CN107056913A - A kind of method for preparing melittin - Google Patents
A kind of method for preparing melittin Download PDFInfo
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- CN107056913A CN107056913A CN201710169095.3A CN201710169095A CN107056913A CN 107056913 A CN107056913 A CN 107056913A CN 201710169095 A CN201710169095 A CN 201710169095A CN 107056913 A CN107056913 A CN 107056913A
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- melittin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
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- Insects & Arthropods (AREA)
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Abstract
The invention discloses a kind of isolation and purification method of melittin, belong to biological technical field.The melittin is that the polypeptide got is extracted from bee venom, using sephadex chromatography, freeze-drying, with reference to centrifugation, the separation method of ethanol precipitation, obtains melittin.The melittin of the present invention, yield is higher, and the production used time is short, and technique is simple, and purity is that electrophoresis is pure, keeps the original structure of melittin and highest physiologically active, can be applied to medicine and other fields.
Description
Technical field
The present invention relates to a kind of method for preparing melittin, belong to biological technical field.
Background technology
Bee venom be worker bee poison gland secretion a kind of transparency liquid with aromatic odor, mainly by polypeptide, active enzyme,
Non- peptides material composition, protein and peptide class material is the main component in bee venom, accounts for the 70%-80% of bee venom.Melittin is
The polypeptide being made up of 26 amino acid residues, molecular weight is 2840Da, accounts for the 50% of bee venom dry weight, its primary structure amino acid
Residue sequence is NH2-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Glu-Leu-Pro-Ala-
Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Glu-Gln-COOH.Melittin has anti-inflammatory, antibacterial, anticancer, resisted
The effects such as radiation, suppression platelet aggregation, can effectively treat scapulohumeral periarthritis, rheumatic arthritis, rheumatoid arthritis etc. many
Disease is planted, there is certain application in field of medicaments.
Melittin is isolated and purified from bee venom and got, with bioactivity it is high, it is free from environmental pollution the features such as, Ke Yiyan
System develops the products such as Organism immunoregulation agent, with the very big prospect of marketing, thus causes academia and widely close
Note.
The method such as it is combined with three column chromatographies with solvent extraction, three column chromatographies, solvent extraction and prepares melittin, its product system
Standby process is cumbersome, and the production cycle is longer, and organic solvent usage amount is big.Kreil（Kreil, G. Structure of
melittin isolated from two species of honey bees [J]. Febs Letters, 1973,33
(2): 241-244.）The method that solvent extraction prepares melittin is reported, bee venom peptide yield is about 20%（Mass fraction）, purity
60.3%（Mass fraction）, preparation method simplicity, extracting 1g melittins needs 200mL n-butanols, and the demand of n-butanol is big,
Cause the severe contamination of environment.Liu Ling and Yang Wenchao（1. Liu Ling, Li Changquan, the purification process of the strong melittins of Huang Xue and external anti-
The Chinese biochemical drug impurity .2003,24 of function of tumor research [J]（4）：16-17 2. Yang Wen surpass, and melittin is isolated and purified
And its radiation resistance study mechanism [D], University Of Agriculture and Forestry In Fujian, 2007.）Report three column chromatographies and prepare melittin, bee venom
Peptide yield is about 48.67%（Mass fraction）, purity is 97.32%（Mass fraction）, purity is higher, solves consumption of organic solvent
Big the problem of, but extraction process is complicated, extraction time is longer.Zhang Wenli and Xu Peng（1. a literary gift, Sun Jing member melittins
Process for separation and purification [P], China, 101089017A.2007-12-19. 2. Xu Peng, Ou Yangyongwei, Huang Jingyao, Zhang Guiying, Xiao
Really, Liu Bo isolate and purify experimental study Chinese herbal medicines [J] 2000,31 (12) of melittin from bee venom: 892-894.）
Report solvent extraction and be combined with chromatography and prepare melittin, bee venom peptide yield is about 47%（Mass fraction）, purity is electrophoresis
It is pure, the time required to shortening three column chromatographies, with Kreil（Kreil, G. Structure of melittin isolated
from two species of honey bees [J]. Febs Letters, 1973,33(2): 241-244.）Preparation
Method compares, and reduces the usage amount of n-butanol, but still needs to substantial amounts of n-butanol in extraction process, is unfavorable for environment guarantor
Shield.
Above-mentioned isolation and purification method complex manufacturing, the production cycle is long, and yield is low, and purity is low, and seriously polluted.
The content of the invention
The invention aims to overcome the shortcomings of the existing separating and purifying technology of melittin, there is provided a kind of quick preparation honeybee
The method of phallotoxins, the melittin prepared by this method, yield is high, and purity is high, and the cycle is short, and energy consumption is low, pollutes small preparation bee venom
The method of peptide.
The recovery rate mass fraction that the present invention obtains melittin is 49%, and purity Coriolis mass fraction is 98%.
A kind of method for preparing melittin of the present invention, step is as follows：
1. the removal of impurities of bee venom
Thick bee venom is added in the cushioning liquid that pH is 4.0-5.5, it is 0.5-1.5mg/mL to make its concentration, with 2500-
3500r/min centrifugations 3-7min removes insoluble impurity, obtains supernatant；
The thick bee venom be by honeybee sting thorn discharge venom dissolved with distilled water, freeze-dried gained；
The buffer solution is NaAc_HAc buffer solution；
Material of the removal of impurities for removing insoluble in cushioning liquid.
2. the separation of melittin
1. the post of sephadex G -25 will be crossed for middle gained supernatant, with distillation water elution, flow control is determined in 25-30mL/h
The molecular weight of each separation component, under sephadex column collect molecular weight freeze-dried must consolidate for 2840Da chromatographic fraction
Body sample；
The sephadex G -25 is that glucan is represented with English alphabet G, the G reflections crosslinking degree of gel, degrees of expansion and
Distribution, 25 absorb water 2.5 grams when being every gram of gel expansion；
The elution is that chromatographic column fixed phase is continued through using distilled water as mobile phase, mobile phase and stationary phase force ratio sample
Product are weak, and sample each component is sequentially washed out from stationary phase；
Being determined as the molecular weight elutes retention time T (min) molecular mass logarithm values corresponding thereto according to standard items
(lgMr) standard curve regression equation, standard items are antibacterial poly saccharide peptide standard product（Mr =1422Da）, GSSG standard items
(Mr=612Da), reductive glutathione standard items (Mr=307.32Da).
The Da is the unit of bee venom peptide molecular weight；
Obtained component is is refrigerated to the below freezing of water by the freeze-drying, in the container for being placed in high vacuum (10-40Pa),
The moisture in material is set directly to be distilled from solid ice as a kind of drying means of steam by heat supply.
3. the purifying of melittin
2. the solid obtained by middle freeze-drying is dissolved with the absolute ethyl alcohol of 2-4 times of volume, through 2500-3500r/min from
Heart 4-6min, deposit is melittin.
The solid matter being precipitated as insoluble in absolute ethyl alcohol.
Using this method produce melittin, simplify the extraction process of melittin, compared with the conventional method the production cycle subtract
Few 1-2 days, yield improves 1-2%, reduces preparation cost, it is pure that melittin purity can reach electrophoresis.
Compared with prior art, the method that the present invention prepares melittin, with following distinguishing feature：
（1）Using the post of sephadex G -25, separating treatment is carried out；
（2）The present invention obtains melittin relative to Liu Ling, Yang Wenchao, Zhang Wenli, Xu Peng（1. Liu Ling, Li Changquan, the strong honeybees of Huang Xue
Purification process and extracorporeal anti-tumor function research China biochemical drug impurity [J] .2003,24 of toxin（4）：The poplars of 16-17 2.
Wen Chao, melittin is isolated and purified and its radiation resistance study mechanism [D], University Of Agriculture and Forestry In Fujian, 2007. 3. literary gifts,
The process for separation and purification of Sun Jing member melittins:China, 101089017A [P] .2007-12-19. 4. Xu Peng, Ou Yangyong
Big, Huang Jingyao, Zhang Guiying, Xiao Cheng, Liu Bo isolate and purify experimental study Chinese herbal medicines [J] 2000 of melittin from bee venom,
31(12): 892-894.）, yield brings up to 1-2%, and preparation time shortens 2-3 days.
（3）The purity that the present invention obtains melittin has reached that electrophoresis is pure.
Embodiment 1：
1. the removal of impurities of bee venom
Thick bee venom is added in the cushioning liquid that pH is 4.75, it is 1.0mg/mL to make its concentration, and 5min is centrifuged with 3000r/min
Insoluble impurity is removed, supernatant is obtained；
The thick bee venom be by honeybee sting thorn discharge venom dissolved with distilled water, freeze-dried gained；
The buffer solution is NaAc_HAc buffer solution；
Material of the removal of impurities for removing insoluble in cushioning liquid.
2. the separation of melittin
1. the post of sephadex G -25 will be crossed for middle gained supernatant, with distillation water elution, flow control determines each point in 28mL/h
From the molecular weight of component, collected under sephadex column molecular weight for 2840Da chromatographic fraction it is freeze-dried solid-like
Product；
The sephadex G -25 is that glucan is represented with English alphabet G, the G reflections crosslinking degree of gel, degrees of expansion and
Distribution, 25 absorb water 2.5 grams when being every gram of gel expansion；
The elution is that chromatographic column fixed phase is continued through using distilled water as mobile phase, mobile phase and stationary phase force ratio sample
Product are weak, and sample each component is sequentially washed out from stationary phase；
Being determined as the molecular weight elutes retention time T (min) molecular mass logarithm values corresponding thereto according to standard items
(lgMr) standard curve regression equation, standard items are antibacterial poly saccharide peptide standard product（Mr =1422Da）, GSSG standard items
(Mr=612Da), reductive glutathione standard items (Mr=307.32Da).
The Da is the unit of bee venom peptide molecular weight；
Obtained component is is refrigerated to the below freezing of water by the freeze-drying, in the container for being placed in high vacuum (10-40Pa),
The moisture in material is set directly to be distilled from solid ice as a kind of drying means of steam by heat supply.
3. the purifying of melittin
2. the solid obtained by middle freeze-drying is dissolved with the absolute ethyl alcohol of 3 times of volumes, 5min is centrifuged through 3000r/min,
Deposit is melittin.
The solid matter being precipitated as insoluble in absolute ethyl alcohol.
The recovery rate mass fraction that present invention obtains melittin is 49%, and purity Coriolis mass fraction is 98%.
Embodiment 2：
1. the removal of impurities of bee venom
Thick bee venom is added in the cushioning liquid that pH is 5.25,1.5mg/mL, removed with 3200r/min centrifugations 6min insoluble
Impurity, obtain supernatant；
The thick bee venom be by honeybee sting thorn discharge venom dissolved with distilled water, freeze-dried gained；
The buffer solution is NaAc_HAc buffer solution；
Material of the removal of impurities for removing insoluble in cushioning liquid.
2. the separation of melittin
1. the post of sephadex G -25 will be crossed for middle gained supernatant, with distillation water elution, flow control determines each point in 30mL/h
From the molecular weight of component, collected under sephadex column molecular weight for 2840Da chromatographic fraction it is freeze-dried solid-like
Product；
The sephadex G -25 is that glucan is represented with English alphabet G, the G reflections crosslinking degree of gel, degrees of expansion and
Distribution, 25 absorb water 2.5 grams when being every gram of gel expansion；
The elution is that chromatographic column fixed phase is continued through using distilled water as mobile phase, mobile phase and stationary phase force ratio sample
Product are weak, and sample each component is sequentially washed out from stationary phase；
Being determined as the molecular weight elutes retention time T (min) molecular mass logarithm values corresponding thereto according to standard items
(lgMr) standard curve regression equation, standard items are antibacterial poly saccharide peptide standard product（Mr =1422Da）, GSSG standard items
(Mr=612Da), reductive glutathione standard items (Mr=307.32Da).
The Da is the unit of bee venom peptide molecular weight；
Obtained component is is refrigerated to the below freezing of water by the freeze-drying, in the container for being placed in high vacuum (10-40Pa),
The moisture in material is set directly to be distilled from solid ice as a kind of drying means of steam by heat supply.
3. the purifying of melittin
2. the solid obtained by middle freeze-drying is dissolved with the absolute ethyl alcohol of 4 times of volumes, 6min is centrifuged through 3200r/min,
Deposit is melittin.
The solid matter being precipitated as insoluble in absolute ethyl alcohol.
The recovery rate mass fraction that present invention obtains melittin is 48.65%, and purity Coriolis mass fraction is 97.21%.
Embodiment 3：
1. the removal of impurities of bee venom
Thick bee venom is added in the cushioning liquid that pH is 4.5,0.5mg/mL, removed with 2800r/min centrifugations 4min insoluble
Impurity, obtain supernatant；
The thick bee venom be by honeybee sting thorn discharge venom dissolved with distilled water, freeze-dried gained；
The buffer solution is NaAc_HAc buffer solution；
Material of the removal of impurities for removing insoluble in cushioning liquid.
2. the separation of melittin
1. the post of sephadex G -25 will be crossed for middle gained supernatant, with distillation water elution, flow control determines each point in 26mL/h
From the molecular weight of component, collected under sephadex column molecular weight for 2840Da chromatographic fraction it is freeze-dried solid-like
Product；
The sephadex G -25 is that glucan is represented with English alphabet G, the G reflections crosslinking degree of gel, degrees of expansion and
Distribution, 25 absorb water 2.5 grams when being every gram of gel expansion；
The elution is that chromatographic column fixed phase is continued through using distilled water as mobile phase, mobile phase and stationary phase force ratio sample
Product are weak, and sample each component is sequentially washed out from stationary phase；
Being determined as the molecular weight elutes retention time T (min) molecular mass logarithm values corresponding thereto according to standard items
(lgMr) standard curve regression equation, standard items are antibacterial poly saccharide peptide standard product（Mr =1422Da）, GSSG standard items
(Mr=612Da), reductive glutathione standard items (Mr=307.32Da).
The Da is the unit of bee venom peptide molecular weight；
Obtained component is is refrigerated to the below freezing of water by the freeze-drying, in the container for being placed in high vacuum (10-40Pa),
The moisture in material is set directly to be distilled from solid ice as a kind of drying means of steam by heat supply.
3. the purifying of melittin
2. the solid obtained by middle freeze-drying is dissolved with the absolute ethyl alcohol of 2 times of volumes, 4min is centrifuged through 2800r/min,
Deposit is melittin.
The solid matter being precipitated as insoluble in absolute ethyl alcohol.
The recovery rate mass fraction that present invention obtains melittin is 48.76%, and purity Coriolis mass fraction is 97.64%.
Claims (10)
1. a kind of method for preparing melittin, step is as follows：
1. the removal of impurities of bee venom
Thick bee venom is added in the cushioning liquid that pH is 4.0-5.5, it is 0.5-1.5mg/mL to make its concentration, with 2500-
3500r/min centrifugations 3-7min removes insoluble impurity, obtains supernatant；
2. the separation of melittin
1. the post of sephadex G -25 will be crossed for middle gained supernatant, with distillation water elution, flow control is determined in 25-30mL/h
The molecular weight of each separation component, under sephadex column collect molecular weight freeze-dried must consolidate for 2840Da chromatographic fraction
Body sample；
3. the purifying of melittin
2. the solid obtained by middle freeze-drying is dissolved with the absolute ethyl alcohol of 2-4 times of volume, through 2500-3500r/min from
Heart 4-6min, deposit is melittin.
2. the method as claimed in claim 1 for preparing melittin, thick bee venom is that honeybee stings to pierce the venom distilled water of discharge
Dissolving, freeze-dried gained.
3. the method as claimed in claim 1 for preparing melittin, buffer solution is NaAc_HAc buffer solution.
4. the method as claimed in claim 1 for preparing melittin, material of the removal of impurities for removing insoluble in cushioning liquid.
5. the method as claimed in claim 1 for preparing melittin, sephadex G -25 is glucan English alphabet G tables
Show, G reflects crosslinking degree, degrees of expansion and the distribution of gel, 25 absorb water 2.5 grams when being every gram of gel expansion.
6. the method as claimed in claim 1 for preparing melittin, elutes to continue through chromatogram as mobile phase using distilled water
Post stationary phase, mobile phase and stationary phase force ratio sample are weak, and sample each component is sequentially washed out from stationary phase.
7. the method as claimed in claim 1 for preparing melittin, being determined as molecular weight elutes retention time T according to standard items
(min) molecular mass logarithm value (lgMr) standard curve regression equation corresponding thereto, standard items are antibacterial poly saccharide peptide standard product（Mr =
1422Da）, GSSG standard items (Mr=612Da), reductive glutathione standard items (Mr=307.32Da).
8. the method as claimed in claim 1 for preparing melittin, Da is the unit of bee venom peptide molecular weight.
9. the as claimed in claim 1 method for preparing melittin, be freeze-dried for by obtained component be refrigerated to the freezing point of water with
Under, in the container for being placed in high vacuum (10-40Pa), make the moisture in material be steam directly from solid ice distillation by heat supply
A kind of drying means.
10. the method as claimed in claim 1 for preparing melittin, is precipitated as the solid matter insoluble in absolute ethyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710169095.3A CN107056913A (en) | 2017-03-21 | 2017-03-21 | A kind of method for preparing melittin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710169095.3A CN107056913A (en) | 2017-03-21 | 2017-03-21 | A kind of method for preparing melittin |
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| CN107056913A true CN107056913A (en) | 2017-08-18 |
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ID=59617855
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| CN201710169095.3A Pending CN107056913A (en) | 2017-03-21 | 2017-03-21 | A kind of method for preparing melittin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107714733A (en) * | 2017-11-28 | 2018-02-23 | 吉安市御美丽健康产业股份有限公司 | A kind of preparation method of antibacterial peptide gynaecologic washing lotion |
| CN109045281A (en) * | 2018-09-28 | 2018-12-21 | 祝国光 | A kind of composition and preparation method thereof and pharmaceutical applications of the melittin containing purification |
| CN112341518A (en) * | 2020-10-30 | 2021-02-09 | 广东丸美生物技术股份有限公司 | Bee venom polypeptide extract and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101088514A (en) * | 2006-06-16 | 2007-12-19 | 张文礼 | Bee venom refining process |
| US20150132780A1 (en) * | 2011-12-01 | 2015-05-14 | Siemens Healthcare Diagnostics Inc. | Melittin peptide conjugates and methods employing same |
-
2017
- 2017-03-21 CN CN201710169095.3A patent/CN107056913A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101088514A (en) * | 2006-06-16 | 2007-12-19 | 张文礼 | Bee venom refining process |
| US20150132780A1 (en) * | 2011-12-01 | 2015-05-14 | Siemens Healthcare Diagnostics Inc. | Melittin peptide conjugates and methods employing same |
Non-Patent Citations (1)
| Title |
|---|
| 杨文超: "蜂毒肽的分离纯化及其抗辐射作用机理研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107714733A (en) * | 2017-11-28 | 2018-02-23 | 吉安市御美丽健康产业股份有限公司 | A kind of preparation method of antibacterial peptide gynaecologic washing lotion |
| CN109045281A (en) * | 2018-09-28 | 2018-12-21 | 祝国光 | A kind of composition and preparation method thereof and pharmaceutical applications of the melittin containing purification |
| CN112341518A (en) * | 2020-10-30 | 2021-02-09 | 广东丸美生物技术股份有限公司 | Bee venom polypeptide extract and preparation method and application thereof |
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