CN103494800A - Application of procyanidine B2 in preparation of drugs for preventing oxidative damage and inhibiting cell apoptosis - Google Patents
Application of procyanidine B2 in preparation of drugs for preventing oxidative damage and inhibiting cell apoptosis Download PDFInfo
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- CN103494800A CN103494800A CN201310489251.6A CN201310489251A CN103494800A CN 103494800 A CN103494800 A CN 103494800A CN 201310489251 A CN201310489251 A CN 201310489251A CN 103494800 A CN103494800 A CN 103494800A
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Abstract
The invention discloses application of procyanidine B2 in preparation of drugs for preventing oxidative damage and inhibiting cell apoptosis. A cell damage model is built by adopting UVB (ultraviolet b) to act on HaCaT cells, protection effect of the procyanidine B2 on UVB-caused HaCaT cell damage is studied, anti-oxidation effect and anti-apoptosis effect of the procyanidine B2 are studied, and findings include that the procyanidine B2 can increase survival rate of cells of UVB-caused cell damage, effectively prevent oxidative damage, reduce active oxygen generation and obviously reduce cell apoptosis, thereby having effect of protecting skin cells and having remarkable treatment efficacy on UVB-caused skin oxidation damage. These results show that the procyanidine B2 can be used for preparing the drugs for preventing oxidative damage, inhibiting cell apoptosis and treating ultraviolet-caused skin oxidation damage, and is a compound having good development prospect.
Description
Technical field
The present invention relates to the medical usage of procyanidin B 2, be specifically related to the application of procyanidin B 2 in preparing anti-oxidative damage and medicaments for inhibiting cell apoptosis.
Background technology
Ultraviolet is the topmost hostile environment factor of skin injury, by its wavelength, can be divided into long wave ultraviolet (UVA, 320-400nm), ultraviolet B radiation (UVB, 280-320nm) and short wave ultraviolet (UVC, 200-280nm).Wherein, most UVC is by the Ozone Absorption in the atmospheric advection layer, and to body generation effect is mainly UVA and UVB.Research shows, UVB is the major reason that causes skin injury, is skin is produced to photochemically reactive most active part, and its energy is greater than UVA, and same dose UVB to the damage of skin approximately than the large 800-1000 of UVA doubly.By UVB irradiated site cutaneous vasodilation, the symptoms such as redness, blister can appear in skin.The permanent skin that irradiates there will be erythema, inflammation, skin aging, and severe patient can cause skin carcinoma.Oxidative damage is the skin injury caused important mechanisms of UVB.Wherein, active oxygen (ROS) plays critical effect.UVB can cause the excessive generation of the ROS in skin, and a large amount of ROS causes the oxidative damage of response to oxidative stress and biomacromolecule (as protein, fat, nucleic acid etc.).Body is subject to the destructive stimulus of UVB, produce a large amount of ROS in body, oxidative system and antioxidant system are unbalance, degree of oxidation exceeds the removing of oxide, and oxidizing process is tended in reaction, and response to oxidative stress occurs, cause the neutrophilic granulocyte inflammatory infiltration, the protease secretion increases, and produces a large amount of intermediate oxidation products, is considered to cause a key factor of senescence and disease.In addition, ROS produces the peroxidating that too much causes unsaturated fatty acid, generates α, beta-unsaturated aldehyde class as 4-hydroxyl nonenyl aldehyde and malonaldehyde, and the secondary product of these ethanol lipid peroxidations is considered to the mark of oxidative stress usually; Protein, with after ROS reacts, excites amino acid radical to shift, and after the protein aminoacyl becomes free radical, free radical can continue to shift along the aminoacid on denatured protein, brings out albuminous degeneration; Due to UVB, DNA damage is except outside the Pass too much having with the ROS generation, and DNA molecular can also directly absorb the UVB photon, causes the coup injury effect of DNA.As can be seen here, the generation of excessive ROS is the key factor that UVB causes skin injury.Serious oxidative stress and the oxidative damage of biomacromolecule can directly cause the apoptosis of cell.UVB acts on cell and causes the activation of a series of signal Signal Transduction Pathways, produces somatomedin or cytokine irritant reaction.Proved that ROS can cause the activation of multiple growth factor receptors and cytokine receptor, such as EGF-R ELISA (EGFR), fibroblast growth factor acceptor (FGFR), TNF-α, IR-1 etc.The wherein activation of EGFR its pivotal role in numerous signal paths, EGFR is the transmembrane signal albumen of a 180kDa, form homology or heterodimer with ligand binding, make specificity tyrosine stump phosphorylation, form again signal conduction complex, think that at present ROS causes the generation of this process.The activation of cell surface cytokine and growth factor receptors causes raising of respective fine intracellular signal albumen, thereby mediation cascade signal transduction, cause the activation of the signal paths such as MAPKs, PI-3 kinases and NF-κ B path, cause a series of inflammatory reactions, immunoreation and apoptosis.
In sum, due to ROS, the oxidative damage of cell is the important mechanisms that UVB causes skin photoage.ROS causes the genesis of photoaging by above several reactions.
Procyanidin compounds, it is a kind of bioflavonoids that special molecular structure is arranged, efficient cofactor, be that generally acknowledge in the world at present, active the strongest, remove the most effective Natural antioxidant of free radical in human body, free radical scavenger, there is very strong activity in vivo.Procyanidin mainly is distributed in following plant: mound dish and Radix Et Rhizoma Rhei etc. wither for Fructus Vitis viniferae, whitethorn, list Fructus Crataegi, Semen arachidis hypogaeae, Semen Ginkgo, the Thujopsis dolabrata of Japan, the arborvitae of North America, osmanli Cacumen Platycladi, Pseudotsuga menziesii (Mirbel) Franco, Bai Yeshu, wild thorn certain herbaceous plants with big flowers, Sirikaya, weeds grape, Japanese Folium illicii Lanceolati, almond, Sorghum vulgare Pers., ear leaf Senna fruit, two paddy Cortex cocois radiciss, cacao bean, Herba Hyperici Monogyni, head Caulis Seu Folium Lespedezae Bicoloris, viscose glue Boswellia carterii, maritime pine, ocean.The free radical resisting oxidability that experiment showed, procyanidin compounds is 50 times of vitamin E, ascorbic 20 times, and absorb rapidly fully, metabolic half life is for 7 hours.Procyanidin extensively is present in plant, by its degree of polymerization, can be divided into procyanidin monomers (being mainly catechin and epicatechin), Oligomeric Proanthocyanidins (monomer two, three, the tetramer) and high poly-procyanidin (more than pentamer).The most basic procyanidin monomers is catechin and epicatechin, and these two kinds of monomer polymerizations form oligomer and polymer.Dimer distributes the widest in all kinds of procyanidins, and antioxidant activity is the strongest.Dimer, because of conformation or the condensation key mapping difference of 2 units, has multiple isomer.Wherein, procyanidin B 2 is polymerized by epicatechin, and molecule is little and flexible, is easy to absorb, and activity is the strongest.Its structure is as follows:
Up to now, there do not is procyanidin B 2 to there is the relevant report of Skin Cell oxidative injury and treatment skin photoage due to anti-UVB.
Summary of the invention
The object of the present invention is to provide the new medical usage of procyanidin B 2, it can be used for preparing anti-oxidative damage, inhibited apoptosis and treatment ultraviolet and causes skin oxidative damage medicine.
Purpose of the present invention is achieved through the following technical solutions:
The present invention, from the angle of anti-light aging damage, adopts UVB to act on the HaCaT cell, sets up the cell injury model, and the research procyanidin B 2 causes the protective effect of HaCaT cell injury to UVB.Found that, procyanidin B 2 can improve the cell survival rate that UVB causes cell injury, effectively anti-oxidative damage.Further having furtherd investigate the ROS level for the cell injury model changes and the apoptosis situation; find that procyanidin B 2 can reduce the generation of ROS; thereby obviously reduce the effect that apoptosis has the protection Skin Cell, for skin oxidative damage due to UVB, there is significant therapeutic efficiency.
The application of procyanidin B 2 in preparing the anti-oxidative damage medicine.
A kind of anti-oxidative damage medicine, comprise procyanidin B 2.
The application of procyanidin B 2 in preparing medicaments for inhibiting cell apoptosis.
A kind of medicaments for inhibiting cell apoptosis, comprise procyanidin B 2.
The application of procyanidin B 2 in preparation treatment ultraviolet causes skin oxidative damage medicine; Described ultraviolet is ultraviolet B radiation.
A kind of ultraviolet for the treatment of causes skin oxidative damage medicine, comprises procyanidin B 2.
The present invention has following advantage and effect with respect to prior art: the present invention has found effectively anti-oxidative damage of procyanidin B 2 first; reduce the generation of active oxygen; thereby obviously reduce the effect that apoptosis has the protection Skin Cell; having significant therapeutic efficiency for skin oxidative damage due to UVB, is a compound with good DEVELOPMENT PROSPECT.
The accompanying drawing explanation
Fig. 1 is that cell survival experiment detects protective effect that procyanidin B 2 causes the HaCaT cell injury to UVB figure as a result.
Fig. 2 is the antioxidation figure as a result that utilizes DCFH-DA to detect procyanidin B 2.
Fig. 3 is the anti-apoptotic effect figure as a result that the two methods of dying of Annexin V-FITC/PI detect procyanidin B 2.
The specific embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.
Embodiment 1 causes the protective effect of HaCaT cell injury by cell survival measuring procyanidin B 2 to UVB
The HaCaT cell is purchased from the triumphant base biology in Nanjing, and cell culture is with in the DMEM culture medium, containing 10%(v/v) hyclone, 100U/mL penicillin, 100 μ g/mL gentamycins, cell is in 5%CO
2, cultivated for 37 ℃.Be grouped into matched group (processing without procyanidin B 2 and UVB), irradiation group (the irradiation group further is grouped into and uses respectively 0,6.25,12.5,25,50 μ Μ procyanidin B 2 processed group).The trophophase HaCaT cell of taking the logarithm, spread into 96 orifice plates every hole 100 μ L with the density of 5000 every hole density.Every group of sample established 3 multiple holes, in 5%CO
2, cultivate 24h for 37 ℃, the irradiation group adds variable concentrations procyanidin B 2 pretreatment 1h, and then UVB processes, and dosage is 50mJ/cm
2, after continuing to cultivate 24h, add MTT(5mg/mL) and the rear 4h of cultivation, to inhale and abandon the culture medium containing MTT, the dissolving crystallized product of 150 μ L DMSO, used microplate reader at 570nm wavelength measurement absorbance.
Cell survival rate (%)=irradiation group absorbance/matched group absorbance * 100%.
Statistical procedures
Experimental data adopts SPSS19.0 software to be added up, and between group, the data ANOVA is analyzed, and experimental result means with the mean value ± variance, and there is significant difference P<0.05 for statistics.
The UVB irradiating cell is set up cell ultraviolet damage model, screening procyanidin B 2 valid density, result as shown in Figure 1: the HaCaT cell is 24h after UVB irradiates, and irradiation group cell survival rate significantly reduces than matched group; And, with procyanidin B 2 pretreatment cell 1h, can reverse the cell survival rate decline that UVB causes.When procyanidin B 2 concentration is greater than 12.5 μ Μ, UVB is caused to the HaCaT cell significant protective effect is arranged; its cell survival rate is significantly higher than does not use the pretreated irradiation group of procyanidin B 2 (0 μ Μ) (P<0.05; * mean that there were significant differences with matched group, # means and does not use procyanidin B 2 (0 μ Μ) there were significant differences for pretreated irradiation group).
UVB measures ROS level in cell immediately after irradiating, 2 ', 7 '-dihydro dichlorofluorescein sodium diacetate (DCFH-DA) is the specificity fluorescent probe that detects ROS, and DCFH-DA has fat-soluble, and it can pass through cell membrane and generate DCFH with intracellular Esterase reaction.Because the latter can not pass cell membrane, can make fluorescent probe be loaded in cell, and be oxidized to the DCF with fluorescence by intracellular ROS.Utilizing DCFH-DA can detect ROS level in cell changes.The grouping situation is: matched group (processing without procyanidin B 2 and UVB), procyanidin B 2 group (processing without UVB), UVB irradiation group (not adding procyanidin B 2), procyanidin B 2+UVB group.By the HaCaT cell of exponential phase with 1 * 10
6every hole density is inoculated in 6 orifice plates, in 5%CO
2, cultivate 24h for 37 ℃, 25 μ Μ procyanidin B 2 pretreatment with the DCFH-DA probe incubated cell of 10 μ Μ 20 minutes, PBS(pH7.4) were washed 3 times after 1 hour, then UVB irradiation, dosage is 50mJ/cm
2, the trypsinization collecting cell.The cells were tested by flow cytometry fluorescence intensity, excitation wavelength is 488nm, wavelength of transmitted light 530nm.As shown in Figure 2, after UVB irradiates, cell ROS level significantly increases result, is 5 times of matched groups; Utilize the procyanidin B 2 pretreatment cell, the ROS level significantly reduces, and is 2.5 times of matched groups, and compared to UVB irradiation group, the ROS level significantly reduces (P<0.05, * means that there were significant differences with matched group, # means that there were significant differences with UVB irradiation group).
Adopt the two methods of dying of Annexin V-FITC/PI to detect apoptosis.Experiment grouping is with embodiment 2, and after UVB irradiates, the HaCaT cell continues to cultivate 24h, and the trypsinization collecting cell PBS(pH7.4) adds the rich biology of sea cowry 400 μ L Bingding Buffer(on after washing 2 times) suspension cell; After adding 5 μ L Annexin V-FITC to mix, add 5 μ L PI, mix; Room temperature lucifuge reaction 15 minutes, flow cytometer detects the apoptotic cell percentage rate.PI hydrion fluorescence excitation, excitation wavelength is 488nm, emission wavelength is greater than 630nm, produces red fluorescence.As shown in Figure 3, cell significantly increases compared to the cellular control unit apoptosis rate result after UVB irradiates, and apoptosis rate increases to 46.37%; And procyanidin B 2 pretreatment 1h can reduce apoptosis rate significantly with respect to UVB irradiation group, apoptosis rate drops to 28.7%, show that procyanidin B 2 has significant anti-apoptotic effect (P<0.05, * mean that there were significant differences with matched group, # means that there were significant differences with UVB irradiation group)
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (7)
1. the application of procyanidin B 2 in preparing the anti-oxidative damage medicine.
2. an anti-oxidative damage medicine, is characterized in that: comprise procyanidin B 2.
3. the application of procyanidin B 2 in preparing medicaments for inhibiting cell apoptosis.
4. a medicaments for inhibiting cell apoptosis, is characterized in that: comprise procyanidin B 2.
5. the application of procyanidin B 2 in preparation treatment ultraviolet causes skin oxidative damage medicine.
6. application according to claim 5 is characterized in that: described ultraviolet is ultraviolet B radiation.
7. a treatment ultraviolet causes skin oxidative damage medicine, it is characterized in that: comprise procyanidin B 2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107056857A (en) * | 2017-03-31 | 2017-08-18 | 湖南中医药大学 | A kind of new flavone compound and preparation method and application |
| CN113476439A (en) * | 2021-06-21 | 2021-10-08 | 广东省农业科学院蚕业与农产品加工研究所 | Application of procyanidine compound in preparation of product for treating skin injury caused by ultraviolet rays |
| CN115252496A (en) * | 2022-08-18 | 2022-11-01 | 云南贝泰妮生物科技集团股份有限公司 | Preparation method of emblic leafflower fruit extract and application of emblic leafflower fruit extract in preventing and treating ultraviolet ray injury |
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| CN101125139A (en) * | 2007-09-28 | 2008-02-20 | 天津市尖峰天然产物研究开发有限公司 | Application of procyanidin B2 in preparing medicine for preventing and treating diabetes and vascular complication |
| CN102688230A (en) * | 2012-06-20 | 2012-09-26 | 浙江萧山医院 | New applciation of procyanidine B2 |
| CN103320490A (en) * | 2013-05-13 | 2013-09-25 | 程树军 | Screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells |
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Patent Citations (3)
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| CN101125139A (en) * | 2007-09-28 | 2008-02-20 | 天津市尖峰天然产物研究开发有限公司 | Application of procyanidin B2 in preparing medicine for preventing and treating diabetes and vascular complication |
| CN102688230A (en) * | 2012-06-20 | 2012-09-26 | 浙江萧山医院 | New applciation of procyanidine B2 |
| CN103320490A (en) * | 2013-05-13 | 2013-09-25 | 程树军 | Screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107056857A (en) * | 2017-03-31 | 2017-08-18 | 湖南中医药大学 | A kind of new flavone compound and preparation method and application |
| CN107056857B (en) * | 2017-03-31 | 2020-02-14 | 湖南中医药大学 | Flavonoid compound and preparation method and application thereof |
| CN113476439A (en) * | 2021-06-21 | 2021-10-08 | 广东省农业科学院蚕业与农产品加工研究所 | Application of procyanidine compound in preparation of product for treating skin injury caused by ultraviolet rays |
| CN115252496A (en) * | 2022-08-18 | 2022-11-01 | 云南贝泰妮生物科技集团股份有限公司 | Preparation method of emblic leafflower fruit extract and application of emblic leafflower fruit extract in preventing and treating ultraviolet ray injury |
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Application publication date: 20140108 |