广藿香油对UV所致小鼠皮肤光老化模型保护作用的实验研究

发布时间：2018-09-11 14:03

【摘要】：目的：光老化是皮肤过早老化的过程,是由于皮肤持续地长时间暴露于紫外线辐射所产生,并以晒斑,粗皱纹,失去弹性和光化性角化病为临床特征。光老化可导致严重后果,其与多种皮肤病甚至于皮肤肿瘤的发生息息相关,约80%以上的暴露部位美容问题均由皮肤光老化引起。中医认为皮肤光老化以外热侵袭为主要病因,以痰浊阻滞为重要病机,可采用活血通络化瘀祛浊法进行治疗。过去几十年来,国内外研究者对皮肤光老化进行了广泛的研究。笔者通过查阅大量文献绘制出皮肤光老化发生的分子机制图,得知皮肤光老化的发生机制与氧化应激和炎症因子有关。广藿香是我国常用的芳香化湿中药之一,具有芳香化浊、和中止呕、发表解暑的功效,不仅用于治疗湿滞中阻、脘痞呕吐等,还被作为古代面部美容方剂的常用中药用于治疗面部疾病。广藿香油是广藿香经过水蒸气蒸馏提取得到的挥发油,是广藿香的主要药用部分,其因具有本草特征及稳定属性已经成为化妆品中最重要的成分之一。广藿香油含有多种生物活性成分,表现出抗过敏、抗菌等皮肤活性及抗氧化和抗炎活性。其中抗氧化和抗炎活性的作用路径均与抗皮肤光老化活性有关。那么其用于面部美容,是否与抗皮肤光老化活性有关呢?广藿香油是否具有抗皮肤光老化活性呢?若具有抗皮肤光老化活性,又是通过何种机制起作用呢?是否与其芳香化浊、发表解暑的功效有关?因此,本文选择广藿香油为研究对象,探讨其对紫外线所致小鼠皮肤光老化模型的保护作用,并从皮肤组织病理学及氧化应激损伤机制探讨其抗皮肤光老化作用的可能机理,并从分子机制探讨广藿香油发挥“芳香化浊,发表解暑”之功拮抗皮肤光老化的现代科学内涵。方法与结果：1.广藿香油的化学成分研究采用GC法测定广藿香油中主要化学成分指标广藿香醇的含量,并采用GC-MS法测定广藿香油的化学组成成分。测得广藿香醇的含量为28.2%(g/g),符合中国药典不低于26%的要求；广藿香油中共鉴别出18种化学成分,占挥发油总成分的88.3%。主要由萜类成分组成,其中单帖和倍半萜占超过50%,广藿香醇占29.3%(峰面积百分比)。通过定性鉴别和定量分析,可确保用于药效学试验的样品质量。2.广藿香油对UV所致小鼠皮肤光老化模型的保护作用研究以KM种小鼠作为实验动物,分为NC、SC、MC、VC、PO-L、PO-H、PO-M、PO-H等七组,每组9只。采用UVA和UVB同时照射小鼠,制备光老化模型。以广藿香油局部涂抹的预给药方式,考察其对皮肤光老化模型的保护作用。实验中,将小鼠背部局部毛发去除使皮肤裸露,用广藿香油处理2小时后于紫外灯下照射。通过宏观评分、提皮试验,以宏观评分值及皮肤恢复时间为指标,探讨其对UV,所致小鼠皮肤光老化模型的保护作用。宏观评分结果：PO-H组给药六周后显现出可抑制UV诱导的广泛红斑和深皱纹的趋势；PO-M组给药八周后显示出对皮肤皱纹形成具有抑制作用；实验结束时,PO-H组和PO-M组分数明显低于VC组,提示PO局部给药可抑制UV诱导的皮肤表观损伤。皮肤弹性试验结果：与VC组比较PO-L组无显著差异,PO-M、PO-H组均显著减少皮肤恢复时间；结果表明,广藿香油局部给药(特别是6mg／只和9mg/只)可促进皮肤弹性。宏观评分及提皮试验结果共同提示,广藿香油局部给药对UV所致小鼠皮肤光老化模型具有显著的保护作用。3.基于皮肤病理学及氧化应激损伤机制探讨广藿香抗皮肤光老化的作用机理采用HE染色法与Gomori's醛品红染色法观察广藿香对光老化皮肤组织结构的影响,通过观察皮肤角质层和表皮厚度变化,胶原纤维及弹性纤维的形态、含量及分布,总体评价广藿香油对光老化皮肤组织结构的影响。表皮厚度测定结果：PO-M和PO-H组可见表皮厚度显著减小,分别减少19.8m和23.7μm；结果提示PO-M、PO-H组均可显著减轻UV诱导的表皮增生。组织特征观察结果：广藿香油局部给药可改善UV照射引起的皮肤结构改变,如表皮异常增厚；表皮真皮连接处扁平化,表皮突与真皮乳头消失；胶原纤维断裂、减少、排列紊乱；弹性纤维变性断裂；提示广藿香油可抑制UV照射引起的异常组织改变。基于氧化应激损伤机制,采用生化指标测定方法考察广藿香油对光老化皮肤中胶原蛋白与丙二醛(MDA)含量的影响,对光老化皮肤中对超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)等活性的影响。实验结果,PO-M和PO-H组均可显著提高胶原蛋白含量分别达21.9%和26.3%,显著降低光老化皮肤的MDA含量,显著增加SOD、GSH-Px活力。PO-L组与PO-H组显著增加CAT活力分别为29.8%和90.7%,PO-M组未对CAT活力有显著影响。结果提示广藿香油可减轻UV诱导的胶原蛋白损坏,抑制UV照射小鼠引起的脂质过氧化反应,减轻UV照射引起的SOD、 GSH-Px、CAT活力降低。4.探讨广藿香发挥“芳香化浊,发表解暑”之功拮抗皮肤光老化的现代科学内涵结合分子机制和中医理论进行探讨：UV照射属“外热”,ROS属“痰浊”,UV照射引起皮肤光老化即为“外热侵袭所致皮肤衰老”,ROS损伤引起皮肤细胞功能障碍即为“痰浊阻滞引起皮肤气血运行失常”。广藿香油抑制UV照射引起的皮肤异常结构改变,即为“发表解暑”,而广藿香油具有芳香属性并能抑制ROS损伤即为“芳香化浊”。因此,广藿香油发挥“芳香化浊,发表解暑”之功,而起抗皮肤光老化作用。结论：根据酶促抗氧化防御系统可调节ROS清除自由基的理解,抗氧化剂治疗被认为是抗皮肤光老化的有效方法。广藿香油作为一种抗氧化剂,局部给药对UV所致小鼠皮肤光老化模型具有显著的保护作用。这种保护作用可能通过减轻表皮增生,抑制皮肤组织结构异常改变,抑制胶原蛋白损坏等机制发生,也可能通过抑制脂质过氧化反应,提高可清除自由基的抗氧化酶活力等机制产生,这可能与其抑制UV诱导的ROS损伤机制有关。本论文首次全面地证明广藿香油局部给药对UV诱导的小鼠皮肤光老化模型具有保护作用,为其可被作为一种的潜在的美容功能产品用于皮肤护理提供证据,并从分子机制诠释广藿香油发挥“芳香化浊,发表解暑”之功拮抗皮肤光老化的现代科学内涵。
[Abstract]:OBJECTIVE: Photoaging is a process of premature skin aging, which is caused by prolonged exposure to ultraviolet radiation and is characterized by sunburn, coarse wrinkles, loss of elasticity and actinic keratosis. Photoaging can lead to serious consequences, which are closely related to a variety of skin diseases and even skin tumors, accounting for more than 80%. The cosmetic problems of exposed areas are caused by photoaging of skin. Traditional Chinese medicine considers that photoaging is the main cause of heat invasion and phlegm blockage is the important pathogenesis. It can be treated by activating blood circulation, removing blood stasis and removing turbidity. Over the past decades, researchers at home and abroad have conducted extensive studies on photoaging of skin. Patchouli Patchouli is one of the most commonly used aromatic moisturizing herbs in China. It has the effects of aromatic turbidity, vomiting and heat relief. It is not only used in the treatment of dampness, blockage, vomiting and so on, but also used as an ancient face. Patchouli oil is a volatile oil extracted by steam distillation. It is the main medicinal part of Patchouli. Patchouli oil has become one of the most important components in cosmetics because of its herbal characteristics and stability properties. Patchouli oil contains a variety of bioactive components and exhibits many biological activities. Anti-allergic, anti-bacterial and anti-oxidant and anti-inflammatory activities of skin. Anti-oxidant and anti-inflammatory activities of the pathways are related to anti-photoaging activities of the skin. Then whether its use in facial beauty is related to anti-photoaging activities of the skin? Is patchouli oil anti-photoaging activity of the skin? If it has anti-photoaging activity of the skin, So, patchouli oil was selected as the research object to explore the protective effect of patchouli oil on UV-induced skin photoaging in mice, and to explore its anti-skin photoaging effect from skin histopathology and oxidative stress injury mechanism. Methods and Results: 1. The chemical constituents of patchouli oil were determined by GC, and the content of patchouli alcohol was determined by GC-MS. The content of patchouli oil was determined by GC-MS. The content of patchouli alcohol was 28.2% (g/g), which met the requirement of Chinese Pharmacopoeia. 18 chemical constituents were identified in patchouli oil, accounting for 88.3% of the total volatile oil. They were mainly composed of terpenoids, including monoterpenoids and sesquiterpenes accounting for more than 50%, patchouli alcohol accounting for 29.3% (peak area percentage). Protective effects of patchouli oil on UV-induced skin photoaging in mice were studied. KM mice were divided into seven groups: NC, SC, MC, VC, PO-L, PO-H, PO-M, PO-H, 9 mice in each group. Photoaging models were prepared by simultaneous irradiation of UVA and UVB. In the experiment, the skin was exposed after removing the local hair from the back of mice. Patchouli oil was treated for 2 hours and then irradiated under ultraviolet lamp. Macroscopic score: PO-H group showed a tendency to inhibit UV-induced extensive erythema and deep wrinkles six weeks after administration; PO-M group showed an inhibitory effect on skin wrinkles eight weeks after administration; at the end of the experiment, PO-H group and PO-M group were significantly lower than VC group, suggesting that PO Bureau The results of skin elasticity test showed that there was no significant difference between PO-L group and VC group, PO-M, PO-H group significantly reduced the skin recovery time; the results showed that patchouli oil topical administration (especially 6mg/only and 9mg/only) could promote skin elasticity. The results of macroscopical score and skin lift test suggested that it was wide. Local administration of Pogostemon oil has a significant protective effect on UV-induced skin photoaging in mice. 3. Based on the mechanism of skin pathology and oxidative stress injury, the anti-photoaging mechanism of Pogostemon Patchouli was studied by HE staining and Gomori's aldehyde fuchsin staining to observe the effect of Pogostemon on the structure of photoaging skin. The changes of cuticle and epidermis thickness, the morphology, content and distribution of collagen fibers and elastic fibers, and the effect of patchouli oil on the structure of photoaging skin were evaluated. V-induced epidermal hyperplasia. Histological characteristics: Patchouli oil topical administration can improve UV-induced skin structural changes, such as abnormal thickening of the epidermis; flattened epidermal junction, epidermal processes and dermal papilla disappeared; collagen fiber breakage, reduction, disordered arrangement; elastic fiber degeneration and rupture; suggesting that patchouli oil can inhibit Based on the mechanism of oxidative stress injury, the effects of patchouli oil on the content of collagen and malondialdehyde (MDA) in photoaging skin were investigated by biochemical methods. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) in photoaging skin were also investigated. The results showed that both PO-M and PO-H groups significantly increased collagen content by 21.9% and 26.3% respectively, significantly decreased MDA content, significantly increased SOD, GSH-Px activity, PO-L and PO-H groups significantly increased CAT activity by 29.8% and 90.7%, respectively. PO-M group had no significant effect on CAT activity. Light UV-induced collagen damage, inhibition of lipid peroxidation induced by UV irradiation in mice, alleviate UV-induced SOD, GSH-Px, CAT activity decreased. 4. To explore the role of Patchouli Patchouli in antagonizing skin photoaging by aromatic turbidity and releasing heat relief. External heat, ROS belongs to "phlegm turbidity". UV irradiation induced skin photoaging is "skin aging caused by exothermic invasion". ROS damage caused by skin cell dysfunction is "phlegm turbidity block caused skin Qi and blood dysfunction". Patchouli oil inhibits the abnormal structural changes of skin caused by UV irradiation, that is, "release heat" and patchouli oil. Therefore, patchouli oil can play the role of "aromatic turbidity, release heat relief" and play an anti-skin photoaging effect. Conclusion: According to the understanding that enzymatic antioxidant defense system can regulate ROS scavenging free radicals, antioxidant therapy is considered to be an effective way to prevent skin photoaging. Patchouli oil, as an antioxidant, has a significant protective effect on UV-induced skin photoaging in mice. This protective effect may be through alleviating epidermal hyperplasia, inhibiting abnormal changes of skin tissue structure, inhibiting collagen damage and other mechanisms. It may also be through inhibiting lipid peroxidation reaction to enhance the removal of UV-induced skin photoaging. In this paper, the protective effect of patchouli oil topical administration on UV-induced skin photoaging in mice was demonstrated for the first time, providing evidence that patchouli oil could be used as a potential cosmetic product for skin care. The modern scientific connotation of patchouli oil in antagonizing skin photoaging was explained by molecular mechanism.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5;R-332

【引证文献】