CN1322014C - Gecko polysaccharide, and its preparing method and pharmaceutical use - Google Patents
Gecko polysaccharide, and its preparing method and pharmaceutical use Download PDFInfo
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Abstract
The invention discloses a gecko polysaccharide from the animal Chinese medicine gecko, and its preparing method and pharmaceutical use for the first time in the world. It has an upper effect for the malignancy: the gecko polysaccharide has a cancer suppressor rate of 70.8% to the human liver cancer cell line Bel-7402 when its concentration is 1 mg/ml, IC[50] <10 mug/ml, showing a high level of cancer suppressor activity; and it can induce the differentiation of the liver cancer cells, with a better curative effect than many other chemotherapy drugs; so it can be used individually for the treatment of malignant tumors, and can also be used for cancer surgery, radiotherapy, chemotherapy and inserting treatment intervention, and adjuvant treatment. Because it contains sulfate, which has extremely bright applications foreground in the prevention and treatment of AIDS and other virus infectious diseases; the polysaccharide usually has a good immune regulation role, and also has a very good therapeutic effect by using the gecko to treat the immunity lowlihead; the gecko can be used as a new immunomodulator, for the treatment of diseases such as malignant tumor, chronic infection; and the toxicity of the gecko on human limits its clinical high-dose application, while the gecko polysaccharide plays a very good attenuation synergistic action.
Description
Technical field
The present invention relates to the traditional Chinese medicine extraction technical field, specifically, relate in a kind of animal Chinese medicine single, activeconstituents that very high pharmaceutical use is arranged, prepare the method for this activeconstituents and the application of this activeconstituents.
Background technology
The traditional Chinese medical science is the quintessence of Chinese culture of China, and Chinese medicinal materials is the material treasure-house of traditional Chinese medical theory, and Chinese medicinal materials is divided into botanical herbs material, animal Chinese medicinal materials and mineral Chinese medicine material; Theory, clinical and the modern study of the tradition traditional Chinese medical science shows that all it is a big characteristic of the traditional Chinese medical science that animal drug is treated malignant tumour, as in Chinese blister beetle, extract Cantharidin, cantharidate is applied to treat malignant tumour and obtains certain effect, and has certain antiviral effect.As patent and applications such as ZL90106365.7 ZL91110970.6, CN1076111A, ZL93110157.3, CN1374310A.
In general, most animals Chinese medicine all has certain toxicity to human body, has limited the dosage of this class medicine when treatment, makes result of treatment be subjected to bigger influence.How to reduce its detrimentally affect to human body when keeping medical active, be the focus that pharmacy men are studied.Simultaneously, the activeconstituents of clear and definite medicine with and the mechanism of action be the top priority of crude drug exploitation.In being arranged, the animal Chinese medicine of antitumous effect extracts that the active substance of antitumous effect is arranged then is the pharmacy man of China and even countries in the world, the focus of work of research and development institution.
House lizard is traditional Chinese medicinal materials, for the Gekkonidae animal does not have web gecko Gekko swinhonis G ū enther or other several geckoes [many warts gecko Gekko japonicus (Dum é ril﹠amp; Bibron); No wart gecko Gekko subpalmatus G ü enther; Webbed toe gecko Gekko chinensis Gray] all, be the traditional salty-cold soft hard medicine of the traditional Chinese medical science.The traditional Chinese medical science is used house lizard treatment scrofula cancerous swelling (" blue or green capsule is assorted usurps ", " must join book on Chinese herbal medicine ", " Sichuan Chinese medicinal herbal ") for a long time, and the tame house lizard treatment malignant tumour of using of domestic more doctor has also obtained better curative effect.In vitro study finds that the house lizard aqueous solution can suppress the breathing of human liver cancer cell, and clear and definite house lizard contains the toxic substance similar to the hornet poison and organizes amine substance, fatty oil, multiple amino acids, trace element and VITAMIN, but mentioned component all is not enough to explain the antitumor action of house lizard, fails to isolate in house lizard its antitumor, antiviral and immunoregulatory activeconstituents both at home and abroad so far.
The tradition traditional Chinese medical science thinks that house lizard is salty-cold slightly poisonous, discovers that house lizard contains the toxic substance similar to the hornet poison and organizes amine substance, makes traditional theory of traditional Chinese medical science obtain checking.And the toxicity of house lizard has limited its clinical heavy dose of application, so the general 1.5～4.5g only of the daily dosage portion of house lizard has limited the medical applications and the medical effect of house lizard greatly.
Known application house lizard mode, except the bulk drug as Chinese materia medica used, Yunnan once was prepared into intramuscular injection with house lizard, and the sixties were once found a good sale in the various places, South East Asia.Its technology is alcohol extracting-water precipitating, the activated carbon adsorption removal of impurities.The problem of its existence is mainly: 1. extracting solution contains multiple composition: once detect crude protein, fat, robust fibre, VITAMIN, trace element etc.; 2. effective constituent is not clear: the antitumor action that above-mentioned substance all can not the good explanation house lizard; 3. need in the alcohol extracting medicinal material was soaked in ethanol 20 days, the problem that this method exists is: I soaks for a long time, the ethanol volatilization, and concentration reduces; The II soak time is long more, and then extract is impure many more; The III soak time is oversize, and medicinal material is apt to deteriorate; The IV soak time is oversize, makes troubles to production, and increases production cost.This injection liquid so can not contain gecko polysaccharide, in fact also fails to detect gecko polysaccharide because its technology is alcohol extracting-water precipitating from this injection liquid.
Polysaccharide is a class natural macromolecular material, extensively is present in animal, plant and the microorganism cells, is one of four big base substances that constitute life.Studies show that polysaccharide has antiviral, antitumor and immunoregulation effect in a large number.Generally speaking, polysaccharide is good immunomodulator.Polysaccharide can activated macrophage, promotes lymphocyte mitotic division, strengthens NK cell (natural killer cell) and LAK cell (killer cell of lymphokineactivation) activity, strengthens T
DCell (delayed hypersensitivity T cell) function induces interleukin-(especially IL-1, IL-2), IFN (Interferon, rabbit), TNF cytokines such as (tumour necrosis factors), and complement activation strengthens erythrocyte immune, regulates nerve-endocrine-immunological network.
At present both at home and abroad separating obtain exhausted in the natural matter medicine is vegetable polysaccharides and microbial polysaccharide mostly.Because mostly polysaccharide is the water soluble component of Chinese medicine or food, general Chinese medicine or food all can be taken by decoction, and its aqueous solution contains a large amount of polysaccharide.Yet isolating both at home and abroad polysaccharide is to separate in the plant of never significantly antitumous effect (most or even do not have antitumous effect) mostly, thereby its polysaccharide that obtains is used for animal or human's body, and its antitumous effect is very limited.
Equally for these reasons, most polysaccharide does not have direct effect to tumour cell.The relevant polysaccharide Antitumor Effects of great majority all is research in the body, yet intravital research often is polysaccharide enhancing body antineoplastic immune and then plays antitumor action indirectly.In fact the immunity system of human body only can be removed less than 10
6-10
9Individual tumour cell is (less than 1cm
3), above-mentioned tumour belongs to utmost point infantile tumour, and not only clinical very difficult discovery even if make a definite diagnosis, is also taked operative treatment and is effected a radical cure.Most of tumour patients are once finding promptly to belong to late period, considerably beyond the removing ability of body immune system.Searching has direct inhibiting medicine to tumour cell and seems particularly important.
Studies show that recently: the polysaccharide of sulfur-bearing acid group has the effect of anti-HIV, thereby sulfated polysaccharide has become the focus of domestic and international research.Because natural sulfated polysaccharide is difficult to obtain, the investigator usually takes the method for synthetic or sulphating to obtain sulfated polysaccharide.At present except that the sulfated polysaccharide that synthetic and chemically modified obtain, only from plant, separate obtaining a few puritan filler polysaccharide, the effect of certain anti-HIV infection is all arranged.
Polysaccharide has unique and complex chemical structure, thereby the research of its chemical structure research and structure activity relationship is quite complicated and difficult, though research in these areas obtains certain progress in recent years, but research of polysaccharide higher structure and structure activity study still have a lot of problems to remain to be illustrated, this also makes us the chemically modified of polysaccharide be existed the influence of numerous factors, usually be difficult to strict its condition of control and accurately illustrate its structure and mechanism, be unfavorable for the research and development of new drug.The for example manually modified sulphating Pachymose that obtains, its complex process not only, condition is difficult to accurate control, and the pachyman sulfuric acid ester that it obtains is in the highest ability 16.2% of inhibiting rate of 2.5mg/ml tumour cell.
In sum, clinically the use of house lizard is had higher requirement, the firstth, reduce its toxic side effect, to improve its usage quantity, increase its curative effect to multiple sufferer; The secondth, the effective ingredient of from the modern medicine development viewpoint, obtaining, understand Chinese medicine for inherit and carry forward motherland's traditional medicine, taking part in international competition has a very important role, and also is the historic mission of the epoch giving us simultaneously; The 3rd is or not very desirable to the treatment for cancer effect in the known medicine, and the effective ingredient of screening treatment cancer is the focus of the medicine scholar research of China in Chinese medicine; Screening simultaneously can be treated the effective constituent of HIV infection and is used for clinical have very great scientific value, medical value and economic worth.
The contriver thinks that by the research to prior art the known method of extracting effective ingredient from Chinese medicine is the poach alcohol precipitation, keeps the aqueous solution and removes precipitation, and be a kind of technical prejudice, be the deviation on the technological line at least; And design a cover extracting method again, and then finish, realized a plurality of goal of the invention of the present invention.
Technology contents
Goal of the invention of the present invention is intended to overcome above-mentioned prior art and lacks limit, and a kind of gecko polysaccharide that is derived from the house lizard of animal Chinese medicine is provided; This gecko polysaccharide to multiple malignant tumour particularly liver cancer cell very strong restraining effect is arranged, cancer suppressing ratio is up to 70.8%, and can induce the liver cancer cell differentiation, simultaneously because this gecko polysaccharide contains sulfate radical virus diseases such as HIV are had tangible antivirus action, gecko polysaccharide also is one to have the biopharmaceuticals of extensive immunoregulation effect; Another goal of the invention of the present invention provides the application at field of medicaments of the preparation method of this gecko polysaccharide and gecko polysaccharide.
Gecko polysaccharide of the present invention, the pure throw out that the poach alcohol deposition method that comprises the steps for employing obtains from house lizard:
Degreasing: house lizard is gone out lipid-soluble substance with the methyl alcohol extracting before water logging is extracted; This is necessary, very important step, because house lizard is a kind of animal, contains a large amount of lipid-soluble substances, and therefore, degreasing has important effect for the recovery rate and the purity of product; Promptly before the flooding of house lizard, go out lipid-soluble substance with the methyl alcohol extracting.
Water is carried: the dregs of a decoction after the degreasing by the 1-8 of house lizard amount water extraordinarily, under 60-120 ℃ of condition lixiviate 1-3 hour, and can carry out repeatedly flooding to same house lizard meal, are merged the gained water extract and use;
Alcohol precipitation: the gradation of water extract adds ethanol to 15%-80% concentration, distinguishes collecting precipitation, will precipitate to be dissolved in water respectively, and the filtering insolubles, filtrate adds ethanol sedimentation again, and precipitation is the subject matter gecko polysaccharide.
Before carrying out above-mentioned defatting step, can pulverize the house lizard raw material of getting ready earlier, be beneficial to the abundant extraction of effective constituent.
Above-mentioned gecko polysaccharide also has features such as following outward appearance and physics and chemistry:
Outward appearance: canescence is to brown ceramic powder;
PH：6-7；
Color reaction: Molish reacting positive, Fehling reacting positive;
Hydrolysis reaction: acid hydrolytic reaction does not have obvious sediment and produces;
The sulfate radical qualitative test is positive.
Above-mentioned gecko polysaccharide is in the application of field of medicaments:
Above-mentioned gecko polysaccharide can be made and meet the oral dosage form that pharmaceutics requires.
Also above-mentioned gecko polysaccharide can be made and meet the injection type that pharmaceutics requires.
Above-mentioned gecko polysaccharide can be applicable to prepare in the medicine for the treatment of malignant tumour.
Above-mentioned gecko polysaccharide also can be applicable to prepare in the medicine for the treatment of liver cancer.
Above-mentioned gecko polysaccharide also can be applicable to prepare in the medicine for the treatment of disease of viral infection.
Above-mentioned gecko polysaccharide also can be applicable to prepare in the medicine of treatment HIV infection.
Above-mentioned gecko polysaccharide also can be applicable to prepare in the immunoregulation druge.
Compare with existing polysaccharide, the scientific value and the significance of application of gecko polysaccharide are: 1, the applicant obtains gecko polysaccharide in the world first, and obtains scientific validation.2, gecko polysaccharide has higher curative effect to malignant tumour: for example primary hepatocarcinoma is commonly called as " cancer king ", be one of China's common cancer, China has 110,000 people to die from liver cancer every year approximately, account for 45% of global PLC mortality number, early hepatocarcinoma (＜3cm), liver transplantation has better curative effect, (need ten thousand yuan of 25-30 approximately yet not only expense is very expensive, and the annual expenses for medicine of keeping that tens thousand of units are still arranged), and the whole world is not enough for the liver source, causes many patients can't go transplantation of liver; Most of liver cancer are once finding promptly to belong to late period, and that no matter the middle and advanced stage patient is operation, radiotherapy, chemotherapy, interventional therapy curative effect is all undesirable.Only a few chemotherapeutic such as Zorubicin, cis-platinum, Tegafur have clear and definite curative effect to liver cancer at present, but because of the lower toxic reaction of its cancer suppressing ratio is bigger, are used for the many no definite curative effects of systemic chemotherapy.Gecko polysaccharide when concentration is 1mg/ml to primary hepatocarcinoma cell strain cancer suppressing ratio just up to 70.8%, IC
50＜10 μ g/ml have shown high tumor-suppression activity, and can induce the liver cancer cell differentiation, and curative effect is better than numerous chemotherapeutics, can be used for the treatment of malignant tumour separately, also can be used for tumor operation, radiotherapy, chemotherapy and interventional therapy.3, traditional traditional Chinese medical science thinks that house lizard becomes cold slightly poisonous, and modern study finds that house lizard contains the toxic substance similar to the hornet poison and organizes amine substance, makes traditional theory of traditional Chinese medical science obtain checking; The toxicity of house lizard has limited its clinical heavy dose of application, so the daily dosage portion of house lizard is 1.5～4.5g only generally, we in the house lizard separation and Extraction malignant tumour and HIV are infected the effective constituent gecko polysaccharide that higher result of treatment is arranged, played good synergism and attenuation.4, owing to contain sulfate radical, gecko polysaccharide has broad application prospects in the control of diseases of viral infection such as acquired immune deficiency syndrome (AIDS).5, polysaccharide is good immunomodulator, and clinical use house lizard treatment immunocompromised also has good result of treatment, and gecko polysaccharide can be used as a kind of new immunomodulator, is used for treatment of diseases such as malignant tumour, chronic infection.
Description of drawings
Fig. 1 is the process flow sheet of preparation gecko polysaccharide;
Fig. 2 is the growth curve of liver cancer Bel-7402 cell after the gecko polysaccharide effect;
Fig. 3 is the cancer suppressing ratio (%) of gecko polysaccharide to liver cancer Bel-7402 cell;
Fig. 4 is the survival rate of liver cancer Bel-7402 cell after the gecko polysaccharide effect;
Fig. 5 is the aspect graph of hepatoma cell strain Bel-7402 negative control group cell;
Fig. 6 is the form change figure of hepatoma cell strain Bel-7402 ATRA positive controls cell;
Fig. 7 is the form change figure of hepatoma cell strain Bel-7402 gecko polysaccharide low dose group cell;
Fig. 8 is the form change figure of dosage group cell in the hepatoma cell strain Bel-7402 gecko polysaccharide;
Fig. 9 is the form change figure of hepatoma cell strain Bel-7402 gecko polysaccharide high dose group cell;
Specific implementation method
Embodiment 1: the preparation of gecko polysaccharide
No web gecko Gekko swinhonis G ū enther 1Kg pulverizes and is thick end, crosses sieve No. 1,60 ℃ of water-bath lixiviates of 4000ml methyl alcohol 3 hours, 2 hours each 1 time, equal 4 layers of filtered through gauze bleed concoction, the dregs of a decoction extracted 3 hours with 90-100 ℃ of water-bath of 8000ml distilled water, 2 hours each 1 time, equal 4 layers of filtered through gauze are extracted in 90-100 ℃ of water-bath of 6000ml distilled water 1 hour 1 time, bleed concoction, merge 3 water and carry soup, 2000 change 20 minutes centrifugal precipitations of removing, and are concentrated into 1500-2000ml, slowly add ethanol to 30% concentration, the limit edged stirs, and leaves standstill precipitation, and soup slowly adds ethanol to 80% concentration again, the limit edged stirs, the results precipitation, precipitation is dissolved with distilled water 400ml, the filtering insolubles, slowly add ethanol to 80% concentration, the limit edged stirs, results precipitation, precipitation absolute ethanol washing 3 times, vacuum-drying, promptly fine ground; The about 3.8-4.2% of yield.
Embodiment 2: the preparation of gecko polysaccharide
Many warts gecko Gekko japonicus (Dum é ril﹠amp; Bibron) 1Kg, pulverizing, the methyl alcohol degreasing, the dregs of a decoction extract 2-3 time with 4-8 times of distilled water 60-80 ℃, the fractional precipitation of 15%-80% ethanol, the results precipitation, water-soluble, use ethanol sedimentation again, precipitation absolute ethanol washing 3 times, drying, promptly fine ground.
Example 1 is identified in checking: the evaluation of gecko polysaccharide
Gecko is pressed the material of the method extraction of the foregoing description 1,2:
Appearance color is canescence to brown ceramic powder;
Water-soluble, be insoluble to organic solvents such as ethanol;
The Molish reaction is positive: get this material 0.1g and be dissolved in 10ml distilled water, get the 1ml aqueous solution and add 3 of 5% α naphthols ethanolic solns, shake up the back and slowly add the vitriol oil along tube wall, have the purple ring to produce between soup and vitriol oil liquid level;
The hydrochloric acid hydrolysis test: get this material aqueous solution 1ml, add 10 in dilute hydrochloric acid, heating hydrolysis is observed after 15 minutes immediately on the spirit lamp, the solution clarification, and not having significantly, precipitation produces;
The Fehling reaction is positive: get this material aqueous solution 1ml, add 10 in dilute hydrochloric acid, heating hydrolysis is after 15 minutes on the spirit lamp, add Fehling reagent 1ml, heating is 10 minutes on the spirit lamp, observes after leaving standstill a few hours, has brick-red precipitation to produce (Fehling reagent: A liquid: 3.45gCuSO
45H
2O is dissolved in the 500ml distilled water, adds the dense H of 50 μ l
2SO
4, mixing stores; B liquid: 6.25gNaOH and 6.85g Seignette salt are dissolved in distilled water, store; Face with preceding A liquid and B liquid equal-volume mixing are got final product).
By provable this material of above-mentioned reaction is polysaccharide, because of it derives from house lizard, is gecko polysaccharide.
Example 2 is identified in checking: the evaluation of gecko polysaccharide sulfate radical
Gecko polysaccharide sulfate radical qualitative test is positive: get gecko polysaccharide aqueous solution 1ml, add 10 in dilute hydrochloric acid, heating hydrolysis dripped chlorination shellfish solution after 15 minutes on the spirit lamp, produced white precipitate.
Prove that this natural gecko polysaccharide contains sulfate radical.
EXPERIMENTAL EXAMPLE 1: the antitumor pharmacology research of gecko polysaccharide (1)
Hepatoma cell strain Bel-7402 is incubated in the RPMI1640 substratum that contains 10% newborn calf serum (56 ℃ of deactivation 30min) 0.25 * 10
4/ ml is inoculated in 12 orifice plates, cultivates in 37 ℃, volume are the constant temperature incubator of 5%CO2, saturated humidity.Inoculate after 24 hours respectively with the PRMI1640 culture medium culturing that contains gecko polysaccharide 1mg/ml, 100 μ g/ml, 10 μ g/ml and equal-volume NS 7 days.30 minutes, 1-6 days difference collecting cell after the dosing, trypan blue dyeing, living cell counting and dead cell are made growth curve, cell survival rate and cancer suppressing ratio and are calculated respectively.Statistical method adopts multiple comparisons, and all (version:11.0, Chicago USA) finish (being right after nextpage) to statistics by statistical package SPSS.
1, gecko polysaccharide is to the growth-inhibiting effect of hepatoma cell strain Bel-7402
Table 1 gecko polysaccharide is to the growth-inhibiting effect (* 10 of liver cancer cell Bel-7402
4/ ml, N=3)
| 1d (30min after the dosing) | 2d | 3d | 4d | 5d | 6d | 7d |
| High 0.375 ± 0.139 dosage group | 0.733± 0.088△ | 0.95± 0.066□ | 1.492± 0.218□ | 1.083± 0.072□ | 4.125 ± 0.573 □ | 4.167± 0.629□ |
| In 0.350 ± 0.090 dosage group | 0.517± 0.063□◆ | 0.50± 0.043□■ | 1.225± 0.175□ | 1.375± 0.225□ | 3.000 ± 0.331 □ | 4.792± 0.191□ |
| Low 0.358 ± 0.063 dosage group | 0.892± 0.138◎ | 1.00± 0.152□ | 1.817± 0.359□ | 1.938± 0.108□○ | 3.917 ± 0.315 □ | 9.750± 0.545 ○□ |
| Shine group to 0.4750 ± 0.2500 | 1.05± 0.090 | 1.65± 0.043 | 2.717± 0.419 | 7.125± 0.534 | 9.667 ± 1.041 | 12.250 ± 0.500 |
Annotate: △ and control group be P＜0.05 relatively; and high dose group be P＜0.05 relatively; ◇ and control group be P＜0.01 relatively;
◆ compare P＜0.01 with high dose group; and control group be P＜0.001 relatively; ■ and high dose group be P＜0.001 relatively; Zero with middle dosage group P＜0.05 relatively; ◎ and middle dosage group be P＜0.01 relatively.
Fig. 1 is the growth curve of liver cancer Bel-7402 cell after the gecko polysaccharide effect;
Table 2 gecko polysaccharide is to the cancer suppressing ratio (%) of liver cancer Bel-7402 cell
| 1d | 2d | 3d | 4d | 5d | 6d | |
| High dose group | 30.5 | 42.4 | 59.9 | 70.8 | 57.3 | 66.0 |
| Middle dosage group | 50.5 | 69.7 | 66.9 | 66.8 | 69.0 | 61.9 |
| Low dose group | 15.2 | 39.4 | 51.1 | 58.8 | 59.5 | 20.4 |
Fig. 2 is the cancer suppressing ratio (%) of gecko polysaccharide to liver cancer Bel-7402 cell
Result of study shows: the high, medium and low dosage group of gecko polysaccharide just can significantly suppress the growth of liver cancer Bel-7402 cell in 24 hours after dosing, cancer suppressing ratio is up to 70.8%, and curative effect is the best with middle dosage group.Gecko polysaccharide is to the IC of liver cancer Bel-7402 cell inhibitory effect
50＜10 μ g/ml.
2, gecko polysaccharide is to the influence of liver cancer cell survival rate
Table 3 gecko polysaccharide is to the influence (n=3) of liver cancer cell Bel-7402 cell survival rate
| 1d (30min after the dosing) | 2d | 3d | 4d | 5d | 6d | 7d | |
| High dose group | 0.886±0.102 | 0.758± 0.065△ | 0.765± 0.006◇ | 0.790± 0.052 | 0.746± 0.021◇ | 0.867± 0.005 | 0.817± 0.035 |
| Middle dosage group | 0.818±0.157 | 0.859± 0.036 | 0.717± 0.084◇ | 0.783± 0.027 | 0.793± 0.042◇ | 0.702± 0.069△ | 0.824± 0.109 |
| Low dose group | 0.941±0.102 | 0.905± 0.065 | 0.758± 0.028◇ | 0.831± 0.033 | 0.783± 0.057◇ | 0.736± 0.041△ | 0.915± 0.023 |
| Control group | 0.970±0.053 | 0.902± 0.076 | 0.913± 0.017 | 0.835± 0.041 | 0.941± 0.016 | 0.867± 0.060 | 0.905± 0.050 |
Annotate: △ and blank group be P＜0.05 relatively;
and high dose group be P＜0.05 relatively;
◇ and blank group be P＜0.01 relatively
Fig. 3 is the survival rate of liver cancer Bel-7402 cell after the gecko polysaccharide effect;
Result of study shows: the high, medium and low dosage group of gecko polysaccharide is all influential to liver cancer Bel-7402 cell survival rate, shows that gecko polysaccharide has certain cytotoxicity to liver cancer Bel-7402 cell.
EXPERIMENTAL EXAMPLE 2: the antitumor pharmacology research of gecko polysaccharide (2)
Hepatoma cell strain Bel-7402 is incubated in the RPMI1640 substratum that contains 10% newborn calf serum (56 ℃ of deactivation 30min) 1 * 10
5/ ml is inoculated in the 20ml culturing bottle, cultivates in 37 ℃, volume are the constant temperature incubator of 5%CO2, saturated humidity.Inoculate and change liquid after 24 hours, respectively with the PRMI1640 culture medium culturing 4 days that contains gecko polysaccharide 1mg/ml, 100 μ g/ml, 10 μ g/ml, all-trans-retinoic acid (ATRA) 3 μ g/ml and equal-volume NS.The observation of cell form, detection of alkaline phosphatase (ALP) and albumin (ALB).See: Fig. 5 is the aspect graph of hepatoma cell strain Bel-7402 negative control group cell;
Fig. 6 is the form change figure of hepatoma cell strain Bel-7402 ATRA positive controls cell;
Fig. 7 is the form change figure of hepatoma cell strain Bel-7402 gecko polysaccharide low dose group cell;
Fig. 7 is the form change figure of dosage group cell in the hepatoma cell strain Bel-7402 gecko polysaccharide;
Fig. 8 is the form change figure of hepatoma cell strain Bel-7402 gecko polysaccharide high dose group cell;
It is the Polygons cell that ATRA can induce the Bel-7402 cytodifferentiation, and 7402 cellular fories after the gecko polysaccharide effect obviously change, and are the spindle shape, and has a large amount of secretory product to exist around the visible cell.Preliminary studies confirm that, the Bel-7402 cell ALP after the gecko polysaccharide effect descends, and ALB secretes rising, and the prompting gecko polysaccharide can induce the Bel-7402 cell toward the differentiation of mature liver cells direction.
Application Example 1: contain the preparation of the injection liquid of gecko polysaccharide
Gecko polysaccharide, dissolved in distilled water filters, and dialyse 3 times, and adjustment concentration is 10mg/ml, degerming, depyrogenation, except that floating preteins, aseptic subpackaged is the 2ml/ bottle.
Application Example 2: gecko polysaccharide capsule
Gecko polysaccharide, the Capsules of packing into, every 50mg seals, gumming, bottling.
Application Example 3: gecko polysaccharide tablet
Gecko polysaccharide adds 2 parts in dextrin, 2 parts of starch, and 1 part of Icing Sugar, the hydroxide gel reaches 3% of total amount, and compressing tablet is the 50mg/ sheet, bottling.
Claims (9)
1, gecko polysaccharide, the pure throw out that the poach alcohol deposition method that comprises the steps for employing obtains from house lizard:
Degreasing: house lizard is gone out lipid-soluble substance with the methyl alcohol extracting before water logging is extracted;
Water is carried: the dregs of a decoction after the degreasing by the 1-8 of house lizard amount water extraordinarily, under 60-120 ℃ of condition lixiviate 1-3 hour, and can carry out repeatedly flooding to same house lizard meal, are merged the gained water extract and use;
Alcohol precipitation: the gradation of water extract adds ethanol to 15%-80% concentration, distinguishes collecting precipitation, will precipitate to be dissolved in water respectively, and the filtering insolubles, filtrate adds ethanol sedimentation again, and precipitation is the subject matter gecko polysaccharide.
2, gecko polysaccharide according to claim 1 is characterized in that: described gecko polysaccharide has following characteristics:
Outward appearance: canescence is to brown ceramic powder;
PH：6-7；
Color reaction: Molish reacting positive, Fehling reacting positive;
Hydrolysis reaction: acid hydrolytic reaction does not have obvious sediment and produces;
The sulfate radical qualitative test is positive.
3, a kind of preparation that contains the described gecko polysaccharide of claim 1 is characterized in that described gecko polysaccharide is made the injection type that meets the pharmaceutics requirement.
4, a kind of preparation that contains the described gecko polysaccharide of claim 1 is characterized in that described gecko polysaccharide is made the oral dosage form that meets the pharmaceutics requirement.
5, the application of the described gecko polysaccharide of claim 1 in the medicine of preparation treatment malignant tumour.
6, the application of the described gecko polysaccharide of claim 1 in the medicine of preparation treatment liver cancer.
7, the application of the described gecko polysaccharide of claim 1 in the medicine of preparation treatment disease of viral infection.
8, the application of the described gecko polysaccharide of claim 1 in the medicine that preparation treatment HIV infects.
9, the application of the described gecko polysaccharide of claim 1 in the preparation immunomodulator.
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| CN1337254A (en) * | 2001-07-11 | 2002-02-27 | 李建生 | Cancer-treating Chinese medicine and its prepn |
| CN1412202A (en) * | 2002-11-20 | 2003-04-23 | 吉林省中医中药研究院 | Ganoderma applanatum polysaccharide, its preparation and medicine composition using said compound as active component |
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| CN1412202A (en) * | 2002-11-20 | 2003-04-23 | 吉林省中医中药研究院 | Ganoderma applanatum polysaccharide, its preparation and medicine composition using said compound as active component |
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