CN1328291C - New chitosan - Google Patents
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- CN1328291C CN1328291C CNB031081835A CN03108183A CN1328291C CN 1328291 C CN1328291 C CN 1328291C CN B031081835 A CNB031081835 A CN B031081835A CN 03108183 A CN03108183 A CN 03108183A CN 1328291 C CN1328291 C CN 1328291C
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Abstract
The present invention provides chitosan. The present invention is characterized in that the weight average molecular weight of the present invention is 50000 to 140000 g/mole, and the degree of deacetylation is 85% to 95%. The present invention also provides a method for preparing the chitosan by fungi and a method for treating breast cancer or cervical cancer with the chitosan.
Description
Technical field
The present invention relates to a kind of chitosan (chitosan) of novelty, prepare the method for chitosan, and use chitosan treatment method for cancer from fungi.
Background technology
Chitin be for a kind of extremely be difficult for molten poly--β-1,4-D-N-second vinegar glycosamine (Poly-β-1.4-N-acetyl-D-glucosamine).Chitosan is the chitin that takes off the acetyl form that dissolves in the acid.Chitin is prevalent in the outer skeleton structure or insect exterior skin of marine invertebrate.Chitin and chitosan all are present in the cell walls of most of zygomycetes (Zygomycetes as.).
Chitosan can be got through deacetylation by chitin, and chitin can and get from the preparation of Carapax Eriocheir sinensis or shrimp shell.Yet this preparation method is difficult to obtain the chitosan of uniform quality.And the one-tenth branch of Carapax Eriocheir sinensis or shrimp shell is along with season or environmental factors and change.Therefore, it is very difficult obtaining the stable chitosan of physico-chemical property from Carapax Eriocheir sinensis or shrimp shell.Chitosan can also be from the filamentous fungus preparation of Mucoraceae (Mucoraceae family).In this preparation process, need not carry out chemical deacetylation.Therefore, the quality of fungi chitosan is comparatively stable.
Summary of the invention
Purpose of the present invention reaches the method for preparing chitosan from fungi for a kind of chitosan of novelty is provided, and use chitosan treatment method for cancer, and chitosan is used for the treatment of the purposes of cancer.
The weight-average molecular weight of chitosan of the present invention is 50,000-140, and 000 gram/mole, and its degree of deacetylation is for being 85%-95%.
Weight-average molecular weight (M
W) be the first part (first moment) of the interior polymer weight of each molecular weight ranges to the curve of molecular weight.Its account form as shown in the formula:
Wherein, summation is to carry out at all different big or small polymer molecules (i=1 to i=∞), and N
iBe that weight is M
iThe quantity (calculating) of molecule with mole.Above-mentioned M
WValue can measure or other methods known in the art record with light scattering technique for example, sedimentation equilibrium.
Deacetylation relates to removes the ethanoyl on the chitin molecular chain, and stays amino (NH
2).Degree of deacetylation (DD) is represented free amino content in the polysaccharide molecule; it can record with any known method, for example: ninhydrin reaction (ninhydrin test), linear potential volumetry, colloid titration method, near infrared light spectrum analysis, nuclear magnetic resonance spectroscopy, hydrogen bromide volumetry, infrared spectrum analysis, ultraviolet spectrophotometry, a subdifferential (first derivative) ultraviolet spectrophotometry, enzyme assay or other means known in the art etc.
Chitosan of the present invention can from, for example, fungi makes.The fungi that is suitable for comprises the fungi of Mucoraceae, for example radiates Mucor (Actinomucor genus).The specific examples of fungi of the present invention is a soy cheese radiation mucormycosis (Actinomucor taiwanensis).Chitosan can followingly obtain: cultivate fungi at 15-24 ℃, that continues isolates chitosan from this cultivation.
Chitosan can be used for the treatment of cancer in comprising the pharmaceutical composition of pharmaceutical acceptable carrier.On the one hand, the invention provides a kind of method that is used for the treatment of mammary cancer (breast cancer).This method comprise to have in requisition for the chitosan of experimenter's administration effective dose, for example weight-average molecular weight is 50,000-140,000 gram/mole, and degree of deacetylation is the chitosan of 85%-95%.On the other hand, the invention provides the method for a kind of treatment uterine neck (cervical) cancer, by to have in requisition for the chitosan of the present invention of experimenter's administration effective dose realize.
The invention provides a kind of chitosan of novelty with antitumour activity.One or more embodiment of the present invention hereinafter is described in detail in detail.Further feature of the present invention, purpose and advantage can be clear that from following explanation and claim.
Embodiment
The present invention is based on following unexpected the discovery: the chitosan of preparing from soy cheese radiation mucormycosis can external (in vitro) suppress human uterus's neck epithelial cancer cells (Human Cervix Epithelioidcarcinoma cell, HeLa) and mankind mastopathy cell's (MCF-1) growth.
Correspondingly, the invention provides a kind of method for preparing chitosan from fungi.This method relates at 15-24 ℃ carries out fungus culture and isolate chitosan from this fungus culture.
Can be used for the fungi that fungi of the present invention comprises Mucoraceae (for example radiating Mucor).These fungies can be obtained by various disclosed fungi preservations storehouse.For example: employed soy cheese radiation mucormycosis can preserve and research centre (Bioresource Collection andResearch Center from Biological resources among the following embodiment, BCRC, BCRC31159), this Biological resources are preserved and the research centre is to be under the jurisdiction of Foodstuff Industrial and Development Inst., and it is positioned at No. 331, the Xinzhu City food road of TaiWan, China.
Can cultivate fungi according to methods known in the art.For example: fungi is inoculated in the YM nutrient agar, inoculation is had the cultivation of fungi cultivated 3 to 6 based on 25-37 ℃.Above-mentioned mycetogenetic spore is suspended in the liquid, obtains every milliliter 10
4-10
7The bacterium liquid of bacterium colony unit (cfu/ml).With this bacterium liquid direct inoculation on fermention medium.
The initial pH value of this fermention medium can be pH3-6, pH3-5 for example, and it can comprise: glucose 5-50g/L (for example 20g/L); Peptone (Peptone) 5-60g/L (for example 10g/L); Yeast extract 0.1-5g/L (for example 1 g/L), and other proper composition.This substratum can further comprise 0.01-30g/L (NH
4)
2SO
40-3g/L K
2HPO
40-3g/L NaCl; 0-15g/LMgSO
47H
2O; 0-0.3g/L CaCl
2Fungi was further cultivated 2 to 4 in 15-24 ℃.
Chitosan can separation and purification from hypha,hyphae goes out with alkaline purification and acid treatment according to the described method of United States Patent (USP) 6255085B1.Cell mass is separated from fermented liquid, cleaned with distilled water again.Then cell is handled with the 0.5N-2N sodium hydroxide solution, this alkali mixed solution is placed 121 ℃ of insulations 15 minutes.Make precipitation of solid material with centrifugation, and clean the centrifugal solid precipitation that draws with distilled water and ethanol.To handle with 2% acetum through this solid sediment that cleans, place 95 ℃ of insulations 12 hours.The suspension that above-mentioned steps is obtained separates with centrifugation, can obtain the acid-solubility supernatant liquor.With the 2N sodium hydroxide solution pH value of this supernatant liquor is adjusted to pH10, makes it possible to chitosan is precipitated and isolated.At last, the chitosan that this is purified into is cleaned postlyophilization with distilled water.
Weight-average molecular weight (the M of chitosan
W) and degree of deacetylation (DD) can be respectively with for example GPC/SEC
3Reach a subdifferential ultraviolet spectrophotometry and record, or utilize any methods known in the art measurement to learn.As described in embodiment hereinafter, utilize the prepared fungi chitosan of aforesaid method, its M
WBe 50,000-140,000 gram/mole, its DD are 85%-95%.
Be 10ppm concentration (about 10 unexpectedly
-7M) fungi chitosan has potent cytotoxic activity to HeLa cell and the growth of MCF-7 cell, and the crust chitosan does not then show this effect.And the fungi chitosan is for human embryos pneumonocyte (MRC-5) or other tumor cell line, for example: human gastric tumor clone (AGS) and human liver cancer clone (Hep G2) is cytotoxic activity not.
Therefore, the invention provides a kind of pharmaceutical composition, it comprises the chitosan of pharmaceutically acceptable carrier and effective dose; wherein the weight-average molecular weight of this chitosan is 50; 000-140,000 gram/mole, and degree of deacetylation is 85%-95%.This pharmaceutical composition can be used for the treatment of or Breast Cancer Prevention or cervical cancer.This pharmaceutically acceptable carrier comprises: solvent, dispersion medium, dressing, antibacterial agent and anti-mycotic agent, isotonic agent (isotonic) and absorption delayer.Above-mentioned effective dose refers to for reaching the required dosage of result of treatment.Be used for animal and human drug dose (every square metre of required milligram quantities of body surface area) as (1996) such as Freireich as described in the Cancer Chemother.Rep.50:219.Body surface area is can be from experimenter's body long and weight is approximate records, and sees ScientificTables, Geigy Pharmaceuticals, and Ardley, New York, 1970 p537 is described.Yet as is known to the person skilled in the art, so-called effective dose also may be different because of factor such as administering mode and vehicle (excipient) usage.
Chitosan of the present invention can use various ordinary methods to be mixed with the medicine type that is suitable for different way of administration.For example, it can be mixed with and be used for oral capsule, glue envelope (gel seal) or tablet.Wherein, capsule can comprise acceptable material on any conventional pharmaceutical, for example gelatin (gelatin) or Mierocrystalline cellulose.Tablet can be according to conventional preparation method, and chitosan and solid carrier and lubricant pressing are formed.The example of solid carrier can be starch or sugared bentonite (sugar bentonite).Chitosan of the present invention can also be with duricrust tablet or capsular form administration, and it comprises: such as wedding agents such as lactose or mannitols, and conventional fillers and sheetization (tableting) agent.This pharmaceutical composition can be via the parenteral route administration.Above-mentioned parenteral formulations can be the aqueous solution of active agents, isotonic saline solution or 5% glucose, or the acceptable excipient of other any known drug.Cyclodextrin (cyclodextrin) or other solvating agent well known in the art all can be used as the drug excipient that is used to transmit the treatment preparation.
The curative effect of pharmaceutical composition of the present invention can (in vivo) and external (in vitro) be assessed in body.Referring to following embodiment.In brief, the effect of described composition inhibition cervical cancer or growth of breast cancers can be tested external.With regard to research in the body, can inject this pharmaceutical composition to animal, estimate the cancer resistant effect of this pharmaceutical composition then.According to these results, can determine route of administration and suitable dosage range.
The present invention also provides a kind of method that is used to prevent and treat mammary cancer or cervical cancer, its to have in requisition for the chitosan (for example above-mentioned fungi chitosan) of experimenter's administration effective dose.The experimenter who accepts this treatment can be the cancer patients, can also be because inherited genetic factors or environmental factors and easily suffer from breast cancer or the individuality of cervical cancer.Method of the present invention can be implemented separately, can also merge with other medicines or treatment to use.
Generally speaking, chitosan is suspended in (for example physiological saline) in the pharmaceutically acceptable carrier, its administering mode can for: oral, vein injects injection or implantation, intratracheal injection or implantation or pulmonary injection or implantation in (intravenous infusion), subcutaneous injection or implantation, intramuscular injection or implantation, intrathecal injection or implantation, peritoneal injection or implantation, internal rectum injection or implantation, intravaginal injection or implantation, nasal injection or implantation, the stomach.The effective dose of aforementioned pharmaceutical compositions be depend on all multifactor: administering mode, formulation properties (thenatore of the formulation), experimenter's healthy state, experimenter's bodily trait (comprising build, body weight, body surface area, age, sex), the other medicines of being given, and responsible doctor's judgement.The dosage range that is fit to is 0.01-100.0mg/kg.Because operable chitosan is varied, and the producible effectiveness of different modes of administration is different, and also there is sizable difference in this effective dose value.For example, the needed dosage of oral dosage form may be than intravenous injection height.Above-mentioned effective dose can be utilized routine known in the art to be used for optimized empirical approach and adjust.Use suitable transport vehicle (for example polymer particle or implantable device) with this pharmaceutical capsuleization, can increase the efficient of transmission, the efficient of especially oral transmission.
Hereinafter described embodiment only is as illustration, is not in order to limit the present invention.Any those skilled in the art, without departing from the spirit and scope of the present invention, institute's a little change of doing and retouching are all within protection domain of the present invention.Document that this paper quotes from is introduced in full with for referencial use.
The preparation of fungi chitosan
(1) preparation of fungi chitosan A
Obtain soy cheese radiation mucormycosis spore suspension from the slant culture of cultivating 4, it direct inoculation is shaken in the bottle in 1 liter that the fresh fermention medium of 400ml is housed.In 22 ℃, with 200rev/min oscillation and fermentation 50 hours.Above-mentioned fermention medium comprises following composition for every liter: 10 gram peptones, 20 gram glucose, 1 gram yeast extract, 5 gram (NH
4)
2SO
4, 1 the gram K
2HPO
4, 1 the gram sodium-chlor, 5 the gram MgSO
47H
2O and 0.1 gram calcium chloride.The initial pH value of this fermention medium is adjusted into pH4.
Cell mass by separating in this fermented liquid, was handled 15 minutes in 121 ℃ with the 1N sodium hydroxide solution again.The material that is insoluble to alkali in this mixed liquid is suspended with 2% acetum, and insulation is 12 hours in 95 ℃, makes chitosan wherein to dissolve.The pH value of this solubility in acid supernatant is adjusted to pH10, makes it possible to the dissolved chitosan by precipitating and isolating.This purifying of throw out (being fungi chitosan A) is cleaned and drying.
(2) preparation of fungi chitosan B
Obtain soy cheese radiation mucormycosis spore suspension from the slant culture of cultivating 4, with it direct inoculation in 5 liters of fermentor tanks that 3 liters of fresh fermention mediums are housed.In 22 ℃, with the air flow fermentation culture of the stirring velocity of 400rpm and 3L/min 50 hours.Above-mentioned fermention medium comprises following composition for every liter: 10 gram peptones, 20 gram glucose, 1 gram yeast extract, 5 gram sulfuric acid amine, 1 gram dipotassium hydrogen phosphate, 1 gram sodium-chlor, 5 gram MgSO
47H
2The calcium chloride of O and 0.1 gram.The initial pH value of this fermention medium is adjusted into pH4.
Cell mass by separating in this fermented liquid, and therefrom is purified into chitosan (fungi chitosan B), and implementation step is above-mentioned as (1).
The antitumour activity of fungi chitosan
HeLa cell (BCRC60005) and MCF-7 (BCRC60436) can be preserved and (Bioresource Collection and Research Center BCRC) obtains in the research centre by the Biological resources of TaiWan, China.Cell suspending liquid is carried out tryptic digestion in the HBSS that contains 0.05% trypsinase and 0.53mM EDTA-4Na (Hank ' s Balanced Salt Solution) handle, inhale repeatedly with suction pipe and to put this cell suspending liquid, and count with the hematimeter pair cell so that cell mass scatters.In MEM substratum (containing 10% serum), the concentration of HeLa cell is every milliliter 8.35 * 10 with cell suspension
3Individual cell, and the concentration of MCF-7 cell is every milliliter 1.67 * 10
4Individual cell.The cell suspending liquid of 180 μ l is inoculated in the tissue culture dish of 96 lattice, in 37 ℃, 5% gas concentration lwevel, cultivated 17 to 20 hours.
Different chitosan is dissolved in contains 5 * 10
-3Among the D-PBS of M acetic acid (Dulbecco ' sphosphate-buffered saline), the chitosan soln of respectively getting 20 μ l adds respectively in each cell lattice of above-mentioned tissue culture dish, and cell was cultivated 3 in 37 ℃, 5% gas concentration lwevel.Contained chitosan final concentration is that 10ppm (is about 10 in the above-mentioned cell cultures
-7M).
With MTT (3-(4,5-dimethyl-thiazol-2-yl) 2,5-phenylbenzene tetrazolium bromide) method analysis of cells vigor.MTT is added in the D-PBS liquid, and making its concentration is 5mg/ml, and this MTT solution of respectively getting 20 μ l adds respectively in each cell lattice of above-mentioned tissue culture dish, cell is cultivated 4 hours in 37 ℃, 5% gas concentration lwevel again.Remove this cell culture medium.In each lattice, add the DMSO of 100 μ l, this tissue culture dish was shaken 5 minutes.Measure the optical density(OD) of each cell lattice, its test wavelength is 540nm, and the contrast wavelength is 690nm.
The chitosan that this test is adopted comprises: fungi chitosan A, fungi chitosan B, crab shell chitosan I, crab shell chitosan II, crab shell chitosan III, crab shell chitosan IV.
The degree of deacetylation of each chitosan utilizes a subdifferential ultraviolet spectrophotometry to learn in this test, and this method is as described in (1998) Talanta 45:713-719 such as Tan.
The molecular characterization of each chitosan is with three detections (triple-dectection) GPC/SEC in this test
3Measure.The concentration of chitosan sample with 2.5mg/ml is formulated in the moving phase (mobile phase), and in 90 degree Celsius, heated 3 hours.This moving phase pH value is pH4.1, and it comprises the sodium-acetate of 0.35N acetic acid and 8.2g/L, and its velocity of flow is 0.7ml/min.The sample injection volume is 100 μ l, SEC
3Separation is with the TSK GPC post GMPW of Japanese Tosoh
XLCarry out.Above-mentioned three used detectors of check and analysis are RI-detector (Waters company), elementary errors viscosity measurement detector (differential viscometer detector), light scattering detector (Viscotek model T60).Use above-mentioned three kinds of detectors simultaneously, collect the detected result data, and detect data with the TriSEC software processes.
Table one is the weight-average molecular weight (M that shows each chitosan in this test
W), number-average molecular weight (M
n), many dispersion indexs (polydispersity index, M
W/ M
n), the section turning radius (radius ofgyration, Rgw), weighted average limiting viscosity (intrinsic viscosity, [η]
w), degree of deacetylation (DD).
Do not reckon with that HeLa cell and MCF-7 cell then do not show responsive effect to fungi chitosan A and B sensitivity to crab shell chitosan (I to IV).As shown in Table 2, fungi chitosan A and B can optionally suppress the growth of tumour cell, and its inhibiting rate is more than 30%.On the other hand, fungi chitosan A and B significantly do not suppress the growth of human embryos pneumonocyte (MRC-5) or other cancerous cell line, and for example: human stomach cancer cell is (AGS) and a human liver cancer clone (Hep G2).
Table 1 is in order to the molecular characterization and the degree of deacetylation of the chitosan of carrying out antitumour activity test
| Chitosan | M w×10 -3(g/mol) | M n×10 -3(g/mol) | M w/M n | Rgw (nm) | [η] w (ml/g) | DD(％) |
| Fungi chitosan A fungi chitosan B crab shell chitosan I crab shell chitosan II crab shell chitosan III crab shell chitosan IV | 116.5 94.4 317.2 163.5 140.7 88.5 | 58.0 89.4 286.3 115.3 80.5 48.6 | 2.01 1.06 1.12 1.42 1.75 1.83 | 18.0 11.0 41.1 28.9 24.7 17.1 | 1.70 0.44 6.40 4.66 3.56 1.90 | 90.1 87.0 79.7 83.5 78.1 78.5 |
The various chitosan of table 2 are for the influence of HeLa cell and the growth of MCF-7 cell
| Cell growth per-cent (mean value S.D.) a，b | |||
| Sample | n＝ | The HeLa cell | The MCF-7 cell |
| Control group (not chitosan-containing) fungi chitosan A fungi chitosan B crab shell chitosan I crab shell chitosan II crab shell chitosan III crab shell chitosan IV | 6 6 6 4 4 4 4 | 100±4 65±3 69±2 98±11 95±7 94±9 99±5 | 100±5 57±6 69±4 96±3 98±7 92±6 98±3 |
A. this value representation average cell growth rate (%), it is the average and the standard deviation thereof of 4 or 6 revision test values.
B. average cell growth rate=(cell count of handling through chitosan)/(cellular control unit number) * 100%.
Other embodiment
Any feature of the present invention described in the specification sheets of the present invention can make up by any way.Any feature of the present invention described in the specification sheets of the present invention can another have similar, the identical or equal means worked as and replace and finish.Therefore, unless state clearly especially, disclosed any technical characterictic only is many similar or equal illustrations when technique means.
Though the present invention with preferred embodiment openly as above; right its is not in order to limit the present invention; the any technician in this area; do not breaking away from the spirit and scope of the invention; when doing a little change and retouching; also in protection scope of the present invention, so protection scope of the present invention defines and is as the criterion when looking accompanying claim.
Claims (5)
1. chitosan, its weight-average molecular weight is 50,000-140,000 gram/mole, and its degree of deacetylation is 85%-95%, this chitosan is from soy cheese radiation mucormycosis preparation and get.
2. pharmaceutical composition, it comprises the described chitosan of claim 1, and pharmaceutically acceptable carrier.
3. the pharmaceutical composition of claim 2, it is the pharmaceutical composition in order to treatment mammary cancer.
One kind in order to the treatment cervical cancer pharmaceutical composition; it comprises the chitosan of effective dose; wherein the weight-average molecular weight of this chitosan is 50; 000-140; 000 gram/mole; and its degree of deacetylation is 85%-95%, and this chitosan is to get from soy cheese radiation mucormycosis preparation.
5. prepare the method for the chitosan of claim 1, this method comprises:
Cultivate described fungi at 15-24 ℃; With
From this fungus culture, isolate chitosan.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031081835A CN1328291C (en) | 2003-03-31 | 2003-03-31 | New chitosan |
| HK05101864A HK1069588A1 (en) | 2003-03-31 | 2005-03-03 | Novel chitosans |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031081835A CN1328291C (en) | 2003-03-31 | 2003-03-31 | New chitosan |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1534047A CN1534047A (en) | 2004-10-06 |
| CN1328291C true CN1328291C (en) | 2007-07-25 |
Family
ID=34283089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031081835A Expired - Lifetime CN1328291C (en) | 2003-03-31 | 2003-03-31 | New chitosan |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1328291C (en) |
| HK (1) | HK1069588A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105147732A (en) * | 2015-10-26 | 2015-12-16 | 北京百生康生物科技有限公司 | Application of crocodile blood polysaccharide to breast cancer treatment |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0531991A2 (en) * | 1991-09-11 | 1993-03-17 | Shin-Etsu Chemical Co., Ltd. | Method for preparing chitosan |
| CN1242377A (en) * | 1998-07-20 | 2000-01-26 | 北京化工大学 | Method for preparing chitosan and low polymerized chitosan |
| US6255085B1 (en) * | 1999-07-08 | 2001-07-03 | Food Industry Research & Development Institute | Production of chitosan and chitin |
-
2003
- 2003-03-31 CN CNB031081835A patent/CN1328291C/en not_active Expired - Lifetime
-
2005
- 2005-03-03 HK HK05101864A patent/HK1069588A1/en not_active IP Right Cessation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0531991A2 (en) * | 1991-09-11 | 1993-03-17 | Shin-Etsu Chemical Co., Ltd. | Method for preparing chitosan |
| CN1242377A (en) * | 1998-07-20 | 2000-01-26 | 北京化工大学 | Method for preparing chitosan and low polymerized chitosan |
| US6255085B1 (en) * | 1999-07-08 | 2001-07-03 | Food Industry Research & Development Institute | Production of chitosan and chitin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1534047A (en) | 2004-10-06 |
| HK1069588A1 (en) | 2005-05-27 |
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