CN102174095B - Malania oleifera lectin and method for preparing same - Google Patents
Malania oleifera lectin and method for preparing same Download PDFInfo
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- CN102174095B CN102174095B CN201110025266.8A CN201110025266A CN102174095B CN 102174095 B CN102174095 B CN 102174095B CN 201110025266 A CN201110025266 A CN 201110025266A CN 102174095 B CN102174095 B CN 102174095B
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- 102000004856 Lectins Human genes 0.000 title claims abstract description 45
- 108090001090 Lectins Proteins 0.000 title claims abstract description 45
- 239000002523 lectin Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 9
- 241000928579 Malania oleifera Species 0.000 title abstract description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 16
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 11
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 6
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 4
- 150000003839 salts Chemical class 0.000 claims abstract description 4
- 238000000227 grinding Methods 0.000 claims abstract description 3
- 240000002234 Allium sativum Species 0.000 claims description 31
- 235000004611 garlic Nutrition 0.000 claims description 31
- 230000004520 agglutination Effects 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 16
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000010612 desalination reaction Methods 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- 238000013016 damping Methods 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 10
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
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- 239000012141 concentrate Substances 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 239000000047 product Substances 0.000 claims 1
- 241000272525 Anas platyrhynchos Species 0.000 abstract description 7
- 241000287828 Gallus gallus Species 0.000 abstract description 7
- 238000001962 electrophoresis Methods 0.000 abstract description 6
- 208000031886 HIV Infections Diseases 0.000 abstract description 2
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- 241000699666 Mus <mouse, genus> Species 0.000 description 6
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 239000003921 oil Substances 0.000 description 5
- 235000019198 oils Nutrition 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000010828 elution Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
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- 241000221014 Olacaceae Species 0.000 description 2
- 241001529246 Platymiscium Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
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- WXCMHFPAUCOJIG-UHFFFAOYSA-N 4'-tert-Butyl-2',6'-dimethyl-3',5'-dinitroacetophenone Chemical compound CC(=O)C1=C(C)C([N+]([O-])=O)=C(C(C)(C)C)C([N+]([O-])=O)=C1C WXCMHFPAUCOJIG-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
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- ALHUZKCOMYUFRB-OAHLLOKOSA-N Muscone Chemical compound C[C@@H]1CCCCCCCCCCCCC(=O)C1 ALHUZKCOMYUFRB-OAHLLOKOSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 238000001042 affinity chromatography Methods 0.000 description 1
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- 238000010322 bone marrow transplantation Methods 0.000 description 1
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- ALHUZKCOMYUFRB-UHFFFAOYSA-N muskone Natural products CC1CCCCCCCCCCCCC(=O)C1 ALHUZKCOMYUFRB-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides malania oleifera lectin and a method for preparing the same. The method comprises the following steps: grinding malania oleifera, stirring and socking the ground malania oleifera into cold water, grading and precipitating by using ammonium sulfate, ultrafiltering to remove salt, concentrating, carrying out ion exchange chromatography, purifying, ultrafiltering to remove salt, concentrating, freezing and drying to obtain the refined malania oleifera lectin. The prepared malania oleifera lectin has the molecular weight of about 60kd, the electrophoresis purity of above 90% and the potency of 1:10<5> or more, namely the HIV infected cell (inhibition concentration 50) IC50 of 1:10<5>, can agglutinate the red blood cells of a rabbit, a pig, a chicken, a duck and a mouse and can be used for preventing HIV infection.
Description
Technical field
The present invention relates to a kind of Olacaceae head of garlic kernel lectin and preparation method thereof, the platymiscium lectin extracts or the fabricating technology field.
Background technology
Lectin is protein or the multivalence carbohydrate-binding protein of a class by animals and plants and non-enzyme of microorganism cells excretory and non-immunity, because of generally being referred to as by some cell of aggegation (as red corpuscle), its family member constantly increases, the function complexity is various, as the virulence decision, aggegation malignant cell, sperm, medullary cell etc. of mitogenesis, virus.Because lectin has the sugared binding specificity of height, research biology and the very useful instrument of clinical medicine have been become, be used for separation and purification and determine sugared complex body, cell fission, Identifying micro-organisms, diagnosing tumor, treatment and prevention bone marrow transplantation etc., some lectin has blocking-up HIV and attacks the cd4 cell ability, (Xu Jin etc.: the Chinese biological goods are learned magazine as the Bacterium lacticum lectin, 2008,9:792-795), sealwort lectin (CN200610005577) etc.The aggegation of different sources have different aggegation spectrums and using value (Liu Xuelan etc.: the biological characteristics of mitten crab serum lectin, Chinese aquatic science 2006,3:335-340), different extracting and preparing technique and method are arranged, as affinity chromatography (CN028243196) etc.
Malania oleifera (
Malana oleifera) have another name called that (Guangxi Zhuang voice), Mi Min behind the kalimeris, Tieghemella heckelii Pierre, mark are thick, mountain bucket fruit etc., belong to the Olacaceae malania oleifera to belong to, be the peculiar autogenus rare plant of China, national second class protection seeds.Mainly be distributed in the lane of western part, Guangxi and southeastern Yunnan, be typical oil crops, plant the benevolence oil length up to 64.5%, grease is edible both, be again synthetic musk ketone (muscone), insect pheromone etc. industrial raw material (Zhao Jingping, Needles is begged in Europe: the applied research of head of garlic nut oil, Chinese oil, 2010,7:12-16).Up to now; forefathers obtain certain achievement in research at aspects such as the development and use of malania oleifera fruit fatty acid oil composition and oleaginousness, reason in imminent danger and protection, fruit fatty acid oil and timber, ecological adaptation feature, tissue culture, but aggregate level also rests on the starting stage.Patent of invention 200510010633 is smashed malania oleifera not being higher than under 4 ℃ the cold condition to pieces, filter, supernatant liquor 30-100% ammonium sulfate precipitation, dialysis, chromatographic apparatus purifying by the protein separation special use again, the 55kd anticancer protein that obtain that the SDS-PAGE electrophoresis is pure, two subunits of molecular weight 32kD and 23kD constitutes.Yet, so far, the report that does not still have the research of head of garlic kernel lectin and use.
Summary of the invention
The purpose of this invention is to provide a kind of lectin that from the malania oleifera platymiscium, extracts and preparation method thereof.
The invention provides a kind of head of garlic kernel lectin, comprise following mass component:
Described head of garlic kernel lectin is used for the red cell agglutination of rabbit, pig, chicken, duck, mouse, HIV (human immunodeficiency virus)-resistant activity.
Another object of the present invention is to provide a kind of preparation method of head of garlic kernel lectin, following each processing step of process:
A. exsiccant head of garlic kernel being pulverized, is that 1 ︰ 3～8 is dissolved in the water by solid-to-liquid ratio, and water temperature is 0～10 ℃, stirs 10～48h under 0～10 ℃ of temperature, then this mixture is filtered, and obtains yellow transparent filtrate;
B. in filtrate, add the ammonium sulfate powder, and stirring and dissolving reaches at 25～40% o'clock and stops until the ammonium sulfate saturation ratio and adds, after leaving standstill 1～2 hour under 0～10 ℃, in 0～10 ℃, rotating speed is to carry out centrifugation under 1000～8000rpm, gets its supernatant liquor and continues to add ammonium sulfate powder to saturation ratio and reach 60～75%, leave standstill 1～2 hour after, in 0～10 ℃, rotating speed is to carry out centrifugation under 1000～8000rpm, remove supernatant liquor, get its throw out, be the lectin crude product;
C. step B gained lectin crude product is carried out ultrafiltration or dialysis desalination after with dissolved in distilled water, concentrated solution;
D. with step C gained concentrated solution process ion exchange chromatography, Tris-HCl damping fluid hanging column with 0.02mol/L, make sample absorption, Tris-HCl damping fluid and concentration with 0.02mol/L are carried out the linear concentration gradient wash-out from the salts solution that the 0.1%NaCl linearity is elevated to 6%NaCl gradually, detect agglutination activity with rabbit erythrocyte, collection has the component of agglutination activity, again through ultrafiltration or dialysis desalination, concentrate, lyophilize, promptly get head of garlic kernel lectin, it comprises following mass component: protein 60～80%, polysaccharide 15～30%.
Exsiccant head of garlic kernel grinding particle size is 40～200 orders in the described steps A.
The film of ultrafiltration or dialysis desalination sees through molecular weight≤10kd among described step C and the D.
Step D gained head of garlic kernel lectin SDS-PAGE sex change electrophoresis is single band (promptly not seeing the foreign protein band), molecular weight 60kd, protein content 80～90% (Kjeldahl determination), sugar content 5～10%(sulfuric acid phynol method), can make the tiring of red cell agglutination of rabbit, pig, chicken, duck, mouse reach 1:10
6More than (μ g/ml promptly≤1), HIV (human immunodeficiency virus)-resistant activity IC
50≤ 50 μ g/ml.
Advantage of the present invention and effect: extraction and purification process is easy fast, particularly ultrafiltration can have desalination, removal of impurities concurrently, concentrate, multiple action such as separation, be easy to linear amplification; Gained head of garlic kernel lectin can make the red cell agglutination of rabbit, pig, chicken, duck, mouse tire up to 1:10
6More than (μ g/ml promptly≤1); Higher HIV (human immunodeficiency virus)-resistant activity is arranged, IC
50≤ 50 μ g/ml; Have a wide range of applications at aspects such as anti-HIV infection, biological detection, medical diagnosis on disease and treatments; Realized the comprehensive high-efficiency utilization of malania oleifera resource.
Description of drawings
Fig. 1 is that head of garlic kernel lectin is through Sepharose XL ion-exchange chromatography elution curve (only the B3 component has agglutination activity);
Fig. 2 is the SDS-PAGE electrophorogram of refining head of garlic kernel lectin;
Fig. 3 is that head of garlic kernel lectin is to HIV-1 Ba-L(R5 virus) the active dose-response curve of inhibition;
Fig. 4 is that head of garlic kernel lectin is to the Cytotoxic dose-response curve of TZM-b1.
Embodiment
Below in conjunction with embodiment the present invention is described further.
A. exsiccant head of garlic kernel is crushed to 100 orders, takes by weighing the 100g powder, be dissolved in the water of 800g, water temperature is 4 ℃, stirs 24h under 4 ℃ of temperature, then this mixture is filtered, and obtains yellow transparent filtrate;
B. in filtrate, add the ammonium sulfate powder, and stirring and dissolving reaches at 40% o'clock and stops until the ammonium sulfate saturation ratio and adds, after leaving standstill 2 hours under 4 ℃, in 0 ℃, rotating speed is centrifugation under the 8000rpm, gets its supernatant liquor and continues to add ammonium sulfate powder to saturation ratio and reach 70%, leave standstill 2 hours after, in 4 ℃, rotating speed is to carry out centrifugation under the 4000rpm, remove supernatant liquor, get its throw out, be the lectin crude product;
C. the desalination of step B gained lectin crude product being dialysed after with dissolved in distilled water, film sees through molecular weight≤10kd, concentrated solution;
D. with step C gained concentrated solution through the ion-exchange column purification, Tris-HCl damping fluid (pH=7.0) hanging column with 0.02mol/L, make sample absorption, carry out linear gradient elution with the Tris-HCl damping fluid (pH=7.0) of 0.02mol/L and the concentration NaCl solution from 0.1～6%, detect agglutination activity, collect the component that agglutination activity is arranged with rabbit erythrocyte, again through the ultrafiltration desalination, film sees through molecular weight≤10kd, and lyophilize promptly gets head of garlic kernel lectin 0.63g.
Gained head of garlic kernel lectin more than 90%, can make the red cell agglutination of rabbit, pig, chicken, duck, mouse through the about 60kd of SDS-PAGE cataphoretic determination molecular weight, electrophoresis purity, and 1:10 tires
6More than (0.478 μ g/ml).Utilize synplasm to suppress the IC that measuring suppresses the HIV virus infected cell
50Be 50 μ g/ml, so can be used for preventing HIV to infect.
Embodiment 2
A. exsiccant head of garlic kernel is crushed to 200 orders, takes by weighing the 100g powder, be dissolved in the water of 300g, water temperature is 0 ℃, stirs 48h under 0 ℃ of temperature, then this mixture is filtered, and obtains yellow transparent filtrate;
B. in filtrate, add the ammonium sulfate powder, and stirring and dissolving reaches at 25% o'clock and stops until the ammonium sulfate saturation ratio and adds, after leaving standstill 1 hour under 0 ℃, in 4 ℃, rotating speed be under the 1000rpm with centrifugation, get its supernatant liquor and continue to add ammonium sulfate powder to saturation ratio and reach 75%, leave standstill 1 hour after, in 0 ℃, rotating speed is to carry out centrifugation under the 1000rpm, remove supernatant liquor, get its throw out, be the lectin crude product;
C. the desalination of step B gained lectin crude product being dialysed after with dissolved in distilled water, film sees through molecular weight≤10kd, concentrated solution;
D. with step C gained concentrated solution through the ion-exchange column purification, Tris-HCl damping fluid (pH=7.0) hanging column with 0.02mol/L, make sample absorption, carry out linear gradient elution with the Tris-HCl damping fluid (pH=7.0) of 0.02mol/L and the concentration NaCl solution from 0.1～6%, detect agglutination activity, collect the component that agglutination activity is arranged with rabbit erythrocyte, again through the dialysis desalination, film sees through molecular weight≤10kd, and lyophilize promptly gets head of garlic kernel lectin 0.59g.
Gained head of garlic kernel lectin more than 95%, can make the red cell agglutination of rabbit, pig, chicken, duck, mouse through the about 60kd of SDS-PAGE cataphoretic determination molecular weight, electrophoresis purity, and 1:10 tires
6More than (0.381 μ g/ml).Utilize synplasm to suppress the IC that measuring suppresses the HIV virus infected cell
50Be 26 μ g/ml, so can be used for preventing HIV to infect.
Embodiment 3
A. exsiccant head of garlic kernel is crushed to 40 orders, takes by weighing the 100g powder, be dissolved in the water of 500g, water temperature is 10 ℃, stirs 10h under 10 ℃ of temperature, then this mixture is filtered, and obtains yellow transparent filtrate;
B. in filtrate, add the ammonium sulfate powder, and stirring and dissolving reaches at 30% o'clock and stops until the ammonium sulfate saturation ratio and adds, after leaving standstill 1.5 hours under 10 ℃, in 10 ℃, rotating speed be under the 4000rpm with centrifugation, get its supernatant liquor and continue to add ammonium sulfate powder to saturation ratio and reach 60%, leave standstill 1.5 hours after, in 10 ℃, rotating speed is to carry out centrifugation under the 8000rpm, remove supernatant liquor, get its throw out, be the lectin crude product;
C. step B gained lectin crude product is carried out the ultrafiltration desalination after with dissolved in distilled water, film sees through molecular weight≤10kd, concentrated solution;
D. with step C gained concentrated solution through the ion-exchange column purification, Tris-HCl damping fluid (pH=7.0) hanging column with 0.02mol/L, make sample absorption, carry out linear gradient elution with the Tris-HCl damping fluid (pH=7.0) of 0.02mol/L and the concentration NaCl solution from 0.1～6%, detect agglutination activity, collect the component that agglutination activity is arranged with rabbit erythrocyte, again through the ultrafiltration desalination, film sees through molecular weight≤10kd, and lyophilize promptly gets head of garlic kernel lectin 0.61g.
Gained head of garlic kernel lectin more than 95%, can make the red cell agglutination of rabbit, pig, chicken, duck, mouse through the about 60kd of SDS-PAGE cataphoretic determination molecular weight, electrophoresis purity, and 1:10 tires
6More than (0.953 μ g/ml).Utilize synplasm to suppress the IC that measuring suppresses the HIV virus infected cell
50Be 41 μ g/ml, so can be used for preventing HIV to infect.
Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be qualification to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give exhaustive to all embodiments.Everyly belong to the row that conspicuous variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (3)
1. head of garlic kernel lectin obtains by following preparation method:
A. exsiccant head of garlic kernel being pulverized, is that 1 ︰ 3～8 is dissolved in the water by solid-to-liquid ratio, and water temperature is 0～10 ℃, stirs 10～48h under 0～10 ℃ of temperature, then this mixture is filtered, and obtains yellow transparent filtrate;
B. in filtrate, add the ammonium sulfate powder, and stirring and dissolving reaches at 25～40% o'clock and stops until the ammonium sulfate saturation ratio and adds, after leaving standstill 1～2 hour under 0～10 ℃, in 0～10 ℃, rotating speed is to carry out centrifugation under 1000～8000rpm, gets its supernatant liquor and continues to add ammonium sulfate powder to saturation ratio and reach 60～75%, leave standstill 1～2 hour after, in 0～10 ℃, rotating speed is to carry out centrifugation under 1000～8000rpm, remove supernatant liquor, get its throw out, be the lectin crude product;
C. step B gained lectin crude product is carried out ultrafiltration or dialysis desalination after with dissolved in distilled water, concentrated solution;
D. with step C gained concentrated solution process ion exchange chromatography, Tris-HCl damping fluid hanging column with 0.02mol/L, make sample absorption, Tris-HCl damping fluid and concentration with 0.02mol/L are carried out the linear concentration gradient wash-out from the salts solution that the 0.1%NaCl linearity is elevated to 6%NaCl gradually, detect agglutination activity with rabbit erythrocyte, collection has the component of agglutination activity, again through ultrafiltration or dialysis desalination, concentrate, lyophilize, promptly get head of garlic kernel lectin, it comprises following mass component: protein 60～80%, polysaccharide 15～30%;
It is characterized in that: this product is used for red cell agglutination and HIV (human immunodeficiency virus)-resistant activity.
2. head of garlic kernel lectin according to claim 1 is characterized in that: exsiccant head of garlic kernel grinding particle size is 40～200 orders in described preparation method's steps A.
3. head of garlic kernel lectin according to claim 1 is characterized in that: the film of ultrafiltration or dialysis desalination sees through molecular weight≤10kd among described preparation method's step C and the D.
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| CN107827965B (en) * | 2017-12-13 | 2020-12-29 | 遵义医科大学 | Method for extracting fruit agglutinin of bread tree |
| CN111909234A (en) * | 2020-08-21 | 2020-11-10 | 南京康齐生物科技有限公司 | Preparation method of allium sativum fruit protein |
| CN117736295B (en) * | 2024-02-21 | 2024-05-07 | 中国科学院烟台海岸带研究所 | Lectin and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410522A (en) * | 2002-11-09 | 2003-04-16 | 广西大学 | Method of extracting essential oil and natural benzaldehyde from fruit skin and fruit pulp of garlic fruit |
| CN1814621A (en) * | 2005-01-31 | 2006-08-09 | 云南大学 | Malania oeifera protein for human cancer treatment |
| CN101817876A (en) * | 2010-03-26 | 2010-09-01 | 山东省花生研究所 | Method for purifying peanut seed agglutinin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410522A (en) * | 2002-11-09 | 2003-04-16 | 广西大学 | Method of extracting essential oil and natural benzaldehyde from fruit skin and fruit pulp of garlic fruit |
| CN1814621A (en) * | 2005-01-31 | 2006-08-09 | 云南大学 | Malania oeifera protein for human cancer treatment |
| CN101817876A (en) * | 2010-03-26 | 2010-09-01 | 山东省花生研究所 | Method for purifying peanut seed agglutinin |
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