CN103405474B - Method for preparing endogenous digitalis factor - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 20
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- 239000012528 membrane Substances 0.000 claims description 31
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 239000000463 material Substances 0.000 claims description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
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Abstract
The invention belongs to the field of biotechnology, and discloses a method for preparing endogenous digitalis factor, which organically combines ultrafiltration technology and antibody technology, can specifically separate endogenous digitalis-like factor from tissues, and solves the problem of difficult separation caused by too low content in the tissues and too many impurities with similar molecular weight for a long time. The method has the advantages of simple and convenient operation, strong specificity and the like.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of Endogenous digitalis like factors.
Background technology
EDF (endogenous digitalis-like factor, EDLF) also claim interior digicitrin or Endoxin element, be a kind of bioactive substance with Folium Digitalis Purpureae activity that produces and secrete in body, this material has good pharmacological action:
(1) have cardiotonic, research shows that EDLF can strengthen guinea pig myocardium contractility, and this effect is not subject to the impact of A or B adrenoceptor blocker.In congestive heart failure patients blood plasma, EDLF level obviously raises, and has dependency with left atrium and pulmonary artery pressure.Total EDLF and free EDLF content are all higher in anemia of pregnant woman and fetus body, and twin pregnancy is apparently higher than singleton pregnancy, and this may be to increase cardiac output by the cardiotonic of EDLF to meet the needs that cardiac load increases.
(2) have sharp sodium, diuresis, Veress etc. respectively to rat injection, find that wherein a kind of non-peptide class fragment effect of micromolecule amount is the strongest by several different composition in blood plasma, and can make to urinate sodium obviously increases.This acts on injection beginning in latter a hour, within 150 minutes, reaches maximum, and urine sodium increases nearly 20 times, finally proves that this micromolecular non-peptide class fragment is EDLF.The sharp sodium of EDLF, diuresis may be not be realized by suppressing aldosterone or affect glomerular filtration rate because the degree that aldehyde changes because of ketone and time phase sharp sodium while expanding with blood volume, the variation of diuresis is not inconsistent.This sharp sodium, diuresis are not subject to the impact of adrenal gland and kidney function of nervous system.Give angiotensin converting enzyme inhibitor (captopril) and also do not affect this effect.And in sharp sodium, diuresis process, whole body and kidney regional blood flow, glomerular filtration rate are all unchanged.Many scholars prove, the sharp sodium of EDLF, diuresis mechanism are mainly to suppress renal tubules heavily absorbing and causing sodium ion.
(3) vasoconstrictive effect, EDLF has received people's concern to the effect of blood vessel in recent years.EDLF, as a kind of natriuretic factor and vaso-excitor material, may play an important role in hypertension generating process.Plasma of Patients with Hypertension suppresses Na
+, K
+ability and the mean arterial pressure of-atpase activity are proportionate.In addition, EDLF can increase the sensitivity of vascular smooth muscle to norepinephrine, angiotensinⅡ.Takahashi observes salt load rat EDLF content obviously to be increased, the EDLF extracting from salt load dog plasma can make Rat Cremaster Muscle small artery strengthen 10% to the reactivity of norepinephrine, inject this material after 50 minutes to normal rat, mean blood pressure rising 10mmHg.
(4) with other vaso-active substance, EDLF and Endothelin, EDLF and atrial natriuretic peptide, EDLF and 5-hydroxy tryptamine have close relationship.
In sum, the preparation method of Study on Endogenous digitalis like factors, significant.
Summary of the invention
For these reasons, applicant is by years of researches, determined a kind of simple to operation, the method for the extraction purification placenta tissue EDF of high specificity.The present invention is by hyperfiltration technique and the organic combination of antibody technique, can EDF be separated from tissue single-mindedly, solved that content is too low in vivo due to it for a long time, and similar molecular weight impurity is too much, and a difficult problem for the separation difficulty causing.
EDF of the present invention, can be for pathological study of preeclampsia or eclamposia etc.Also can be for other pharmacological action.
The present invention is achieved through the following technical solutions.
A kind of method of preparing Endogenous digitalis like factors:
(1) get Placenta Hominis, be cut into bulk, with liquid nitrogen freezing, be stored in-80 ℃;
(2) after the section of freezing Placenta Hominis, put into homogenizer, homogenizer was put into after liquid nitrogen container 50-70 second, take out homogenizer, under 1400 revs/min-1600 revs/min, homogenate 4-6 minute;
(3) take out homogenate tissue, add isopyknic organic solvent, mix homogeneously, centrifugal 4-6 minute under 1400 revs/min-1600 revs/min, leaves standstill;
(4) get supernatant, reclaim organic solvent to the greatest extent, use ultrafilter membrane centrifugal ultrafiltration, centrifugal 8-12 minute under 2800 revs/min-3200 revs/min, retains permeate;
(5) in permeate, add DigiTAb, mix homogeneously, places 25-35 minute under room temperature, uses ultrafilter membrane centrifugal ultrafiltration, at 800 revs/min of-1200 revs/min of lower centrifugal ultrafiltration 8-12 minute, retains and does not see through material, and permeate discards;
(6) do not see through material and add glycine solution, mix homogeneously, uses ultrafilter membrane centrifugal ultrafiltration, centrifugal 8-12 minute under 800 revs/min-1200 revs/min, and permeate is concentrated, obtains Endogenous digitalis like factors.
Or get blood and add isopyknic organic solvent, mix homogeneously, centrifugal 4-6 minute under 1400 revs/min-1600 revs/min, leaves standstill; Get supernatant, reclaim organic solvent to the greatest extent, use ultrafilter membrane centrifugal ultrafiltration, centrifugal 8-12 minute under 2800 revs/min-3200 revs/min, retains permeate; In permeate, add DigiTAb, add volume and the permeate volume equivalent of DigiTAb, mix homogeneously, under room temperature, place 25-35 minute, use ultrafilter membrane centrifugal ultrafiltration, at 800 revs/min of-1200 revs/min of lower centrifugal ultrafiltration 8-12 minute, retain and do not see through material, permeate discards; Do not see through material and add glycine solution, the concentration of glycine is 0.3 mol/L, volume with do not see through material volume equivalent, mix homogeneously, use ultrafilter membrane centrifugal ultrafiltration, centrifugal 8-12 minute under 800 revs/min-1200 revs/min, permeate is concentrated, vacuum concentration, vacuum is-0.1MPa, thickening temperature is 40-50 ℃, and time 30-40 minute, obtains Endogenous digitalis like factors.
Organic solvent described above is methanol or ethanol.
Ultrafilter membrane described above is to intercept the ultrafilter membrane that molecular weight is 10000.
In above-mentioned steps (5), add volume and the permeate volume equivalent of DigiTAb.
The concentration that adds glycine in above-mentioned steps (6) is 0.3 mol/L, volume with do not see through material volume equivalent.
Simmer down to vacuum concentration described above, vacuum is-0.1MPa that thickening temperature is 40-50 ℃.
Placenta Hominis of the present invention, all derives from puerpera, multipara's informed consent, and sign Informed Consent Form.
An anti digoxin antibody of the present invention has another name called anti digoxin antibody purchased from GlaxoSmithKline(), two anti-mouse-anti sheep IgG are purchased from Jackson ImmunoResearch Laboratories, Inc,
3h labelling divaricoside purchased from Perkin Elmer, polyethylene glycol 6000 purchased from phosphate buffer (PBS buffer) autogamy of Calbiochem, pH=7.4,
3h scintillation solution purchased from National Diagnostics, divaricoside (obtain for divaside separates, divaside is the mixture of divaricoside and divaside second) purchased from the TRIS buffer (Tris buffer) of Sigma, pH=7.4 purchased from Sigma.
Accompanying drawing explanation
Fig. 1: represent embodiment 1 method, A represents the concentration of normal pregnancies blood sample Endogenous digitalis like factors, B represents the concentration of eclamposia anemia of pregnant woman blood sample Endogenous digitalis like factors.EDF concentration, unit receives and rubs/liter.
Fig. 2: represent standard curve of the present invention (obtaining according to Specification Curve of Increasing method), abscissa representative
3h scintillation solution, the μ L of unit; Vertical coordinate represents EDF concentration, unit receives and rubs/liter.
Preparation Example
Embodiment 1
Get anemia of pregnant woman's blood sample, add isopyknic methanol, mix homogeneously, under 1500 revs/min centrifugal 5 minutes, leaves standstill;
Get supernatant, reclaim methanol to the greatest extent, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 3000 revs/min centrifugal 10 minutes, reservation permeate;
In permeate, add isopyknic DigiTAb, mix homogeneously, places under room temperature 30 minutes, and the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, 1000 revs/min of lower centrifugal ultrafiltrations 10 minutes, retains and do not see through material, and permeate discards;
Do not see through material and add 0.3 mol/L, volume with do not see through material volume equivalent glycine solution, mix homogeneously, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 1000 revs/min centrifugal 10 minutes, permeate is vacuum concentration at 45 ℃ in-0.1MPa, temperature, 35 minutes time, obtains Endogenous digitalis like factors.
Get 100 microlitre Endogenous digitalis like factors solution, take out DigiTAb in test kit, two anti-, radioactive label divaricosides, equivalent adds respectively, fully mixes incubated at room; Add again the PEG4000 solution of equivalent, centrifugal, be precipitated thing; Precipitate is resuspended with the PBS of 500 microlitre pH7.4, adds
3 h scintillation solution 200 μ L; Measuring samples exit dose, calculates contained EDF concentration in sample according to standard curve.
Test kit: anti digoxin antibody 12 mcg/ml, two anti-mouse-anti sheep IgG5 × 10
-7mol/L,
3h labelling divaricoside concentration is that 10.0Ci/mmol, Macrogol 4000 concentration are 30%, the phosphate buffer (PBS buffer) of pH=7.4,
3the TRIS buffer (Tris buffer) of H scintillation solution 200 μ L, pH=7.4.
Test kit of the present invention needs to measure
3the radioactive instrument and equipment of H is measured.(equipment is called scintillation counter.Any laboratory that does radioactivity experiment has, and operating process is for to put in sample, and operation software, directly goes out result.）
The drafting of standard curve:
(1) gather eclamposia Placenta Hominis (signature Informed Consent Form), be cut into the piece of tissue of 2cm × 2cm × 2cm size, liquid nitrogen freezing, is stored in-80 ℃;
(2) freezing placenta tissue piece is cut into slices, be placed in homogenizer, put into liquid nitrogen container freezing 1 minute; With the speed of 1500rpm by tissue slice homogenate 5 minutes;
(3) collect homogenate tissue, add the methanol of equivalent, mix, with the speed of 2000rpm centrifugal 5 minutes, removal albumen; Collect centrifuged supernatant, vacuum concentration, 4 ℃ of preservations;
(4) TRIS buffer (Tris buffer) of DigiTAb, two anti-mouse-anti sheep IgG, 2nM divaricoside, pH=7.4 in taking-up test kit, equivalent adds respectively, fully mixes incubated at room;
(5) add again the PEG6000 solution of equivalent, centrifugal, be precipitated thing;
(6) precipitate is resuspended with the PBS of 500 microlitre pH7.4, adds
3h scintillation solution 2 μ L;
(7) measuring samples exit dose, obtains contained EDF concentration in sample;
(8) repeat aforesaid operations, step (4) divaricoside replaces with 5nM successively, 10nM, and 20nM, 50nM, 0.1M and 0.2M, add step (6) meanwhile
3h scintillation solution replaces to 5 μ L, 10 μ L, 20 μ L, 50 μ L, 100 μ L, 200 μ L successively, records numerical value and is depicted as standard curve.
Embodiment 2
(1) get Placenta Hominis, be cut into bulk, with liquid nitrogen freezing, be stored at-80 ℃ stand-by;
(2) after section, put into homogenizer, homogenizer was put into liquid nitrogen container after 50 seconds, take out homogenizer, under 1450 revs/min, homogenate 6 minutes;
(3) take out homogenate tissue, add isopyknic methanol, mix homogeneously, under 1450 revs/min centrifugal 6 minutes, standing;
(4) get supernatant, reclaim methanol to the greatest extent, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 2800 revs/min centrifugal 12 minutes, reservation permeate;
(5) in permeate, add isopyknic DigiTAb, mix homogeneously, places under room temperature 25 minutes, and the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, 800 revs/min of lower centrifugal ultrafiltrations 12 minutes, retains and do not see through material, and permeate discards;
(6) do not see through material and add 0.3 mol/L, volume with do not see through material volume equivalent glycine solution, mix homogeneously, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 850 revs/min centrifugal 12 minutes, permeate is vacuum concentration at 45 ℃ in-0.1MPa, temperature, 30 minutes time, obtains Endogenous digitalis like factors.
Embodiment 3
(1) get Placenta Hominis, be cut into bulk, with liquid nitrogen freezing, be stored at-80 ℃ stand-by;
(2) after section, put into homogenizer, homogenizer was put into liquid nitrogen container after 70 seconds, take out homogenizer, under 1600 revs/min, homogenate 4 minutes;
(3) take out homogenate tissue, add isopyknic methanol, mix homogeneously, under 1600 revs/min centrifugal 4 minutes, standing;
(4) get supernatant, reclaim methanol to the greatest extent, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 3000 revs/min centrifugal 10 minutes, reservation permeate;
(5) in permeate, add isopyknic DigiTAb, mix homogeneously, places under room temperature 35 minutes, and the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, 1200 revs/min of lower centrifugal ultrafiltrations 8 minutes, retains and do not see through material, and permeate discards;
(6) do not see through material and add 0.3 mol/L, volume with do not see through material volume equivalent glycine solution, mix homogeneously, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 1200 revs/min centrifugal 12 minutes, permeate is vacuum concentration at 45 ℃ in-0.1MPa, temperature, 40 minutes time, obtains Endogenous digitalis like factors.
Embodiment 4
(1) get Placenta Hominis, be cut into bulk, with liquid nitrogen freezing, be stored at-80 ℃ stand-by;
(2) after section, put into homogenizer, homogenizer was put into liquid nitrogen container after 60 seconds, take out homogenizer, under 1500 revs/min, homogenate 5 minutes;
(3) take out homogenate tissue, add isopyknic ethanol, mix homogeneously, under 1500 revs/min centrifugal 5 minutes, standing;
(4) get supernatant, reclaim ethanol to the greatest extent, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 3000 revs/min centrifugal 10 minutes, reservation permeate;
(5) in permeate, add isopyknic DigiTAb, mix homogeneously, places under room temperature 30 minutes, and the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, 1000 revs/min of lower centrifugal ultrafiltrations 10 minutes, retains and do not see through material, and permeate discards;
(6) do not see through material and add 0.3 mol/L, volume with do not see through material volume equivalent glycine solution, mix homogeneously, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 1000 revs/min centrifugal 10 minutes, permeate is vacuum concentration at 45 ℃ in-0.1MPa, temperature, 35 minutes time, obtains Endogenous digitalis like factors.
Get 100 microlitre Endogenous digitalis like factors, take out DigiTAb in test kit, two anti-, radioactive label divaricosides, equivalent adds respectively, fully mixes incubated at room; Add again the PEG4000 solution of equivalent, centrifugal, be precipitated thing; Precipitate is resuspended with the PBS of 500 microlitre pH7.4, adds
3 h scintillation solution 200 μ L; Measuring samples exit dose, calculates contained EDF concentration in sample according to standard curve.
Test kit: anti digoxin antibody 12 mcg/ml, two anti-mouse-anti sheep IgG5 × 10
-7mol/L,
3h labelling divaricoside concentration is that 10.0Ci/mmol, Macrogol 4000 concentration are 30%, the phosphate buffer (PBS buffer) of pH=7.4,
3the TRIS buffer (Tris buffer) of H scintillation solution 200 μ L, pH=7.4.
Test kit of the present invention needs to measure
3the radioactive instrument and equipment of H is measured.(equipment is called scintillation counter.Any laboratory that does radioactivity experiment has, and operating process is for to put in sample, and operation software, directly goes out result.）
The drafting of standard curve:
(1) gather eclamposia patient's Placenta Hominis (signature Informed Consent Form), be cut into the piece of tissue of 2cm × 2cm × 2cm size, liquid nitrogen freezing, is stored in-80 ℃;
(2) freezing placenta tissue piece is cut into slices, be placed in homogenizer, put into liquid nitrogen container freezing 1 minute; With the speed of 1500rpm by tissue slice homogenate 5 minutes;
(3) collect homogenate tissue, add the methanol of equivalent, mix, with the speed of 2000rpm centrifugal 5 minutes, removal albumen; Collect centrifuged supernatant, vacuum concentration, 4 ℃ of preservations;
(4) TRIS buffer (Tris buffer) of DigiTAb, two anti-mouse-anti sheep IgG, 2nM divaricoside, pH=7.4 in taking-up test kit, equivalent adds respectively, fully mixes incubated at room;
(5) add again the PEG6000 solution of equivalent, centrifugal, be precipitated thing;
(6) precipitate is resuspended with the PBS of 500 microlitre pH7.4, adds
3h scintillation solution 2 μ L;
(7) measuring samples exit dose, obtains contained EDF concentration in sample,
(8) repeat aforesaid operations, step (4) divaricoside replaces with 5nM successively, 10nM, and 20nM, 50nM, 0.1M and 0.2M, add step (6) meanwhile
3h scintillation solution replaces to 5 μ L, 10 μ L, 20 μ L, 50 μ L, 100 μ L, 200 μ L successively, records numerical value and is depicted as standard curve.
Embodiment 5
Get anemia of pregnant woman's blood sample, add isopyknic ethanol, mix homogeneously, under 1450 revs/min centrifugal 6 minutes, leaves standstill;
Get supernatant, reclaim ethanol to the greatest extent, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 2800 revs/min centrifugal 12 minutes, reservation permeate;
In permeate, add isopyknic DigiTAb, mix homogeneously, places under room temperature 25 minutes, and the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, 800 revs/min of lower centrifugal ultrafiltrations 12 minutes, retains and do not see through material, and permeate discards;
Do not see through material and add 0.3 mol/L, volume with do not see through material volume equivalent glycine solution, mix homogeneously, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 850 revs/min centrifugal 12 minutes, permeate is vacuum concentration at 45 ℃ in-0.1MPa, temperature, obtains Endogenous digitalis like factors.
Embodiment 6
Get anemia of pregnant woman's blood sample, add isopyknic ethanol, mix homogeneously, under 1600 revs/min centrifugal 4 minutes, leaves standstill;
Get supernatant, reclaim ethanol to the greatest extent, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 3000 revs/min centrifugal 10 minutes, reservation permeate;
In permeate, add isopyknic DigiTAb, mix homogeneously, places under room temperature 35 minutes, and the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, 1200 revs/min of lower centrifugal ultrafiltrations 8 minutes, retains and do not see through material, and permeate discards;
Do not see through material and add 0.3 mol/L, volume with do not see through material volume equivalent glycine solution, mix homogeneously, the ultrafilter membrane centrifugal ultrafiltration that is 10000 with intercepting molecular weight, under 1200 revs/min centrifugal 12 minutes, permeate is vacuum concentration at 45 ℃ in-0.1MPa, temperature, 35 minutes time, obtains Endogenous digitalis like factors.
The test of cardiac diuretic reason
(1) diuretic test
The healthy male mice of experimental animal: body weight 20g left and right.
Test method: get healthy male mice, random packet, 10 every group, and weigh.Before test, make it in advance to adapt to metabolic cage environment 1 day, whether the urine amount under free conditions of water drinking of observing is stablized.Test fasting in first 18 hours and can't help water, before medication, fill with normal saline 5ml/100g by body weight, as water load, collect 2 hours urine amounts, urine amount exceedes 40% water bearer, can proceed formal experiment.
By qualified mice random packet: normal saline group, embodiment 1-6 group, every group 10, before test, fasting be can't help water 18 hours, measuring every Mouse Weight gives normal saline by the dosage of every mice 5ml/l00g and loads as water, test 1-6 group abdominal cavity gives digitalis like factors solution, dosage 0.1ml Endogenous digitalis like factors/100g, matched group gives physiologic saline for substitute medicinal liquid.The light mice lower abdomen of pressing before administration, drains the dirty interior remaining urine of wing.Record respectively the urine amount in mice 3 hours.
Result of the test is in table 1.
The impact of table 1 on mouse retention amount
Note: with relatively * P<0.05 of matched group.
(2) cardiotonic
Get the mice after 3 hours urine amounts of laboratory observation, obtain every mice 0.5-1.0ml blood with plucking eyeball blood taking method, put in people's centrifuge tube and (in every centrifuge tube, put in advance the heparin 0.05ml that people diluted), then centrifuge tube is put to people's centrifuge centrifugal 15 minutes.After 15 minutes, the supernatant of getting in centrifuge tube is that blood plasma moves in prior ready test tube, carries out changes in plasma aldosterone levels mensuration with putting the method for exempting from.
Result of the test is in table 2.
Table 2 is to mice plasma aldosterone level
Note: with relatively * * P<0.01 of matched group.
Conclusion (of pressure testing): above-mentioned test shows, the Endogenous digitalis like factors solution that the inventive method obtains has the effect of good heart tonifying diuresis.
Embodiment includes but not limited to above-mentioned.
Claims (4)
1. a method of preparing Endogenous digitalis like factors, is characterized in that:
(1) get Placenta Hominis, be cut into bulk, with liquid nitrogen freezing, be stored in-80 ℃;
(2) after the section of freezing Placenta Hominis, put into homogenizer, homogenizer was put into after liquid nitrogen container 50-70 second, take out homogenizer, under 1400 revs/min-1600 revs/min, homogenate 4-6 minute;
(3) take out homogenate tissue, add isopyknic organic solvent, mix homogeneously, centrifugal 4-6 minute under 1400 revs/min-1600 revs/min, leaves standstill;
(4) get supernatant, reclaim organic solvent to the greatest extent, use ultrafilter membrane centrifugal ultrafiltration, centrifugal 8-12 minute under 2800 revs/min-3200 revs/min, retains permeate;
(5) in permeate, add DigiTAb, mix homogeneously, places 25-35 minute under room temperature, uses ultrafilter membrane centrifugal ultrafiltration, at 800 revs/min of-1200 revs/min of lower centrifugal ultrafiltration 8-12 minute, retains and does not see through material, and permeate discards;
(6) do not see through material and add glycine solution, mix homogeneously, uses ultrafilter membrane centrifugal ultrafiltration, centrifugal 8-12 minute under 800 revs/min-1200 revs/min, and permeate is concentrated, obtains Endogenous digitalis like factors;
Wherein said organic solvent is methanol or ethanol;
Wherein said ultrafilter membrane is to intercept the ultrafilter membrane that molecular weight is 10000.
2. a kind of method of preparing Endogenous digitalis like factors according to claim 1, wherein adds volume and the permeate volume equivalent of DigiTAb in step (5).
3. a kind of method of preparing Endogenous digitalis like factors according to claim 1, wherein in step (6), adding the concentration of glycine is 0.3 mol/L, volume with do not see through material volume equivalent.
4. a kind of method of preparing Endogenous digitalis like factors according to claim 1, wherein said simmer down to vacuum concentration, vacuum is-0.1MPa that thickening temperature is 40-50 ℃, time 30-40 minute.
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