CN111569044A - Composition containing porphyridium polypeptide and preparation method thereof - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A—HUMAN NECESSITIES
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- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
The invention discloses a composition containing porphyridium polypeptide and a preparation method thereof, wherein the composition comprises the following effective components in parts by weight: 0.1-0.9 parts of porphyridium polypeptide; 99.1-99.9 parts of matrix; the preparation method comprises the following steps: the acquisition of porphyridium polypeptide comprises the following steps: repeatedly freezing and thawing porphyridium, then carrying out ultrasonic crushing, centrifuging the obtained algae liquid, and taking supernatant; separating by vertical electrophoresis to obtain polypeptide with Kda below 5, i.e. porphyridium polypeptide; or homogenizing porphyridium, sterilizing, filtering, centrifuging, collecting supernatant, ultrafiltering with ultrafiltration membrane system with 0.001-0.1 μm microporous membrane to obtain porphyridium polypeptide with molecular weight cutoff of 1 KDa; mixing: mixing porphyridium polypeptide and a matrix according to parts by weight; obtaining a composition comprising porphyridium polypeptides; the composition can promote wound healing, reduce wound opening time, and promote rehabilitation after body surface injury.
Description
Technical Field
The invention relates to a composition containing porphyridium polypeptide and a preparation method thereof, belonging to the technical field of biological pharmaceutical preparations.
Background
Porphyridium (Porphyridium) as a relatively primitive unicellular alga of Rhodophyta can produce a plurality of bioactive substances such as phycobiliprotein, polyunsaturated fatty acid and exopolysaccharide substances, and is mainly used as a bait for juvenile shrimps at present, but research shows that the Porphyridium has high medicinal value at present. In recent years, porphyridium preparation has special effect in preventing and treating cardiovascular and cerebrovascular diseases and resisting cancer. Research on porphyridium at the present stage mostly focuses on improving immunity, resisting cancer and resisting thrombus, but medicinal development on porphyridium is not carried out yet. With the development of molecular biology and separation and purification means, the active substances in porphyridium are to be further developed.
Disclosure of Invention
In order to overcome the defects of the prior art, the first object of the invention is to provide a composition containing porphyridium polypeptide, wherein the composition can promote wound healing, reduce wound opening time and help body surface recovery after damage.
The second purpose of the invention is to provide a preparation method of the composition containing the porphyridium polypeptide, the preparation method is used for separating specific polypeptide from the porphyridium, and the preparation efficiency and the repeatability are high.
The first purpose of the invention can be achieved by adopting the following technical scheme: a composition containing porphyridium polypeptide comprises the following effective components in parts by weight:
0.1-0.9 parts of porphyridium polypeptide;
99.1-99.9 parts of matrix.
Further, the porphyridium polypeptide includes at least one of a 6 peptide and a 17 peptide; the sequence of the 6 peptide is shown as SEQ ID NO.1, and the sequence of the 17 peptide is shown as SEQ ID NO. 2.
Further, porphyridium polypeptides include 6 and 17 peptides; the mass ratio of the 6 peptide to the 17 peptide is 1 (0.1-10).
Further, the mass ratio of the 6 peptide to the 17 peptide was 1: 4.
Further, the composition comprises the following effective components in parts by weight:
further, the composition is a spreadable composition.
The second purpose of the invention can be achieved by adopting the following technical scheme: a method of making a composition comprising porphyridium polypeptides, comprising:
the acquisition of porphyridium polypeptide comprises the following steps: repeatedly freezing and thawing porphyridium, then carrying out ultrasonic crushing, centrifuging the obtained algae liquid, and taking supernatant; separating by vertical electrophoresis to obtain polypeptide below 5KDa, i.e. porphyridium polypeptide;
or homogenizing porphyridium, sterilizing, filtering, centrifuging, collecting supernatant, ultrafiltering with ultrafiltration membrane system with 0.001-0.1 μm microporous membrane to obtain porphyridium polypeptide with molecular weight cutoff of 1 KDa;
mixing: the porphyridium polypeptide and the matrix are mixed according to the following weight portions: 0.1-0.9 part of porphyridium polypeptide and 99.1-99.9 parts of matrix are mixed; obtaining the composition containing the porphyridium polypeptide.
Further, in the step of obtaining porphyridium polypeptide, separation is performed by using a trihydroxymethyl glycine gel vertical electrophoresis.
Further, in the step of obtaining the porphyridium polypeptide, the operating pressure difference of ultrafiltration is 0.1-0.5 MPa.
In the formula, the porphyridium polypeptide can repair body surface damage in a targeted manner, has no protein immunogenicity, avoids anaphylactic reaction of people with allergic constitution, can cause severe damage to skin soft tissue due to the fact that the body surface damage is damage caused by physical, biological, chemical and other factors and acts on the skin soft tissue of a human body, and the damaged skin can generate open wound according to the body surface to induce bacterial infection to cause septicemia.
Compared with the prior art, the invention has the beneficial effects that:
1. the composition containing the porphyridium polypeptide can promote wound healing, reduce the wound opening time, help the recovery after the body surface is damaged, and develop a new direction for the medical use of the porphyridium;
2. the preparation method of the composition containing the porphyridium polypeptide separates specific polypeptide from the porphyridium, and has high preparation efficiency and good repeatability.
Drawings
FIG. 1 is a graph of the wound healing rate of example 4;
FIG. 2 is a bar graph of scab removal time for example 4;
FIG. 3 is a bar graph of hydroxyproline content for example 4.
Detailed Description
The invention will be further described with reference to the accompanying drawings and the detailed description below:
the specific embodiment provides a composition containing porphyridium polypeptide, which comprises the following effective components in parts by weight:
wherein the porphyridium polypeptide comprises at least one of 6 peptide and 17 peptide; the sequence of the 6 peptide is shown as SEQ ID NO.1, and the sequence of the 17 peptide is shown as SEQ ID NO. 2; the mass ratio of the 6 peptide to the 17 peptide is 1 (0.1-10).
The preparation method of the composition containing the porphyridium polypeptide comprises the following steps:
the acquisition of porphyridium polypeptide comprises the following steps: repeatedly freezing and thawing porphyridium, then carrying out ultrasonic crushing, centrifuging the obtained algae liquid, and taking supernatant; separating polypeptide below 5KDa by vertical electrophoresis with trihydroxymethyl glycine (Tricine) gel to obtain porphyridium polypeptide;
or homogenizing porphyridium, sterilizing, filtering, centrifuging, collecting supernatant, ultrafiltering with ultrafiltration membrane system with microporous membrane of 0.001-0.1 μm, with cut-off molecular weight of 1KDa and operation pressure difference of 0.1-0.5MPa to obtain porphyridium polypeptide;
mixing: mixing porphyridium polypeptide with a matrix; obtaining the composition containing the porphyridium polypeptide.
Example 1:
preparation of porphyridium polypeptide: adding sterile distilled water into porphyridium, repeatedly freezing and thawing (overnight at-20 deg.C and repeated at room temperature for 3 times), then ultrasonically crushing for 30min, stopping for 5min, and repeating for 5 times; centrifuging the obtained algae liquid and taking supernatant; separating with trihydroxymethyl glycine (Tricine) gel vertical electrophoresis, and collecting polypeptide band below 5KDa as porphyridium polypeptide; and cutting a target band, and detecting the cut target band, and analyzing by liquid chromatography-mass spectrometry to obtain two polypeptides: 6 peptide (720.87Da), the sequence IFNLSK is shown as SEQ ID NO.1, 17 peptide (1862.18Da), and the sequence FTRPREVAAALPPPSPR is shown as SEQ ID NO. 2.
Example 2:
preparation of porphyridium polypeptide: adding 1-6 times of sterile water into porphyridium, homogenizing, sterilizing, filtering, centrifuging, collecting supernatant, ultrafiltering with an ultrafiltration membrane system with 0.001-0.1 μm microporous membrane, wherein the molecular weight cutoff is 1KDa, the operating pressure difference is 0.1-0.5MPa, and the two polypeptides are obtained by liquid chromatography-mass spectrometry: 6 peptide (720.87Da), the sequence IFNLSK is shown as SEQ ID NO.1, 17 peptide (1862.18Da), and the sequence FTRPREVAAALPPPSPR is shown as SEQ ID NO. 2.
Example 3:
preparation of the composition: the porphyridium polypeptide obtained in example 1 was mixed with the matrix in the parts by weight in table 1:
TABLE 1 compositions containing porphyridium polypeptides
Obtaining the composition containing the porphyridium polypeptide.
Example 4:
the composition obtained in example 3 was tested:
1) after SPF-level KM mice with the weight of 21.0 +/-1.8 g are selected and bred for one week, the mice are randomly divided into 3 groups, namely a control group, a porphyridium polypeptide group and a positive drug group. The mice are anesthetized with 350mg/kg chloral hydrate, after the back is unhaired and disinfected, the whole skin layer is cut on the back of the mice by using a circular medical puncher with the diameter of 8 mm to form a whole skin layer skin wound animal model, and the mice are raised in cages.
Porphyridium polypeptide group: preparing the mixture into paste according to the mass ratio of 2:1 of the mixture of the embodiment 3 and water, and smearing the paste on a wound until the paste is full;
positive drug group: the Yunnan white drug powder is used as a positive control drug and is also smeared on wounds;
control group: no administration was given.
Taking a fixed-height photograph while dosing once every day, analyzing and calculating the wound healing rate by using Image J software, and calculating a formula: rate of wound healing (S)0－SN)/S0In the formula S0Represents the wound surface area of 0 day, SNRepresents the wound area of N days, unit: square millimeter, where N is 0,1,3, 4, …, 14. The healing of the wound surfaces in each group is shown in fig. 1: the wound area of each group of mice gradually decreased with the time after injury prolonged, and soft scabs covered the wounds 1 day after model creation. The wound healing rate of the porphyridium polypeptide group is obviously higher than that of a control group from 1 day to 14 days after the modeling, the wound healing rate of the positive drug group starting from 2 days after the modeling is obviously higher than that of the control group, and the porphyridium polypeptide group has no obvious difference compared with the positive drug group from 1 to 7 days. But the healing rate of the porphyridium polypeptide group is higher than that of the positive medicament group at the 8 th day, the healing rate of the wound surfaces of each group at the 8 th day is that the porphyridium polypeptide group: (75.07 ± 3.53)%, positive drug group: (61.32. + -. 3.01)% and pairAccording to the group: (42.35 ± 2.08)%; the healing rate of wound surfaces of each group at day 14, porphyridium peptide group (95.70 +/-0.80)%, positive drug group: (88.77 ± 4.50)% and control: (65.68. + -. 3.52)%.
FIG. 2 shows the scab removal times for each group, respectively: porphyridium polypeptide group: day (13.2 ± 0.6), positive drug group: day (14.8 ± 0.7), control group: (18.0. + -. 1.1) days. The porphyridium peptide group and the positive drug group have significantly reduced scab removal time compared with the control group.
2) The effect of the composition containing porphyridium polypeptides on the hydroxyproline content in wound tissue was examined, as shown in fig. 3. The hydroxyproline content in the wound surface tissues of each group tends to increase along with the increase of time. On the 7 th day, the contents of wound hydroxyproline in the control group, the porphyridium peptide group and the positive medicament group are respectively (4025 +/-238) mu g/mg, (5018 +/-281) mu g/mg and (7862 +/-312) mu g/mg, and the contents of the porphyridium peptide group and the positive medicament group are obviously higher than those of the control group. On the 14 th day, the hydroxyproline content of the control group, the porphyridium peptide group and the positive drug group is (6873 +/-389) mu g/mg, (8341 +/-428) mu g/mg and (8512 +/-486) mu g/mg respectively, and the porphyridium peptide group and the positive drug group are obviously higher than the control group, which shows that the porphyridium peptide and the positive control drug Yunnan Baiyao have similar effects and can promote the deposition of collagen on wound surfaces.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
<110> ocean medicine institute of Zhanjiang province of Guangdong
<120> a composition containing porphyridium polypeptide and preparation method thereof
<130>2020
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>6
<212>PRT
<213> Porphyridium (Porphyridium)
<400>1
Ile Phe Asn Leu Ser Lys
1 5
<210>2
<211>17
<212>PRT
<213> Porphyridium (Porphyridium)
<400>2
Phe Thr Arg Pro Arg Glu Val Ala Ala Ala Leu Pro Pro Pro Ser Pro
1 5 10 15
Arg
Claims (9)
1. The composition containing porphyridium polypeptide is characterized by comprising the following effective components in parts by weight:
0.1-0.9 parts of porphyridium polypeptide;
99.1-99.9 parts of matrix.
2. The porphyridium-polypeptide-containing composition of claim 1, wherein the porphyridium polypeptide comprises at least one of a 6 peptide and a 17 peptide; the sequence of the 6 peptide is shown as SEQ ID NO.1, and the sequence of the 17 peptide is shown as SEQ ID NO. 2.
3. The composition of claim 1, wherein the porphyridium polypeptide comprises 6 and 17 peptides; the mass ratio of the 6 peptide to the 17 peptide is 1 (0.1-10).
4. The porphyridium-polypeptide-containing composition of claim 3, wherein the mass ratio of 6 peptides to 17 peptides is 1: 4.
5. The porphyridium-polypeptide-containing composition of claim 1, wherein the composition comprises the following active ingredients in parts by weight:
6. the porphyridium-polypeptide-containing composition of claim 1, wherein the composition is a spread composition.
7. A method for preparing a composition comprising porphyridium polypeptides, comprising:
the acquisition of porphyridium polypeptide comprises the following steps: repeatedly freezing and thawing porphyridium, then carrying out ultrasonic crushing, centrifuging the obtained algae liquid, and taking supernatant; separating by vertical electrophoresis to obtain polypeptide with Kda below 5, i.e. porphyridium polypeptide;
or homogenizing porphyridium, sterilizing, filtering, centrifuging, collecting supernatant, ultrafiltering with ultrafiltration membrane system with 0.001-0.1 μm microporous membrane to obtain porphyridium polypeptide with molecular weight cutoff of 1 KDa;
mixing: the porphyridium polypeptide and the matrix are mixed according to the following weight portions: 0.1-0.9 part of porphyridium polypeptide and 99.1-99.9 parts of matrix are mixed; obtaining the composition containing the porphyridium polypeptide.
8. The method of claim 7, wherein said porphyridium polypeptide is obtained by separation using vertical electrophoresis on trimethylol glycine gel.
9. The method of claim 7, wherein the step of obtaining the porphyridium polypeptide comprises an operating pressure differential of ultrafiltration of 0.1 to 0.5 MPa.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114225100A (en) * | 2021-12-22 | 2022-03-25 | 浙江清荣生物科技发展有限公司 | Porphyridium-based liquid dressing and preparation method thereof |
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| US20090069213A1 (en) * | 2007-07-18 | 2009-03-12 | Solazyme, Inc. | Compositions for Improving the Health and Appearance of Skin |
| CN102961733A (en) * | 2012-11-16 | 2013-03-13 | 福建省水产研究所 | Application of seaweed polypeptide in preparing health-care product for reducing blood fat and blood sugar as well as beverage containing polypeptide for reducing blood fat and blood sugar |
| CN108785144A (en) * | 2018-09-04 | 2018-11-13 | 广东医科大学 | A kind of skin matrix and its application |
| CN110041413A (en) * | 2019-03-04 | 2019-07-23 | 齐鲁工业大学 | A kind of method of atomizing freeze drying machine assisted extraction phycoerythrin |
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2020
- 2020-05-31 CN CN202010481473.3A patent/CN111569044B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090069213A1 (en) * | 2007-07-18 | 2009-03-12 | Solazyme, Inc. | Compositions for Improving the Health and Appearance of Skin |
| CN102961733A (en) * | 2012-11-16 | 2013-03-13 | 福建省水产研究所 | Application of seaweed polypeptide in preparing health-care product for reducing blood fat and blood sugar as well as beverage containing polypeptide for reducing blood fat and blood sugar |
| CN108785144A (en) * | 2018-09-04 | 2018-11-13 | 广东医科大学 | A kind of skin matrix and its application |
| CN110041413A (en) * | 2019-03-04 | 2019-07-23 | 齐鲁工业大学 | A kind of method of atomizing freeze drying machine assisted extraction phycoerythrin |
Non-Patent Citations (3)
| Title |
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| 孙利芹等: "紫球藻细胞破碎方法研究", 《海洋通报》 * |
| 郑志鸿等: "方格星虫酶解物对小鼠皮肤创伤修复的作用", 《广东海洋大学学报》 * |
| 陈晓清等: "紫球藻抗菌蛋白的纯化及其部分性质研究", 《湖北农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114225100A (en) * | 2021-12-22 | 2022-03-25 | 浙江清荣生物科技发展有限公司 | Porphyridium-based liquid dressing and preparation method thereof |
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