CN1695733A - Wood louse analgesia peptide, preparation method and application - Google Patents
Wood louse analgesia peptide, preparation method and application Download PDFInfo
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- CN1695733A CN1695733A CN 200510016787 CN200510016787A CN1695733A CN 1695733 A CN1695733 A CN 1695733A CN 200510016787 CN200510016787 CN 200510016787 CN 200510016787 A CN200510016787 A CN 200510016787A CN 1695733 A CN1695733 A CN 1695733A
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Abstract
An antalgic peptide in the form of aqueous solution for suppressing pain is prepared from pillbug through extracting to obtain liquid extract, ion exchange chromatography, and post-treating. It can be used to prepare antalgic medicines.
Description
Technical field
The invention belongs to organic chemistry filed, effective ingredient Armadillidium analgesic peptide and extracting method thereof and the application in analgesic in particularly a kind of Chinese medicine medical material.
Background technology
Having an intense pain of treatment cancer pain one class is still important research project in the medical research field at present, although psychotropic substances such as morphine, dolantin can be brought into play analgesic effect preferably, but follow the addiction of its drug effect and stop using after withdrawal symptom, brought greatly harm for the use of analgesic.Because there are shortcomings such as cost height, complex process, productive rate be low in synthetic chemical analgesic.Therefore, it is little to seek a kind of toxic and side effects, and processing simple product becomes one of main direction of study of pharmacy.
The document record is arranged, and Armadillidium has the effect of antipyretic analgesic, removing blood stasis diuretic.The domestic analgesic Chinese traditional patent formulation that is used for contains Armadillidium, can be used for the later period of hepatocarcinoma pain relieving.The applying biological method is extracted the research of the analgesia peptide aspect in the Armadillidium and is not seen bibliographical information yet.
Summary of the invention
The technical problem to be solved in the present invention is, extracts the analgesic effective component in the Chinese medicine Armadillidium, and carries out analgesic experiment, investigates its analgesic effect, makes it become the main component of analgesic drug; Provide the preparation method of the Armadillidium analgesic peptide effective, that technology is easy simultaneously.
A kind of Armadillidium analgesic peptide is to be the analgesia peptide that exists with the aqueous solution form that raw material makes with the oven dry Armadillidium, and its molecular weight is 1000～5000Da; Its protein content is 0.1～0.8mg/ml; PH value of water solution is 5～7; Have typical alpha-helix conformation, still have analgesic activity after the heated and boiled.
Wherein, molecular weight is through gel chromatography and polyacrylate hydrogel electrophoresis detection; Protein content adopts biuret method to measure; After the heated and boiled, still has analgesic activity through the animal experiment proof.
Further extract, can obtain two components of Armadillidium analgesic peptide, molecular weight is respectively 1000～2000Da, 3000～5000Da.
The preparation method of Armadillidium analgesic peptide of the present invention is to be raw material with the oven dry Armadillidium, makes the analgesia peptide that exists with the aqueous solution form, through extracting, produce the technical process of water extract, ion-exchange chromatography, post processing;
Said extractive process is to get the oven dry Armadillidium and be ground to Powdered; Press Armadillidium powder quality and distilled water volume 1g: the mixed of 2～8ml, at room temperature stir and got extract in 50～80 minutes;
The said water extract of producing is, with extract under 8000～16000g/min centrifugal 20～60 minutes, abandons precipitation, gets supernatant and gets water extract;
Said ion-exchange chromatography is that water extract is through the DEAE-52 ion-exchange chromatography; Balance liquid is the phosphate buffer of 0.01～0.08mol/L, pH7.2, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 0.5～1.5mol/LNaCl, collects the vigor peak with analgesic activity;
Said post processing is to obtain 24～48 hours desalinations of active component dialysis; Distilling under reduced pressure again concentrates, and obtains a component, and promptly molecular weight is that 1000～5000Da, protein content are the Wood louse analgesia peptide solution of 0.1～0.8mg/ml.
In order to obtain two components of Armadillidium analgesic peptide, can after last handling process, carry out gel filtration chromatography.That is, through Sephadex G-50 gel filtration chromatography, just obtaining molecular weight is respectively the Wood louse analgesia peptide solution of two components of 1000～2000Da, 3000～5000Da with a component obtaining.Wherein elution requirement is 0.01～0.05mol/L, pH6.2 phosphate buffer or distilled water.
The preparation method of preferred Armadillidium analgesic peptide, extractive process are to get the every 100g of oven dry Armadillidium powder to add the 300ml distilled water, at room temperature stir 55～65 minutes; The condition of producing water extract be with extract under 10000g/min, centrifugal 28～32 minutes; Balance liquid is the phosphate buffer of 0.05mol/L, pH7.2 in the ion-exchange chromatography, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 1mol/LNaCl; Last handling process is 35～38 hours desalinations of dialysis.
The method of producing Armadillidium analgesic peptide of the present invention is effective, and technical process is easy, is of value to pharmaceuticals industry production.
The purposes of Armadillidium analgesic peptide of the present invention is that the raw material of making analgesic or analgesic uses.
Because the Armadillidium analgesic peptide that extracted exists with the aqueous solution form, can directly oral or injection, to slight illness, grieved, comprise the good restraining effect that had an intense pain that cancer causes.The analgesic drug that the Armadillidium analgesic peptide aqueous solution can be prepared various dosage forms with other conventional adjuvant.
Description of drawings
Fig. 1 is that molecular weight of the present invention is the circular dichroism spectrogram of 1000-2000Da Armadillidium analgesic peptide.
Fig. 2 is that molecular weight of the present invention is the circular dichroism spectrogram of 3000-5000Da Armadillidium analgesic peptide.
The specific embodiment
Implementing the present invention can be undertaken by following embodiment, but and does not mean that limitation of the scope of the invention.
Embodiment 1
1, gets the every 100g of oven dry Armadillidium powder and add the 300ml distilled water, at room temperature stirred 60 minutes, get extract;
2, above-mentioned extract is under 10000g/min, and centrifugal 30 minutes, abandon precipitation, get supernatant;
3, supernatant is through the DEAE-52 ion-exchange chromatography, and balance liquid is the phosphate buffer of 0.05mol/L, pH7.2, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 1mol/LNaCl, collects the vigor peak with analgesic activity;
4, DEAE-52 is obtained 36 hours desalinations of active component dialysis; With the dialysis solution distilling under reduced pressure, concentrate, obtain the Wood louse analgesia peptide solution of a component.
Can also be with Sephadex G-50/G-25 gel filtration chromatography on the Armadillidium analgesic peptide concentrated solution of a component, elution requirement is a distilled water, obtains having two component Wood louse analgesia peptide solutions of analgesic activity.
Fig. 1 is that molecular weight of the present invention is the circular dichroism spectrogram of 1000-2000Da Armadillidium analgesic peptide.Wherein molecular weight is that the 1000-2000Da Armadillidium analgesic peptide has two paddy at 208nm and 222nm place, is typical alpha-helix conformation, accounts for 20.6%.
Fig. 2 is that molecular weight of the present invention is the circular dichroism spectrogram of 3000-5000Da Armadillidium analgesic peptide.Wherein molecular weight is that the 3000-5000Da Armadillidium analgesic peptide has two paddy at 208nm and 222nm place, is typical alpha-helix conformation, accounts for 15.2%.
Adopting biuret method to measure protein content is 0.8mg/ml.
Present embodiment is an optimal process routes.
1, gets the every 100g of oven dry Armadillidium powder and add the 200ml distilled water, at room temperature stirred 80 minutes;
2, above-mentioned extract is under 8000g/min, and centrifugal 40 minutes, abandon precipitation, get supernatant;
3, supernatant is through the DEAE-52 ion-exchange chromatography, and balance liquid is the phosphate buffer of 0.08mol/L, pH7.2, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 1.5mol/LNaCl, collects the vigor peak with analgesic activity;
4, DEAE-52 is obtained 24 hours desalinations of active component dialysis;
5, with the dialysis solution distilling under reduced pressure, concentrate;
6, Sephadex G-50/G-25 gel filtration chromatography on the concentrated solution, elution requirement is 0.01mol/L, pH6.2 phosphate buffer, obtains two components, has analgesic activity.
The circular dichroism spectrogram of two components of resulting Armadillidium analgesic peptide is identical with embodiment 1, sees Fig. 1, Fig. 2.
It is 0.5mg/ml that resulting Wood louse analgesia peptide solution adopts biuret method to measure protein content.
1, gets the every 100g of oven dry Armadillidium powder and add the 500ml distilled water, at room temperature stirred 50 minutes;
2, above-mentioned extract is under 16000g/min, and centrifugal 20 minutes, abandon precipitation, get supernatant;
3, supernatant is through the DEAE-52 ion-exchange chromatography, and balance liquid is the phosphate buffer of 0.01mol/L, pH7.2, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 0.5mol/LNaCl, collects the vigor peak with analgesic activity;
4, DEAE-52 is obtained 24 hours desalinations of active component dialysis;
5, with the dialysis solution distilling under reduced pressure, concentrate;
6, Sephadex G-50/G-25 gel filtration chromatography on the concentrated solution, elution requirement is 0.05mol/L, pH6.2 phosphate buffer, obtains two components, has analgesic activity.
The circular dichroism spectrogram of two components of resulting Armadillidium analgesic peptide is identical with embodiment 1, sees Fig. 1, Fig. 2.
It is 0.3mg/ml that resulting Wood louse analgesia peptide solution adopts biuret method to measure protein content.
Embodiment 4
Analgesia peptide of the present invention has following characteristic:
1, physicochemical property
(1) aesthetic appearance: colourless solution;
(2) pH value: 5-7;
(3) molecular weight: 1000Da～5000Da
(4) amino acid content
Molecular weight is that to record amino acid content behind acid hydrolysis be aspartic acid 9.30%, glutamic acid 8.64%, serine 7.93%, histidine 10.67%, glycine 8.17%, threonine 4.45%, arginine 3.08%, alanine 5.64%, tyrosine 8.32%, valine 4.52%, methionine 3.62%, phenylalanine 8.24%, isoleucine 2.65%, leucine 4.05%, lysine 4.90%, proline 5.82% to the 1000-2000Da Armadillidium analgesic peptide.
Molecular weight is that to record amino acid content behind acid hydrolysis be aspartic acid 12.64%, glutamic acid 12.73%, serine 6.41%, histidine 4.47%, glycine 6.23%, threonine 6.76%, arginine 4.50%, alanine 6.58%, tyrosinase 15 .65%, valine 5.66%, methionine 2.02%, phenylalanine 4.37%, isoleucine 4.34%, leucine 6.85%, lysine 6.26%, proline-4 .53% to the 3000-5000Da Armadillidium analgesic peptide.
The present invention compares with the chemosynthesis analgesic drug has little, the cold-resistant good heat resistance of toxic and side effects, and no addiction is easy to clinical expansion.
Embodiment 5
Present embodiment provides the activity of Armadillidium analgesic peptide biology, adopts the acetic acid twisting method to investigate its analgesic activity.
1.1 causing pain model sets up
To body weight is the Kunming kind mice by intraperitoneal injection 1.0% acetic acid 0.1ml/10g of 18～22 grams, causes that because of maincenter causes the pain effect mouse writhing phenomenon occurs, and pain Shaoxing opera is strong, and it is high more to turn round body frequency.
1.2 analgesia investigation method
1.2.1 gastric infusion: get an amount of Armadillidium analgesic peptide with the mouse stomach device, directly irritate stomach, inject acetic acid behind the 60min, write down and turn round the body number of times in 20 minutes, in 20 minutes, turn round the body number of times relatively by blank group and administration group.
1.2.2 subcutaneous injection administration: get an amount of Armadillidium analgesic peptide with injector for medical purpose, in the mouse back subcutaneous injection, inject acetic acid behind the 20min, write down and turn round the body number of times in 20 minutes, the body number of times of turning round in 20 minutes compares by blank group and administration group, calculates the suppression ratio of medicine to pain:
1.3 experimental result
1.3.1 this Armadillidium analgesic peptide is through gastric infusion 40mg/kg, the pain suppression ratio is 53.55%;
1.3.2 dolantin is through subcutaneous administration 45mg/kg, the pain suppression ratio is 100%;
1.3.3 drug administration by injection 42.53mg/kg under this Armadillidium analgesic peptide percutaneous, pain suppression ratio are 95.60%;
1.3.4 experimental data
Irritate the administration of harmonization of the stomach subcutaneous injection and investigate Armadillidium analgesic peptide analgesic effect
| Group | Administering mode | ?n | Dosage | Turn round body number of times (X ± SD) | Suppression ratio |
| The blank group of blank group (normal saline) Armadillidium analgesic peptide (normal saline) dolantin Armadillidium analgesic peptide | Irritate stomach and irritate gastrodermis injection subcutaneous injection subcutaneous injection down | ?10 ?10 ?10 ?10 ?10 | ?10ml/kg ?40mg/kg ?10ml/kg ?45mg/kg ?42.53mg/kg | ?28.20±3.05 ?13.10±4.48 **?25.20±4.76 ?0 **?1.11±1.45 ** | ??- ??53.55％ ??- ??100％ ??95.60％ |
Contrast with the blank group: Δ P＞0.05
*P＜0.05
*P＜0.001
1.4 addiction experiment
1.4.1 addiction is surveyed in the perpendicular tail experiment of mice
Get healthy mice 18～22g, male and female are not limit, and are divided into three groups at random, 7 every group:
(1) blank group;
(2) dolantin group 7.5mg/kg;
(3) Armadillidium analgesic peptide group 40mg/kg
Difference subcutaneous injection morphine and Armadillidium analgesic peptide, successive administration 15 days, blank gives water for injection.
15min after administration every day observes the phenomenon of walking about whether white mice perpendicular tail occurs and do not stop.
Mice is erected the tail experimental result
| Group | The blank group | The morphine group | The Armadillidium analgesic peptide group |
| Perpendicular tail number of animals (only) | ??????0 | ????7 | ??????0 |
Do not see the Armadillidium analgesic peptide group in this experiment using dosage scope addiction is arranged.
1.4.2 addiction is surveyed in the mouse jump experiment
Select healthy mice 18～22g for use, male, be divided into three groups at random, 7 every group:
(1) blank group;
(2) dolantin group 7.5mg/kg;
(3) Armadillidium analgesic peptide group 40mg/kg
Every 12h subcutaneous injection morphine and Armadillidium analgesic peptide, totally 14 times.
Last injection back 8h, subcutaneous injection Allylnoroxymorphone 0.06mg/kg urges addiction, immediately white mice is placed on high 35cm, on the round platform of diameter 30cm, observes the jump number of animals in the Allylnoroxymorphone injection back 30min.
The mouse jump experimental result
| Group | The blank group | The morphine group | The Armadillidium analgesic peptide group |
| Perpendicular tail number of animals (only) | ??????0 | ????5 | ??????0 |
Do not see the Armadillidium analgesic peptide group in this experiment using dosage scope addiction is arranged.
Claims (6)
1, a kind of Armadillidium analgesic peptide is characterized in that, is to be the analgesia peptide that exists with the aqueous solution form that raw material makes with the oven dry Armadillidium, and its molecular weight is 1000～5000Da; Its protein content is 0.1-0.8mg/ml; PH value of water solution is 5～7; Have typical alpha-helix conformation, still have analgesic activity after the heated and boiled.
According to the described Armadillidium analgesic peptide of claim 1, it is characterized in that 2, said Armadillidium analgesic peptide has two components, molecular weight is respectively 1000～2000Da, 3000～5000Da.
3, a kind of preparation method of Armadillidium analgesic peptide is to be raw material with the oven dry Armadillidium, makes the analgesia peptide that exists with the aqueous solution form, through extracting, produce the technical process of water extract, ion-exchange chromatography, post processing;
Said extractive process is to get the oven dry Armadillidium and be ground to Powdered; Press Armadillidium powder quality and distilled water volume 1g: the mixed of 2～8ml, at room temperature stir and got extract in 50～80 minutes;
The said water extract of producing is, with extract under 8000～16000g/min centrifugal 20～60 minutes, abandons precipitation, gets supernatant and gets water extract;
Said ion-exchange chromatography is that water extract is through the DEAE-52 ion-exchange chromatography; Balance liquid is the phosphate buffer of 0.01～0.08mol/L, pH7.2, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 0.5～1.5mol/LNaCl, collects the vigor peak with analgesic activity;
Said post processing is to obtain 24～48 hours desalinations of active component dialysis; Distilling under reduced pressure again concentrates, and obtains a component, and promptly molecular weight is that 1000～5000Da, protein content are the Wood louse analgesia peptide solution of 0.1～0.8mg/ml.
4, according to the preparation method of the described Armadillidium analgesic peptide of claim 3, it is characterized in that, after last handling process, carry out gel filtration chromatography, that is, with a component obtaining through Sephadex G-50 gel filtration chromatography; Elution requirement is 0.01-0.05mol/L, pH6.2 phosphate buffer or distilled water; Obtaining molecular weight is respectively the Wood louse analgesia peptide solution of two components of 1000～2000Da, 3000～5000Da.
According to the preparation method of claim 3 or 4 described Armadillidium analgesic peptide, it is characterized in that 5, said extractive process is to get the every 100g of oven dry Armadillidium powder to add the 300ml distilled water, at room temperature stirs 55～65 minutes; The condition of producing water extract be with extract under 10000g/min, centrifugal 28～32 minutes; Balance liquid is the phosphate buffer of 0.05mol/L, pH7.2 in the ion-exchange chromatography, and eluent is 0.05mol/L, the pH6.2 phosphate buffer of 1mol/LNaCl; Last handling process is 35～38 hours desalinations of dialysis.
6, a kind of application of Armadillidium analgesic peptide is characterized in that, is to be the analgesia peptide that exists with the aqueous solution form that raw material makes with the oven dry Armadillidium; Its molecular weight is 1000～5000Da, and its protein content is 0.1-0.8mg/ml, and pH value of water solution is 5～7, has typical alpha-helix conformation; Purposes is that the raw material of making analgesic or analgesic uses.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526239A (en) * | 2012-02-15 | 2012-07-04 | 常炜 | Femoral healing |
| CN105732789A (en) * | 2016-03-03 | 2016-07-06 | 中国科学院昆明动物研究所 | Haemadipsa sylvestris analgesic peptide mh2620 and gene and application thereof |
| CN110127642A (en) * | 2018-02-09 | 2019-08-16 | 杨建中 | Containing orgon compound and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1130213C (en) * | 2001-02-26 | 2003-12-10 | 丁铁岭 | Chinese herbal plaster for anti-cancer and analgesic |
| CN1164309C (en) * | 2002-03-21 | 2004-09-01 | 张金清 | Ningtongling as a pain killer |
| CN1528433A (en) * | 2003-10-17 | 2004-09-15 | 吉林大学 | Method for extracting active rpinciple for analgesia from fillworm using water |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526239A (en) * | 2012-02-15 | 2012-07-04 | 常炜 | Femoral healing |
| CN105732789A (en) * | 2016-03-03 | 2016-07-06 | 中国科学院昆明动物研究所 | Haemadipsa sylvestris analgesic peptide mh2620 and gene and application thereof |
| CN110127642A (en) * | 2018-02-09 | 2019-08-16 | 杨建中 | Containing orgon compound and its preparation method and application |
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