Trichosanthes kirilowii anti-tumor activity protein as well as preparation method and application thereof

An anti-tumor activity and anti-tumor technology, applied in the field of preparation and application of plant protein, can solve the problems of fever, trichosanthin protein is easy to cause allergy, etc., and achieve the effect of good activity

Active Publication Date: 2014-10-29
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ribosome-inactivating protein represented by trichosanthin is mainly reported, but because trichosanthin is easy to cause adverse reactions such as allergies and fever in clinical application, its clinical application has gradually decreased

Method used

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  • Trichosanthes kirilowii anti-tumor activity protein as well as preparation method and application thereof
  • Trichosanthes kirilowii anti-tumor activity protein as well as preparation method and application thereof
  • Trichosanthes kirilowii anti-tumor activity protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Preparation of Trichosanthes antitumor active protein

[0020] (1) Take 20g whole Trichosanthes, grind and pulverize, add 100mL protein extract (phosphate buffer solution with pH7.4, 1L buffer solution contains 8g NaCl, 0.2g KCl, 3.63g Na 2 HPO4.12H 2 O, 0.24g KH 2 PO 4 ), placed on a stirrer, soaked in a refrigerator at 4°C and stirred for 12-16 hours, centrifuged at 11,000 rpm at 4°C for 10 minutes, and then the supernatant was taken, which was the crude extract of Trichosanthes protein.

[0021] (2) Slowly add 200mL of acetone pre-cooled at -20°C to the crude extract of Trichosanthes protein obtained in (1), stir while adding, put it in a refrigerator at 4°C for 40min to precipitate, 4°C, 11000rpm Centrifuge for 10 min, discard the supernatant, collect the precipitate, place the precipitate at -20°C for 1 h to completely volatilize the acetone, freeze-dry the precipitate, and obtain the crude protein of Trichosanthes chinensis.

[0022] (3) Dissolve th...

Embodiment 2

[0026] Example 2: Effect of Trichosanthes antitumor active protein on tumor cell survival

[0027] DLD1, T24, MCF-7, HeLa, NCM460 cells in the logarithmic growth phase were mixed with 3×10 3 per well into a 96-well culture plate. 37°C with 5% CO 2 After incubating in the incubator for 24 hours, the anti-tumor active protein of Trichosanthes chinensis in Example 1 was added respectively, the final concentrations were 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and six duplicate holes were set up, and used The medium containing the same volume of phosphate buffer was used as a control. After incubation for 24 hours, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation was continued for 4 hours before the culture was terminated. Carefully aspirate and discard the culture supernatant in the wells, add 150 μL DMSO to each well, and shake for 10 minutes to fully dissolve the crystals. At a wavelength of 570 nm on a microplate reader, the light absorbance o...

Embodiment 3

[0029] Example 3: Identification of Trichosanthes antitumor active protein

[0030] The purified anti-tumor active pure protein of Trichosanthes chinensis was sent to Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for mass spectrometric identification. The identification results show that the anti-tumor protein of Trichosanthes is 100% homologous to the cucumber in Luo Han Guo (such as image 3 shown).

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Abstract

The invention provides a trichosanthes kirilowii anti-tumor activity protein as well as a preparation method thereof and application of the trichosanthes kirilowii anti-tumor activity protein in preparing anti-tumor drugs. The preparation method comprises the following steps: crushing full trichosanthes kirilowii, adding a protein extracting solution, stirring at 4 DEG C overnight, subsequently adding precooled acetone low-temperature precipitated protein, centrifugalizing, and then dissolving by using the protein extracting solution to obtain trichosanthes kirilowii crude protein; screening, separating and purifying anti-tumor activity components contained in the trichosanthes kirilowii crude protein by adopting fractional precipitation, anion-exchange chromatography and gel filtration chromatography to obtain and trichosanthes kirilowii anti-tumor activity protein. The trichosanthes kirilowii anti-tumor activity protein provided by the invention can be used for obviously inhibiting the survival of colon cancer DLD1 cells, breast cancer MCF-7 cells, bladder cancer T24 cells and cervical carcinoma HeLa cells and can be applied to preparing the anti-tumor drugs.

Description

technical field [0001] The invention relates to the preparation and application of plant protein, in particular to a trichomona antitumor active protein and its preparation method, as well as the application of the protein in the preparation of antitumor drugs. Background technique [0002] Trichosanthes kirilowii Maxim is a perennial vine belonging to Cucurbitaceae. Also known as melon basket, medicine melon, hanging melon. Trichosanthes are highly adaptable, easy to survive, and relatively cold-resistant. In my country, they are mainly distributed in Shandong, Henan, Shanxi and other provinces. It is mainly used as medicine by its fruit and root, and it is a commonly used traditional Chinese medicinal material with a long history in our country. [0003] The medicinal parts of Trichosanthes mainly include Trichosanthes, Trichosanthes seeds and Trichosanthes Fen. Gualou is the dried fruit of Trichosanthes chinensis. It is a commonly used Chinese medicinal material. It wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K1/36C07K1/30C07K1/18A61K38/16A61P35/00
CPCA61K38/00C07K14/415
Inventor 宋莉常姣李卓玉李汉卿晋晓婷
Owner SHANXI UNIV
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