CN103980355B - The preparation method and application of Semen Abutili activated protein - Google Patents
The preparation method and application of Semen Abutili activated protein Download PDFInfo
- Publication number
- CN103980355B CN103980355B CN201410238318.3A CN201410238318A CN103980355B CN 103980355 B CN103980355 B CN 103980355B CN 201410238318 A CN201410238318 A CN 201410238318A CN 103980355 B CN103980355 B CN 103980355B
- Authority
- CN
- China
- Prior art keywords
- semen abutili
- protein
- supernatant liquor
- centrifugal
- extract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention provides the preparation method of a kind of herbal medicine Semen Abutili activated protein, and the application of this albumen in antitumor drug. Preparation method: ground by Chinese medicine Semen Abutili, adds protein extract PBS lysate and stirs immersion of spending the night, add the acetone low-temperature sludge albumen of precooling subsequently, obtain Semen Abutili crude protein. This Semen Abutili crude protein 100 DEG C of water are boiled 30min, after low-temperature centrifugation, supernatant liquor is crossed anion-exchange chromatography post, collect and penetrate liquid, namely obtain the active pure protein of Semen Abutili. This albumen detects through anti tumor activity in vitro, has the activity of good inhibitor against colon carcinoma cells cell, colon cancer cell propagation is had obvious restraining effect, and normal cell is not had obvious toxic-side effects.
Description
Technical field
The present invention relates to the Synthesis and applications of vegetable-protein, specifically belong to the preparation method of a kind of Chinese medicine Semen Abutili activated protein, and this albumen is in the application prepared in antitumor drug.
Background technology
Semen Abutili, has another name called certain herbaceous plants with big flowers, certain herbaceous plants with big flowers vegetable seeds. It is Mongols's conventional crude drugs, it is the dry mature fruit of Malvaceae plant winter certain herbaceous plants with big flowers. Former plant distributions in northeast, North China, northwest, Japan, Korea, Mongolia, the Soviet Union, Europe with North America also have distribution. Main product in Heilungkiang, Jilin, Liaoning, the Inner Mongol, Hebei, Shanxi, the ground such as Ningxia. Gather when summer, autumn two season fruit maturation, remove impurity, dry in the shade.
Semen Abutili begins to be loaded in " recognizing medicine Bai Jingjian " as anaesthetic " Sha's day wood lattice-account for bar ", and successive dynasties documents such as rear " errorless anaesthetic mirror " all has record. Current Mongolian medicine is clinical and anaesthetic manufacturing enterprise uses " Sha's day wood lattice-account for bar " is Semen Abutili. Semen Abutili has effect of clearing away heat and promoting diuresis, detumescence, can promote blood circulation, diuresis, clear dry purulence, consumer edema. Cure mainly kidney heat, bladder heat, gonorrhoea, renal shutdown, vesical calculus, thirsty, sore etc.
In recent years, Semen Abutili progressively obtains more application in clinical. The literature search of prior art finds, the domestic and international report of the research about Semen Abutili is still less at present, more has no the report about Semen Abutili anti-tumor active protein.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of Chinese medicine Semen Abutili activated protein, and the application of this albumen in antitumor drug.
The preparation method of a kind of Semen Abutili activated protein provided by the invention, comprises the steps:
The first step: get Chinese medicine Semen Abutili, after grinding, adds the protein extract of precooling, soaks and stir 12-16 hour at 4 DEG C, get supernatant liquor after centrifugal, be Semen Abutili protein crude extract; Filter residue after centrifugal can extract 1-2 time by this method again;
Described protein extract is PBS, containing 8gNaCI, 0.2gKCI, 3.63gNa in 1L buffer solution system2HPO4.12H2O��0.24gKH2PO4, pH7.4;
The solid-liquid ratio (weightmeasurement ratio) of described Semen Abutili and protein extract is 1:4-6, it is preferable that 1:5;
2nd step: the acetone adding-20 DEG C of precoolings of 1.5-2 times of volume in Semen Abutili protein crude extract, protein precipitation 0.5-1 hour, 4 DEG C are centrifugal, collecting precipitation, places 30-40min at-20 DEG C, and acetone is volatilized completely, lyophilize, obtains Semen Abutili crude protein;
Supernatant liquor 100 DEG C of water are boiled 30min by the 3rd step: dissolved on ice by the PBS of Semen Abutili crude protein pH7.4, get supernatant liquor after centrifugal, 4 DEG C centrifugal after get supernatant liquor;
4th step: above-mentioned supernatant liquor is crossed Q anion-exchange chromatography post, in Ultraviolet Detector 280nm wavelength place, collection penetrates peak and obtains Semen Abutili activated protein, preserves under 4 DEG C of conditions.
Adopt PAGE gel electrophoretic technique, by contrasting with standard molecular weight, it is contemplated that the molecular weight of Semen Abutili anti-tumor active protein is 14kDa.
Through anti tumor activity in vitro detection display, this Semen Abutili albumen can the propagation of suppression colon cancer cell of specificity. Processing colorectal carcinoma, bladder cancer, mammary cancer, liver cancer, cervical cancer and normal cell 48 hours respectively with this albumen, after wherein processing colorectal carcinoma and transitional cell bladder carcinoma cell line, median lethal rate is about 33%, 24% respectively, and to the almost non-toxic side effect of normal cell. This albumen has good antitumor drug application prospect.
Compared with prior art, the Semen Abutili albumen that the present invention obtains detects through anti tumor activity in vitro, has the activity of good inhibitor against colon carcinoma cells cell, colon cancer cell propagation is had obvious restraining effect, and normal cell is not had obvious toxic-side effects.
Accompanying drawing explanation
The SDS-PAGE of Semen Abutili activated protein prepared by Fig. 1 embodiment 1 (after Q column purification gained pure protein sample, applied sample amount is 10 �� g; D: target protein; Marker: standard molecular weight)
Fig. 2 Semen Abutili crude protein is on the impact of five kinds of cancer cells and two kinds of normal cell growth
The impact that colon cancer cell is grown by Fig. 3 Semen Abutili anti-tumor active protein
Embodiment
The preparation of Semen Abutili activated protein
Embodiment 1:
(1) getting 50g Chinese medicine Semen Abutili, after grinding, (PBS of pH7.4, containing 8gNaCI, 0.2gKCI, 3.63gNa in 1L damping fluid to add the protein extract of 250mL precooling2HPO4.12H2O��0.24gKH2PO4). Soak in 4 DEG C of refrigerators and stir 12-16 hour, 4 DEG C, get supernatant liquor after the centrifugal 10min of 11,000rpm, be Semen Abutili protein crude extract. Filter residue after centrifugal adds 250mL protein extract again, the same operation, merges supernatant liquor and is Semen Abutili crude protein extracting solution.
(2) by the Semen Abutili protein crude extract obtained in (1) slowly adds the acetone of-20 DEG C of precoolings of 500mL, stirring while adding, it is placed on 4 DEG C of refrigerators and places 1 hour precipitation. 4 DEG C, the centrifugal 10min of 11,000rpm, abandons supernatant, collecting precipitation, and precipitation is placed in-20 DEG C of 30min, and acetone is volatilized completely. Lyophilize will be precipitated further, obtain the Semen Abutili crude protein of 120mg.
(3) being dissolved by the PBS of the pH7.4 of the Semen Abutili crude protein 50mL of extraction in (2), the centrifugal 10min of 11,000rpm, gets supernatant. Precipitation is dissolved with the PBS of 50mL again, after centrifugal, gets supernatant. Merge supernatant liquor. By this supernatant liquor in water-bath 100 DEG C boil 30min, get supernatant liquor after 12,000rpm is centrifugal.
(4) supernatant liquor in (3) is carried out Q anion exchange chromatography, with the PBS wash-out of pH7.4, Ultraviolet Detector detects in 280nm wavelength place, collection penetrates peak, obtaining 0.98mg/mL Semen Abutili anti-tumor active protein 2.94mg after concentrated, SDS-PAGE is shown in Fig. 1.
Embodiment 2:
(1) getting 50g Chinese medicine Semen Abutili, after grinding, (PBS of pH7.4, containing 8gNaCI, 0.2gKCI, 3.63gNa in 1L damping fluid to add the protein extract of 200mL precooling2HPO4.12H2O��0.24gKH2PO4). Soak in 4 DEG C of refrigerators and stir 12-16 hour, 4 DEG C, get supernatant liquor after the centrifugal 10min of 11,000rpm, be Semen Abutili protein crude extract. Filter residue after centrifugal adds 200mL protein extract again, the same operation, merges supernatant liquor and is Semen Abutili crude protein extracting solution.
(2) by the Semen Abutili protein crude extract obtained in (1) slowly adds the acetone of-20 DEG C of precoolings of 400mL, stirring while adding, it is placed on 4 DEG C of refrigerators and places 1 hour precipitation. 4 DEG C, the centrifugal 10min of 11,000rpm, abandons supernatant, collecting precipitation, and precipitation is placed in-20 DEG C of 30min, and acetone is volatilized completely. Lyophilize will be precipitated further, obtain the Semen Abutili crude protein of 100mg.
(3) being dissolved by the PBS of the pH7.4 of the Semen Abutili crude protein 40mL of extraction in (2), the centrifugal 10min of 11,000rpm, gets supernatant. Precipitation is dissolved with the PBS of 40mL again, after centrifugal, gets supernatant. Merge supernatant liquor. By this supernatant liquor in water-bath 100 DEG C boil 30min, get supernatant liquor after 12,000rpm is centrifugal.
(4) supernatant liquor in (3) is carried out Q anion exchange chromatography, with the PBS wash-out of pH7.4, Ultraviolet Detector, in the detection of 280nm wavelength place, is collected and is penetrated peak, obtains 0.78mg/mL Semen Abutili anti-tumor active protein 2.34mg after concentrated.
Embodiment 3:
(1) getting 50g Chinese medicine Semen Abutili, after grinding, (PBS of pH7.4, containing 8gNaCI, 0.2gKCI, 3.63gNa in 1L damping fluid to add the protein extract of 300mL precooling2HPO4.12H2O��0.24gKH2PO4). Soak in 4 DEG C of refrigerators and stir 12-16 hour, 4 DEG C, get supernatant liquor after the centrifugal 10min of 11,000rpm, be Semen Abutili protein crude extract. Filter residue after centrifugal adds 300mL protein extract again, the same operation, merges supernatant liquor and is Semen Abutili crude protein extracting solution.
(2) by the Semen Abutili protein crude extract obtained in (1) slowly adds the acetone of-20 DEG C of precoolings of 600mL, stirring while adding, it is placed on 4 DEG C of refrigerators and places 1 hour precipitation. 4 DEG C, the centrifugal 10min of 11,000rpm, abandons supernatant, collecting precipitation, and precipitation is placed in-20 DEG C of 30min, and acetone is volatilized completely. Lyophilize will be precipitated further, obtain the Semen Abutili crude protein of 125mg.
(3) being dissolved by the PBS of the pH7.4 of the Semen Abutili crude protein 50mL of extraction in (2), the centrifugal 10min of 11,000rpm, gets supernatant. Precipitation is dissolved with the PBS of 50mL again, after centrifugal, gets supernatant. Merge supernatant liquor. By this supernatant liquor in water-bath 100 DEG C boil 30min, get supernatant liquor after 12,000rpm is centrifugal.
(4) supernatant liquor in (3) is carried out Q anion exchange chromatography, with the PBS wash-out of pH7.4, Ultraviolet Detector, in the detection of 280nm wavelength place, is collected and is penetrated peak, obtains 0.89mg/mL Semen Abutili anti-tumor active protein 2.67mg after concentrated.
Embodiment 4: the impact that seven kinds of cells are grown by Semen Abutili crude protein
By DLD1, T24, MCF-7, HepG2, HeLa, FHC, HL-7702 cell of logarithmic phase, with 8 �� 10396 well culture plates are imported in individual hole into. 37 DEG C, after CO2 incubator containing 5% hatches 24h, the final concentration adding embodiment 1 respectively is respectively the Semen Abutili crude protein 20 �� L of 0.24 �� g/ �� L, 0.32 �� g/ �� L, 0.60 �� g/ �� L, 0.80 �� g/ �� L, 1.0 �� g/ �� L, if six multiple holes, and with the PBS of the pH7.4 of same volume in contrast. Inhaling after hatching 48h and abandon liquid in hole, PBS adds new substratum after washing 2 times, every hole adds MTT solution (5mg/mL) 20 �� L, terminates cultivating after continuing to hatch 4h. Careful suction abandons culture supernatant in hole, every hole adds 150 �� LDMSO, and vibration 10min, makes crystallisate fully melt. 492nm wavelength place on enzyme linked immunological monitor, measures each hole absorbance value.
The Semen Abutili anti-tumor activity crude protein of gained of the present invention, carries out above-mentioned anti tumor activity in vitro detection to several people sources cancer cells. Result shows: when final concentration of protein is 1.0 �� g/ �� L, the inhibiting rate of DLD1 cell growth is about 33%, T24 inhibitory rate of cell growth is about 24%, to MCF-7, HepG2, HeLa cell without obvious restraining effect, on normal intestinal cells FHC, HL-7702 liver cell growth substantially without impact (as shown in Figure 2).
Embodiment 5: the impact that DLD1 cell is grown by Semen Abutili anti-tumor activity pure protein
By the DLD1 cell of logarithmic phase, with 8 �� 10396 well culture plates are imported in individual hole into. 37 DEG C, containing the CO of 5%2After incubator hatches 24h, the final concentration adding embodiment 1 respectively is respectively the Semen Abutili anti-tumor activity pure protein 20 �� L of 0.24 �� g/ �� L, 0.32 �� g/ �� L, 0.60 �� g/ �� L, 0.80 �� g/ �� L, 1.0 �� g/ �� L. If six multiple holes, and with the PBS of the pH7.4 of same volume in contrast. Inhaling after hatching 48h and abandon liquid in hole, PBS adds new substratum after washing 2 times, every hole adds MTT solution (5mg/mL) 20 �� L, terminates cultivating after continuing to hatch 4h. Careful suction abandons culture supernatant in hole, every hole adds 150 �� LDMSO, and vibration 10min, makes crystallisate fully melt. 492nm wavelength place on enzyme linked immunological monitor, measures each hole absorbance value.
The Semen Abutili anti-tumor activity pure protein of gained of the present invention, human colon carcinoma DLD1 is carried out above-mentioned anti tumor activity in vitro detection, result shows: when final concentration of protein is 1.0 �� g/ �� L, and the inhibiting rate of DLD1 cell growth was about 33% (as shown in Figure 3).
Claims (2)
1. the preparation method of a Semen Abutili activated protein, it is characterised in that, comprise the steps:
The first step: get Semen Abutili, after grinding, adds the protein extract of precooling, soaks and stir 12-16 hour at 4 DEG C, get supernatant liquor after centrifugal, be Semen Abutili protein crude extract; Filter residue after centrifugal can extract 1-2 time by this method again;
Described protein extract is PBS, containing 8gNaCl, 0.2gKCl, 3.63gNa in 1L buffer solution system2HPO4.12H2O��0.24gKH2PO4, pH7.4;
The solid-liquid ratio of described Semen Abutili and protein extract is 1:4-6;
2nd step: the acetone adding-20 DEG C of precoolings of 1.5-2 times of volume in Semen Abutili protein crude extract, protein precipitation 0.5-1 hour, 4 DEG C are centrifugal, collecting precipitation, places 30-40min at-20 DEG C, and acetone is volatilized completely, lyophilize, obtains Semen Abutili crude protein;
Supernatant liquor 100 DEG C of water are boiled 30min by the 3rd step: dissolved on ice by the PBS of Semen Abutili crude protein pH7.4, get supernatant liquor after centrifugal, 4 DEG C centrifugal after get supernatant liquor;
4th step: above-mentioned supernatant liquor is crossed Q anion-exchange chromatography post, in Ultraviolet Detector 280nm wavelength place, collection penetrates peak and obtains Semen Abutili activated protein, preserves under 4 DEG C of conditions; Described Semen Abutili activated protein is applied in be prepared in inhibitor against colon carcinoma cells and bladder cancer medicine.
2. the preparation method of a kind of Semen Abutili activated protein according to claim 1, it is characterised in that, described Semen Abutili and the solid-liquid ratio of protein extract are 1:5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410238318.3A CN103980355B (en) | 2014-05-30 | 2014-05-30 | The preparation method and application of Semen Abutili activated protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410238318.3A CN103980355B (en) | 2014-05-30 | 2014-05-30 | The preparation method and application of Semen Abutili activated protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103980355A CN103980355A (en) | 2014-08-13 |
| CN103980355B true CN103980355B (en) | 2016-06-01 |
Family
ID=51272543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410238318.3A Expired - Fee Related CN103980355B (en) | 2014-05-30 | 2014-05-30 | The preparation method and application of Semen Abutili activated protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103980355B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117357567B (en) * | 2023-11-23 | 2024-03-05 | 河南中医药大学第一附属医院 | Abelmoschus manihot seed extract, preparation method thereof and application thereof in treatment of respiratory diseases |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1713909B1 (en) * | 2004-02-02 | 2009-08-05 | E.I. Du Pont De Nemours And Company | Altering root structure during plant development |
| CN102286450B (en) * | 2011-06-13 | 2013-03-27 | 南京大学连云港高新技术研究院 | Technique for extracting active proteins from seashore mallow seeds and application thereof |
| CN103059112A (en) * | 2013-01-22 | 2013-04-24 | 山西大学 | Method of extracting anti-intestinal cancer active protein from nostoc commune |
-
2014
- 2014-05-30 CN CN201410238318.3A patent/CN103980355B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103980355A (en) | 2014-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105801690B (en) | A kind of trypsin inhibitor and its preparation method and application | |
| CN102274341A (en) | Extracting and refining process for medicinal components of figwort root | |
| CN107266400B (en) | Method for extracting lactuca sativa bitter principle from chicory | |
| Zhu et al. | Structural characterisation and ACE-inhibitory activities of polysaccharide from Gastrodia elata Blume | |
| CN107371960A (en) | A kind of implantation methods of fertile mandarin orange | |
| CN105085703A (en) | Method for separating and purifying polysaccharides from winter jujubes | |
| CN109232760A (en) | Phellinus protect liver polysaccharide PPB-2 and preparation method thereof | |
| CN103837486A (en) | Method for determining desert alga biomass in biological crusts | |
| CN102180938A (en) | Method for preparing capilliposide | |
| CN106110214A (en) | A kind of harvesting and processing method of Rhizoma Curcumae | |
| CN103980355B (en) | The preparation method and application of Semen Abutili activated protein | |
| CN104292355B (en) | A kind of extracting method of platycodon root polysaccharide | |
| CN102617718A (en) | Bran coat antitumor active protein, as well as preparation method and applications thereof | |
| CN104119433B (en) | A kind of snakegourd anti-tumor active protein and its preparation method and application | |
| CN104844721B (en) | Extraction and separation method of Agrocybe aegirit polysaccharides | |
| CN102988457A (en) | Total flavone extract of lonicera macranthoides leaves, and preparation method and application thereof | |
| CN103013937B (en) | Method for extracting polyphenol oxidase from lotus seedpod | |
| CN103113489B (en) | Method of purifying polysaccharide of Xinjiang jun dates | |
| CN103468553B (en) | Method for producing cordyceps sinensis health-preserving liquor | |
| CN103059112A (en) | Method of extracting anti-intestinal cancer active protein from nostoc commune | |
| CN106883306A (en) | A kind of extraction separation method of the sunflower stem core polysaccharide with antitumor activity | |
| CN106074237A (en) | A kind of plant extract type whitening water | |
| CN103588893B (en) | A kind of preparation method of southern Anhui Umbilicaria esculenta polysaccharide | |
| CN102698116A (en) | Application of lophatherum gracile total flavonoids in preparation of medicine and functional food for preventing and treating myocardial ischemia disease | |
| CN105153320A (en) | Laminarin extraction method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160601 Termination date: 20190530 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |