CN101693737A - Cornu-like polypeptide with antioxidant activity, separation method and application thereof - Google Patents
Cornu-like polypeptide with antioxidant activity, separation method and application thereof Download PDFInfo
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- CN101693737A CN101693737A CN200910233784A CN200910233784A CN101693737A CN 101693737 A CN101693737 A CN 101693737A CN 200910233784 A CN200910233784 A CN 200910233784A CN 200910233784 A CN200910233784 A CN 200910233784A CN 101693737 A CN101693737 A CN 101693737A
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 61
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- 238000000926 separation method Methods 0.000 title claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 9
- PZIBBFAIJOAECN-UHFFFAOYSA-N Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn Natural products CC(C)CC(NC(=O)C(CCC(=O)O)NC(=O)C(CC(=O)N)NC(=O)C(C)NC(=O)C(CC(=O)N)NC(=O)C(CC(=O)O)NC(=O)C(C)NC(=O)C(C)N)C(=O)NC(Cc1ccccc1)C(=O)N2CCCC2C(=O)N3CCCC3C(=O)NC(CC(=O)N)C(=O)O PZIBBFAIJOAECN-UHFFFAOYSA-N 0.000 claims abstract description 7
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- 238000013375 chromatographic separation Methods 0.000 claims description 6
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
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Abstract
The invention relates to the technical field of medicines, in particular to cornu-like polypeptide with antioxidant activity and a separation method and application thereof. The polypeptide of the invention has following sequential structures: Gln-Tyr-Asp-Gln-Gly-Val; Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn; Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn. The polypeptide of the three sequences is extracted and separated from buffalo cornus. Pharmacological tests prove that the polypeptide has fine antioxidant activity.
Description
Technical field
The present invention relates to technical field of pharmaceuticals, possess and relate to angle class polypeptide, its separation method and the purposes that a class has anti-oxidant activity.
Background technology
Angle class animal drug is the important component part of motherland's medical treasure-house, and its exact efficacy acts on conspicuous characteristics.Just put down in writing the nature and flavor and the effect of rhinoceros horn (Cornu Rhinoceri Asiatici) and sheep's horn (Cornu Saigae Tataricae) in the existing known herbal works Shennong's Herbal the earliest of China, then occurred record in Han dynasty " Mingyi Bielu " about Cornu Bubali (CornuBubali) nature and flavor and effect.Over the past thousands of years, rhinoceros (extensively) angle, sheep's horn, Cornu Bubali isogonism class medicinal material are the indispensable key medicines of tcm clinical practice always, are mainly used in the treatment heat symptom-complex.Yet at present law has been prohibited use and conclude the business rhinoceros horn and goods thereof, and sahilite is as endangered species, and the use at its angle also is restricted, and has only Cornu Bubali extensive, cheap because of drawing materials, determined curative effect and being used till today.From the sixties in 20th century, the scientific research personnel just begins multiple angle class medicinal material is compared research and estimates, the result thinks that Cornu Bubali is in aspect and exact efficacy similar to rhinoceros horn such as amino acid composition, macro micro element composition, pharmacological effect effects, therefore the traditional Chinese medical science generally adopts Cornu Bubali and Pulvis Cornus Bubali Concentratus to substitute rhinoceros (extensively) angle as medicine industry and clinical adjustment at present, obtain good effect, to alleviate the dependency that tcm clinical practice used for rhinoceros (extensively) angle over thousand.
Cornu Bubali Cornu Bubali is two angles of bovid Bubalus bubalis Linnaeus, is that 2005 editions Pharmacopoeias of the People's Republic of China record kind, and the nature and flavor hardship is cold, the thoughts of returning home, Liver Channel.Cornu Bubali mainly contains materials such as Keratin sulfate, polypeptide, amino acid, cholesterol, effects such as clearing heat and detoxicating, the cool blood of tool, arresting convulsion, and tcm clinical practice is directly to fry in shallow oil soup or to go into prescription and fry in shallow oil soup and take, be mainly used in the high heat of treatment warm disease, unconsciousness and delirium is sent out the spot dermexanthesis, hematemesis and epistaxis, infantile convulsion, the demented card that waits.
Though rhinoceros (extensively) angle, sheep's horn, Cornu Bubali isogonism class medicinal material applicating history exceed thousand, but because of singularity such as crude drug source position, material, moietys, its effector substance basis is also comparatively weak with Its Mechanisms, and is almost nil about the report of its activeconstituents.Therefore, how to make full use of the modern biotechnology means, separation and purification active polypeptide and carry out structure and identify from the class medicinal material of angle carries out to study class medicinal material effector substance basis, angle and study on mechanism in a deep going way, is that this field scientific research technician makes great efforts one of research direction of seeking breakthrough for many years.
Summary of the invention
The present invention adopts modern biochemical technology means, follows the tracks of having anti-oxidant activity polypeptide composition in the Cornu Bubali, carries out progressively separation, purifying, and the structure of this active ingredient is carried out Analysis and Identification.
The invention discloses the polypeptide of following 3 kinds of structural formulas:
Gln-Tyr-Asp-Gln-Gly-Val(I)；
Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn(II)；
Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn(III)。
They all separate from Cornu Bubali, but also synthetic.
Comprise from the isolating method of Cornu Bubali:
A, water intaking horn powder are used the pure water heating and refluxing extraction, filter, and filtrate concentrated frozen drying gets crude extract;
B, crude extract is carried out gel filtration chromatography separate, under 254nm, detect and flow out sample, collect each component, measure anti-oxidant activity according to the sample peak of detector;
C, the active component of high anti-oxidation is further utilized DEAE Sepharose Fast Flow anionresin column separating purification, ion exchange column is earlier with 50mM phosphate buffered saline buffer (PBS) pH6.5, with 1ml/min flow velocity pre-treatment 3h; Behind the last sample, ion exchange column with 1ml/min flow velocity wash-out 2h, is the PBS damping fluid 50mM of 0-0.3M NaCl with 50mM PBS pH6.5 with the linear gradient then, the pH6.5 wash-out, and elution curve detects under 254nm; Collect each component, lyophilize according to elution peak;
D, with the lyophilized powder that c obtains, pure water is dissolved into higher concentration, carries out desalination by the automatic purifying HPLC of Waters 2545-2489-2676 system, measures anti-oxidant activity, obtains better anti-oxidant activity component D2 and D3;
E, with component D2 and D3, again by the further separation and purification of the automatic purifying HPLC of Waters 2545-2489-2676 system; With the linear gradient is the 2%-60% methanol-eluted fractions, wherein contains 0.1% trifluoroacetic acid, detects under 230nm; During D2 component reversed-phase HPLC chromatographic separation, collect two chromatographic peaks that retention time is about 4.6min and 7.3min, be respectively polypeptide I and II; During D3 component reversed-phase HPLC chromatographic separation, collect the chromatographic peak that retention time is about 5.4min, be polypeptide III; The sample that collection obtains, after concentrating under reduced pressure is removed methyl alcohol, lyophilize; Obtain polypeptide I respectively, the lyophilized powder of II and III.
Active polypeptide I, the II of above-mentioned separation purification method purifying, the molecular weight of III are respectively 708.090Da, 1018.296Da and 1271.379Da.Structural formula:
Gln-Tyr-Asp-Gln-Gly-Val(I)；
Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn(II)；
Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn(III)。
3 polypeptide are dissolved in the cell culture fluid, are made into 0.5 μ M respectively through dilution, 5 μ M and 50 μ M solution, incubate 12h in advance with rat brain capillary endothelium (rCMECs) after, remove the cell culture fluid that contains polypeptide, wash twice with PBS, add and contain 500 μ M H
2O
2Cell culture fluid, incubate 2h altogether after, carry out MTT experiment, the cell conditioned medium of sucking-off simultaneously liquid is used to measure LDH content.The result shows that 3 polypeptide can increase the rCMECs cell survival rate when 5 μ M and 50 μ M concentration, reduces supernatant LDH burst size, to H
2O
2Due to rCMECs damage provide protection is all arranged.Wherein, obviously be better than polypeptide I and polypeptide II, can significantly improve cell survival rate, and reduce the release of LDH with anti-oxidant the best of living of polypeptide III.The results are shown in Figure 1 and Fig. 2.
The present invention follows the tracks of the polypeptide composition that has anti-oxidant activity in the Cornu Bubali, progressively separates, purifying, and pure polypeptide is carried out amino acid sequence analysis, obtains 3 angle class polypeptide with anti-oxidant activity.Technology of the present invention is simple, and applied range is other angle class medicinal material basic substance research, and the separation and purification of activeconstituents provides thinking and method.
The characteristics of 3 active polypeptide are to have good antioxidant activity, to H
2O
2Due to the rCMECs peroxide injury have provide protection, embodied anti-oxidant activity; 3 active polypeptide molecular weight characteristics all are lower than 2kDa, and the polypeptide that molecular weight is more little facilitates penetration of the body semi-permeable membranes more and arrives site of action; The amino acid compositing characteristic is all to contain hydrophobic amino acid and protophobe amino acid, hydrophobic amino acid residues can promote combining of polypeptide and biosystem inner lipid, protophobe amino acid can provide proton to participate in redox reaction, therefore 3 active polypeptide is fat-soluble better, and has good antioxidant activity.It is little that the present invention separates the polypeptide molecular weight that obtains, active outstanding, aminoacid sequence is clear and definite, for further synthetic active polypeptide provides necessary base, carries out product research exploitations such as medicine, healthcare products and lays a good foundation for developing angle class anti-oxidant activity polypeptide from now on.
Active polypeptide of the present invention can be mixed and made into various formulations with pharmaceutically acceptable carrier, also can be mixed and made into protective foods with foodstuff additive.Polypeptide of the present invention can also form the form existence of pharmacy acceptable salt with pharmaceutical salts.
Description of drawings
Fig. 1 is purified polypeptide I, II and the anti-rCMECs oxidative damage of III MTT experiment synoptic diagram.
Fig. 2 is purified polypeptide I, II and the anti-rCMECs oxidative damage of III LDH release experiment synoptic diagram.
Fig. 3 is the chromatography synoptic diagram that Sephadex G-25 gel filtration chromatography separates active polypeptide in the Cornu Bubali extract.
Fig. 4 is the chromatography synoptic diagram of DEAE Sepharose Fast Flow anion exchange chromatography isolating active component G3.
Fig. 5 is the chromatography synoptic diagram of the purifying activeconstituents D2 of the automatic purifying HPLC of Waters system.
Fig. 6 is the chromatography synoptic diagram of the purifying activeconstituents D3 of the automatic purifying HPLC of Waters system.
From the beginning Fig. 7 calculates synoptic diagram for tandem mass spectrum and the aminoacid sequence of purified polypeptide I.
From the beginning Fig. 8 calculates synoptic diagram for tandem mass spectrum and the aminoacid sequence of purified polypeptide II.
From the beginning Fig. 9 calculates synoptic diagram for tandem mass spectrum and the aminoacid sequence of purified polypeptide III.
Embodiment
(1) preparation of Cornu Bubali extract:
Water intaking horn powder 500g, mix with the 5000ml pure water, heat little twice of refluxing extraction of boiling, each 8 hours, decoction finishes the back merging filtrate, filters, and filtrate is carried out lyophilize after 50 ℃ of decompression rotary evaporations are concentrated in right amount, can get the crude extract lyophilized powder, be used for separation and each component anti-oxidant activity evaluation experimental of back different components.
(2) gel filtration chromatography to crude extract separates:
The crude extract that step (1) is made mixes with pure water, fully stirs down at 4 ℃, and centrifugal 20min behind 4 ℃ of following 12000rpm collects supernatant liquor, adopts drop-burette that sample carefully is added to Sephadex G-25 post 2.0 * 100cm top.Carry out wash-out with pure water, flow velocity is 0.15ml/min, and the detection wavelength is 254nm, and automatic fraction collector is collected the elutriant sample, and every 2ml collects 1 pipe.Behind the G-25 gel filtration chromatography, obtain G1 altogether, G2, four components of G3 and G4, its chromatography synoptic diagram is seen Fig. 3.Collecting this four components, after the lyophilize, carry out cell experiment, is index with tetrazolium bromide (MTT) experiment and serum lactic dehydrogenase (LDH) release experiment, observes four components to H
2O
2Due to the provide protection of rCMEC damage, estimate its anti-oxidant activity.The result shows that G3 has the highest anti-oxidative damage activity, carries out further anion-exchange chromatography.
(3) anion exchange chromatography to separated portion separates:
The active ingredient that the separation of process step (2) obtains is earlier with an amount of 50mM PBS pH6.5 damping fluid dissolving, last sample is to DEAE Sepharose Fast Flow anion-exchange column 2.0 * 20cm, and ion exchange column is used the abundant balance of 50mM PBS pH6.5 damping fluid in advance.Behind the last sample, with the washing of 1ml/min flow velocity, after waiting peak D1 to occur penetrating, adopting linear gradient is the 50mM PBS pH6.5 buffer solution elution of 0-0.3M NaCl, two components of D2 and D3 occur with 50mM PBS pH6.5, and its chromatography synoptic diagram is seen Fig. 4.Collect this three component lyophilizes, lyophilized powder with a small amount of dissolved in distilled water after, carry out desalination by the automatic purifying HPLC of Waters2545-2489-2676 system, separator column is a SunFire C18 reversed-phase column, 19mm * 50mm; Behind the sample introduction, adopt 2% methanol-eluted fractions 2min earlier, 60% meoh eluate is collected with 60% methanol-eluted fractions 10min in the back, after the decompression rotary evaporation concentrates and removes methyl alcohol, and lyophilize; Lyophilized powder carries out cell experiment, is index with MTT experiment and LDH release experiment, observes three components to H
2O
2Due to the provide protection of rCMEC damage, estimate its anti-oxidant activity.The result shows that D2 and D3 all have good anti-oxidative damage activity, further carry out purifying by reversed-phase HPLC.
(4) reversed-phase HPLC chromatography purification:
Separate the better anti-oxidant activity component that has that the back obtains through anion exchange chromatography, pass through the further separation and purification of the automatic purifying HPLC of Waters2545-2489-2676 system again; With the linear gradient is the 2%-60% methanol-eluted fractions, wherein contains 0.1% trifluoroacetic acid (TFA), detects under 230nm; During D2 component reversed-phase HPLC chromatographic separation, collect two chromatographic peaks that retention time is about 4.6min and 7.3min, be respectively polypeptide I and II, its chromatography synoptic diagram is seen Fig. 5; During D3 component reversed-phase HPLC chromatographic separation, collect the chromatographic peak that retention time is about 5.4min, be polypeptide III, its chromatography synoptic diagram is seen Fig. 6; Collect sample according to elution peak, after concentrating under reduced pressure is removed methyl alcohol, lyophilize; Obtain 3 angle class active polypeptide lyophilized powders after the freeze-drying, be respectively I, II and III.The sequence of polypeptide I is Gln-Tyr-Asp-Gln-Gly-Val, and the sequence of polypeptide II is Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn, and the sequence of polypeptide III is Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn.Polypeptide I and II obtain from the D2 partial purification, and polypeptide III obtains from the D3 partial purification.
(5) sequential analysis of purifying active polypeptide:
In this research, we adopt molecular weight and aminoacid sequence thereof through substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS) and 3 active polypeptide of tandem mass spectrum (MALDI-TOF/TOF-MS) analysis.The last sample matrix of MALDI-TOF is selected alpha-cyano-4-hydroxycinnamic acid for use, and behind the sample and 1 μ l matrix thorough mixing with about 1 μ l, point sample is in last sample target, and is dry under the room temperature.It is 337nm that MALDI-TOF/TOF-MS analyzes optical maser wavelength, and one-level mass spectrum acceleration voltage is set to 19kV, and the final acceleration voltage of tandem mass spectrum is 29kV, and tandem mass spectrum is approximately added up by 1000 bombardments and forms.The result obtains 3 polypeptide [M+H]
+Be respectively 709.090Da, 1019.296Da and 1272.379Da; 3 amino acid sequence of polypeptide are respectively: Gln-Tyr-Asp-Gln-Gly-Val, Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn and Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn, from the beginning the tandem mass spectrum of 3 polypeptide and aminoacid sequence calculate synoptic diagram and see Fig. 7, Fig. 8 and Fig. 9.
The peptide sequence table
＜110〉full name of applicant: Nanjing University of Traditional Chinese Medicine
＜120〉denomination of invention: a class has angle class polypeptide, its separation method and the purposes of anti-oxidant activity
＜160〉number of sequence in the sequence table: 3
＜210〉the corresponding sequence identifier of sequence: 1
＜211〉sequence length: 6 amino acid
＜212〉type of sequence: PRT
＜213〉organism: Asia buffalo kind (Bubalus bubalis)
＜400〉sequence identifier:
Gln-Tyr-Asp-Gln-Gly-Val
3 6
＜210〉the corresponding sequence identifier of sequence: 2
＜211〉sequence length: 9 amino acid
＜212〉type of sequence: PRT
＜213〉organism: Asia buffalo kind (Bubalus bubalis)
＜400〉sequence identifier:
Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn
3 6 9
＜210〉the corresponding sequence identifier of sequence: 3
＜211〉sequence length: 12 amino acid
＜212〉type of sequence: PRT
＜213〉organism: Asia buffalo kind (Bubalus bubalis)
＜400〉sequence identifier:
Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn
3 6 9 12
Claims (4)
1. the polypeptide or its pharmacy acceptable salt that have formula I, II or III structure:
Gln-Tyr-Asp-Gln-Gly-Val(I)；
Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn(II)；
Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn(III)。
2. the preparation method of the polypeptide of claim 1 comprises:
A, water intaking horn powder are used the pure water heating and refluxing extraction, filter, and filtrate concentrated frozen drying gets crude extract;
B, crude extract is carried out gel filtration chromatography separate, under 254nm, detect and flow out sample, collect each component, measure anti-oxidant activity according to the sample peak of detector;
C, the active component of high anti-oxidation is further utilized DEAE Sepharose Fast Flow anionresin column separating purification, ion exchange column is earlier with 50mM phosphate buffered saline buffer pH6.5, with 1ml/min flow velocity pre-treatment 3h; Behind the last sample, ion exchange column with 1ml/min flow velocity wash-out 2h, is the phosphate buffered saline buffer 50mM of 0-0.3MNaCl with 50mM phosphate buffered saline buffer pH6.5 with the linear gradient then, the pH6.5 wash-out, and elution curve detects under 254nm; Collect each component, lyophilize according to elution peak;
D, with the lyophilized powder that c obtains, pure water is dissolved into higher concentration, carries out desalination by the automatic purifying HPLC of Waters 2545-2489-2676 system, measures anti-oxidant activity, obtains better anti-oxidant activity component D2 and D3;
E, with component D2 and D3, again by the further separation and purification of the automatic purifying HPLC of Waters 2545-2489-2676 system; With the linear gradient is the 2%-60% methanol-eluted fractions, wherein contains 0.1% trifluoroacetic acid, detects under 230nm; During D2 component reversed-phase HPLC chromatographic separation, collect two chromatographic peaks that retention time is about 4.6min and 7.3min, be respectively polypeptide I and II; During D3 component reversed-phase HPLC chromatographic separation, collect the chromatographic peak that retention time is about 5.4min, be polypeptide III; The sample that collection obtains, after concentrating under reduced pressure is removed methyl alcohol, lyophilize; Obtain polypeptide I respectively, the lyophilized powder of II and III.
3. pharmaceutical composition wherein contains polypeptide or its pharmacy acceptable salt and the pharmaceutically acceptable carrier of claim 1.
4. the polypeptide of claim 1 or its pharmacy acceptable salt are used to prepare the purposes of anti-oxidation medicine.
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| CN 201110278430 Division CN102304170B (en) | 2009-10-26 | 2009-10-26 | Horn polypeptide, and separation method and antioxidation application thereof |
| CN 201110278428 Division CN102321157B (en) | 2009-10-26 | 2009-10-26 | Hexapeptide with antioxidant activity and separation method as well as application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266530A (en) * | 2017-07-03 | 2017-10-20 | 大连理工大学 | Anti-oxidant ultrashort peptide, preparation method and applications |
| CN112098579A (en) * | 2020-09-01 | 2020-12-18 | 南京中医药大学 | Deer-derived characteristic peptide fragment and detection method thereof |
| CN117462650A (en) * | 2023-10-08 | 2024-01-30 | 南京中医药大学 | Application of polypeptide A in promoting bone regeneration or bone repair |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101485686A (en) * | 2009-02-18 | 2009-07-22 | 南京同仁堂药业有限责任公司 | Method for preparing extract of antelope's horn |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266530A (en) * | 2017-07-03 | 2017-10-20 | 大连理工大学 | Anti-oxidant ultrashort peptide, preparation method and applications |
| CN107266530B (en) * | 2017-07-03 | 2020-08-14 | 大连理工大学 | Antioxidant ultrashort peptide, preparation method and application thereof |
| CN112098579A (en) * | 2020-09-01 | 2020-12-18 | 南京中医药大学 | Deer-derived characteristic peptide fragment and detection method thereof |
| CN112098579B (en) * | 2020-09-01 | 2021-11-23 | 南京中医药大学 | Characteristic peptide segment for distinguishing deerhorn glue or deerhorn glue and detection method thereof |
| CN117462650A (en) * | 2023-10-08 | 2024-01-30 | 南京中医药大学 | Application of polypeptide A in promoting bone regeneration or bone repair |
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