ???10.04 | ???0.16 | ??903.24 | ????6.59 | ||
| ????4 | ???8.00 | ???0.24 | ?? ***＜0.01 | ??242.33 | ????3.10 | ??? ***＜0.01 |
Remarks:
*Represent that the 2nd group and the 6th group is compared.
*Represent that the 1st group and the 5th group is compared.
* *Represent that the 1st group and the 4th group is compared.
Table 2
| Developing medium | ?????????????????ConA | ????????????????ConA | ||||
| Grouping | IL-4 (pg/ml) (mean) | IL-4 (standard deviation) | IL-4 (P value) | IFN-γ (pg/ml) (mean) | IFN-γ (standard deviation) | IFN-γ (P value) |
| ????6 | ???19.33 | ????0.92 | ???8534.60 | ????110.64 | ||
| ????2 | ???328.62 | ????8.35 | ?? *＜0.01 | ???11532.73 | ????316.35 | ?? *＜0.01 |
| ????5 | ???78.38 | ????0.62 | ???2402.98 | ????44.31 | ||
| ????1 | ???446.13 | ????19.84 | ?? **＜0.01 | ???4717.03 | ????451.04 | ?? **＜0.01 |
| ????3 | ???191.11 | ????3.95 | ???7788.60 | ????230.09 | ||
| ????4 | ???98.45 | ????4.15 | ?? ***＜0.01 | ???2899.21 | ????340.12 | ?? ***＜0.01 |
Remarks:
*Represent that the 2nd group and the 6th group is compared.
*Represent that the 1st group and the 5th group is compared.
* *Represent that the 1st group and the 4th group is compared.
Table 3
| Developing medium | ????????????????ConA | ||
| Grouping | IL-6 (pg/ml) (mean) | IL-6 (standard deviation) | IL-6 (P value) |
| ????6 | ????608.76 | ????7.14 | |
| ????2 | ????1051.98 | ????16.14 | ?? *＜0.01 |
| ????5 | ????109.62 | ????0.58 | |
| ????1 | ????450.09 | ????6.76 | ?? **＜0.01 |
| ????3 | ????298.01 | ????11.48 | |
| ????4 | ????179.09 | ????9.28 | ?? ***＜0.01 |
Remarks:
*Represent that the 2nd group and the 6th group is compared.
*Represent that the 1st group and the 5th group is compared.
* *Represent that the 1st group and the 4th group is compared.
As can be seen from the above results: matrimony vine AGPs can induce normal mouse to produce IL-4, IL-3, IL-6 and IFN-γ.Matrimony vine AGPs also can induce endoxan chemotherapy mouse boosting cell to produce IL-2, IL-4, IL-3, IL-6 and IFN-γ.And growing amount is apparently higher than isodose LBP.
Embodiment 11
Matrimony vine AGPs improves S-180 tumor-bearing mice spleen nk cell activity, stimulates S-180 lotus knurl
Mouse boosting cell produces interleukin II (IL-2).
The Balb/c male mice, body weight 18-22g, random packet, 5 every group.1. normal control group.2.S-180 control group.3. matrimony vine AGPs administration group.4.LBP administration group.5. polyporusum bellatus administration group (positive control).First day, except that the 1st group, each mouse oxter intradermal injection 1 * 10
6/ ml S-180 cell 0.2ml.Next day the 3rd group of subcutaneous injection matrimony vine AGPs, the 4th group of subcutaneous injection LBP, the 5th group of subcutaneous injection polyporusum bellatus, dosage is 100mg/kg/d, continuous use 7 days.The 8th day, put to death all mouse, the preparation splenocyte suspension.
1.NK cell activity is measured:, add each 100 μ l (final concentration of cells: 5 * 10 of splenocyte suspension respectively with 96 porocyte culture plates
5/ ml) and
3The target cell 100 μ l (final concentration of cells: 5 * 10 of H-TdR mark
5/ ml), only add the target cell 200 μ l (final concentration of cells: 5 * 10 of 3H-Tymidine mark
5/ ml) hole is a blank.At 5%CO
2, hatched 4 hours in 37 ℃ of incubators, collect with cell harvestor, read the cpm value at the γ liquid scintillation instrument, calculate specific killing per-cent.
0. the active mensuration of interleukin II (IL-2):, add each 1ml of splenocyte suspension (final concentration of cells: 5 * 10 respectively with 24 porocyte culture plates
6/ ml), add ConA 1ml (final concentration is 2.5 μ g/ml), 5%CO
2, cultivated 48 hours for 37 ℃, centrifugal, get supernatant liquor, detect the content of IL-2 with the ELASA method.
The results are shown in following table:
| Grouping | The NK cell-specific kills and wounds % | The IL-2 activity | ||||
| Mean | Standard deviation | The P value | Mean (pg/ml) | Standard deviation | The P value | |
| ????1 | ????4.10 | ????0.8 | ???533.13 | ????0.67 | ||
| ????2 | ????4.10 | ????5.4 | ???475.45 | ????2.06 | ||
| ????3 | ????12.75 | ????1.2 | ??? *＜0.01 | ???619.53 | ????5.42 | ??? *＜0.01 |
| ????4 | ????6.1 | ????3.8 | ??? **＜0.01 | ???519.47 | ????6.45 | ??? **＜0.01 |
| ????5 | ????8.4 | ????2.2 | ???517.58 | ????7.81 | ||
*Represent that the 2nd group and the 3rd group is compared.
*Represent that the 3rd group and the 4th group is compared.
As can be seen from the above results: matrimony vine AGPs has improved S-180 tumor-bearing mice spleen nk cell activity, stimulates S-180 tumor-bearing mice splenocyte to produce interleukin II (IL-2).And effect is apparently higher than isodose LBP.
Matrimony vine AGPs can be used for the treatment of neutrophilic granulocytopenia, anaemia, thrombocytopenia separately and as anticancer adjuvant according to above result.Or with astragalus polysaccharides, Radix Angelicae Sinensis polysaccharide or G-CSF combination, to play the effect of synergy.
Claims (12)
1. the Arabinogalactan-Protein (Arabinogalactan proteins) that extraction from lycium barbarum (Lycium barbarum L.), separation, purifying obtain is called for short matrimony vine AGPs.
2. the weight-average molecular weight of the described matrimony vine AGPs of claim 1 is 30,000-100,000 dalton.
3. claim 1 and 2 described matrimony vine AGPs is characterized in that possessing the chemical characteristic of higher plant AGPs: 1. polysaccharide: albumen=80-95%:5-20%.2. sugared compositing characteristic: mainly contain pectinose (Ara) and semi-lactosi (Gal), both molar percentage sum＞50%.3. sugar chain: Ara is mainly 5-, 2,5-and 3, and 5-connects.Gal is mainly 6-and 3, and 6-connects.4. amino acid (AA) compositing characteristic: oxyproline (Hyp): 15-20%, Serine (Ser): 10-20%, Threonine (Thr): 5-10%, L-glutamic acid (Glu): 5-10%, glycine (Gly): 5-10%, aspartic acid (Asp): 5-10%, L-Ala (Ala): 5-10%.5. weight-average molecular weight: 30,000-100,000 dalton.6. solvability: water-soluble.
4. the described matrimony vine AGPs of claim 1 to 3, content＞80%.
5. extraction, separation, the purification process of the described matrimony vine AGPs of claim 1 to 4, it is characterized in that may further comprise the steps: wolfberry fruit water is carried, the 30-45% ethanol sedimentation, collect supernatant liquor, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 10K, keep liquid through cationic exchange coloum and anion-exchange column, buffer solution elution with pH5-6,30-50mM, collect effluent liquid, effluent liquid is the ultra-filtration membrane ultrafiltration of 5K through molecular weight cut-off, gained keeps liquid through 70% ethanol sedimentation twice after the ultrafiltration, and the gained throw out is described matrimony vine AGPs.
6. the described method of claim 5 is characterized in that ion exchange resin used in preparation process is SP Sepharose and DEAE Sepharose ion exchange resin.
7. according to claim 5 or 6 described methods, it is characterized in that: preparing matrimony vine AGPs post precipitation, add water, obtain purified matrimony vine AGPs through spraying or lyophilize with resolution of precipitate through 2 times 70% alcohol precipitation processes.
8. containing the described matrimony vine AGPs of arbitrary claim injection among the with good grounds claim 1-7, is to add one or more pharmacy acceptable auxiliary materials by described matrimony vine AGPs to make, and contains matrimony vine AGPs 30mg in each injected dose unit at least.
According to the described matrimony vine AGPs of arbitrary claim among the claim 1-7 at the medicine of preparation treatment neutrophilic granulocytopenia, anaemia, thrombocytopenia with prepare purposes in the anticancer ancillary drug.
10. purposes according to claim 9, it is characterized in that described anticancer ancillary drug for put, the chemotherapy adjuvant drug.
11. a composition wherein contains the described matrimony vine AGPs of with good grounds claim 1-4, and is selected from astragalus polysaccharides, the Radix Angelicae Sinensis polysaccharide any one, the weight proportion of matrimony vine AGPs and described vegetable polysaccharides is 1: 0.5-2.
12. a composition wherein contains described matrimony vine AGPs of with good grounds claim 1-4 and G-CSF, the weight proportion of matrimony vine AGPs and G-CSF is 1: 0.000010-0.000030.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020719B (en) * | 2006-02-14 | 2011-01-12 | 北京美迪克斯生物技术有限公司 | Composite angelica polysaccharide and its preparation process and use |
| CN108976311A (en) * | 2018-08-06 | 2018-12-11 | 南京林业大学 | A method of separating the arabogalactan of different glucuronic acid base contents |
-
2004
- 2004-03-29 CN CN 200410029817 patent/CN1676530A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020719B (en) * | 2006-02-14 | 2011-01-12 | 北京美迪克斯生物技术有限公司 | Composite angelica polysaccharide and its preparation process and use |
| CN108976311A (en) * | 2018-08-06 | 2018-12-11 | 南京林业大学 | A method of separating the arabogalactan of different glucuronic acid base contents |
| CN108976311B (en) * | 2018-08-06 | 2020-06-30 | 南京林业大学 | Method for separating arabinogalactans with different glucuronic acid group contents |
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