CN109464434A - Application of the disodium ethylene diamine tetraacetate as treatment jellyfish dermatitis drug - Google Patents
Application of the disodium ethylene diamine tetraacetate as treatment jellyfish dermatitis drug Download PDFInfo
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- 241000242583 Scyphozoa Species 0.000 title claims abstract description 26
- 201000004624 Dermatitis Diseases 0.000 title claims abstract description 19
- 229940079593 drug Drugs 0.000 title claims abstract description 16
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 title abstract description 5
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 title abstract description 5
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 title abstract 3
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 claims abstract description 53
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 7
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract
The present invention relates to field of marine biotechnology, and in particular to a kind of application of disodium ethylene diamine tetraacetate (non-specific metal protease inhibitors) as treatment jellyfish dermatitis drug.Application of the EDETATE SODIUM as treatment jellyfish dermatitis drug.Dead and inflammatory factor mRNA the expression quantity of two kinds of cells (Human keratinocytes HaCaT and human embryonic fibroblast CCC-ESF-1) caused by medusocongestin can be effectively suppressed using disodium ethylene diamine tetraacetate by the present invention, and then the application range of EDETATE SODIUM can have been widened as the drug for being used to prepare jellyfish dermatitis.
Description
Technical field:
The present invention relates to field of marine biotechnology, and in particular to a kind of disodium ethylene diamine tetraacetate (non-specific metal
Protease inhibitors) as the application for treating jellyfish dermatitis drug.
Background technique:
Medusa Coelenterata has the ocean aquatic animal of cnidophore, and when people touches jellyfish, the toxin in cnidophore is with pole
Fast speed, which is pierced into skin, to be caused to sting.Main jellyfish of hurting sb.'s feelings has the sea of sand to bite and rosy clouds jellyfish in China.In recent years, global range
Jellyfish, which breaks out, not only causes marine ecosystems disorder, also brings serious influence to human being's production, life.Jellyfish stings
Through becoming the most common injuries from marine creature.Estimate according to U.S. National Institutes, the whole world is bitten there are about 1.5 hundred million person-times by jellyfish every year
Wound, wherein being no lack of death.The jellyfishes dermatitis symptoms such as jellyfish red and swollen erythema caused by stinging, bubble and scratchy, numb pain
It is cardinal symptom, influences routine work and life, especially sleep.Currently, the drug for female dermatitis of not harnessing the river specially both at home and abroad, greatly
It continues to use civil local method, proved recipe or the dermatitis with reference to other types to treat, effect is poor, even the exacerbation state of an illness more.
Medusocongestin is the material base that jellyfish stings, and is the basic reason of morbidity.The main active of medusocongestin
For polypeptide and albumen, there are a variety of lifes such as haemolysis, enzymatic activity, cardiac toxic, neurotoxicity, musclar toxicity, cutaneous necrosis effect
Object activity.Transcript profile combine protein science to medusocongestin component resolving find, metalloproteinases be medusocongestin it is main at
Point, conventional biochemical analysis also demonstrates medusocongestin with metal proteinase activity.Metalloprotein enzyme component in snake venom is at it
Bite caused wound pain, play an important role in the more than and intoxicating phenomenons such as fester of bleeding.Therefore, the gold in medusocongestin
Proteases may be an important factor for leading to jellyfish dermatitis.It is treated so needing to be developed according to the bioactivity of medusocongestin
The drug of jellyfish dermatitis.
Summary of the invention:
The object of the present invention is to provide a kind of EDETATE SODIUM (non-specific metal protease inhibitors) as preparation jellyfish
The application of dermatitis drug.
To achieve the above object, the invention adopts a technical scheme as
A kind of application of EDETATE SODIUM as preparation jellyfish dermatitis drug.
Application of the EDETATE SODIUM shown in the formula I after dissolved dilution as preparation jellyfish dermatitis drug；
After the sterilized water dissolution of EDETATE SODIUM shown in the formula I, then with 10mM phosphate buffer (NaH2PO4-
Na2HPO4, pH 7.4) and dilution.
EDETATE SODIUM inhibits medusocongestin to Human keratinocytes HaCaT and human embryonic fibroblast CCC-ESF-1
The experimental method of the death rate are as follows:
After EDETATE SODIUM is dissolved with aqua sterilisa, then with 10mM phosphate buffer (NaH2PO4-Na2HPO4, pH 7.4))
Dilution obtains EDETATE SODIUM solution.
Then the mixing of EDETATE SODIUM solution is added into medusocongestin, make EDETATE SODIUM and medusocongestin in mixed liquor (with
Protein refractometer) mass ratio be equal to or less than 1.49:1, mix, react stand-by after 30min at 37 DEG C.
To being covered with 1 × 105It is added 100 μ L's in 96 orifice plates of a/mL cell (cell can be HaCaT or CCC-ESF-1)
EDETATE SODIUM and medusocongestin mixed liquor, 100 μ L's is by volume the medusocongestin and cell culture medium mixed liquor of 1:1 mixing
The cell culture medium (blank) of (control) and 100 μ L, with the survival rate of mtt assay measurement cell.
EDETATE SODIUM inhibits Human keratinocytes HaCaT and human embryonic fibroblast CCC- caused by medusocongestin
The experimental method of ESF-1 inflammatory factor mrna expression amount are as follows:
After EDETATE SODIUM is dissolved with aqua sterilisa, then with 10mM phosphate buffer (NaH2PO4-Na2HPO4, pH 7.4))
Dilution obtains EDETATE SODIUM solution.
Then the mixing of EDETATE SODIUM solution is added into medusocongestin, make EDETATE SODIUM and medusocongestin in mixed liquor (with
Protein refractometer) mass ratio be equal to or less than 1.49:1, mix, react stand-by after 30min at 37 DEG C.
To being covered with 1 × 1051mL EDTA is added in the culture dish of a/mL cell (cell can be HaCaT or CCC-ESF-1)
The mixed liquor of disodium and medusocongestin, 1mL are that the medusocongestin of 1:1 mixing (compares) with cell culture medium mixed liquor by volume
And 1mL cell culture medium (blank), after reacting for 24 hours in 37 DEG C of incubators, extracts the RNA of cell and reverse transcription is at cDNA, according to
QRT-PCR method (Pfaffl, M.W. (2001) .A new mathematical model for relative
quantification in real-time RT-PCR.Nucleic Acids Research,29(9),45e–
45.https://doi.org/10.1093/nar/29.9.e45) measurement cell in inflammatory factor mRNA expression quantity.
Advantages of the present invention:
Two kinds of cell (Human keratinocytes caused by medusocongestin can be effectively suppressed using EDETATE SODIUM in the present invention
HaCaT and human embryonic fibroblast CCC-ESF-1) death and inflammatory factor mRNA expression quantity, and then can be as
It is used to prepare the drug of jellyfish dermatitis, has widened the application range of EDETATE SODIUM.
Detailed description of the invention:
Fig. 1 compares condition in different quality for EDETATE SODIUM provided in an embodiment of the present invention and medusocongestin (with protein refractometer)
Under, influence diagram of the EDETATE SODIUM to human embryonic fibroblast (CCC-ESF-1) survival rate.
Fig. 2 compares condition in different quality for EDETATE SODIUM provided in an embodiment of the present invention and medusocongestin (with protein refractometer)
Under, influence diagram of the EDTA to Human keratinocytes (HaCaT) survival rate.
Fig. 3 is that EDETATE SODIUM provided in an embodiment of the present invention inhibits medusocongestin to human embryonic fibroblast (CCC-
ESF-1) the expression figure of inflammatory factor mRNA.
Fig. 4 is that the EDETATE SODIUM provided in the embodiment of the present invention inhibits medusocongestin to Human keratinocytes (HaCaT)
The expression figure of inflammatory factor mRNA.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Following solution used in the examples are respectively as follows:
The preparation method of EDETATE SODIUM solution are as follows: take 372.24mg EDETATE SODIUM, dissolved with 100mL aqua sterilisa, is made
The EDETATE SODIUM solution of 10mM；Then by the EDETATE SODIUM solution of 10mM 10mM phosphate buffer (NaH2PO4-Na2HPO4,
PH 7.4) dilution, respectively compound concentration be 10 μM, 50 μM, 100 μM, 200 μM of EDETATE SODIUM solution.
The preparation method of the medusocongestin solution of 50 μ g/mL are as follows: the jellyfish tentacle tissue of frost takes out from -80 DEG C of refrigerators
Afterwards, defrosting, self-dissolving 2-4 days in 4 DEG C of refrigerators are placed in.Period is to accelerate the dissolution of jellyfish tentacle, every other day carefully topples over upper layer
Fresh seawater is added after seawater, ecthoaeum bladder cell is accelerated to fall off from tentacle.After jellyfish organizes complete self-dissolving, slowly outwell
Layer is collected lower layer with precipitating from solution, is then filtered respectively with the sub-sieve of 60 mesh and 100 mesh from solution.Collect filtering
Liquid, 3000g are centrifuged 15min, collect lower sediment, then are washed 2-3 times and be centrifuged with clean seawater to get more pure ecthoaeum is arrived
Capsule.Ecthoaeum bladder cell is placed in -80 DEG C of refrigerators and is saved backup.The 20mM phosphate buffer that will be pre-chilled in nematocyst and 4 DEG C
(NaH2PO4-Na2HPO4, pH 7.4) and it is extracted according to the ratio of 1:3 (w/v).4 DEG C after the completion of broken, 15000g freeze from
Heart 30min, supernatant are medusocongestin solution.The measurement of medusocongestin uses Forint phenol method (Zhang Lei, Liu Yu, Jiang Dahe, poplar
Bright garden, the low-priced Biochemistry Experiment guidance of the Cao Zhi Wuhan [M]: publishing house, Wuhan University, 2011.95~98.), pure with ox blood
Albumen (BSA) is standard.Again by toxin with the 20mM phosphate buffer (NaH being pre-chilled in 4 DEG C2PO4-Na2HPO4, pH 7.4)
It is diluted to 50 μ g/mL.
The preparation of Human keratinocytes (HaCaT) culture medium: 55.6mL is added into 500mL MEM/EBSS culture medium
Fetal calf serum and 5.6mL it is dual anti-.
The preparation of Human embryo fiber formation cell (CCC-ESF-1) culture medium: add into 500mL DMEM high glucose medium
Enter 125mL fetal calf serum and 6.3mL it is dual anti-.
The preparation of cell effect terminate liquid: after weighing SDS 20g 200mL distilled water dissolution, 0.176mL HCl is added.
Embodiment 1:
Above-mentioned 10 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1,37
After reacting 30min at DEG C, as mixed liquor A, for use；
To being covered with 100 μ L 1 × 105It is added in 96 orifice plates of a/mL human embryonic fibroblast (CCC-ESF-1) cell
The mixed liquor A of 100 μ L, 100 μ L by volume for medusocongestin and the cell culture medium mixed liquor (control) of 1:1 mixing and
The cell culture medium (blank) of 100 μ L after reacting for 24 hours, removes supernatant, and after MTT reaction 4h is added, terminate liquid is added, interior for 24 hours
A is measured with microplate reader490nm, reaction setting three is parallel, calculates cell survival rate according to the following formula.
Experimental result is shown (referring to Fig. 1), when the mass ratio of EDETATE SODIUM and medusocongestin (with protein refractometer) is 0.074:1
When, the survival rate of cell is respectively 64.23%, 65.12%, 63.99%, and average value is 64.45 ± 0.59%, is higher than control group
Cell survival rate 55.32% shows that EDETATE SODIUM is able to suppress human embryonic fibroblast (CCC-ESF- caused by medusocongestin
1) death.
Embodiment 2:
50 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1, at 37 DEG C
After reacting 30min, as mixed liquid B, for use；
To being covered with 100 μ L 1 × 105It is added in 96 orifice plates of a/mL human embryonic fibroblast (CCC-ESF-1) cell
The mixed liquid B of 100 μ L, 100 μ L by volume for medusocongestin and the cell culture medium mixed liquor (control) of 1:1 mixing and
The cell culture medium (blank) of 100 μ L after reacting for 24 hours, removes supernatant, and after MTT reaction 4h is added, terminate liquid is added, interior for 24 hours
A is measured with microplate reader490nm, reaction setting three is parallel, calculates cell survival rate according to the following formula.
Experimental result is shown (referring to Fig. 1), when the mass ratio of EDETATE SODIUM and medusocongestin (with protein refractometer) is 0.37:1
When, cell survival rate is respectively 75.41%, 75.78%, 68.73%, and average value is 73.31 ± 3.97%, and it is thin to be higher than control group
Born of the same parents' survival rate 55.32% shows that EDETATE SODIUM is able to suppress human embryonic fibroblast caused by medusocongestin (CCC-ESF-1)
Death.
Embodiment 3:
50 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1, at 37 DEG C
After reacting 30min, as mixed liquid B, for use；
To being covered with 100 μ L 1 × 105It is added 100 μ L's in 96 orifice plates of a/mL Human keratinocytes (HaCaT) cell
Mixed liquid B, 100 μ L are by volume the thin of medusocongestin and the cell culture medium mixed liquor (control) of 1:1 mixing and 100 μ L
Born of the same parents' culture medium (blank) after reaction for 24 hours, removes supernatant, after MTT reaction 4h is added, terminate liquid is added, for 24 hours interior microplate reader
Measure A490nm, reaction setting three is parallel, calculates cell survival rate according to the following formula.
Experimental result is shown (referring to fig. 2), when the mass ratio of EDETATE SODIUM and medusocongestin (with protein refractometer) is 0.37:1
When, the survival rate of cell is respectively 56.81%, 53.52%, 64.46%, and average value is 58.26 ± 5.61%, is higher than control group
Cell survival rate 50.59% shows that EDETATE SODIUM is able to suppress the dead of Human keratinocytes caused by medusocongestin (HaCaT)
It dies.
Embodiment 4:
100 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1, at 37 DEG C
After lower reaction 30min, as mixed liquor C, for use；
To being covered with 100 μ L 1 × 105It is added 100 μ L's in 96 orifice plates of a/mL Human keratinocytes (HaCaT) cell
Mixed liquor C, 100 μ L are by volume the thin of medusocongestin and the cell culture medium mixed liquor (control) of 1:1 mixing and 100 μ L
Born of the same parents' culture medium (blank) after reaction for 24 hours, removes supernatant, after MTT reaction 4h is added, terminate liquid is added, for 24 hours interior microplate reader
Measure A490nm, reaction setting three is parallel, calculates cell survival rate according to the following formula.
Experimental result is shown (referring to fig. 2), when the mass ratio of EDETATE SODIUM and medusocongestin (with protein refractometer) is 0.74:1
When, the survival rate of cell is respectively 59.69%, 60.56%, 61.31%, and average value is 60.52 ± 0.81%, is higher than control group
Cell survival rate 50.59% shows that EDETATE SODIUM is able to suppress the dead of Human keratinocytes caused by medusocongestin (HaCaT)
It dies.
Embodiment 5:
200 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1, at 37 DEG C
After lower reaction 30min, as mixed liquor D, for use；
To being covered with 100 μ L 1 × 105It is added 100 μ L's in 96 orifice plates of a/mL Human keratinocytes (HaCaT) cell
The medusocongestin and Human keratinocytes (HaCaT) culture medium mixed liquor of mixed liquor D, 100 μ L mixed by volume for 1:1
Human keratinocytes (HaCaT) culture medium (blank) of (control) and 100 μ L after reaction for 24 hours, remove supernatant, MTT are added
After reacting 4h, terminate liquid is added, it is interior for 24 hours to measure A with microplate reader490nm, reaction setting three is parallel, calculates according to the following formula thin
Born of the same parents' survival rate.
Experimental result is shown (referring to fig. 2), when the mass ratio of EDETATE SODIUM and medusocongestin (with protein refractometer) is 1.49:1
When, the survival rate of cell is respectively 60.13%, 60.66%, 61.31%, and average value is 60.70 ± 0.59%, is higher than control group
Cell survival rate 50.59% shows that EDETATE SODIUM is able to suppress the dead of Human keratinocytes caused by medusocongestin (HaCaT)
It dies.
Embodiment 6:
50 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1, at 37 DEG C
After reacting 30min, as mixed liquid B, for use；
To being covered with 1mL 1 × 1051mL is added in the Tissue Culture Dish of a/mL human embryonic fibroblast (CCC-ESF-1)
Mixed liquid B, 1mL by volume for 1:1 mixing medusocongestin and the cell of cell culture medium mixed liquor (control) and 1mL
Culture medium (blank), after reaction for 24 hours, removes supernatant, measures human embryonic fibroblast (CCC-ESF- according to qRT-PCR method
1) mrna expression amount of inflammatory factor IL-6 and TNF-α in, reaction setting three are parallel.
As shown in figure 3, when 50 μM EDETATE SODIUM solution and 50 μ g/mL medusocongestin isometric hybrid reaction after, energy
Enough reduce the expression quantity of inflammatory factor mRNA in human embryonic fibroblast (CCC-ESF-1).
Embodiment 7:
50 μM of EDETATE SODIUM solution and the medusocongestin of 50 μ g/mL is taken to be uniformly mixed according to volume ratio 1:1, at 37 DEG C
After reacting 30min, as mixed liquid B, for use；
To being covered with 1mL 1 × 105The mixing of 1mL is added in the Tissue Culture Dish of a/mL Human keratinocytes (HaCaT)
Liquid B, 1mL's is by volume the medusocongestin of 1:1 mixing and the cell culture medium of cell culture medium mixed liquor (control) and 1mL
(blank), after reaction for 24 hours, removes supernatant, measures inflammatory factor in Human keratinocytes (HaCaT) according to qRT-PCR method
The mrna expression amount of IL-6 and TNF-α, reaction setting three are parallel.
As shown in figure 4, when 50 μM EDETATE SODIUM solution and 50 μ g/mL medusocongestin isometric hybrid reaction after, energy
Enough reduce the expression quantity of inflammatory factor mRNA in Human keratinocytes (HaCaT).
Claims (3)
1. a kind of application of EDETATE SODIUM as treatment jellyfish dermatitis drug.
2. application of the EDETATE SODIUM according to claim 1 as treatment jellyfish dermatitis drug, which is characterized in that the formula I
Application of the shown EDETATE SODIUM after dissolved dilution as preparation jellyfish dermatitis drug；
3. application of the EDETATE SODIUM as described in claim 2 as preparation jellyfish dermatitis drug, which is characterized in that the formula I
After the sterilized water dissolution of shown EDETATE SODIUM, then with 10mM phosphate buffer (NaH2PO4-Na2HPO4, pH 7.4) and dilution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5788952A (en) * | 1993-02-11 | 1998-08-04 | Beiersdorf Ag | Cosmetic and dermatological photoprotective formulations containing inorganic micropigments |
| CN102002094A (en) * | 2010-10-10 | 2011-04-06 | 中国科学院海洋研究所 | Method for protecting bioactivity of cyanea nozakii toxin |
-
2018
- 2018-11-08 CN CN201811324640.2A patent/CN109464434A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5788952A (en) * | 1993-02-11 | 1998-08-04 | Beiersdorf Ag | Cosmetic and dermatological photoprotective formulations containing inorganic micropigments |
| CN102002094A (en) * | 2010-10-10 | 2011-04-06 | 中国科学院海洋研究所 | Method for protecting bioactivity of cyanea nozakii toxin |
Non-Patent Citations (4)
| Title |
|---|
| CHANGKEUN KANG等: "Protective Effect of Tetracycline against Dermal Toxicity Induced by Jellyfish Venom", 《PLOS ONE》 * |
| E. H. BAXTER等: "SEA WASP (CHIRONEX FLECKERI) VENOM: LETHAL, HAEMOLYTIC AND DERMONECROTIC PROPERTIES", 《TOXLCON》 * |
| RENEE GENDRON等: "Inhibition of the Activities of Matrix Metalloproteinases 2, 8,and 9 by Chlorhexidine", 《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》 * |
| 李荣锋: "水母毒素的分离纯化及蛋白组学研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
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