CN103570814A - Technique for extracting hemaglutinin with antitumor action from Canavalia ensiformis - Google Patents

Abstract

The invention discloses a technique for extracting hemaglutinin with an antitumor action from Canavalia ensiformis. The technique comprises the following steps: 1) removing impurities from a dry product of the Canavalia ensiformis, to be specific, firstly, rinsing by running water for three times, and removing dust, soil and the like; 2) rinsing by distilled water for 3-5 times, soaking by 75-80% ethanol for 30 minutes, and then putting into a constant-temperature oven of 35-37 DEG C to dry for later use; 3) crushing the dried Canavalia ensiformis into 100-150 meshes of fine powder under a sterile condition, and adding 2-3 folds of normal saline to stand for 24-48 hours at 5 DEG C; 4) carrying out centrifugal separation, removing the slag, taking centrifugate, adding solid ammonium sulfate to the centrifugate, refrigerating for 12 hours below 5 DEG C, centrifuging, separating out supernate and removing sediments; and 5) separating the supernate on a gel column, eluting and separating by 1M of NaCl solution, collecting eluent, forming the required ConA after the test is qualified, and preparing freeze-dried powder from the obtained ConA, wherein the freeze-dried powder is clinically applied to treatment of tumors.

Description

From Canavalia ensiformis, extract the processing method of the homo agglutinin with antitumor action

(i) technical field: the invention belongs to extract drugs technique, specifically a kind of processing method of extracting the homo agglutinin with antitumor action from Canavalia ensiformis.

(ii) background technology: Canavalia ensiformis Canavalia ensiformis (L.) DC., originate in western India, also there is minority cultivation in China, and torrid areas is born in happiness.Its seed is also used as medicine on an equal basis.Canavalia ensiformis is very similar on plant each several part organ morphology to native country sword bean, both distinctive points: sword bean is prehensile draft, and hilum is about as 3/4 of seed, and Canavalia ensiformis is orthostatic draft, and hilum is about as 1/2 of seed.Modern study shows: Canavalia ensiformis includes the multiple sphaeroprotein such as Canavalia ensiformis homo agglutinin (Concanavalin A is called for short ConA).Other still have urease, glucosidase, turn aminosuccinic acid enzyme, Cyklokapren enzyme, canclin, canavanine, L-homoserine etc.Recently because Canavalia ensiformis homo agglutinin comes into one's own to special the doing evil of tumour cell.

Research shows, Canavalia ensiformis homo agglutinin is a kind of of phytoh(a)emagglutinin, tool antitumor action.Canavalia ensiformis homo agglutinin can cause the distortion (being that lymphocyte is stimulated and changes lymphoblast into) of human lymphocyte, but does not produce corresponding cytotoxin, also can suppress the cytotoxin that other PHA cause.With golden vole embryo, test, Canavalia ensiformis homo agglutinin is greater than Normocellular toxicity the toxicity of the amoebocyte obtaining after processing with virus or chemical carcinogens.Canavalia ensiformis homo agglutinin (through trypsin treatment) can also make tumour cell (the mouse fibroblast after distortion) be restored to Normocellular growth conditions.Glycoprotein in Canavalia ensiformis homo agglutinin and Fructus Hordei Germinatus, soybean to YAC cell all can aggegation it, but only have Canavalia ensiformis homo agglutinin to have significant toxicity to YAC cell.In test, with the Canavalia ensiformis homo agglutinin of 125 micrograms, plant therewith cell and incubate altogether 24 hours in vitro.Can make 95% cytolysis; In vivo in test, give 1 mouse after adult mice abdominal injection YAC cell, 2 days, 5 days 1 milligram of abdominal injection Canavalia ensiformis homo agglutinin again, can suppress respectively tumour 70%, 50% and 20%.Canavalia ensiformis homo agglutinin can aggegation by the caused amoebocyte of various carcinogens, and to normal cell, only with after trypsin treatment, can aggegation.This kind of aggegation all can be by Alpha-Methyl-D-MANNOSE glucoside competitive antagonism in addition.Therefore imagination, be combined and work in the position of the glucose of Canavalia ensiformis homo agglutinin on amoebocyte surface film or seminose sample, and at normal cell, this position is being covered.

Therefore, find the processing method of homo agglutinin in the separated Canavalia ensiformis of a kind of effective extraction, improve recovery rate, there is very important value.

(iii) summary of the invention: object of the present invention is just to provide a kind of processing method of extracting the homo agglutinin with antitumor action from Canavalia ensiformis; Present method technique is simple, easy to operate, and yield is high.

The inventive method comprises following processing step:

1) Canavalia ensiformis dry product is chosen to decon, first with tap water, rinse 3 times dust earth;

2), with distilled water flushing 3-5 time, by 75-80% alcohol immersion, after 30 minutes, put 24-28 hour in 35-37 ℃ of thermostat container, dry for standby;

3) Canavalia ensiformis of oven dry is ground into 100-150 order fine powder under aseptic condition, adds 2-3 times of physiological saline and place 24-28 hour under 5 ℃ of low temperature;

4) centrifugation, removes slag, extracting centrifugal liquid; In centrifugate, add solid ammonium sulfate, refrigerate 12 hours at 5 ℃, centrifugal, separate supernatant liquor, remove precipitation;

5) on supernatant liquor, gel column is separated, separated with 1M NaCl eluant solution, collects elutriant, detects after qualified and is needed ConA.

Resulting ConA can make injection lyophilized powder, clinically for oncotherapy.

(iv) embodiment:

Embodiment 1

1) Canavalia ensiformis dry product is chosen to decon, first with tap water, rinse 3 times dust earth;

2), with distilled water flushing 5 times, by 75% alcohol immersion after 30 minutes, put in 37 ℃ of thermostat containers dry for standby 24 hours;

3) Canavalia ensiformis of oven dry is ground into 100 order fine powders under aseptic condition, adds 2 times of physiological saline and at 5 ℃, place 28 hours;

4) centrifugation, removes slag, extracting centrifugal liquid; In centrifugate, add solid ammonium sulfate, refrigerate 12 hours at 5 ℃, centrifugal, separate supernatant liquor, remove precipitation;

5) on supernatant liquor, gel column is separated, separated with 1M NaCl eluant solution, collects elutriant, detects after qualified and is needed ConA.

Yield can reach 75%, than the high 3%-5% of existing technique.

Claims (1)

1. from Canavalia ensiformis, extract the processing method of the homo agglutinin with antitumor action, comprise the following steps:

1) Canavalia ensiformis dry product is chosen to decon, first with tap water, rinse 3 times dust earth;

2), with distilled water flushing 3-5 time, by 75-80% alcohol immersion, after 30 minutes, put 24-28 hour in 35-37 ℃ of thermostat container, dry for standby;

3) Canavalia ensiformis of oven dry is ground into 100-150 order fine powder under aseptic condition, adds 2-3 times of physiological saline and place 24-28 hour at 5 ℃;

4) centrifugation, removes slag, extracting centrifugal liquid; In centrifugate, add solid ammonium sulfate, refrigerate 12 hours at 5 ℃, centrifugal, separate supernatant liquor, remove precipitation;

5) on supernatant liquor, gel column is separated, separated with 1M NaCl eluant solution, collects elutriant, detects after qualified and is needed ConA.