CN105769913A - Method of extracting active substance in Cornu cervi pantotrichum - Google Patents
Method of extracting active substance in Cornu cervi pantotrichum Download PDFInfo
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- CN105769913A CN105769913A CN201610164593.4A CN201610164593A CN105769913A CN 105769913 A CN105769913 A CN 105769913A CN 201610164593 A CN201610164593 A CN 201610164593A CN 105769913 A CN105769913 A CN 105769913A
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a method of extracting active substance in Cornu cervi pantotrichum. The method includes extracting active substance in a solution and residue of Cornu cervi pantotrichum after hydrolysis. The method is simple, unharsh in condition, high in active substance extraction rate and can be used in industry for extracting the active substance.
Description
Technical field
The present invention relates to active substance extractive technique.More particularly, to a kind of method extracting active substances in cornua cervi pantotrichum.
Background technology
Cornu Cervi Pantotrichum refers to that the stag of Cervus nippon Temminck or Cervus elaphus linnaeus is unossified and young horn with fine hair.Cervus nippon Temminck, Cervus elaphus linnaeus be the male fawn at 8-10 monthly age after giving birth to, and forehead starts projection, forms long fine and soft basis, and after 2 actual ages, Cornu Cervi Pantotrichum diverges, and Cornu Cervi Pantotrichum is with being preferred that 3-6 gives birth to.Cornu Cervi Pantotrichum is rare medicinal herbs.Cornu Cervi Pantotrichum is warm in nature and not dry, has and rouses oneself and improve body function, to patient after asthenia universalis, prolonged illness, has good Tonic Action.Pharmaceutical analysis shows, containing the multiple material with important physiologically active in Cornu Cervi Pantotrichum, female dimacteric syndrome is had preventive and therapeutic action by the estrogen wherein contained, progesterone etc.；Hypoxanthine, uracil, cholesterol, hydroxy benzaldehyde etc. have the anti-aging effects suppressing monoamine oxidase, MAO；Insulin-like growth factor-i (IGF-1) is the one of somatomedin, no matter is in vivo or tissue and cell to isolated culture all can show certain biological function, and diabetes and complication etc. thereof mainly have special curative effect.Cornu Cervi Pantotrichum closes and has the compositions such as phospholipid, glycolipid, glue fat, hormone, fatty acid, aminoacid, protein and calcium, phosphorus, magnesium, sodium, the chemical composition of Cornu Cervi Pantotrichum: rich in aminoacid, cholesterol myristinate, cholesteryl oleate, cholesterol palm fibre phase acid esters, gallbladder from materials such as alcohol stearate, protein, LYSO-PHOSPHATIDYLCHOLINE LYSOPC, myristic acid and pentadecanoic acids.
The Cornu Cervi Pantotrichum extensive backwardness of Product processing mode that traditional processing method obtains, easily makes some active ingredient loss in Cornu Cervi Pantotrichum be not fully utilized.Therefore, adopt advanced person to efficiently separate technology and the effective ingredient in Cornu Cervi Pantotrichum is carried out extracting and developing and activity analysis thereof, to the precious Chinese medicine Cornu Cervi Pantotrichum resource scientifically utilizing China, improve its added value and promote that modernization of Chinese medicine development is significant.
Summary of the invention
It is an object of the invention to provide a kind of method extracting active substances in cornua cervi pantotrichum, the method makes the percent hydrolysis height of Cornu Cervi Pantotrichum, can effectively keep the active component of Cornu Cervi Pantotrichum, and Cornu Cervi Pantotrichum liposoluble ingredient and water soluble ingredient can be obtained simultaneously, it addition, the method condition is simple, it is easy to implement.
For reaching above-mentioned purpose, the present invention adopts following technical proposals:
A kind of method extracting active substances in cornua cervi pantotrichum, comprises the steps:
1) by fresh Cornu Cervi Pantotrichum unhairing, clean, cut into serving pieces；
2) to step 1) gained piece of antler adds the mixed liquor of ethanol and water, in mixed liquor, ethanol is 0.8-1.1～0.8-1.1 with the volume ratio of water, extracts under 40-60MPa pressure, filters, collect supernatant liquid and residue respectively, and save backup under 4 DEG C of conditions；
3) by step 2) lyophilization at-50 DEG C～-70 DEG C temperature of gained supernatant liquid, the lyophilized powder obtained is in Cornu Cervi Pantotrichum in water insoluble ingredients active substance effective component extracts.
Further, a kind of method extracting active substances in cornua cervi pantotrichum of the present invention, also comprise the steps:
4) to step 2) in residue obtained in add water, the volume ratio of residue and water is 1:15～1:20, stirs at 35～40 DEG C of temperature, obtain residue solution, keep temperature-resistant, in residue solution, add alkaline protease, adding KOH to PH is 7.5～8.5, carries out enzyme digestion reaction；
5) enzyme-deactivating: to step 4) gains add HCl, to PH less than 7, at 40 DEG C of temperature, keep 25～30min, centrifugal filtration takes supernatant liquid, by liquid lyophilizing, obtains surfactant protein polypeptide extract；
Preferably, step 2) in, the volume ratio of piece of antler and mixed liquor is 1:20～1:30；The time extracted is 5～15min.
Preferably, step 2) in, the mass ratio of gained supernatant liquid and residue is 3:1～4:1.
Preferably, step 4) in, the time of described stirring is 20～25min.
Preferably, step 4) in, alkaline protease is selected from Alcalase or trypsin；The addition of described alkaline protease is the 0.8%～1% of mass of residue.
Preferably, step 4) in, the time of described enzyme digestion reaction is 2～3h.
Further, unless there are specified otherwise, the alkaline protease in the present invention such as Alcalase or trypsin are commercially available, as included but not limited to purchased from lucky precious (Qingdao) bio tech ltd.
Further, extracting under 40～60MPa in the inventive method refers to the press device that can use any suitable pressure up to 40～60MPa.
Further, the pressure in the inventive method can carry out preferably under 50MPa.
Beneficial effects of the present invention is as follows:
A kind of method extracting active substances in cornua cervi pantotrichum of the application, its preparation method is simple, condition needed for preparation is also simpler, easily implement, after adding the mixed liquor of ethanol and water of volume ratio 0.8-1.1～0.8-1.1 in piece of antler, the addition ethanol comparing simple or water, extracting required pressure can reduce greatly, percent hydrolysis is higher so that extract safer and easy to implement.Additionally, in the application, residue after Cornu Cervi Pantotrichum is separated has been also carried out enzymatic isolation method and has extracted active substance, by to the selection of enzyme, the determination of pH value, the component of each material addition and the determination in response time, active matter mass-energy in residue is extracted fully so that the extraction ratio of extraction of substance is higher.Comparing prior art, the extracting method of the present invention can make active substances in cornua cervi pantotrichum obtain more extracting fully.
Detailed description of the invention
In order to be illustrated more clearly that the present invention, below in conjunction with preferred embodiment, the present invention is described further.It will be appreciated by those skilled in the art that following specifically described content is illustrative and be not restrictive, should not limit the scope of the invention with this.
Embodiment 1
A kind of method extracting active substances in cornua cervi pantotrichum, step is as follows:
1) by fresh Cornu Cervi Pantotrichum unhairing, the sheet cleaning up, being cut into 5～6mm thickness, standby；
2) the above-mentioned gained piece of antler of 100g is taken, and in piece of antler, add the ethanol of its volume 20 times and the mixed liquor of water, wherein, in mixed liquor, ethanol is 1:1 with the volume ratio of water, pressurize 15min under the pressure of 40MPa, extracting solution filters, and collects supernatant liquid and residue respectively, the mass ratio of supernatant liquid and residue is 3:1, and saves backup under 4 DEG C of conditions；
3) by step 2) lyophilization under-50 DEG C of conditions of gained supernatant liquid, obtain water-insoluble active substance in Cornu Cervi Pantotrichum；
4) to step 2) in residue obtained in add water, wherein, the volume ratio of residue and water is 1:15, and at 35 DEG C of temperature stir 20min, obtain residue solution, keep 35 DEG C temperature-resistant, Alcalase is added in residue solution, the quality of Alcalase is the 1% of residue, and adding a certain amount of KOH to solution PH is 7.5, enzyme digestion reaction 3h；
5) to step 4) to add a certain amount of HCl to gains PH in gains after enzyme digestion reaction be 6, and at 40 DEG C of temperature, keep 25min, centrifugal filtration, take supernatant liquid, and by liquid lyophilizing, obtain surfactant protein polypeptide extract.
In the present embodiment, to step 3) non-water-soluble active substance in Cornu Cervi Pantotrichum can be obtained, to step 5) available active substance is protein polypeptide extract.After measured, in the present embodiment, the percent hydrolysis of Cornu Cervi Pantotrichum reaches 75%, and the quality of gained water-insoluble active substance lyophilized powder is 75g, and in water-insoluble active substance lyophilized powder, the content of fresh pantotrichum insulin-like growth factor I GF-1 is 4602ng/g.The quality of gained protein polypeptide extract is 18.4g.
Embodiment 2
A kind of method extracting active substances in cornua cervi pantotrichum, step is as follows:
1) by fresh Cornu Cervi Pantotrichum unhairing, the sheet cleaning up, being cut into 5～6mm thickness, standby；
2) the above-mentioned gained piece of antler of 100g is taken, and in piece of antler, add the ethanol of its volume 30 times and the mixed liquor of water, wherein, in mixed liquor, ethanol is 0.8:1.1 with the volume ratio of water, pressurize 5min under the pressure of 60MPa, extracting solution filters, and collects supernatant liquid and residue respectively, the mass ratio of supernatant liquid and residue is 4:1, and saves backup under 4 DEG C of conditions；
3) by step 2) lyophilization under-70 DEG C of conditions of gained supernatant liquid, obtain water-insoluble active substance in Cornu Cervi Pantotrichum；
4) to step 2) in residue obtained in add water, wherein, the volume ratio of residue and water is 1:20, and at 40 DEG C of temperature stir 25min, obtain residue solution, keep 40 DEG C temperature-resistant, trypsin is added in residue solution, tryptic quality is the 0.8% of residue, and adding a certain amount of KOH to solution PH is 8.5, enzyme digestion reaction 3h；
5) to step 4) to add a certain amount of HCl to gains PH in gains after enzyme digestion reaction be 5, and at 40 DEG C of temperature, keep 30min, centrifugal filtration, take supernatant liquid, and by liquid lyophilizing, obtain surfactant protein polypeptide extract.
In the present embodiment, to step 3) non-water-soluble active substance in Cornu Cervi Pantotrichum can be obtained, to step 5) available active substance is protein polypeptide extract.After measured, in the present embodiment, the percent hydrolysis of Cornu Cervi Pantotrichum reaches 80%, and the quality of gained water-insoluble active substance lyophilized powder is 78g, and in water-insoluble active substance lyophilized powder, the content of fresh pantotrichum insulin-like growth factor I GF-1 is 4642ng/g.The quality of gained protein polypeptide extract is 14.1g.
Embodiment 3
Repeat embodiment 1, be distinctive in that, by step 2) in the volume ratio of ethanol and water change 1.1:0.8 into, other condition is constant, and the content extracting the active substance obtained in Cornu Cervi Pantotrichum is close with embodiment 1.
Embodiment 4
Repeat embodiment 1, be distinctive in that, by step 2) in pressure change 50MPa into, other condition is constant, extracts the content obtaining active substance in Cornu Cervi Pantotrichum close with embodiment 1.
Comparative example 1
A kind of method extracting active substances in cornua cervi pantotrichum, step is as follows:
1) by fresh Cornu Cervi Pantotrichum unhairing, the sheet cleaning up, being cut into 5～6mm thickness, standby；
2) take the above-mentioned gained piece of antler of 100g, and in piece of antler, add the ethanol of its volume 20 times, wherein, pressurize 15min under the pressure of 40MPa, extracting solution filters, and collects supernatant liquid and residue respectively, the mass ratio of supernatant liquid and residue is 3:1, and saves backup under 4 DEG C of conditions；
3) by step 2) lyophilization under-50 DEG C of conditions of gained supernatant liquid, obtain water-insoluble active substance in Cornu Cervi Pantotrichum；
4) to step 2) in residue obtained in add water, wherein, the volume ratio of residue and water is 1:15, and at 35 DEG C of temperature stir 20min, obtain residue solution, keep 35 DEG C temperature-resistant, Alcalase is added in residue solution, the quality of Alcalase is the 1% of residue, and adding a certain amount of KOH to solution PH is 7.5, enzyme digestion reaction 3h；
5) to step 4) to add a certain amount of HCl to gains PH in gains after enzyme digestion reaction be 6, and at 40 DEG C of temperature, keep 25min, centrifugal filtration, take supernatant liquid, and by liquid lyophilizing, obtain surfactant protein polypeptide extract.
In the present embodiment, to step 3) non-water-soluble active substance in Cornu Cervi Pantotrichum can be obtained, to step 5) available active substance is protein polypeptide extract.After measured, in the present embodiment, the percent hydrolysis of Cornu Cervi Pantotrichum reaches 60%, and the quality of gained water-insoluble active substance lyophilized powder is 58g, and in water-insoluble active substance lyophilized powder, the content of fresh pantotrichum insulin-like growth factor I GF-1 is 4014ng/g.The quality of gained protein polypeptide extract is 9.3g.
Comparative example 2
A kind of method extracting active substances in cornua cervi pantotrichum, step is as follows:
1) by fresh Cornu Cervi Pantotrichum unhairing, the sheet cleaning up, being cut into 5～6mm thickness, standby；
2) take the above-mentioned gained piece of antler of 100g, and in piece of antler, add the water of its volume 20 times, wherein, pressurize 15min under the pressure of 60MPa, extracting solution filters, and collects supernatant liquid and residue respectively, the mass ratio of supernatant liquid and residue is 3:1, and saves backup under 4 DEG C of conditions；
3) by step 2) lyophilization under-50 DEG C of conditions of gained supernatant liquid, obtain water-insoluble active substance in Cornu Cervi Pantotrichum；
4) to step 2) in residue obtained in add water, wherein, the volume ratio of residue and water is 1:15, and at 35 DEG C of temperature stir 20min, obtain residue solution, keep 35 DEG C temperature-resistant, Alcalase is added in residue solution, the quality of Alcalase is the 1% of residue, and adding a certain amount of KOH to solution PH is 7.5, enzyme digestion reaction 3h；
5) to step 4) to add a certain amount of HCl to gains PH in gains after enzyme digestion reaction be 6, and at 40 DEG C of temperature, keep 25min, centrifugal filtration, take supernatant liquid, and by liquid lyophilizing, obtain surfactant protein polypeptide extract.
In the present embodiment, to step 3) non-water-soluble active substance in Cornu Cervi Pantotrichum can be obtained, to step 5) available active substance is protein polypeptide extract.After measured, in the present embodiment, the percent hydrolysis of Cornu Cervi Pantotrichum reaches 65%, and the quality of gained water-insoluble active substance lyophilized powder is 62g, and in water-insoluble active substance lyophilized powder, the content of fresh pantotrichum insulin-like growth factor I GF-1 is 4107ng/g.The quality of gained protein polypeptide extract is 9.7g.
Obviously; the above embodiment of the present invention is only for clearly demonstrating example of the present invention; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot all of embodiment be given exhaustive, every belong to apparent change that technical scheme extended out or the variation row still in protection scope of the present invention.
Claims (7)
1. the method extracting active substances in cornua cervi pantotrichum, it is characterised in that comprise the steps:
1) by fresh Cornu Cervi Pantotrichum unhairing, clean, cut into serving pieces；
2) to step 1) gained piece of antler adds the mixed liquor of ethanol and water, in mixed liquor, ethanol is 0.8-1.1～0.8-1.1 with the volume ratio of water, extracts under 40～60MPa pressure, filters, collect supernatant liquid and residue respectively, and save backup under 4 DEG C of conditions；
3) by step 2) lyophilization at-50 DEG C～-70 DEG C temperature of gained supernatant liquid, the lyophilized powder obtained is in Cornu Cervi Pantotrichum water-insoluble active substance.
2. method according to claim 1, it is characterised in that also comprise the steps:
4) to step 2) in residue obtained in add water, the volume ratio of residue and water is 1:15～1:20, stirs at 35～40 DEG C of temperature, obtain residue solution, keep temperature-resistant, in residue solution, add alkaline protease, adding KOH to PH is 7.5～8.5, carries out enzyme digestion reaction；
5) enzyme-deactivating: to step 4) gains add HCl, to PH less than 7, at 40 DEG C of temperature, keep 25～30min, centrifugal filtration takes supernatant liquid, by liquid lyophilizing, obtains surfactant protein polypeptide extract；
3. method according to claim 1, it is characterised in that step 2) in, the volume ratio of piece of antler and mixed liquor is 1:20～1:30；The time extracted is 5～15min.
4. method according to claim 1, it is characterised in that step 2) in, the mass ratio of gained supernatant liquid and residue is 3:1～4:1.
5. method according to claim 2, it is characterised in that step 4) in, the time of described stirring is 20～25min.
6. method according to claim 2, it is characterised in that step 4) in, alkaline protease is selected from Alcalase or trypsin；The addition of described alkaline protease is the 0.8%～1% of mass of residue.
7. method according to claim 2, it is characterised in that step 4) in, the time of described enzyme digestion reaction is 2～3h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107375331A (en) * | 2017-08-15 | 2017-11-24 | 吉林睿康生物科技有限公司 | A kind of extracting method of pilose antler |
| CN109303789A (en) * | 2017-07-28 | 2019-02-05 | 中国科学院大连化学物理研究所 | A kind of pilose antler active component and preparation and application with prolyl inscribe enzyme inhibition activity |
| CN111850075A (en) * | 2020-07-21 | 2020-10-30 | 长春中医药大学 | Cervus elaphus linnaeus soluble protein extract and extraction method and application thereof |
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| CN109303789B (en) * | 2017-07-28 | 2021-10-15 | 中国科学院大连化学物理研究所 | Cornu Cervi Pantotrichum active component with prolyl endonuclease inhibiting activity, and its preparation method and application |
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| CN111850075A (en) * | 2020-07-21 | 2020-10-30 | 长春中医药大学 | Cervus elaphus linnaeus soluble protein extract and extraction method and application thereof |
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Inventor after: Kong Qingjun Inventor after: Long Hongzhi Inventor after: Long Hongyang Inventor after: Xing Yunfeng Inventor before: Jiang Furu Inventor before: Xin Jingwei Inventor before: Jiang Xin Inventor before: Ge Jing Inventor before: Wei Lijing |
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Effective date of registration: 20180503 Address after: 134000 four groups of river village, Ju Xin Economic Development Zone, Tonghua, Tonghua, Jilin Applicant after: Tonghua Jinhui Biological Engineering Co., Ltd. Address before: 130041 Jilin Road, Changchun, Changchun, Jilin Applicant before: Jilin Biaoda Technology Co., Ltd. |
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Application publication date: 20160720 |
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| RJ01 | Rejection of invention patent application after publication |