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Ginkgo protein GAPIIa and its preparing method and use

A technology of ginkgo and protein, applied in the field of plant protein separation and purification, can solve the problems of low extraction rate, easy inactivation of protein, unsuitable for separation and purification of ginkgo protein, etc.

Inactive Publication Date: 2004-07-14
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing methods are complicated in process, low in extraction rate, and protein is easily inactivated during operation
[0012] There is no report about the separation and purification of ginkgo active protein, and the existing methods are not suitable for the separation and purification of ginkgo protein

Method used

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  • Ginkgo protein GAPIIa and its preparing method and use
  • Ginkgo protein GAPIIa and its preparing method and use
  • Ginkgo protein GAPIIa and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: Separation and purification of ginkgo protein GAPIIa

[0072] 1. Purification of Albumin

[0073] At 4°C, add 0.14mol / L NaCl solution at a material-to-liquid ratio of 1:3 (w / v), thoroughly homogenize 500g ginkgo defatted powder three times, and centrifuge the homogenate at 10,000r / min for 15min. Take the supernatant and add solid ammonium sulfate to reach 35 (w / w)% saturation. Stir and place for more than 1h, centrifuge at 10000r / min for 15min, and take the supernatant. Then add solid ammonium sulfate to reach 90 (w / w)% saturation, stir for more than 1 hour, and centrifuge at 10000r / min for 15 minutes to collect crude protein. The crude protein precipitate was redissolved in distilled water and dialyzed against distilled water with a 1.0 kDa dialysis membrane. As the salt concentration decreases, the globulin will slowly precipitate, and the dialyzed protein solution is centrifuged at 10000r / min for 15min. Get supernatant, freeze-dry to obtain 18.6g gi...

Embodiment 2

[0081] Embodiment 2: Identification of ginkgo protein GAPIIa

[0082] 1. Purity identification of DEAE-Sephadex-A50 GAPIIa

[0083] The GAPIIa sample purified in embodiment (1) was subjected to another DEAE-Sephadex-A50 column chromatography (as shown in Figure (8)). As can be seen from the attached figure, there is only a single symmetrical peak. Therefore, it can be considered that GAPIIa is homogeneous in chromatographic properties, and its purity reaches chromatographic purity.

[0084] 2. Conventional-polyacrylamide gel electrophoresis and SDS-PAGE purity identification of GAPIIa

[0085] The purity of GAPIIa was identified by the most commonly used conventional polyacrylamide gel electrophoresis method with higher resolution. GAPIIa was made into a solution with a concentration of 2 mg / mL, and the injection volume was 20 uL for spotting, and the electrophoresis was performed at 100V for 6 hours. After electrophoresis, the gel was first fixed with a fixative solution ...

Embodiment 3

[0136] Embodiment 3: the mensuration of ginkgo protein biological activity

[0137] 1. The scavenging effect of ginkgo protein on free radicals (OH)

[0138] Using the 2-deoxy-D-ribose (DR) method (Halliwell B, Gutteridge J MC, Aruoma O I. Thedeoxyribose method: a simple "test-tube" assay for determination of rate contents for reactions of hydroxyl radicals. Anal Biochem., 1987 , 165:215-219): Add 0.4mL50mmol / L KH at pH7.5 to a clean test tube 2 PO 4 -KOH buffer solution, certain concentration sample, 0.1mL 1.04mmol / L EDTA solution, 0.1mL10mmol / L H 2 o 2 , 0.1mL 60mmol / L DR (without control), 0.1mL 2mmol / L vitamin C, and 0.1mL 1mmol / L FeCl 3 , the final volume of each tube is 1.0 mL. Take it out after incubating at 37°C for 1 hour, add 1mL of 25% HCl to terminate the reaction, add 1.0ml of 1% TBA solution, mix the solutions in each test tube, boil in a boiling water bath for 15min, cool immediately and then centrifuge (3000rpm). Measure the absorbance at a wavelength of ...

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Abstract

The present invention belongs to the field of plant protein separating and purifying technology, and is ginkgo protein GAP IIa separated from gingko and with functions of delaying senility and resisting oxidation and its preparation process and use. The present invention via the process of salt separation, salt dissolution, two-step ion exchanging chromatography and other steps, new protein GAP IIa of molecular weight 29248 Da is separated from gingko and purified. The primary structure and spatial structure detection shows that GAP IIa consists of two peptide chains, GAP IIa-1 and GAP IIa-2, with similar molecular weight and similar amino acid sequence and connected via disulfide bond and with a few glycosyl via O-glycosidic bond. Each of GAP IIa-1 and GAP IIa-2 contains peptide segments of peptide mass number of 911, 1-11 and 2470. The ginkgo protein may be used in producing health food and medicine for resisting oxidation and delaying senility.

Description

technical field [0001] The invention belongs to the technical field of plant protein separation and purification, and in particular relates to a new protein ginkgo protein GAPIIa isolated from ginkgo (gingko biloba) with anti-aging and anti-oxidation properties, its preparation method and application. Background technique [0002] Ginkgo biloba is a gymnosperm phylum Ginkgo biloba (GinKgo bilba L.), a special plant of one family and one genus, also known as ginkgo and gongsun tree. The kernel of ginkgo seed is commonly known as ginkgo, and its kernel is the edible part. Because of its rich nutrition, it has been regarded as the top grade for health preservation and longevity since ancient times, and it has become a traditional export product in modern times. The main medicinal effects are to restrain lung qi, calm asthma and cough, stop turbidity, and reduce stool. In addition, it is also used to treat skin diseases and surgical infections s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P39/06A61P43/00C07K1/14C07K14/415
Inventor 黄文谢笔钧王益杨尔宁邓乾春凌智群
Owner HUAZHONG AGRI UNIV
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