CN1834108B - Acanthopanax giraldii Harms compound polysaccharide, preparation technique, uses and its compound polysaccharide composition - Google Patents
Acanthopanax giraldii Harms compound polysaccharide, preparation technique, uses and its compound polysaccharide composition Download PDFInfo
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- CN1834108B CN1834108B CN2005100534940A CN200510053494A CN1834108B CN 1834108 B CN1834108 B CN 1834108B CN 2005100534940 A CN2005100534940 A CN 2005100534940A CN 200510053494 A CN200510053494 A CN 200510053494A CN 1834108 B CN1834108 B CN 1834108B
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Abstract
This invention relates to a kind of composite polysaccharide that is extracted, separated and purified from the stem skin of acanthopanax giraldii harms, has a weight-average molecular weight of 20000~40000 and mainly comprises Arabinogalactan proteins (AGPs), Rhamnogalacturonan Is (RGI), Homogalacturonans (HGs) and Arabinogalactans (AGs). A proper amount of acanthopanax giraldii harms composite polysaccharide is taken and one or more species of pharmacologically permitted agent are added to produce injections. Each injection unit contains at least 30mg of the substance in this invention. It is applicable in the therapy of neutropenia, anemia, pancytopenia and virus-infected diseases and can be used as anti-cancer agent. It can be taken independently or coupled with other polysaccharides or G-CSF.
Description
The present invention relates to field of traditional Chinese medicine pharmacy.Be specifically related to a kind ofly from girald acanthopanax bark stem skin, extract, the weight-average molecular weight that purifying obtains is 20,000-40, between 000 dalton, mainly by Arabinogalactan-Protein (AGPs), rhamnosyl galacturonic acid glycan I (RG I), complex polysaccharide and preparation technology thereof that galacturonic acid glycan (HGs) and arabogalactan (AGs) are formed, preparation and uses thereof, the application also provides described girald acanthopanax bark complex polysaccharide and Radix Astragali extract simultaneously, Radix Angelicae Sinensis extract, the composition of Radix Glycyrrhizae extract or G-CSF can make each component synergy by combination.
Girald acanthopanax bark (Acanthopanax giraldii, Harms) be the perennial machaka of Araliaceae Acanthopanax, grow in the bushes of height above sea level 1300-3500 rice, mainly be distributed in provinces such as Shaanxi, Gansu, Sichuan, Ningxia, Qinghai, Henan, Hubei, medicinal part is the stem skin.
The traditional Chinese medical science is thought: Cortex Acanthopanacis Giraldii nature and flavor suffering, temperature, effect are that wind dispelling wets, gets round by bribery, strengthening the bones and muscles, are used for the treatment of contraction pain, arthralgia due to wind-cold-dampness pathogen BI syndrome.Along with modern medicine, pharmacy and development of molecular biology, people have also had further understanding for the pharmacological action of girald acanthopanax bark stem skin.Guo Hui etc. (northwest pharmaceutical journal .1996.3 (11) .117-119), (Chinese materia medica research .14. (1) .8-9) shows the pharmacological research of girald acanthopanax bark stem skin aqueous extract: it is low that girald acanthopanax bark stem skin aqueous extract can improve the immune function of mice that endoxan causes, and improves the level of tumor-bearing mice blood plasma tumour necrosis factor (TNF).Open and to arrive gorge etc. (Chinese TCM basis medical journal .1999.5 (3) .25-27) girald acanthopanax bark stem skin Crude polysaccharides has been carried out the research of antiviral activity aspect, found that it is to vesicular stomatitis virus (VSV), herpes simplex virus I-type (HSV-1) and Coxsackie virus B
3Type (CB
3V) the obvious suppression effect is all arranged.Chen Ping etc. (Pharmacology and Clinics of Chinese Materia Medica .1994. (3) .17-19) have carried out antagonism Zidovodine (AZT) to girald acanthopanax bark stem skin Crude polysaccharides and suppressed mouse hemopoietic function and the discovery of immunoreactive pharmacological research: girald acanthopanax bark stem skin aqueous extract Crude polysaccharides can resist mouse hematopoiesis and the immunosuppressant toxic reaction that AZT causes, its peripheral blood leucocyte, marrow hemopoietic stem cells and grain, monosystem progenitor cell number are increased, the splenic T lymphopoiesis, the content of interleukin II (IL-2) raises.Therefore, discussion is used for green blood with acanthopanax giraldii harms polysaccharose as efficient part, improves immunologic function and anti-virus infection etc., and prospect is arranged very much.
The invention provides and a kind ofly from girald acanthopanax bark stem skin, extract, the weight-average molecular weight that purifying obtains is 20,000-40, between 000 dalton, mainly by Arabinogalactan-Protein (AGPs), rhamnosyl galacturonic acid glycan I (RGIs), the complex polysaccharide that galacturonic acid glycan (HGs) and arabogalactan (AGs) are formed, its sugar is formed (Mole%): pectinose (Ara): 28.9, rhamnosyl (Rha): 8.93, wood sugar (Xyl): 1.57, galacturonic acid (GalA): 36.0, semi-lactosi (Gal): 19.1, glucose (Glc): 4.31,4-methoxyl group glucuronic acid (4-o-me-GlcA): 1.20.Polysaccharide content is more than 95%., detect with molecular sieve gel filtration high performance liquid chromatography-RI-HPLC as the polysaccharide standard reference material with the blue polysaccharide series in general Shandong, its weight-average molecular weight is 20,000-40,000 dalton.
The main technique flow process is as follows: after girald acanthopanax bark stem skin drying is cleaned, add water 100 ℃ of extractions (amount of water is 4-5 times, extracts 1.5 hours, extracts altogether 3 times) at every turn.Extracting solution 60-65 ℃ is evaporated to rare thick paste shape, carries out the 30-40% alcohol precipitation after the cooling, and 4 ℃ left standstill 8-12 hour.Collect supernatant liquor, spraying drying promptly gets girald acanthopanax bark complex polysaccharide crude product.With girald acanthopanax bark complex polysaccharide 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.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 the girald acanthopanax bark complex polysaccharide, and polysaccharide content is more than 95%.
Or collect the supernatant liquor of girald acanthopanax bark water extract behind alcohol precipitation, reclaiming behind the ethanol with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 10K and later step, obtains the girald acanthopanax bark complex polysaccharide equally.
The present invention also provides the preparation method of girald acanthopanax bark complex polysaccharide injection: get an amount of girald acanthopanax bark complex polysaccharide, 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.
Girald acanthopanax bark complex polysaccharide provided by the invention is used for the treatment of neutrophilic granulocytopenia, anaemia, thrombocytopenia, disease of viral infection and as anticancer adjuvant.Can be independent medication, also can with medication combined medications such as other polysaccharide or G-CSF.
Girald acanthopanax bark complex polysaccharide provided by the invention in the pharmacological action of green blood, enhance immunity function aspects is: the result of experiment in vitro shows, when using the girald acanthopanax bark complex polysaccharide separately, its concentration can stimulate the peripheral blood lymphocytes with phytohaemagglutinin (PHA) activatory people to produce interleukin 1 β (IL-1 β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), grain/macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) when 50-400ug/ml effectively.
After girald acanthopanax bark complex polysaccharide and astragalus polysaccharides, Radix Angelicae Sinensis polysaccharide, licorice polysaccharide or the G-CSF combination, can bring into play synergistic function.Hot hair slender acanthopanax complex polysaccharide and astragalus polysaccharides (with reference to sealing Silan etc. the astragalus polysaccharides process study. the technology among Chinese patent medicine .1997.19 (12) .5-6. is also improved the back and is extracted, purifying and getting, polysaccharide content is more than 95%) drug combination, girald acanthopanax bark complex polysaccharide concentration is 100ug/ml, when astragalus polysaccharides concentration is 100ug/ml, with independent use girald acanthopanax bark complex polysaccharide or astragalus polysaccharides, concentration is that 200ug/ml compares, and stimulates the ability enhancing that produces IL-6, GM-CSF and G-CSF with PHA activatory people's peripheral blood lymphocytes.Hot hair slender acanthopanax complex polysaccharide and G-CSF drug combination, girald acanthopanax bark complex polysaccharide consumption is 100mg/kg/d, the G-CSF consumption is 100ug/kg/d, with independent use girald acanthopanax bark complex polysaccharide, consumption is 100mg/kg/d, or use the G-CSF consumption to compare separately as 100ug/kg/d, 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, the Balb/C mouse of accepting radiotherapy is used the girald acanthopanax bark complex polysaccharide separately, when dosage is 50-200mg/kg/d, the subcutaneous injection administration, used 7 days continuously, compare with placebo, 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 the splenic T lymphopoiesis.Hot hair slender acanthopanax complex polysaccharide and astragalus polysaccharides drug combination, dosage is girald acanthopanax bark complex polysaccharide 100mg/kg/d, astragalus polysaccharides 100mg/kg/d, when usage is the same, with independent use girald acanthopanax bark complex polysaccharide or astragalus polysaccharides, dosage is that the 200mg/kg/d group is compared, and leukocytic recovery has shifted to an earlier date 2-4 days.Hot hair slender acanthopanax complex polysaccharide 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. also improve the back extraction, purifying and getting.Polysaccharide content is more than 95%) during drug combination, dosage is girald acanthopanax bark complex polysaccharide 100mg/kg/d, Radix Angelicae Sinensis polysaccharide 100mg/kg/d, when usage is the same, with independent use girald acanthopanax bark complex polysaccharide or Radix Angelicae Sinensis polysaccharide, dosage is that the 200mg/kg/d group compares, and white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date.Hot hair slender acanthopanax complex polysaccharide and G-CSF drug combination, dosage is girald acanthopanax bark complex polysaccharide 200mg/kg/d, G-CSF 1-3ug/kg/d, when usage is the same, with independent use G-CSF, dosage is 1-3ug/kg/d or uses the girald acanthopanax bark complex polysaccharide separately, dosage be the 200mg/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 the girald acanthopanax bark complex polysaccharide separately, when dosage is 50-200mg/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.Hot hair slender acanthopanax complex polysaccharide and astragalus polysaccharides drug combination, dosage is girald acanthopanax bark complex polysaccharide 100mg/kg/d, astragalus polysaccharides 100mg/kg/d, when usage is the same, with independent use girald acanthopanax bark complex polysaccharide or astragalus polysaccharides, dosage is that the 200mg/kg/d group is compared, and leukocytic recovery has shifted to an earlier date 2-4 days.Hot hair slender acanthopanax complex polysaccharide and Radix Angelicae Sinensis polysaccharide drug combination, dosage is girald acanthopanax bark complex polysaccharide 100mg/kg/d, Radix Angelicae Sinensis polysaccharide 100mg/kg/d, when usage is the same, with independent use girald acanthopanax bark complex polysaccharide or Radix Angelicae Sinensis polysaccharide, dosage is that the 200mg/kg/d group compares, white corpuscle, and thrombocyte and erythrocytic recovery have all shifted to an earlier date.Hot hair slender acanthopanax complex polysaccharide and G-CSF drug combination, dosage is girald acanthopanax bark complex polysaccharide 200mg/kg/d, G-CSF 1-3ug/kg/d, when usage is the same, with independent use G-CSF, when dosage is 1-3ug/kg/d or use separately girald acanthopanax bark complex polysaccharide, dosage be 200mg/kg/d group relatively, white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date 3-4 days.
Girald acanthopanax bark complex polysaccharide provided by the invention in the pharmacological action of anti-virus aspect is: the result of experiment in vitro shows, when using the girald acanthopanax bark complex polysaccharide separately, its concentration is when 50-200ug/ml, to vesicular stomatitis virus (VSV), herpes simplex virus I-type (HSV-1) and Coxsackie B virus
3Type (CB
3V) the obvious suppression effect is all arranged.Hot hair slender acanthopanax complex polysaccharide and licorice polysaccharide (with reference to Zhou Rong etc. the separation and purification of licorice polysaccharide and high performance capillary electrophoresis analysis. the method that analytical chemistry .1999.27 (2) .245. provides also improve the back extraction, purifying and getting.Polysaccharide content is more than 95%) drug combination, its concentration is when 50-200ug/ml, except to vesicular stomatitis virus (VSV), herpes simplex virus I-type (HSV-1) and Coxsackie B virus
3Type (CB
3V) all have outside the obvious suppression effect, adenovirus III type (ADVIII) and CA
16Type (CA
16V) the obvious suppression effect is also arranged.Result of experiment shows that attack malicious chick to bursal disease virus of chickens and use the girald acanthopanax bark complex polysaccharide separately, consumption used 5 days continuously at 20mg/ days in the body, continues to observe 14 days.Compare 100% survival of medication treated animal with control animals dead 60%.Suffer from dog for canine parvovirus and hundstaupe disease and use the girald acanthopanax bark complex polysaccharide separately, consumption used 3-7 days when 75-150mg/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.
Girald acanthopanax bark complex polysaccharide provided by the invention in the pharmacological action of auxiliary anticancer aspect is: give S-180 tumor-bearing mice subcutaneous injection girald acanthopanax bark complex polysaccharide, when dosage is 100-300mg/kg/d, used 14 days continuously, can significantly improve the lethality of tumor-bearing mice spleen NK cell, P＜0.01.Simultaneously, induce the tumor-bearing mice spleen cell to generate cytokines such as IL-2 and IFN-γ.The girald acanthopanax bark complex polysaccharide is added in the cell suspension of human tumor cell lines such as COlO205 and HT-29, concentration is 25-500ug/ml, at 5%CO
2Incubator was cultivated 48 hours for 37 ℃, did not see the effect of growth such as the human tumor cell line that stimulates COlO205 and HT-29.
Embodiment
Embodiment 1
The preparation of acanthopanax giraldii harms polysaccharose crude product
Girald acanthopanax bark stem skin 40kg, extracting in water three times adds water 160kg at every turn, extracts 1.5 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 10 hours for 4 ℃, collect supernatant liquor, spraying drying gets acanthopanax giraldii harms polysaccharose crude product 690g.Polysaccharide content is 16.8%.
Embodiment 2
The preparation of acanthopanax giraldii harms polysaccharose elaboration
With acanthopanax giraldii harms polysaccharose crude product 400g water dissolution, concentration is 4%, is the ultra-filtration membrane ultrafiltration of 10K with molecular weight cut-off, retaining liquid is long-pending to be 8L, and last 200 * 300mm SP Sepharose 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 6L, add 95% ethanol to final concentration 70%, placed the centrifugal 5min of 5000 * g, collecting precipitation 10 hours for 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 acanthopanax giraldii harms polysaccharose elaboration 97g (girald acanthopanax bark complex polysaccharide), and polysaccharide content is 97%.
Embodiment 3
The preparation of girald acanthopanax bark complex polysaccharide lyophilized injectable powder
Get embodiment 2 gained girald acanthopanax bark complex polysaccharide 30g, with containing 1.0% N.F,USP MANNITOL, 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 4
The girald acanthopanax bark complex polysaccharide is induced PHA activatory people's peripheral blood
Monocyte generates GM-CSF, G-CSF and IL-6
It is 10 groups that experiment is divided into: the 1st group of reagent is embodiment 1, and concentration is 50ug/ml.The 2nd group of reagent is embodiment 1, and concentration is 100ug/ml.The 3rd group of reagent is embodiment 1, and concentration is 200ug/ml.The 4th group of reagent is embodiment 1, and concentration is 400ug/ml.The 5th group of reagent is embodiment 2, and concentration is 50ug/ml.The 6th group of reagent is embodiment 2, and concentration is 100ug/ml.The 7th group of reagent is embodiment 2, and concentration is 200ug/ml.The 8th group of reagent is embodiment 2, and concentration is 400ug/ml.The 9th group of reagent is polyporusum bellatus injection (Lianyun Harbour east wind pharmaceutical factory), and concentration is 400ug/ml.As positive control.The 10th 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 4ug/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 acanthopanax giraldii harms polysaccharose of different purity, different concns
Induce and generate GM-CSF, the quantity of G-CSF and IL-6
| The reagent title | Reagent concentration (ug/ml) | G-CSF (S/C) | GM-CSF (S/C) | IL-6 (S/C) |
| Embodiment 1 | 50 | 3.9 | 0.9 | 39.1 |
| Embodiment 1 | 100 | 4.6 | 0.8 | 67.1 |
| Embodiment 1 | 200 | 8.9 | 1.9 | 87.4 |
| Embodiment 1 | 400 | 11.7 | 2.7 | 125.7 |
| Embodiment 2 | 50 | 10.8 | 2.8 | 176.4 |
| The reagent title | Reagent concentration (ug/ml) | G-CSF (S/C) | GM-CSF (S/C) | IL-6 (S/C) |
| Embodiment 2 | 100 | 16.0 | 5.0 | 330.6 |
| Embodiment 2 | 200 | 19.7 | 7.7 | 380.6 |
| Embodiment 2 | 400 | 24.7 | 10.7 | 508.1 |
| Polyporusum bellatus | 400 | 9.2 | 5.2 | 16.0 |
As can be seen from the above results: the crude product of acanthopanax giraldii harms polysaccharose and elaboration (girald acanthopanax bark complex polysaccharide) all 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 activity of elaboration is significantly higher than crude product.
Embodiment 5
Girald acanthopanax bark complex polysaccharide 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 2, and concentration is 200ug/ml.The 2nd group of reagent is astragalus polysaccharides, and concentration is 200ug/ml.The 3rd group of reagent is embodiment 2+ astragalus polysaccharides, and concentration respectively is 100ug/ml.The 4th group of reagent is polyporusum bellatus injection (Lianyun Harbour east wind pharmaceutical factory), and concentration is 400ug/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 4ug/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 girald acanthopanax bark complex polysaccharide and astragalus polysaccharides share reinforcement and induce generation
G-CSF, GM-CSF and IL-6
| The reagent title | Reagent concentration (ug/ml) | G-CSF (S/C) | GM-CSF (S/C) | IL-6 (S/C) |
| Embodiment 2 | 200 | 19.7 | 7.7 | 380.6 |
| Astragalus polysaccharides | 200 | 14.8 | 8.5 | 410.2 |
| Embodiment 2+ astragalus polysaccharides | 100+ 100 | 25.1 | 12.8 | 690.8 |
| Polyporusum bellatus | 400 | 7.4 | 4.4 | 23.9 |
As can be seen from the above results: when hot hair slender acanthopanax complex polysaccharide 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 6
Girald acanthopanax bark complex polysaccharide 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 2, consumption 100mg/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 100ug/kg/d.The 3rd group. embodiment 2+G-CSF, consumption are embodiment 2100mg/kg/d, G-CSF 100ug/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 girald acanthopanax bark complex polysaccharide 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: hot hair slender acanthopanax complex polysaccharide and G-CSF share the ability that stimulates the Balb/c mouse to produce GM-CFC and BFU-E obviously to be strengthened.
Embodiment 7
The girald acanthopanax bark complex polysaccharide 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 2, and consumption is 50mg/kg/d.The 2nd group is embodiment 2, and consumption is 100mg/kg/d.The 3rd group is embodiment 2, and consumption is 200mg/kg/d.The 4th group is placebo.The 5th group is the normal control group.
Each is organized mouse and accepted x-ray bombardment at 0 day, and 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.
Following table shows peripheral blood WBC (* 10
9/ L) variation
Following table shows peripheral blood RBC (* 10
12/ L) variation
Following table shows peripheral blood PLT (* 10
9/ L) variation
As can be seen from the above results: the recovery of the 1st group of RBC has shifted to an earlier date 3 days than 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 has shifted to an earlier date 3 days than 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 has shifted to an earlier date 3 days than 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.
As can be seen from the above results: the consumption of hot hair slender acanthopanax complex polysaccharide can promote to accept the recovery of radiotherapy mouse peripheral hemogram when 50-200mg/kg/d, particularly promote the recovery of PLT and WBC, and situation and the dosage recovered are proportionate.
Embodiment 8
Girald acanthopanax bark complex polysaccharide 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 2, and consumption is 200mg/kg/d.The 2nd group is Radix Angelicae Sinensis polysaccharide, and consumption is 200mg/kg/d.The 3rd group is embodiment 2+ Radix Angelicae Sinensis polysaccharide, and consumption is each 100mg/kg/d.The 4th group is placebo.The 5th group is the normal control group.
Each is organized mouse and accepted x-ray bombardment at 0 day, and 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.
Following table shows peripheral blood WBC (* 10
9/ L) variation
Following table shows peripheral blood RBC (* 10
12/ L) variation
Following table shows peripheral blood PLT (* 10
9/ L) variation
Found that: the recovery of the 1st group of RBC has shifted to an earlier date 4 days than 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 girald acanthopanax bark complex polysaccharide and Radix Angelicae Sinensis polysaccharide, has complementary action, shift to an earlier date time of recovery than simple RBC with the girald acanthopanax bark complex polysaccharide, shift to an earlier date the time of recovery than the WBC, the PLT that use Radix Angelicae Sinensis polysaccharide separately, and the peripheral hemogram of irradiation back mouse is recovered as early as possible comprehensively.
Embodiment 9
Girald acanthopanax bark complex polysaccharide 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 2, and consumption is 200mg/kg/d.The 2nd group is astragalus polysaccharides, and consumption is 200mg/kg/d.The 3rd group is embodiment 2+ astragalus polysaccharides, and consumption is each 100mg/kg/d.The 4th group is placebo.The 5th group is the normal control group.
Each is organized mouse and 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.
Following table shows peripheral blood WBC (* 10
9/ L) variation
Following table shows peripheral blood RBC (* 10
12/ L) variation
Following table shows peripheral blood PLT (* 10
9/ L) variation
Found that: the recovery of the 1st group of RBC has shifted to an earlier date 3 days than 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 4 days than control group, P＜0.05.The recovery of the 3rd group of RBC has shifted to an earlier date 3 days than 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 girald acanthopanax bark complex polysaccharide 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 10
Girald acanthopanax bark complex polysaccharide 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 2, and consumption is 200mg/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 3ug/kg/d.The 3rd group is embodiment 2+G-CSF, and consumption is embodiment 2 200mg/kg/d, G-CSF 3ug/kg/d.The 4th group is placebo.The 5th group is the normal control group.
Each is organized mouse and 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.
Following table shows peripheral blood WBC (* 10
9/ L) variation
Following table shows peripheral blood RBC (* 10
12/ L) variation
Following table shows peripheral blood PLT (* 10
9/ L) variation
As can be seen from the above results: hot hair slender acanthopanax complex polysaccharide and G-CSF drug combination, compare with independent use G-CSF or girald acanthopanax bark complex polysaccharide, have synergism, white corpuscle, thrombocyte and erythrocytic recovery have all shifted to an earlier date 3-4 days.
Embodiment 11
Girald acanthopanax bark complex polysaccharide list with or share with licorice polysaccharide
At external antivirus action
Selected virus has 5 kinds: vesicular stomatitis virus (VSV), herpes simplex virus I-type (HSV-1), Coxsackie B virus
3Type (CB
3V), adenovirus III (ADVIII) and CA
16Type (CA
16V).Wherein, the host of VSV, HSV-1 and ADVIII selects L929 cell, CA for use
16The host of V selects Vero cell, CB for use
3The host of V selects the Hela cell for use.Reagent has 7 kinds: the 1st group is embodiment 1, and concentration is 200ug/ml.The 2nd group is embodiment 2, and concentration is 100ug/ml.The 3rd group is embodiment 2, and concentration is 200ug/ml.The 4th group is licorice polysaccharide, and concentration is 100ug/ml.The 5th group is licorice polysaccharide, and concentration is 200ug/ml.The 6th group is embodiment 2+ licorice polysaccharide, and concentration respectively is 100ug/ml.The 7th group is ribavirin, and concentration is 200ug/ml.Establish the contrast of virus control and cell simultaneously.
96 porocyte culture plate infective virus added above-mentioned soup after 2 hours, put 5%CO
2In the incubator, 37 ℃ of cultivations.Every day is the observation of cell pathology under inverted microscope.Observed 3 days continuously, write down each hole pathology situation.
Following table show girald acanthopanax bark complex polysaccharide list with or share with licorice polysaccharide
Virus is caused cytopathic restraining effect
"+" expression has pathology.The degree of how much representing pathology of its quantity.Obvious pathology is not observed in "-" expression.
As can be seen from the above results: girald acanthopanax bark complex polysaccharide concentration is when 100-200ug/ml, to VSV, HSV-1 and CB
3The cytopathy that V causes has the obvious suppression effect.And the activity of elaboration is significantly higher than crude product.Licorice polysaccharide concentration is when 100-200ug/ml, to ADVIII and CA
16The cytopathy that V causes has the obvious suppression effect.After both share, antiviral spectrum broadened.
Embodiment 12
The girald acanthopanax bark complex polysaccharide 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, 20 every group, male and female half and half.The 1st group: control group, not administration.The 2nd group: the oral administration group, embodiment 2 adds in the drinking-water, 20mg/ days.Successive administration 5 days.The 3rd group, the administered intramuscular group, embodiment 2 consumptions are 20mg/ 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 the general state of the 14th day medication group and control animals,
Mortality ratio and fabricius bursa weight
| Group | General state | Mortality ratio | Fabricius bursa weight |
| Control group | Become thin poor growth | 60％ | 5-7g |
| The oral administration group | Well, weight increase | 0 | 1-2g |
| Group | General state | Mortality ratio | Fabricius bursa weight |
| 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 the girald acanthopanax bark complex polysaccharide, and consumption can obviously alleviate the infection and the pathology of the fabricius bursa in the time of 20mg/ days, prevents that bursal disease virus of chickens from attacking the death of malicious animal.
Embodiment 13
The girald acanthopanax bark complex polysaccharide 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 1000-5000g, and vein is given embodiment 2.The animal 75mg/d of body weight 1000-2500g, the animal 150mg/d of body weight 2500-5000g, 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 1000-5000g, and therapy for treating routinely, in contrast.The 3rd group: clinical definite is 37 of the small-sized dogs of canine distemper, body weight 1000-5000g, intravenously administrable.Usage is the same.Continuous use 3-7 days.The 4th group: clinical definite is 18 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 | 17％/0 | 17％/0 |
| 2 | 35％/0 | 49％/0 | 49％/13％ |
| 3 | 19％/0 | 23％/0 | 23％/0 |
| 4 | 31％/0 | 46％/0 | 46％/16％ |
Canine distemper and Canine parvovirus infection are two kinds of serious Animal diseases, without treatment mortality ratio＞95%.Girald acanthopanax bark complex polysaccharide consumption is when 75-150mg/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.
The girald acanthopanax bark complex polysaccharide can be used for the treatment of neutrophilic granulocytopenia, anaemia, thrombocytopenia, disease of viral infection separately and as anticancer adjuvant according to above result.Or with astragalus polysaccharides, Radix Angelicae Sinensis polysaccharide, licorice polysaccharide or G-CSF combination, to play the effect of synergy.
Claims (10)
1. one kind from girald acanthopanax bark (Acanthopanax giraldii, Harms) extract in the stem skin, separate, the method of purifying complex polysaccharide, it is characterized in that may further comprise the steps: girald acanthopanax bark stem skin, water is carried, the 30-40% ethanol sedimentation, collect supernatant liquor, with molecular weight cut-off is the ultra-filtration membrane ultrafiltration of 10K, keep liquid through cationic exchange coloum, use pH5-6, the buffer solution elution of 30-50mM is collected effluent liquid, and 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 girald acanthopanax bark complex polysaccharide.
2. the described method of claim 1, elutriant is citric acid-sodium citrate damping fluid, Sodium phosphate dibasic-citrate buffer solution or acetate-sodium acetate buffer.
3. method according to claim 1 and 2 is characterized in that: preparing the complex polysaccharide post precipitation through 2 times 70% alcohol precipitation processes, add water with resolution of precipitate, obtain the purified complex polysaccharide through spraying or lyophilize.
4. the complex polysaccharide that obtains with claim 1 or 2 described methods, it is characterized in that this complex polysaccharide is made up of Arabinogalactan-Protein, rhamnosyl galacturonic acid glycan I, galacturonic acid glycan and arabogalactan, polysaccharide content is more than 95%, with the blue polysaccharide series in general Shandong as the polysaccharide standard reference material, detect with molecular sieve gel filtration high performance liquid chromatography-RI-HPLC, its weight-average molecular weight is 20,000-40,000 dalton.
5. the injection that contains the described girald acanthopanax bark complex polysaccharide of arbitrary claim among the with good grounds claim 1-4, be to add one or more pharmacy acceptable auxiliary materials by described girald acanthopanax bark complex polysaccharide to make, contain girald acanthopanax bark complex polysaccharide 30mg in each injected dose unit at least.
According to the described girald acanthopanax bark complex polysaccharide of arbitrary claim among the claim 1-4 at the medicine of preparation treatment neutrophilic granulocytopenia, anaemia, thrombocytopenia, disease of viral infection with prepare purposes in the anticancer ancillary drug.
7. purposes according to claim 6, it is characterized in that described anticancer ancillary drug for put, the chemotherapy adjuvant drug.
8. purposes according to claim 6 is characterized in that described disease of viral infection is vesicular stomatitis virus, herpes simplex virus I-type, adenovirus III type, Coxsackie B virus
3Type, CA
16Disease that type, bursal disease virus of chickens, Canine parvovirus infection cause and hundstaupe disease.
9. a composition wherein contains with good grounds right and wants 4 described girald acanthopanax bark complex polysaccharides, and is selected from astragalus polysaccharides, Radix Angelicae Sinensis polysaccharide, the licorice polysaccharide any one, and the weight proportion of girald acanthopanax bark complex polysaccharide and described vegetable polysaccharides is 1: 1.
10. a composition wherein contains girald acanthopanax bark complex polysaccharide according to claim 4 and granulocyte colony-stimulating factor, and the weight proportion of girald acanthopanax bark complex polysaccharide and granulocyte colony-stimulating factor is 1: 0.000015.
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| CN1073599A (en) * | 1992-09-26 | 1993-06-30 | 中国中医研究院基础理论研究所 | The preparation method of acanthopanax giraldii harms polysaccharose and preparation thereof |
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| CN1073599A (en) * | 1992-09-26 | 1993-06-30 | 中国中医研究院基础理论研究所 | The preparation method of acanthopanax giraldii harms polysaccharose and preparation thereof |
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