Process of extracting and preparing deer nerve growth factor (DEER NGF)

A nerve growth factor and deer antler technology, applied in the directions of nerve growth factor, growth factor/inducing factor, nervous system diseases, etc., can solve the problems of no elution peak map, complex production process, harsh conditions, etc.

Active Publication Date: 2007-08-22
CHENGDU RUNXINTANG PHARMA
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  • Abstract
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Problems solved by technology

For example, Chinese patent CN1133651C discloses a method for extracting NGF from snake venom. After pretreatment, three-step chromatography is required, the production process is complicated and the conditions are relatively harsh; Chinese patent CN1104095A discloses a kind of

Method used

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  • Process of extracting and preparing deer nerve growth factor (DEER NGF)

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, the pretreatment of deer antler

[0026] Take 0.5kg of fresh velvet antler, use a cryostat to cut the velvet antler into slices with a thickness of 1-2mm, then add an appropriate amount (200mL) of pre-cooled deionized water to pulverize with a homogenizer at 4°C, and perform the homogenization operation until homogenized End when no tissue clumps are visible in the slurry. The homogenate was centrifuged at 5000 revolutions per minute (rpm) for 20 minutes, and the supernatant was collected. Add 200 mL of pre-cooled deionized water to the pellet, resuspend the pellet, centrifuge again at 5000 rpm for 20 minutes, and take the supernatant. Then, repeat the above resuspension and centrifugation process once, and take the supernatant. Combine the supernatants obtained from each centrifugation, put them into a dialysis bag with a molecular weight cut-off of 10,000 Da (purchased from Sigma Company), and dialyze against 0.02mol / L potassium phosphate buffer (pH6.8...

Embodiment 2

[0027] Example 2, Chromatographic Purification of Antler Nerve Growth Factor and Preparation of Freeze-dried Agent

[0028] Add 95mL 0.5mol / L HAc-NaAc damping fluid (pH4.0) to the velvet antler extract solution that obtains in 850mL embodiment 1, make the pH value of solution down to 4.0, then add solid NaCl to wherein again, make in solution The final concentration of NaCl is 0.4 mol / L, mix well and let stand for 10 minutes, then centrifuge at 5000rpm for 5 minutes. Take 900mL supernatant and load it on CM-Cellulose32 column (5.5cm×10cm) (purchased from Pharmacia Company), and then carry out stepwise gradient elution. During stepwise gradient elution, use 400 mL of 0.5 mol / L HAc-NaAc buffer solution (pH4.0) containing 0.8 mol / L NaCl and 350 mL of 0.5 mol / L potassium phosphate containing 0.8 mol / L NaCl at a flow rate of 10 mL / min. Buffer (pH7.0) and 500mL 0.5mol / L potassium phosphate buffer (pH7.0) containing 1mol / L NaCl were eluted and detected at a wavelength of 280nm by an...

Embodiment 3

[0029] Embodiment 3, the detection of nerve growth factor

[0030] Antler nerve growth factor was detected according to the "Molecular Cloning Test Guide" (Science Press, 2002) and the test method of Zhai Lei et al. (Progress in Microbiology and Immunology, 1999, 27(1): 43-46). The antler nerve growth factor solution obtained in Example 2 is carried out to SDS-PAGE (15% polyacrylamide separating gel, 4% stacking gel, 10mA constant current electrophoresis), after the electrophoresis is finished, it is stained with Coomassie brilliant blue, and the photo of the electrophoresis gel is as follows: As shown in Figure 2, there is a single band of nerve growth factor at a molecular weight of about 14kD, and its purity reaches 96% through image analysis. The biological activity of NGF is detected by the growth of the dorsal root ganglion of the chicken embryo: in the blank control group with buffer solution, the dorsal root ganglion of the chicken embryo has no process growth; In the...

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Abstract

The present invention relates to process of extracting nerve growth factor (Deer NGF) from pilose antler. The process includes the further cation exchanging chromatography on pilose antler extracting liquid and the stepped gradient elution. In addition, the present invention also relates to the pilose antler extract rich in the nerve growth factor and the application of the pilose antler extract in medicine, food, health product, etc.

Description

technical field [0001] The invention belongs to the field of medical or edible products of deer antler extract. More specifically, the present invention relates to a method for extracting nerve growth factor (DEER NGF) from deer antler and antler extract rich in nerve growth factor prepared by the method. In addition, the present invention also relates to the application of the velvet antler extract in medicine, food, health products and the like. Background technique [0002] Antler (the young antlers of deer that have not yet been ossified) and antlers, as traditional Chinese medicinal materials, have the functions of strengthening muscles and bones, tonifying kidney and strengthening yang, dredging blood vessels, etc., and have been widely used to prepare various nourishing medicines, food, and health care products since ancient times and cosmetics. Through the analysis of modern medicinal chemistry research, velvet antler contains many kinds of physiologically active i...

Claims

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Application Information

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IPC IPC(8): C07K14/48A61K38/18A61P25/00A61K35/32
Inventor 朱成钢王利忠
Owner CHENGDU RUNXINTANG PHARMA
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