CN1371920A

CN1371920A - Persimmon leaf polysaccharide and biological activity thereof

Info

Publication number: CN1371920A
Application number: CN 01105483
Authority: CN
Inventors: 方积年; 陈林祥
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2001-02-28
Filing date: 2001-02-28
Publication date: 2002-10-02

Abstract

The present invention provides a polysaccharide dextracted from persimmon leaf and its extraction method. Said polysaccharide possesses immunological enhancement and sugar-reducing action for diabetic of type II.

Description

Persimmon leaf polysaccharide and biological activity thereof

The present invention relates to vegetable polysaccharides extraction and its biological activity, more specifically say so persimmon polysaccharide and its hypoglycemic activity.

Polysaccharide such as heparin has blood coagulation resisting function in blood system be early found, it as medicinal application in clinical.Along with to the going deep into of sugared function understanding, the polysaccharide of progressively finding a fixed structure has significant effect to the immunity system of body after the fifties: as antitumor action, and antivirus action particularly anti-AIDS effect, anti-inflammatory action and radiation resistance.These polysaccharide also have three kinds as medicine formally use clinically at home and abroad (the also having several of unofficial use).Certainly the polysaccharide of also finding some structure also has effect as emesis, antiulcer agent etc. to gi system.All are applied to the maximum characteristics of clinical polysaccharide as medicine be that toxic side effect is little.Have some polysaccharide to have the report of hypoglycemic activity in addition, but effect is not remarkable yet.Because diabetes have become a kind of common disease, have a strong impact on patient's quality of life, even life, have great social significance and using value so seek the hypoglycemic drug of high-efficiency low-toxicity.

It is widely known that persimmon extensively is distributed in its fruit of northern province, persimmons such as China Henan, and its leaf acts as a fuel mostly and wastes, and the present invention extracts polysaccharide and carries out various pharmacological testings and shows that it has significant hypoglycemic activity and immunologic enhancement from the persimmon leaf.

The object of the invention provides a kind of effective ingredient of extracting to hypoglycemic little polysaccharide medicine or the healthcare products of tool side effect with obvious effect from plant.

The present invention implements like this:

Fresh or exsiccant persimmon leaf soaked in ethanol more than 72 hours, took out air-dryly, took by weighing dried persimmon leaf, add 10-15 doubly (w/w) deionized water calorify under the boiling situation 3-8 hour, cross the Lu residue, again with 8-10 doubly (w/w) deionized water as above method extract again once, merge secondary Lu liquid, be evaporated to 1/15-/20 of original volume, add 3-5 times of ethanol and stir precipitation, throw out is through centrifugal, vacuum-drying, obtain the raw product of polysaccharide, this raw product promptly can be used as healthcare products and uses.Raw product to water dialysis 2 days, sees through 3 times of ethanol sedimentations of 1/10 adding that liquid is evaporated to original volume after water dissolution, taking precipitate is soluble in water by DEAE-cellulose column, water and 0.2M NaCl stepwise elution, and effluent liquid detects through the sulfuric acid phynol method, gets partly positive, merge dialysis, lyophilize, the lyophilize thing is soluble in water, be splined on Sephacrgl S-300 gel column, 0.1M NaCl opens up layer, the differential detector detects, and merges the main peak part, dialysis, lyophilize.This sample detects through HPLC, and the result is a single symmetrical peak, proves that this polysaccharide is pure product (promptly all a part of), and these pure product can be used as medicine and use.

Adopt the separation of polysaccharides method, we at first obtain the raw product (doing health care uses) and the pure product (doing medicinal application) of polysaccharide at home from the persimmon leaf.This polysaccharide not only has immunologic enhancement, the more important thing is the significant hypoglycemic activity of tool, and crude product can be used as diabetic subject's healthcare products, and its pure product promptly can be used as diabetic subject's treatment medicine.

In biological activity test, with the PTP1B expression and purification, the enzyme that obtains exists with the form of gst fusion protein.The phosphatide key of PTP1B hydrolysis substrate PNPP, the product that obtains has very strong photoabsorption at the 410nm place.The variation that therefore can directly detect the photoabsorption of 410nm place is with the activity change of observation enzyme and the compound inhibition situation (Zhang, ZY., Dixon, JE. (1994) Adv.Enzymol.68:1-36) to it.It is as follows that standard is surveyed live body: 10mM Tris.Cl, pH 7.6,10mM PNPP, 2%DMSO, 200nM PTP1B.The result is that 100 μ g/m (crude product) and 10 μ g/m (pure product) reach more than 95% protein tyrosine phosphatase esterase PTPIB inhibiting rate in concentration respectively, protein tyrosine phosphatase esterase PTPIB can make the insulin receptor generation dephosphorylation of phosphorylation, thereby in vivo regulating effect is played in the effect of Regular Insulin, therefore, type ii diabetes and obesity had therapeutic action.

Immunocompetence test: lymphocyte is at mitogen such as PHA, ConA, and under the stimulation of LPS etc., a series of variation can take place in the form of cell and metabolism, is converted into parent cell, and differentiation and proliferation.Propagation with mtt assay quantitative assay cell.This experiment adds LPS (lipopolysaccharides) 10 μ g/ml and induces bone-marrow-derived lymphocyte propagation.The result shows: concentration is respectively 0.01mg/ml (crude product) and its all genus remarkable (P＜0.01) to the proliferative response of bone-marrow-derived lymphocyte of 0.01mg/ml (pure product), above-mentioned polysaccharide crude and pure product are tested the oral maximum tolerated dose of its mouse all greater than 8g/kg through mouse LD50, conclusion thus, the not only equal tool notable biological activity of crude product and pure product, and have no side effect, can the independent or compound healthcare products of doing, or be further used as medicinal application.

Advantage of the present invention:

1. persimmon leaf wide material sources are turned waste into wealth, by abandoning originally or becoming healthcare products or medicine as fuel.

2. extraction process is easy, and is with low cost.

Embodiment:

Fresh persimmon leaf soaked in ethanol 72 hours, take out, natural draft drying, take by weighing the persimmon leaf of 1Kg drying, add the 10l deionized water, ebuillition of heated 4 hours, filter Lu liquid, the Lu slag adds the 10l deionized water again, and ebuillition of heated was crossed the Lu in 4 hours and got Lu liquid for the second time, merges secondary Lu liquid, concentrating under reduced pressure is to 800ml, under agitation, add 3 times of amount 90%7 alcohol slowly, obtain precipitation, throw out is through centrifugal, (＜60 ℃) drying is 24 hours in vacuum drying oven, takes out, and pulverizes promptly to obtain making the polysaccharide 28g that healthcare products are used.

Get above-mentioned raw product polysaccharide 10g and be dissolved in the deionized water (100ml),, take out and do not see through liquid in the dialysis tubing deionized water dialysis 2 days, be concentrated on the 100ml DEAE-cellulose chromatography, 0.2MNaCl wash-out, get effluent liquid sugar reacting positive part, merge, water dialysis two days are stayed in steaming, see through liquid and be concentrated into upward Sephacrgl S-300 post of 10ml, 0.1M NaCl opens up layer, merges efficient part, the 2 days concentrating under reduced pressure of dialysing, lyophilize then gets pure product 254mg.Hypoglycemic test:

It is soluble in water respectively to get above-mentioned raw product and pure product, making into concentration is 100 μ g/ml and 10 μ g/ml, observe its inhibiting rate and found that its inhibiting rate is respectively 99% and 95% the protein tyrosine phosphatase ester, the used phosphoric acid ester PTPIB of this experiment is from expression in escherichia coli and purifying, and adopting positive in the experiment is the sodium vanadate of 2 μ M with reference to medicine.

Claims

1. polysaccharide that extracts by the persimmon leaf, it is characterized in that in ethanol, soaking by the persimmon leaf, air-dry then, add deionized water, be heated to boiling 3-8 hour, filter, residue again with deionized water extraction once, merging filtrate, concentrating under reduced pressure adds 3-5 times of ethanol and stirs to small volume, precipitation, throw out is centrifugal, soluble in water again to water dialysis 48 hours, do not see through liquid and be evaporated to 1/10 of original volume, add ethanol sedimentation again, throw out is soluble in water by the DEAE-cellulose column, water and rare NaCl stepwise elution, stream place liquid detects through the sulfuric acid phynol method, gets positive part, merges the dialyzate lyophilize.The lyophilize thing is soluble in water, is splined on the Sephacrgl gel column, and with rare NaCl exhibition layer, the differential detector detects, and merges the main peak part, dialysis, and lyophilize, sample HPLC detects, and gets the polysaccharide of single symmetrical peak.

2. according to the described polysaccharide that extracts by the persimmon leaf of claim 1, it is characterized in that throw out passes through DEAE-cellulose column water and 0.1-0.3M NaCl stepwise elution.

3. according to the described polysaccharide that is extracted by the persimmon leaf of claim 1, it is characterized in that the lyophilize thing is splined on the Sephacrgl gel column, developping agent is a 0.1-0.3M NaCl solution.

4. according to the described polysaccharide that extracts by the persimmon leaf of claim 1, it is characterized in that getting the polysaccharide of single symmetrical peak by the HPLC test sample.

5. the polysaccharide that the persimmon leaf extracts is characterized in that it is to using in the treatment type ii diabetes medicine.

6. the polysaccharide that the persimmon leaf extracts is characterized in that using in its treatment to obesity.