CN109646424B - Preparation method and application of phenolic compound - Google Patents
Preparation method and application of phenolic compound Download PDFInfo
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- CN109646424B CN109646424B CN201811175921.6A CN201811175921A CN109646424B CN 109646424 B CN109646424 B CN 109646424B CN 201811175921 A CN201811175921 A CN 201811175921A CN 109646424 B CN109646424 B CN 109646424B
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Abstract
The invention provides a preparation method and application of a phenolic compound with the function of promoting wound healing, and the structures of the phenolic compound are (E) -4- (3,4-dihydroxyphenyl) but-3-en-2-one, 2- (3,4-dihydroxyphenyl) -2-oxoacetate and methyl 2- (4-dihydroxyphenyl) acetate respectively. The invention also provides an extraction and separation method of the compound. The invention shows that the compounds have the effect of promoting wound healing.
Description
Technical Field
The invention belongs to the field of chemical drugs, and relates to a phenolic compound and application thereof in healing of wound surfaces including ulcer.
Background
The causes, hazards and treatments of wounds, including ulcers, have been described in the prior application CN 105434450A. The tissue remodeling process of wound at least includes proliferation, migration and vascularization of cells, and HUVEC (human umbilical vein endothelial cells) has become an important model for studying the promotion of wound healing. The invention adopts human skin fibroblast (HDFs) and HUVEC tests to find that the active ingredients for promoting wound repair are provided.
Periplaneta americana (Periplaneta americana) is an insect of the genus Periplaneta of the family Blattaria, the class Insecta, class Pterobia, and the clinical treatment of various wounds including ulcers is confirmed by the rehabilitation new liquid prepared therefrom, indicating that an active ingredient or a drug that promotes wound healing is expected to be found from the liquid, but this research is still relatively deficient. Our studies show that various components in periplaneta americana play a role in promoting wound healing from different angles.
Disclosure of Invention
The invention provides application of a phenolic compound in preparation of a medicine for promoting wound healing, wherein the wound is an ulcer wound.
Further, the compound is one of structural formulas i-iii.
Further, the compound is obtained by extracting from periplaneta americana or artificially synthesizing.
Further, the compound has effects of promoting cell proliferation, cell migration and angiogenesis.
The invention also provides a preparation method of the phenolic compound, which comprises the following preparation steps:
taking dried Periplaneta americana bodies, crushing, extracting with hot water under reflux for 3 times, each time for 2 hours, concentrating the extract under reduced pressure to obtain a total extract, subjecting the total extract to reverse phase chromatography MCI gel CHP 20P column and gradient elution with methanol-water according to the proportion of 0-100% to obtain 11 components, namely F1-F11, subjecting F9 to Sephadex LH-20 gel to obtain 4 components, namely F9.1-F9.4, subjecting F9.3 to MCI gel CHP 20P column and gradient elution with methanol-water according to the proportion of 10-100% to obtain 7 components, namely F9.3.1-F9.3.7, subjecting F9.3.4 to RP-18 column and gradient elution with methanol-water according to the proportion of 15-50%, subjecting F9.3.4.1-F9.3.4.5, subjecting F9.3.4.4 to semi-preparative HPLC to obtain compounds (E) -4- (3,4-dihydroxyphenyl) but-3-en-2-one and 2- (3,4-dihydroxyphenyl) -2-oxo-acetate, mobile phase for semi-preparative HPLC was methanol-water, methanol volume: 25:75, F9.3.4.5, and obtaining the compound methyl 2- (4-hydroxyphenyl) acetate by semi-preparative HPLC, wherein the mobile phase of the semi-preparative HPLC is methanol-water, and the volume of the methanol is: the volume of water is 20: 80.
The invention has the beneficial effects that:
1. the invention discloses a method for extracting and separating the phenolic compounds from the periplaneta americana for the first time.
2. The phenolic compound is obtained by separating and identifying from the periplaneta americana for the first time.
3. The phenolic compound provided by the invention has the effects of remarkably promoting human fibroblast proliferation, HUVEC cell migration and angiogenesis.
Drawings
The compound of fig. 1 promotes the proliferation activity of HDFs (n-3). P <0.05, p <0.01, p < 0.001.
The compounds of fig. 2a and 2b promote the migration activity of HUVECs (n-3). P <0.05, p <0.01, p < 0.001.
The results of the angiogenesis promoting activity test of the compounds of figures 3a and 3b (n-3). P <0.05, p <0.01, p < 0.001.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1 extraction separation and Structure identification of the phenolic Compounds of the invention
The periplaneta americana is from the periplaneta americana GAP base of the western pharmaceutical industry llc by the good doctor of Sichuan, and the periplaneta americana extract is the main component of the rehabilitation new liquid.
Pulverizing Periplaneta americana dry body 25kg, extracting with hot water under reflux for 3 times (2 hr each time), concentrating the extractive solution under reduced pressure to obtain total extract 2kg, subjecting the total extract to reversed phase chromatography MCI gel CHP 20P column with methanolWater is eluted according to a proportion of 0 to 100 percent in a gradient manner to obtain 11 components, namely F1 to F11, F9(40g) is subjected to Sephadex LH-20 gel to obtain 4 components, namely F9.1 to F9.4, F9.3(5g) is subjected to MCI gel CHP 20P chromatographic column and is subjected to gradient elution by adopting methanol-water according to a proportion of 10 to 100 percent to obtain 7 components, namely F9.3.1 to F9.3.7, F9.3.4(500mg) is subjected to RP-18 chromatographic column and is subjected to methanol-water according to a proportion of 15 to 50 percent to obtain 5 components, namely F9.3.4.1 to F9.3.4.5, and F9.3.4.4(40mg) is subjected to semi-preparative HPLC to obtain a compound (E) -4- (3,4-dihydroxyphenyl) but-3-en-2-one (10.0mg, t) andR10.5 min; corresponding numbers qzl-10) and 2- (3,4-dihydroxyphenyl) -2-oxoethyl acetate (3.5mg, tR18.1 min; corresponding to code qzl-12a), the mobile phase for semi-preparative HPLC was methanol-water, methanol volume: the compound methyl 2- (4-hydroxyphenyl) acetate (5.2mg, t) was obtained by semi-preparative HPLC with a water volume of 25:75, F9.3.4.5R14.8 min; corresponding to code qyzl-104), the mobile phase for semi-preparative HPLC was methanol-water, methanol volume: the volume of water is 20: 80.
Compound results were identified as follows:
the compound (E) -4- (3,4-dihydroxyphenyl) but-3-en-2-one is pale yellow solid, ESI-MS M/z is 177[ M-H]–,1H NMR(600MHz,CD3OD)δ:7.51(1H,d,J＝16.2Hz,H-7),7.08 (1H,brs,H-2),6.99(1H,d,J＝7.5Hz,H-6),6.79(1H,d,J＝7.5Hz,H-5),6.54(1H, d,J＝16.2Hz,H-8),2.33(3H,s,H-10)；13C NMR(methanol-d4,150MHz)δC 201.6 (C-9),150.0(C-4),146.9(C-3),146.9(C-7),127.7(C-1),124.7(C-8),123.6(C-6), 116.5(C-2),115.3(C-5),27.0(C-10)。
The compound 2- (3,4-dihydroxyphenyl) -2-oxoethyl acetate as a pale yellow solid, ESI-MS M/z 209[ M-H]–,1H NMR(400MHz,CD3OD)δ:7.40(1H,dd,J＝8.2,1.8Hz,H-6), 7.38(1H,d,J＝1.8Hz,H-2),6.84(1H,d,J＝8.2Hz,H-5),5.32(2H,s,H-8),2.16 (3H,s,H-OAc)。
The compound methyl 2- (4-hydroxypentyl) acetate, a pale yellow solid, ESI-MS M/z 165[ M-H ]]–,1H NMR(400MHz,CD3OD)δ:7.07(2H,d,J＝8.5Hz,H-2,H-6),6.72(2H,d, J＝8.5Hz,H-3,H-5),3.66(3H,s,H-OCH3),3.52(2H,s,H-7)。
Example 2 test method for the Activity of phenolic Compounds to promote the proliferation of HDFs cells
Human skin fibroblasts (HDFs) were purchased from American Type Culture Collection (ATCC). DMEM medium and fetal bovine serum were purchased from Gibco. The logarithmic phase HDFs cells are adjusted to the cell suspension concentration of about 3X 103Perml, inoculated in a 96-well plate at 100mL per well, incubated at 37 ℃ with 5% CO2After the cells are cultured for 24h under the condition and attached to the wall, a compound solution (with a final concentration of 20 mu M) is added for culturing 100mL, and an equal amount of fresh culture medium is added into a blank control group. Each group was set with 3 parallel wells, incubated in an incubator for 48h, and 20mL of 5mg/mL MTT solution was added to each well, incubated at 37 ℃ with 5% CO2Incubating for 4h under the condition, terminating the culture, removing supernatant, adding 150mL DMSO into each hole, shaking for 15min, fully dissolving the blue-violet crystal, measuring absorbance value at 490nm wavelength of an enzyme labeling instrument, averaging each group, and repeating the experiment for 3 times.
Cell growth rate (%) (OD value in experiment group-OD value in blank control group/OD value in blank control group) × 100%
Example 3 Experimental method for promoting HUVECs migration by phenolic compound
Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from Shenzhen university department of medicine and presented by professor Liubaohua. M199 medium and fetal bovine serum were purchased from Gibco. HUVECs at log phase were collected and plated in 24-well plates at 14X 10/well4And (4) cells. Placing at 37 ℃ and 5% CO2After cells were attached to the wall after 24h of culture under the conditions, they were washed 3 times with 1 × PBS and then starved for 6 hours with M199 medium containing 0.5% FBS. After the end of hunger, the tip of P10 was used for scratching, M199 medium containing the drug dissolved therein (20. mu.M final concentration) was added to the experimental group, and fresh M199 medium was added to the blank group, and photographs were taken. At 37 deg.C, 5% CO2After incubation for 6h under conditions, wash 3 times with 1 × PBS, add fresh M199 medium, and take pictures. The scratch area calculation was performed using Image pro plus 6.
Migration area% ═ 100% (test group 0h scratch area-test group 6h scratch area/blank control group 0h scratch area-blank group 6h scratch area) ×
Example 4 assay of the angiogenic Activity of phenolic Compounds for HUVECs
Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the department of medicine of Shenzhen university and presented by professor Liubaohua; m199 medium and fetal bovine serum were purchased from Gibco; matrigel gum was purchased from Corning corporation. Uniformly spreading Matrigel gel in a pre-cooled 96-well plate, incubating at 37 deg.C for 40min with 50 μ L of Matrigel per well, and adding 1 × 10 gel per well after the Matrigel is solidified4HUVECs cells, test group treated with test compound (20 μ M final concentration) and control group without drug. At 37 deg.C, 5% CO2Under the conditions, after 6h of incubation, photographs were observed under a microscope and Image analysis was performed using Image J software, 3 replicates for each experiment.
Angiogenesis rate (%) - (length of drug-treated component tube/length of control component tube × 100%
As can be seen from the figure, the phenolic compound shows obvious promotion effects on human fibroblast proliferation (figure 1), human umbilical vein endothelial cell migration (figures 2a and 2b) and angiogenesis (figures 3a and 3b) respectively, and shows that the phenolic compound has the effect of promoting wound healing.
Claims (3)
1. An application of a phenolic compound in preparing a medicine for promoting wound healing is characterized in that: the wound surface is an ulcer wound surface; the structural formula of the phenolic compound is one of ii-iii;
2. the use of a phenolic compound as claimed in claim 1 in the manufacture of a medicament for promoting wound healing, wherein: the compound is obtained by extracting from periplaneta americana or artificially synthesizing.
3. Use of a phenolic compound as claimed in claim 1 or claim 2 in the manufacture of a medicament for promoting wound healing, wherein: the compounds have the effects of promoting cell proliferation, cell migration and angiogenesis.
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| KR100998920B1 (en) * | 2008-08-21 | 2010-12-09 | 한밭대학교 산학협력단 | A skin external composition for wound healing |
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