CN1321469A - Pinctada martensii visceral organs glycoprotein and glycosaminoglycan with tumor-resisting activity and their extraction process - Google Patents
Pinctada martensii visceral organs glycoprotein and glycosaminoglycan with tumor-resisting activity and their extraction process Download PDFInfo
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- CN1321469A CN1321469A CN00105196A CN00105196A CN1321469A CN 1321469 A CN1321469 A CN 1321469A CN 00105196 A CN00105196 A CN 00105196A CN 00105196 A CN00105196 A CN 00105196A CN 1321469 A CN1321469 A CN 1321469A
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Abstract
The present invention discloses the application of glucoprotein and osamine polysaccharide compound extracted from all-organs of pearl oyster as medicine composite. Said medicine composite possesses obvious action of inhibiting tumor and immuno suppressive medicine action for resisting coagulation and chemicotherapy. Said invention also provides a preparation process for extrating glucoprotein and osamine polysaccharide compound from all-organs of pearl oyster.
Description
The present invention relates to from glycoprotein, the glycosaminoglycans of pinctada martensii visceral organs extraction, and extraction process, above-mentioned glycoprotein, glycosaminoglycans chemical compound have antitumor, anticoagulant isoreactivity, can be used as the medical composition of antineoplastic agent.
Malignant tumor serious harm human beings'health, and the anticancer chemotherapy medicine all presents certain toxicity.Extract some and have the native compound of anti-tumor activity from animal and plant, the antitumor drug that is used to produce high-efficiency low-toxicity is a direction of antitumor drug research.Margarita is a rare Chinese medicine, has the report Margarita layer powder that murine sarcoma S-180 is had inhibitory action, as the parent of breeding Margarita and nacreous layer-Pinctada margaritifera whole viscera, whether also contains glycoprotein, the glycosaminoglycans chemical compound of same or similar effect, and the research report is not arranged.This seminar has confirmed that the extract of pinctada martensii visceral organs has significant antitumaous effect, it uses the proliferation function that remarkable inhibition tumor cell is arranged separately, share the significantly tumor-inhibiting action of enhanced sensitivity chemotherapeutic with other chemotherapeutic, resist the untoward reaction that it suppresses immunity simultaneously.
The objective of the invention is to extract a kind of or several have the native compound of active anticancer, determine its chemical nature and architectural feature, understand its physicochemical property, for clinical medicine provides a kind of new antitumor drug from pinctada martensii visceral organs.
The present invention relates to from glycoprotein, glycosaminoglycans two compounds of pinctada martensii visceral organs extraction.This two compounds is to extract from pinctada martensii visceral organs, and purification is after chemical composition mensurations, gas chromatographic analysis infrared spectrum analysis, efficient liquid phase chromatographic analysis are identified its chemical nature of confirmation and architectural feature.
This seminar from the technology that pinctada martensii visceral organs extracts glycoprotein, glycosaminoglycans is: pinctada martensii visceral organs freeze thawing, salt are molten, organic solvent deposit or with equal-volume acetone soak degreasing, the dry grinding of filtering acetone solution after bake, with gastric enzyme, pancreatin or pancreatin, bacillus subtilis neutral proteinase enzymolysis, with kieselguhr, activated carbon (2: 3) decolouring, be dried to powder behind the enzyme denaturing through organic solvent (acetone, ethanol, ethyl acetate) post precipitation; Above-mentioned powder decolours with 1.0% activated carbon and 0.5% kieselguhr, pH2.0 and pH7.0 remove foreign protein and dialysis desalting, dialysis solution precipitates and the 5%CTAB complexation through 60% ethanol-sodium acetate with 60% ethanol-sodium acetate precipitation and 5%CTAB precipitation gained fraction again, gets a series of chemical compounds.Main level lease making chemical composition is measured: aminohexose content is 52.5%, and wherein aminogalactose content is 34.3%, and hexuronic acid content is 7.3%; Be shown as single district band, R through cellulose acetate electrophoresis
fBetween 0.3～0.4; Show through the antitumor result of the test: S-180 sarcoma and ehrlich carcinoma have the tumor-inhibiting action of remarkable enhanced sensitivity positive control drug in to body, and tumour inhibiting rate reaches 61.96% and 46.3% respectively; In vitro culture HL-60 cell there is certain lethal effect; Simultaneously, the anticoagulation experimental result shows: extract has certain anticoagulation, and the anticoagulant effect is one of two percentages of heparin.
Above-mentioned two kinds of chemical compounds are as medical composition, and antineoplastic pharmacologically active particularly is by following experiment confirm.
Embodiment one:
1, materials and methods:
Pinctada martensii visceral organs glycosaminoglycans: extract and get by the described technology of claims.
Platform is expected orchid, 5-Fu, ftorafur.
KM mice: regular grade, body weight 20 ± 2g, male and female half and half are provided by experimental unit of Guangdong Medical College center.
Move growing property tumor: S
180Sarcoma and ehrlich carcinoma (EAC) are provided by Zhongshan Medical Univ. tumor research center, the cultivation of going down to posterity of tumor research group of Pharmacological Teaching and Research Section, Guangdong Medical College routine.
Under the aseptic condition, S is extracted in twice experiment respectively
180And ehrlich carcinoma (EAC) ascites fluid, (platform expected that blue dyeing counting is 8.0 * 10 with dilution in 1: 1 with N.S
6Individual/ml and 6.5 * 10
6Individual/ml), aseptic inoculation (every Mus 0.2ml) is in the right oxter of KM mice.Inoculation back be divided at random next day normal saline group, positive controls (5-FU or ftorafur), low dosage be subjected to reagent group (pinctada martensii visceral organs glycosaminoglycans), high dose be subjected to reagent group (pinctada martensii visceral organs glycosaminoglycans), and positive control drug be subjected to totally five groups of reagent combination group.Every group of mice difference gastric infusion (0.1ml/10g body weight), continuous 10 days, put to death in the 11st day, to weigh, dissection is got sarcoma and is weighed.
2, experimental result
The pinctada martensii visceral organs glycosaminoglycans is to S
180The tumor-inhibiting action of sarcoma and ehrlich carcinoma (EAC) sees Table 1,2.
Table 1. pinctada martensii visceral organs glycosaminoglycans is to S
180Tumor-inhibiting action group dosage number of animals
Body weightTumor heavily presses down tumor
(mg/kg) rate (%) (1) N.S matched group 13 21.6 ± 2.5 30.6 ± 4.7 0.92 ± 0.4 (2) 5-FU 25 10 22.3 ± 2.4 27.9 ± 3.4 0.60 ± 0.43 34.5 after the preceding administration of administration
*(3) complete 200 12 22.0 ± 2.5 28.3 ± 3.7 0.79 ± 0.47 14.1 internal organs glycosaminoglycans (5) (2)+(3) 12 22.2 ± 2.7 26.6 ± 1.8 0.35 ± 0.16 61.96 of complete 50 11 21.3 ± 2.7 29.1 ± 4.5 0.86 ± 0.36 6.5 internal organs glycosaminoglycans (4) pteria martensiis of pteria martensii
* *P＜0.05
*P＜0.001 table 2. pinctada martensii visceral organs glycosaminoglycans is to the tumor-inhibiting action group dosage number of animals of ehrlich carcinoma (EAC)
Body weightTumor heavily presses down tumor
(mg/kg) complete 10 10 25.9 ± 1.8 31.1 ± 3.5 1.89 ± 0.57 10.1 internal organs glycosaminoglycans (5) (2)+(3) 10 24.3 ± 3.0 29.1 ± 4.8 1.14 ± 0.70 46.3 of complete 5 10 24.8 ± 3.2 29.6 ± 2.9 1.60 ± 0.77 24.5 internal organs glycosaminoglycan (4) pteria martensiis of rate (%) (1) N.S control group 10 24.6 ± 2.2 30.7 ± 4.1 2.12 ± 1.16 (2) Tegafur 5 10 24.3 ± 2.7 32.8 ± 6.8 1.58 ± 0.89 25.6 (3) pteria martensii after the front administration of administration* *P＜0.05
Experimental result shows: pinctada martensii visceral organs glycosaminoglycans (50mg/kg) is to S
180Though sarcoma tumor-inhibiting action weak (tumour inhibiting rate 6.5%), the remarkable tumor-inhibiting action of enhanced sensitivity positive control drug 5-FU (25mg/kg, inhibitory rate 34.5%, P＜0.05), inhibitory rate 61.9% (P＜0.001); Pinctada martensii visceral organs glycosaminoglycans (5mg/kg) has tumor-inhibiting action to ehrlich carcinoma (EAC), inhibitory rate 24.5%, and with positive control drug ftorafur (5mg/kg, inhibitory rate 25.6%, P＜0.05) synergism is arranged, inhibitory rate 46.3% (P＜0.01).And twice experiment front and back respectively organize the mice body weight change and the normal saline matched group does not have significant difference, the illustrative experiment reliable results.3, conclusion
The pinctada martensii visceral organs glycosaminoglycans is to S in the body
180Sarcoma and ehrlich carcinoma (EAC) have the tumor-inhibiting action of remarkable enhanced sensitivity positive control drug, and tumour inhibiting rate reaches 61.9% and 46.3% respectively.
Embodiment two
1, materials and methods
Pinctada martensii visceral organs glycosaminoglycans: extract and get by the described technology of claims.
HL-60 cell: the cultivation of going down to posterity of tumor research group of Pharmacological Teaching and Research Section, Guangdong Medical College routine is provided by Zhongshan Medical Univ. tumor research center.
In 24 well culture plates, inoculation HL-60 cell suspension, the inoculating cell number is 5.0 * 10
4Individual/ml, establish 3 parallel holes for every group, add the 5-FU and the pinctada martensii visceral organs glycosaminoglycans of variable concentrations respectively, blank group adds the culture medium of equivalent, places 37 ℃, 5%CO
2Incubator is cultivated 24h, expects blue dyeing counting living cells with platform.Result of the test repeats twice.
2, experimental result
The pinctada martensii visceral organs glycosaminoglycans sees Table 3 to the lethal effect of HL-60 cell.Table 3, pinctada martensii visceral organs glycosaminoglycans are to the lethal effect of HL-60 cell
Group dosage example number becomes viable count kill rate (%) IC
50(1) N.S matched group culture medium 9 7.06 (2) 5-FU 0.001 9 4.67 ± 2.1 33.85 (3) pinctada martensii visceral organs 1.0 9 4.33+1.7 38.67 1.72 glycosaminoglycans (4) pinctada martensii visceral organs 0.1 9 5.25 ± 2.1 25.64 glycosaminoglycans
(become viable count to be: * 10
4Individual/ml)
Experimental result shows: (1.0mg/ml, 0.1mg/ml) kill rate to the HL-60 cell is respectively 38.7% and 25.6%, IC to the pinctada martensii visceral organs glycosaminoglycans when medication 24h
50Be 1.72mg/ml.5-FU (0.01mg/ml) is 33.85% to the kill rate of HL-60 cell, and experimental result is reliable.Prompting: the pinctada martensii visceral organs glycosaminoglycans has certain lethal effect to In vitro culture HL-60 cell.
3, conclusion
When pinctada martensii visceral organs glycosaminoglycans dosage is 1.0mg/ml and 0.1mg/ml In vitro culture HL-60 cell killing rate is reached 38.7% and 25.6% respectively.
Embodiment three:
With preceding method pinctada martensii visceral organs glycoprotein is studied, this chemical compound of also preliminary proof has similar anti-tumor activity.
Embodiment four:
Carried out the research of other pharmacology aspect with pinctada martensii visceral organs glycosaminoglycans, glycoprotein, proved that tentatively it has certain anticoagulation, the anticoagulant effect is one of two percentages of heparin; Also there is the antagonism anticancer chemotherapeutic agent to suppress the effect of the side effect of immunity.
Claims (9)
1, a kind of medical composition with medicine effects such as antitumor and anticoagulants is characterized in that said composition contains the extract of pteria martensii (Pinctada martensi (Dunker)) whole viscera.
2, medical composition as claimed in claim 1 is characterized in that wherein the chemical nature of pinctada martensii visceral organs extract is the contained glycoprotein of pteria martensii, glycosaminoglycans.
3, the contained pinctada martensii visceral organs extract of medical composition as claimed in claim 1, it is characterized in that extraction process is raw material with the pinctada martensii visceral organs,, organic solvent (acetone, ethanol, ethyl acetate) molten through freeze thawing or salt precipitate pinctada martensii visceral organs glycoprotein; Through pepsin, trypsin, bacillus subtilis neutral proteinase hydrolysis, behind decolouring, organic solvent deposit, use method remove impurity such as absorption, isoelectric precipitation, dialysis, precipitate with ethanol, CTAB precipitation, crude extract gets pinctada martensii visceral organs glycosaminoglycans goods again through precipitate with ethanol, CTAB complexometry purification.
4, extraction process as claimed in claim 3 is characterized in that raw material is pteria martensii (Pinctada martensi (Dunker)) whole viscera.
5, extraction process as claimed in claim 3 is characterized in that extracting the pharmacologically actives such as pinctada martensii visceral organs glycoprotein, glycosaminoglycans tool anti-tumor activity and anticoagulant of gained.
6, extraction process as claimed in claim 3 is characterized in that having some fraction to have anti-tumor activity in a series of chemical compounds by this technology extraction gained.
7, extraction process as claimed in claim 3 is characterized in that the used enzyme of hydrolysis is pepsin, trypsin, bacillus subtilis neutral proteinase.
8, extraction process as claimed in claim 3, it is characterized in that freeze thawing, salt is molten or enzymolysis after with organic solvent (acetone, ethanol, ethyl acetate) precipitation, use 1.0% activated carbon and 0.5% diatomite adsorption then, PH2.0 and PH7.0 isoelectric point, IP are removed albumen and dialysis desalting, dialysis solution precipitates with 60% ethanol-sodium acetate precipitation and 5%CTAB, the gained fraction through 60% ethanol-sodium acetate precipitation and 5%CTAB complexation, gets a series of chemical compounds again.
9, extraction process as claimed in claim 3 is characterized in that dissociating with 1.0ml Nacl in the CTAB complexometry in the purification.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011047518A1 (en) * | 2009-10-23 | 2011-04-28 | 中国科学院南海海洋研究所 | Enteral nutrition comprising marine bioactive polysaccharide, preparation method and use thereof |
| CN103130904A (en) * | 2013-01-07 | 2013-06-05 | 天津科技大学 | High-valued utilization method for patinopecten yessoensis offal |
| CN104177484A (en) * | 2014-05-29 | 2014-12-03 | 浙江海洋学院 | Prostatic cancer-resistant mussel glycoprotein and preparation and application thereof |
| WO2016067008A1 (en) * | 2014-10-30 | 2016-05-06 | University Of Salford Enterprises Limited | Pharmaceutical extracts and uses thereof |
| CN115212236A (en) * | 2022-09-19 | 2022-10-21 | 广东医科大学附属医院 | Application of exosome-like nano vesicle derived from pinctada martensii mucus in antitumor drugs |
-
2000
- 2000-04-30 CN CN00105196A patent/CN1321469A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011047518A1 (en) * | 2009-10-23 | 2011-04-28 | 中国科学院南海海洋研究所 | Enteral nutrition comprising marine bioactive polysaccharide, preparation method and use thereof |
| CN103130904A (en) * | 2013-01-07 | 2013-06-05 | 天津科技大学 | High-valued utilization method for patinopecten yessoensis offal |
| CN104177484A (en) * | 2014-05-29 | 2014-12-03 | 浙江海洋学院 | Prostatic cancer-resistant mussel glycoprotein and preparation and application thereof |
| WO2016067008A1 (en) * | 2014-10-30 | 2016-05-06 | University Of Salford Enterprises Limited | Pharmaceutical extracts and uses thereof |
| CN115212236A (en) * | 2022-09-19 | 2022-10-21 | 广东医科大学附属医院 | Application of exosome-like nano vesicle derived from pinctada martensii mucus in antitumor drugs |
| CN115212236B (en) * | 2022-09-19 | 2022-12-20 | 广东医科大学附属医院 | Application of exosome-like nano vesicle derived from pinctada martensii mucus in antitumor drugs |
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