【摘要】：目的：光老化是皮膚過早老化的過程,是由于皮膚持續(xù)地長時間暴露于紫外線輻射所產(chǎn)生,并以曬斑,粗皺紋,失去彈性和光化性角化病為臨床特征。光老化可導致嚴重后果,其與多種皮膚病甚至于皮膚腫瘤的發(fā)生息息相關(guān),約80%以上的暴露部位美容問題均由皮膚光老化引起。中醫(yī)認為皮膚光老化以外熱侵襲為主要病因,以痰濁阻滯為重要病機,可采用活血通絡(luò)化瘀祛濁法進行治療。過去幾十年來,國內(nèi)外研究者對皮膚光老化進行了廣泛的研究。筆者通過查閱大量文獻繪制出皮膚光老化發(fā)生的分子機制圖,得知皮膚光老化的發(fā)生機制與氧化應(yīng)激和炎癥因子有關(guān)。廣藿香是我國常用的芳香化濕中藥之一,具有芳香化濁、和中止嘔、發(fā)表解暑的功效,不僅用于治療濕滯中阻、脘痞嘔吐等,還被作為古代面部美容方劑的常用中藥用于治療面部疾病。廣藿香油是廣藿香經(jīng)過水蒸氣蒸餾提取得到的揮發(fā)油,是廣藿香的主要藥用部分,其因具有本草特征及穩(wěn)定屬性已經(jīng)成為化妝品中最重要的成分之一。廣藿香油含有多種生物活性成分,表現(xiàn)出抗過敏、抗菌等皮膚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徑均與抗皮膚光老化活性有關(guān)。那么其用于面部美容,是否與抗皮膚光老化活性有關(guān)呢?廣藿香油是否具有抗皮膚光老化活性呢?若具有抗皮膚光老化活性,又是通過何種機制起作用呢?是否與其芳香化濁、發(fā)表解暑的功效有關(guān)?因此,本文選擇廣藿香油為研究對象,探討其對紫外線所致小鼠皮膚光老化模型的保護作用,并從皮膚組織病理學及氧化應(yīng)激損傷機制探討其抗皮膚光老化作用的可能機理,并從分子機制探討廣藿香油發(fā)揮“芳香化濁,發(fā)表解暑”之功拮抗皮膚光老化的現(xiàn)代科學內(nèi)涵。方法與結(jié)果：1.廣藿香油的化學成分研究采用GC法測定廣藿香油中主要化學成分指標廣藿香醇的含量,并采用GC-MS法測定廣藿香油的化學組成成分。測得廣藿香醇的含量為28.2%(g/g),符合中國藥典不低于26%的要求；廣藿香油中共鑒別出18種化學成分,占揮發(fā)油總成分的88.3%。主要由萜類成分組成,其中單帖和倍半萜占超過50%,廣藿香醇占29.3%(峰面積百分比)。通過定性鑒別和定量分析,可確保用于藥效學試驗的樣品質(zhì)量。2.廣藿香油對UV所致小鼠皮膚光老化模型的保護作用研究以KM種小鼠作為實驗動物,分為NC、SC、MC、VC、PO-L、PO-H、PO-M、PO-H等七組,每組9只。采用UVA和UVB同時照射小鼠,制備光老化模型。以廣藿香油局部涂抹的預給藥方式,考察其對皮膚光老化模型的保護作用。實驗中,將小鼠背部局部毛發(fā)去除使皮膚裸露,用廣藿香油處理2小時后于紫外燈下照射。通過宏觀評分、提皮試驗,以宏觀評分值及皮膚恢復時間為指標,探討其對UV,所致小鼠皮膚光老化模型的保護作用。宏觀評分結(jié)果：PO-H組給藥六周后顯現(xiàn)出可抑制UV誘導的廣泛紅斑和深皺紋的趨勢；PO-M組給藥八周后顯示出對皮膚皺紋形成具有抑制作用；實驗結(jié)束時,PO-H組和PO-M組分數(shù)明顯低于VC組,提示PO局部給藥可抑制UV誘導的皮膚表觀損傷。皮膚彈性試驗結(jié)果：與VC組比較PO-L組無顯著差異,PO-M、PO-H組均顯著減少皮膚恢復時間；結(jié)果表明,廣藿香油局部給藥(特別是6mg／只和9mg/只)可促進皮膚彈性。宏觀評分及提皮試驗結(jié)果共同提示,廣藿香油局部給藥對UV所致小鼠皮膚光老化模型具有顯著的保護作用。3.基于皮膚病理學及氧化應(yīng)激損傷機制探討廣藿香抗皮膚光老化的作用機理采用HE染色法與Gomori's醛品紅染色法觀察廣藿香對光老化皮膚組織結(jié)構(gòu)的影響,通過觀察皮膚角質(zhì)層和表皮厚度變化,膠原纖維及彈性纖維的形態(tài)、含量及分布,總體評價廣藿香油對光老化皮膚組織結(jié)構(gòu)的影響。表皮厚度測定結(jié)果：PO-M和PO-H組可見表皮厚度顯著減小,分別減少19.8m和23.7μm；結(jié)果提示PO-M、PO-H組均可顯著減輕UV誘導的表皮增生。組織特征觀察結(jié)果：廣藿香油局部給藥可改善UV照射引起的皮膚結(jié)構(gòu)改變,如表皮異常增厚；表皮真皮連接處扁平化,表皮突與真皮乳頭消失；膠原纖維斷裂、減少、排列紊亂；彈性纖維變性斷裂；提示廣藿香油可抑制UV照射引起的異常組織改變�；谘趸瘧�(yīng)激損傷機制,采用生化指標測定方法考察廣藿香油對光老化皮膚中膠原蛋白與丙二醛(MDA)含量的影響,對光老化皮膚中對超氧化物歧化酶(SOD)、谷胱甘肽過氧化物酶(GSH-Px)、過氧化氫酶(CAT)等活性的影響。實驗結(jié)果,PO-M和PO-H組均可顯著提高膠原蛋白含量分別達21.9%和26.3%,顯著降低光老化皮膚的MDA含量,顯著增加SOD、GSH-Px活力。PO-L組與PO-H組顯著增加CAT活力分別為29.8%和90.7%,PO-M組未對CAT活力有顯著影響。結(jié)果提示廣藿香油可減輕UV誘導的膠原蛋白損壞,抑制UV照射小鼠引起的脂質(zhì)過氧化反應(yīng),減輕UV照射引起的SOD、 GSH-Px、CAT活力降低。4.探討廣藿香發(fā)揮“芳香化濁,發(fā)表解暑”之功拮抗皮膚光老化的現(xiàn)代科學內(nèi)涵結(jié)合分子機制和中醫(yī)理論進行探討：UV照射屬“外熱”,ROS屬“痰濁”,UV照射引起皮膚光老化即為“外熱侵襲所致皮膚衰老”,ROS損傷引起皮膚細胞功能障礙即為“痰濁阻滯引起皮膚氣血運行失�！�。廣藿香油抑制UV照射引起的皮膚異常結(jié)構(gòu)改變,即為“發(fā)表解暑”,而廣藿香油具有芳香屬性并能抑制ROS損傷即為“芳香化濁”。因此,廣藿香油發(fā)揮“芳香化濁,發(fā)表解暑”之功,而起抗皮膚光老化作用。結(jié)論：根據(jù)酶促抗氧化防御系統(tǒng)可調(diào)節(jié)ROS清除自由基的理解,抗氧化劑治療被認為是抗皮膚光老化的有效方法。廣藿香油作為一種抗氧化劑,局部給藥對UV所致小鼠皮膚光老化模型具有顯著的保護作用。這種保護作用可能通過減輕表皮增生,抑制皮膚組織結(jié)構(gòu)異常改變,抑制膠原蛋白損壞等機制發(fā)生,也可能通過抑制脂質(zhì)過氧化反應(yīng),提高可清除自由基的抗氧化酶活力等機制產(chǎn)生,這可能與其抑制UV誘導的ROS損傷機制有關(guān)。本論文首次全面地證明廣藿香油局部給藥對UV誘導的小鼠皮膚光老化模型具有保護作用,為其可被作為一種的潛在的美容功能產(chǎn)品用于皮膚護理提供證據(jù),并從分子機制詮釋廣藿香油發(fā)揮“芳香化濁,發(fā)表解暑”之功拮抗皮膚光老化的現(xiàn)代科學內(nèi)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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R285.5;R-332

【引證文獻】

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本文編號：2236898