CN109966304A - Garden burnet active constituent is preparing the purposes in wound healing drug - Google Patents
Garden burnet active constituent is preparing the purposes in wound healing drug Download PDFInfo
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- CN109966304A CN109966304A CN201910322823.9A CN201910322823A CN109966304A CN 109966304 A CN109966304 A CN 109966304A CN 201910322823 A CN201910322823 A CN 201910322823A CN 109966304 A CN109966304 A CN 109966304A
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- wound healing
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- xyloside
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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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- 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
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
Abstract
The invention belongs to field of medicaments, specifically disclose garden burnet active constituent 3, and 8- dimethylellagic acid -2- xyloside (3,8--dimethyl ellagic acid -2- xylycoside, DEAX) is preparing the purposes in wound healing drug.3,8- dimethylellagic acid -2- xyloside is specifically disclosed in the present invention, cell secretion TGF-β 1 and I-type collagen are promoted by the proliferation and migration of promotion NIH3T3 cell, while cell mRNA being promoted to be expressed as exposure basis, thus wound healing.
Description
Technical field
The invention belongs to field of medicaments, and in particular to garden burnet active constituent 3,8- dimethylellagic acid -2- xyloside are being made
Purposes in standby wound healing drug.
Background technique
Garden burnet (Sanguisorba officinalis L.) is rosaceae garden burnet platymiscium, has cooling blood and hemostasis, removing toxic substances
The effect of sore, it is conventionally used to treatment hematochezia, scald carbuncle sore tumefacting virus, modern pharmacology research shows garden burnet also and have anti-
Bacterium, it is anti-inflammatory, promote hematopoiesis and it is anti-oxidant the effects of.Treatment scald, pressure sore are used clinically for by the preparation of raw material of garden burnet
Etc. (referring to following non-patent literature 1-2) significant in efficacy.Clinical research discovery Rhizoma sanguisorbae preparation can be obviously promoted postoperative patient granulation
The hyperplasia of tissue (referring to following non-patent literatures 3).
Wound healing is a complicated process, is related to various kinds of cell, cell factor and extracellular matrix interaction
Dynamic process mainly includes inflammatory phase, proliferation period and reconstruct phase.Several seconds wound repair then starts to start after skin injury.However,
Blood platelet, horn cell, immunocyte, microvascular endothelial cells and fibroblast play important in processes of wound repair
Effect.As the important member of wound healing, the growth of fibroblastic proliferation and migration energy promotion granulation tissue.It is some thin
Intracellular cytokine such as transforming growth factor (TGF-β 1), basic fibroblast growth factor (bFGF) etc. can be obviously promoted into fiber finer
The proliferation and migration of born of the same parents promotes granulation tissue growth, promotes wound healing.In the wound healing later period, fibroblast is changed into
Myofibroblast, it can be synthesized and deposition of cells epimatrix, especially collagen (referring to following non-patent literatures 4).It can
See, the synthesis of fibroblastic proliferation and collagen plays an important role in wound healing process.TGF-β 1 and collagen egg
It is white to play an important role in wound healing.In wound healing process, TGF-β 1 is by raising fibroblast and stimulation collagen
Albumen, proteoglycans, fibronectin and others ECM synthesis promote progression of fibrosis.
There has been no the active constituents that document report garden burnet promotees wound healing at present.Also have no that garden burnet active constituent can pass through
To promote fibroblastic proliferation and migration, promote cell secretion TGF-β 1 and I-type collagen, while promoting cell mRNA
It is expressed as the relevant report of two wound healing of exposure basis.
Non-patent literature list
[1] effect observation [J] All-round nursing of Chinese medicine garden burnet oil Local out dressing treatment II phase pressure sore, 2011 (13):
1133-1134.
[2] Yang Fan, Wang Teng, Qin Yiming, the observation of curative effect for waiting garden burnet pulvis rhei the to scald rat light degree Ⅱ Asia-Pacific [J] pass
System medicine, 2016,12 (18): 10-12.
[3] Lu Dan, Cao Bo, Li Yunfei wait stilbene elm cream sliver to be applied with the clinical research for promoting more analgesic to anal fistula postoperative wound
[J] the south of Guizhou Province nationality cures special journal, 2016,29 (2): 106-108,111.
[4] along clear, Yu Ye, the interaction of Zhang Yiming fibroblast and extracellular matrix in skin ultrastructure
And its regulation [J] China wound magazine, 2000,16 (6): 345-346.
Summary of the invention
The purpose of the present invention is to provide garden burnet active constituent 3,8- dimethylellagic acid -2- xyloside (3,8--
Dimethyl ellagic acid-2-xylycoside, DEAX) preparing the application in wound healing drug.
Specifically include also applying for following aspect:
3,8- dimethylellagic acid -2- xyloside is preparing the purposes in wound healing drug.
Preferably, the application concentration of 3,8- dimethylellagic acid -2- xyloside at least 5 μ g/ml or preferred concentration be at least
10 μ g/ml, more preferable concentration at least 20 μ g/ml, preferred concentration at least 40 μ g/ml.
Further, the drug of the wound healing is by promoting fibroblastic migration generation effect.
Further, the drug of the wound healing is by promoting fibroblastic proliferation generation effect.
Further, the drug of the wound healing is by promoting 1 generation of fibroblasts to secrete TGF-β effect.
Further, the drug of the wound healing is by promoting fibroblasts to secrete collagen generation effect.
Further, the table that the drug of the wound healing passes through I-type collagen mRNA in promotion fibroblast
It is acted on up to generation.
It is demonstrated experimentally that 3, the 8 dimethylellagic acid -2- xyloside dosage of 40 μ g/ml, 20 μ g/ml can remarkably promote mouse
The proliferation of embryo fibroblast NIH3T3, and there are the correlations of regular hour and dosage.Compared with testing blank group, 3,
8 dimethylellagic acid -2- xylosides can also be obviously promoted the migration of NIH3T3 cell.3,8- dimethylellagic acid -2- wood
Glucosides can promote NIH3T3 cell secretion TGF-β 1 and collagen simultaneously, and there are certain dosage correlation, collagens
It is reconstructed tissue and the regenerated two kinds of important adjusters of granulation tissue with ECM protein, they play weight in wound
It acts on, TGF-β 1 is to adjust the important cytokine that fibrin is formed in wound healing process.In addition, 3,8- dimethyl tans
After spending acid -2- xyloside to intervene NIH3T3 cell 10h, TGF-β 1mRNA expression is significantly increased, and I-type collagen mRNA is expressed
Obvious up-regulation.To sum up, garden burnet active constituent 3,8- dimethylellagic acid -2- xyloside by promote NIH3T3 cell proliferation and
Migration promotes cell secretion TGF-β 1 and I-type collagen, while promoting the expression of its mRNA, thus wound healing.
Further, the drug of the wound healing is topical agent, such as paste, gelling agent, emulsion, liniment, patch
Deng.
Further, the medicament of the wound healing is sustained release preparation.
Detailed description of the invention:
Fig. 1 shows influence of the 3,8- dimethylellagic acid -2- β xyloside to NIH3T3 cell migration in example 2.
Fig. 2 shows 3,8- dimethylellagic acid -2- xylosides in example 4 to the mRNA of NIH3T3 cell TGF-β 1
The influence of expression.
Fig. 3 shows 3,8- dimethylellagic acid -2- xyloside in example 4 and expresses the mRNA of I-type collagen
Influence.
Fig. 4 shows in embodiment 5 3,8- dimethylellagic acid -2- xyloside to the shadow of normal rat surface of a wound area
It rings.
Specific embodiment
Below with reference to test example and specific embodiment, the present invention is described in further detail.But this should not be understood
It is all that this is belonged to based on the technology that the content of present invention is realized for the scope of the above subject matter of the present invention is limited to the following embodiments
The range of invention.
Main agents and instrument in embodiment:
MEM culture medium (Hyclone, lot number: AC10232463), penicillin and streptomysin mixed liquor (Beyotime, goods
Number: C0222), fetal calf serum (fetal bovine serum, Zhejiang Tian Hang biotinylated biomolecule Science and Technology Co., Ltd., lot number:
11011-8611), pancreatin cell dissociation buffer (Beyotime, article No.: C0201), total RNA extraction reagent box (TIANGEN, lot number:
Q5615)、TransScript All-in-One First-Strand cDNA Synthesis SuperMix for PCR
(Trans, lot number: L10324), PowerUpTM Green Master Mix(Themo Fisher
Scientific, lot number: 1707039) carbon dioxide constant incubator (LabServ), inverted phase contrast microscope (NiKon), poly-
Synthase chain reaction (polymerase chain reaction, PCR) instrument (BIO-RAD), 96 0 R Aqueous of CellTiter
One Solution Cell Proliferation Assay (Promega, lot number: 0000251174), and Mouse
Hydroxyproline (HYP) ELISA assay kit (biotechnology is built up in Nanjing, lot number: 201710), and Mouse TGF-β
1ELISA assay kit (Excell Bio, lot number: 21G125), Mouse bFGF/FGF2Protein (Sino
Biological), TGF-β 1, Collegen I, the equal designed, designed of β-actin primer, in Invitrogen Invitrogen Shanghai
Trading company's synthesis.
Cell: mouse embryonic fibroblasts (NIH3T3) are purchased from China typical culture collection center Wuhan cell bank.
1 3,8- dimethylellagic acid -2- xyloside of embodiment promotes mouse embryonic fibroblasts (NIH3T3) proliferation to make
With
NIH3T3 in logarithmic growth phase is cells trypsinised, cell is counted after digestion, and adjusts
Whole cell concentration is inoculated in 96 orifice plates with 3000 every holes, gives up periphery hole.After cell is adherent, after removing culture medium, to contain
The MEM culture medium assimilation cell of 0.5%FBS is for 24 hours.Experiment is divided into 6 groups, every group of 6 multiple holes, and 40ng/ is added in positive controls
The bFGF of ml.3,8- dimethylellagic acid -2- xyloside sets 5,10,20,40 μ g/ml, tetra- dosage groups.Various concentration is added
Drug containing fresh culture intervenes cell 36h, 48h, 60h, cell supernatant is removed, after MTS and MEM culture medium is mixed with 1:9
100 μ l are added with every hole, after being incubated for 2h in incubator, its absorbance is detected at 490nm.
Experimental data carries out statistical analysis using SPSS21.0 software.Statistical data is indicated with " x ± SD ".Measurement data
When meeting normal distribution and neat variance, select one-way analysis of variance (One-Way ANOVA) examine, be unsatisfactory for normal distribution or
When heterogeneity of variance, using non-parametric test.Insolation level α=0.05.
Experimental result is shown in Table 1, MTS experimental result and illustrates that 3,8 dimethylellagic acid -2- xylosides can promote NIH3T3 cell
Proliferation, and there are the correlations of regular hour and dosage.And the drug dose of 40 μ g/ml, 20 μ g/ml can remarkably promote
The proliferation of NIH3T3 cell.
1 3,8 dimethylellagic acid -2- β of table-xyloside promotes NIH3T3 cel l proliferation
Note: compared with blank group, * indicates that P < 0.05, * * indicate P < 0.01；
3,8- dimethylellagic acid -2- xyloside (3,8--dimethyl ellagic acid-4-xylycoside,
DEAX), similarly hereinafter.
2 3,8- dimethylellagic acid -2- xyloside of embodiment promotes NIH3T3 cell migration effect
Carry out the scratch Healing Experiments of NIH3T3 cell.Before plating cells, at the 6 orifice plates back side with Maker uniform stroke 3
Road horizontal line.By cell with 2 × 105After a/ml is inoculated in 6 orifice plates, culture medium is removed after cell is adherent after 6h, with PBS rinse
Twice.Ruler compares, and does vertical scratch, PBS cleaning in every hole cell growth single layer same position with the pipette tips of 200 sterile μ l
Twice to remove the cell scratched, the pastille culture medium for containing 0.5% of various concentration is then added, bFGF concentration is 40ng/ml,
Blank group is arranged in final concentration of 40,20,10, the 5 μ g/ml of 3,8- dimethylellagic acid -2- xylosides.Straight line and scratch intersection
As scratch width measurement point, 40 × take pictures, every hole 3 is opened.It takes pictures after pharmaceutical intervention 26h, method is same as above.Using Image Pro
Plus calculates scratch width, and average scratches width of every group of the mean value as the group is repeated 3 times.
Experimental result is shown in Fig. 1 and table 2, and compared to the blank group, bFGF group and pharmaceutical intervention group can be obviously promoted NIH3T3
The migration (P < 0.01) of cell, this explanation 3,8- dimethylellagic acid -2- xyloside can be fibroblastic by promoting
Migrate wound healing.
Influence of the 2 3,8- dimethylellagic acid -2- β xyloside of table to NIH3T3 cell migration
Compared with blank group, * indicates that P < 0.05, * * indicate P < 0.01
The expression of 3 3,8- dimethylellagic acid -2- xyloside of embodiment promotion TGF-β 1 and hydroxyl proline
It is 5 × 10 by density4For the NIH3T3 cell inoculation of a/ml in 24 orifice plates, experiment is divided into 6 groups, and every group 3 multiple
Hole is repeated 3 times.The bFGF of 40ng/ml is added in bFGF control group, and containing for 5,10,20,40 μ g/ml final concentrations is then added in medicine group
Medicine culture medium.37 DEG C, 5%CO2After cultivating 48h in incubator, cell supernatant is collected, for detecting TGF-β 1 and hydroxyl proline
Content.
Hydroxyproline is the peculiar product of collagen, collagen and ECM protein be reconstructed tissue and granulation tissue again
Two kinds of raw important adjusters, they play a significant role in wound, and TGF-β 1 is to adjust wound healing process
The important cytokine that middle fibrin is formed.3,8- dimethylellagic acid -2- xyloside can promote NIH3T3 cell to secrete
TGF-β 1 and hydroxyl proline, and there are certain dosage correlations.The results are shown in Table 3.
3 3,8- dimethylellagic acid -2- xyloside of table promotes NIH3T3 cell secretion TGF-β 1 and hydroxyl proline
Compared with blank group, * indicates that P < 0.05, * * indicate P < 0.01
Embodiment 43, -2 β of 8- dimethylellagic acid-xyloside promote TGF-β 1, the expression of I-type collagen mRNA
By NIH3T3 cell with 2 × 105A/ml is inoculated in 6 orifice plates, removes culture medium after cell is adherent, with containing
After the assimilation of 0.5%FBS culture medium, culture medium is removed after the pastille culture medium intervention 10h of various concentration is added, after PBS cleaning, use
Trizol method traditional extraction total serum IgE, using Trans Script Reverse Transcriptase kit by total serum IgE reverse transcription at cDNA, with β-
Actin is internal reference, carries out fluorescence quantitative polymerase chain reaction (qPCR) detection.With 2-ΔΔCTIt indicates experimental result, is calculated as and blank
Group be compared after relative value, blank group is set as 1.Primer information is shown in Table 4.
Experimental result is as Figure 2-3, after pharmaceutical intervention NIH3T3 cell 10h, bFGF medicine group TGF-β 1mRNA expression
(P < 0.01) is significantly increased compared with blank group, I-type collagen mRNA expression declines compared with blank group.Medicine group TGF-β 1mRNA expression
It significantly increases (P < 0.01), and the obvious up-regulation of I-type collagen mRNA expression.
Melting curve analysis parameter setting
| Initial temperature | Recurring number | Every circulating temperature gradient | Final temperature |
| 65℃ | 60 | 0.5℃ | 95.0℃ |
The setting of q-PCR response parameter
4 primer information table of table
In conclusion inventor probes into the garden burnet activity for promoting wound healing by establishing rush NIH3T3 proliferation
Ingredient, the results showed that 3,8- dimethylellagic acid -2- β xylosides can be obviously promoted the proliferation and migration of NIH3T3 cell, promote
The secretion of TGF-β 1 and collagen and synthesis wound healing, can be used for preparing the medicine of the surface of a wound caused by treatment a variety of causes
Object.
The effect of 5 3,8- dimethylellagic acid -2- xyloside of embodiment promotion normal rat wound healing
Reagent and instrument: the purchase of SD rat tests Company of Animals Ltd., experimental animal quality certification number: SYXK from Chengdu up to rich fruit
(river) 2015-030.Southwestern pharmaceutical college, medical university SPF animal house, experimental animal use credit number: SYXK (river) 2018-
213.BFGF-ESSEX (contains Biology Pharmacy Co., Ltd in Zhuhai hundred million.Batch number: 01180803).
Purchase SPF grades SD rat 34, preferably 30, weight 180g-220g, 6-7 week old, half male and half female.According to weight
It is divided into 3 groups: control group, 3,8- dimethylellagic acid -2- xyloside group, bFGF-ESSEX group, every group 10.It tests the 1st day, will own
After the anesthesia of rats by intraperitoneal injection 30mg/kg yellow Jackets, using shaver in rat back for hair, skin of back uses Iodophor
Thimerosal routine disinfection cuts the round surface of a wound for being deep to fascia that 1 diameter is 1.8cm along position among backbone with operating scissors,
Anti-infective therapy is carried out using penicillinase-fast penicillin.It tests the 1st day, catches for 9 points of every morning and hold fixed rat, 3,8- dimethyl tans flower
Acid -2- xyloside group only (is prepared into 2mg/ml using 3,8- dimethylellagic acid -2- xyloside solution spraying surface of a wound 0.12mg/
Solution is contained in bFGF-ESSEX spray bottle, is firmly pressed 2 times, apart from rat surface of a wound 2-3cm), control group sprays equivalent solvent,
BFGF-ESSEX group sprays bFGF-ESSEX 300AU/ daily and only (firmly presses 2 times, apart from rat surface of a wound 2-3cm), and successive administration 7 days, often
Cameras record surface of a wound area is used before its administration, and uses imageJ software reference area.
It is analyzed using SPSS17.0 software.So data withIt indicates, two groups are compared using paired t-test in groups
Etc. being analyzed.Experimental result is as shown in table 5 and Fig. 4.
Influence of the 5 3,8- dimethylellagic acid -2- xyloside of table to normal rat surface of a wound area
Note:*Compared with the control group, P < 0.05；**Compared with the control group, P < 0.05.
It tests the 1st day, three groups of rat surface of a wound areas are almost the same, show that experiment modeling method is feasible.BFGF-ESSEX group is from the 3rd
It starts, and surface of a wound area is substantially less than control group (P < 0.05)；3,8- dimethylellagic acid -2- xyloside group was opened from the 4th day
Begin, surface of a wound area is substantially less than control group (P < 0.05), this explanation 3,8- dimethylellagic acid -2- xyloside can promote rat
Wound healing.
Claims (8)
1.3,8- dimethylellagic acid -2- xyloside is preparing the purposes in wound healing drug.
2. purposes according to claim 1, which is characterized in that the drug of the wound healing is by promoting into fiber
The migration generation of cell acts on.
3. purposes according to claim 1, which is characterized in that the drug of the wound healing is by promoting into fiber
The proliferation generation of cell acts on.
4. purposes according to claim 1, which is characterized in that the drug of the wound healing is by promoting into fiber finer
1 generation of intracrine TGF-β effect.
5. purposes according to claim 1, which is characterized in that the drug of the wound healing is by promoting into fiber finer
Intracrine collagen generation effect.
6. purposes according to claim 1, which is characterized in that the drug of the wound healing is by promoting into fiber finer
The expression generation effect of I-type collagen mRNA in born of the same parents.
7. -6 purposes according to claim 1, which is characterized in that the drug of the wound healing is topical agent, such as
Paste, gelling agent, emulsion, liniment, patch etc..
8. purposes according to claim 7, which is characterized in that the medicament of the wound healing is sustained release preparation.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810369046 | 2018-04-23 | ||
| CN2018103690469 | 2018-04-23 |
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| CN109966304A true CN109966304A (en) | 2019-07-05 |
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ID=67085558
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105012294A (en) * | 2015-06-18 | 2015-11-04 | 苏州禾研生物技术有限公司 | New uses of ellagic acid compound in preparation of hyperuricemia treating drug |
-
2019
- 2019-04-22 CN CN201910322823.9A patent/CN109966304A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105012294A (en) * | 2015-06-18 | 2015-11-04 | 苏州禾研生物技术有限公司 | New uses of ellagic acid compound in preparation of hyperuricemia treating drug |
Non-Patent Citations (2)
| Title |
|---|
| 林家祥: "地榆炭治疗小面积烫伤", 《江苏中医》 * |
| 柏冲飞等: "地榆成分 3，3"-二甲氧基逆没食子酸- 4-木糖苷对 NIH3T3 细胞促增殖作用及其机制研究", 《天然产物研究与开发》 * |
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