TWI702960B - Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing - Google Patents
Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing Download PDFInfo
- Publication number
- TWI702960B TWI702960B TW107123164A TW107123164A TWI702960B TW I702960 B TWI702960 B TW I702960B TW 107123164 A TW107123164 A TW 107123164A TW 107123164 A TW107123164 A TW 107123164A TW I702960 B TWI702960 B TW I702960B
- Authority
- TW
- Taiwan
- Prior art keywords
- wound
- extract
- osmanthus fragrans
- osmanthus
- composition
- Prior art date
Links
Images
Landscapes
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
本發明提供一種桂花萃取物用於製備促進毛髮增長組合物及傷口癒合組合物之用途。The present invention provides a use of Osmanthus fragrans extract for preparing hair growth promoting composition and wound healing composition.
桂花 (Osmanthus fragrans (Thunb) Lour.),為木犀科(Oleaceae) 木犀屬(Osmanthus)常綠灌木,花芳香,可萃取其精油,製備桂花浸膏,並可 用於食品及化粧品的生產,在醫療上,具有溫肺化飲及散寒止痛作用。在藥用 上,其根或根皮可用於治胃痛、牙痛、風濕麻木及筋骨疼痛;其花可用於化痰、 散瘀、治痰飲咳喘、腸風血痢、疝瘕、牙痛及口臭;而其果實可用於暖胃、平肝、益腎、散寒及治肝胃氣痛。Osmanthus ( Osmanthus fragrans (Thunb) Lour.) is an evergreen shrub of Oleaceae (Oleaceae) Osmanthus (Osmanthus). The flower is fragrant. Its essential oil can be extracted to prepare osmanthus extract. It can be used in the production of food and cosmetics. Above, it has the effects of warming the lung, dispelling cold and relieving pain. In medicinal use, its root or root bark can be used to treat stomach pain, toothache, rheumatism, numbness, and bone pain; its flower can be used to resolve phlegm, dispel blood stasis, treat phlegm, cough, intestinal wind and blood dysentery, hernia, toothache and bad breath ; And its fruit can be used to warm the stomach, calm the liver, invigorate the kidney, dispel cold and treat liver and stomach pain.
曾有中華民國(臺灣)專利公告第I341205號(專利申請案第 096137207號),揭示一種由桂花中萃取抗氣喘物質之方法,以乙醇萃取得具抗 氣喘生理功效的物質,可減緩呼吸道及肺部的慢性發炎症狀,達到舒緩氣喘症狀,及預防與減少氣喘之發作,具有明顯的保健作用。中華民國(臺灣)專利公告第I442929號(專利申請案第101118954號),揭示一種桂花水萃取的產物可被用來治療肺纖維化的用途。中華民國(臺灣)專利公告第I411440號(專利申請案第100119436號),揭示一種桂花萃取物在製備預防或治療憂鬱症藥物或保健食品之用途。There has been a Republic of China (Taiwan) Patent Publication No. I341205 (Patent Application No. 096137207), which discloses a method of extracting anti-asthmatic substances from Osmanthus fragrans, and extracting substances with anti-asthmatic effects with ethanol, which can relieve the respiratory tract and lung The chronic inflammatory symptoms in the lower part of the body can relieve the symptoms of asthma, prevent and reduce the onset of asthma, and have obvious health care effects. The Republic of China (Taiwan) Patent Publication No. I442929 (Patent Application No. 101118954) discloses that a product extracted from Osmanthus fragrans water can be used to treat lung fibrosis. The Republic of China (Taiwan) Patent Publication No. I411440 (Patent Application No. 100119436) discloses the use of an Osmanthus fragrans extract in the preparation of drugs or health foods for the prevention or treatment of depression.
另有中國專利公開第CN103768152 A號(專利申請案第201410043501.8號),揭示一種桂花苯乙醇苷提取物製備防治糖尿病的食品、藥物或保健品的應用。中國專利公開第CN103767975 A號(專利申請案第201410043486.7號),揭示一種桂花苯乙醇苷提取物製備美白化妝品的應用。中國專利公開第CN103768151 A號(專利申請案第201410043488.6號),揭示一種桂花苯乙醇苷提取物製備抗衰老藥物或保健品的應用。中國專利公開第CN103223018 A號(專利申請案第201310169455.1號),揭示一種桂花多酚類提取物在製備抗腫瘤藥物或保健品的應用。In addition, Chinese Patent Publication No. CN103768152 A (Patent Application No. 201410043501.8) discloses the application of an Osmanthus fragrans phenylethanol glycoside extract to prepare food, medicine or health care products for preventing and treating diabetes. Chinese Patent Publication No. CN103767975 A (Patent Application No. 201410043486.7) discloses the application of an Osmanthus fragrans phenylethanol glycoside extract to prepare whitening cosmetics. Chinese Patent Publication No. CN103768151 A (Patent Application No. 201410043488.6) discloses an application of an Osmanthus fragrans phenylethanol glycoside extract to prepare anti-aging drugs or health products. Chinese Patent Publication No. CN103223018 A (Patent Application No. 201310169455.1) discloses the application of an Osmanthus fragrans polyphenol extract in the preparation of anti-tumor drugs or health products.
然而並無任一相關文獻曾報導桂花或其萃取物具有促進毛髮增長及傷口癒合之用途。本發明非可預期的發現一種桂花萃取物用於促進毛髮增長及傷口癒合的新穎用途。However, there is no related literature that has reported that Osmanthus fragrans or its extract has the function of promoting hair growth and wound healing. The present invention unexpectedly finds a novel use of Osmanthus fragrans extract for promoting hair growth and wound healing.
本發明一方面提供一種桂花萃取物用於製備促進毛髮增長組合物之用途。One aspect of the present invention provides a use of Osmanthus fragrans extract for preparing a hair growth promoting composition.
本發明另一方面提供一種桂花萃取物用於製備促進傷口癒合組合物之用途。Another aspect of the present invention provides a use of Osmanthus fragrans extract for preparing a composition for promoting wound healing.
在本發明實施例中,本發明桂花萃取物可促進血管新生、及促進膠原蛋白生成。In the embodiment of the present invention, the Osmanthus fragrans extract of the present invention can promote angiogenesis and promote collagen production.
在本發明實施例中,本發明桂花萃取物其中該桂花萃取物係先以純水萃取後再以乙醇溶液萃取而得。In the embodiment of the present invention, the Osmanthus fragrans extract of the present invention is obtained by first extracting with pure water and then extracting with an ethanol solution.
本發明之該等及其它方面,可藉由以下之較佳具體實施例之描述以及圖式,得以更為明晰;即便其中可能會有變化或修飾,但不背離本發明所揭露之新穎觀念的精神及範疇。These and other aspects of the present invention can be made clearer by the following descriptions and drawings of the preferred embodiments; even though there may be changes or modifications, they do not deviate from the novel concepts disclosed by the present invention Spirit and category.
除非另有定義,所有本文所用之技術性及科學性術語,對於屬於本發明領域之具有通常知識者而言,皆具有與其所習知者相同意義。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those who are acquainted with in the field of the present invention.
除非文中有清楚指明者,於本文中所使用之單數形式「一」、「一種」、及「該」之涵義均為包括「至少一種」的複數形式。因此,例如,當提及「一成分」時,包括複數個該等成分及對該領域具有通常知識者所知之同等物。Unless clearly specified in the text, the meanings of the singular forms "one", "one", and "the" used in this article all include the plural form of "at least one". Therefore, for example, when referring to "an ingredient", it includes a plurality of such ingredients and equivalents known to those with ordinary knowledge in the field.
此處所使用的「萃取物」指針對一材料進行萃取所得之產物,通常是藉由將所欲萃取的材料浸泡或混合於溶劑中而獲得的溶液或濃縮製劑。典型地,萃取物係製備自新鮮植物或經研磨或乾燥之植物樣本。The "extract" used here refers to the product obtained by extracting a material, usually a solution or concentrated preparation obtained by immersing or mixing the material to be extracted in a solvent. Typically, the extract is prepared from fresh plants or ground or dried plant samples.
此處所使用的「傷口」包括感染性傷口或非感染性傷口,係指是對於表皮的傷害(例如皮膚的真皮層)的傷害,包括皮膚撕裂、切斷、或刺破。傷口包括但不限於開放性傷口,例如切口、砍傷、割傷、擦傷、刺傷、外傷性皮膚損傷、滲透性創傷,燒傷;疾病或其它慢性組織損傷導致的傷口;由外來傷害或外科手術導致的傷口;醫療過程引起的傷口,例如皮膚科手術。傷口亦可由皮質類固醇反應所以引起皮膚症狀,例如過敏性皮膚炎。"Wound" as used herein includes infectious wounds or non-infectious wounds, and refers to injuries to the epidermis (such as the dermis layer of the skin), including skin tears, cuts, or punctures. Wounds include but are not limited to open wounds, such as incisions, cuts, cuts, abrasions, stab wounds, traumatic skin injuries, penetrating wounds, burns; wounds caused by disease or other chronic tissue damage; caused by external injuries or surgical operations Wounds; wounds caused by medical procedures, such as dermatological surgery. Wounds can also cause skin symptoms such as allergic dermatitis due to corticosteroid reactions.
根據本發明,於動物實驗中發現桂花萃取物具有促進毛髮增長之功效。因此,本發明提供一種桂花萃取物用於製備促進毛髮增長組合物之用途。該促進毛髮增長組合物可製成藥物、洗髮精、潤絲精、護髮劑、染髮劑、髮油或噴劑的形式。According to the present invention, it has been found in animal experiments that Osmanthus fragrans extract has the effect of promoting hair growth. Therefore, the present invention provides a use of Osmanthus fragrans extract for preparing hair growth promoting composition. The hair growth promoting composition can be made into the form of medicine, shampoo, conditioner, hair conditioner, hair dye, hair oil or spray.
另一方面,本發明又提供一種桂花萃取物促進傷口癒合之功效。因此,本發明提供一種桂花萃取物用於製備促進傷口癒合組合物之用途。該促進傷口癒合組合物可製成藥物、護膚劑、防蟲劑或噴劑的形式。On the other hand, the present invention also provides an Osmanthus fragrans extract to promote wound healing. Therefore, the present invention provides a use of Osmanthus fragrans extract for preparing a composition for promoting wound healing. The wound healing promoting composition can be made into the form of medicine, skin care agent, insect repellent or spray.
根據本發明,該傷口可為感染性傷口或非感染性傷口。在本發明一特定實施例中,其中該感染性傷口係為金黃色葡萄球菌所感染。According to the present invention, the wound may be an infectious wound or a non-infectious wound. In a specific embodiment of the present invention, the infectious wound is infected by Staphylococcus aureus.
如本發明實施例所載,本發明桂花萃取物可促進血管新生、及促進膠原蛋白生成。As described in the examples of the present invention, the Osmanthus fragrans extract of the present invention can promote angiogenesis and promote collagen production.
根據本發明之一實施態樣,該桂花萃取物係先以純水萃取後再以乙醇溶液萃取而得。其中一實施例中,該桂花萃取物係取桂花(Osmanthus fragrans )以純水萃取過濾濃縮後,再以乙醇溶液進行萃取,再經過濾且濃縮後而得。According to an embodiment of the present invention, the osmanthus extract is obtained by first extracting with pure water and then extracting with an ethanol solution. In one embodiment, the Osmanthus fragrans extract is obtained by extracting and filtering Osmanthus fragrans with pure water, then extracting with an ethanol solution, and then filtering and concentrating.
為實施本發明,該桂花萃取物可製成各種類型之組合物的形式使用。其中該組合物可為一種溶液、水凝膠、噴霧劑、滴劑或軟膏之形式。此外,亦可進一步包含其他抗菌劑、消炎藥、清潔或護膚之組成份。To implement the present invention, the Osmanthus fragrans extract can be used in the form of various types of compositions. The composition may be in the form of a solution, hydrogel, spray, drops or ointment. In addition, it may further contain other antibacterial agents, anti-inflammatory agents, cleansing or skin care components.
依本發明,其藥品型式可依任何習知技術配合常用的賦形劑或填充劑。亦可依一般醫藥上常用之技術或方法,含有醫療有效量之本發明桂花萃取物,佐以醫藥上可接受之載劑調製成藥品。According to the present invention, the pharmaceutical form can be combined with commonly used excipients or fillers according to any conventional technology. It can also be formulated into medicines according to common techniques or methods commonly used in medicine, containing a medically effective amount of the Osmanthus fragrans extract of the present invention, together with a pharmaceutically acceptable carrier.
以上述發明說明以及下列實施例說明本發明,但並非用以限制本發明之範圍。The above description of the invention and the following examples illustrate the present invention, but are not intended to limit the scope of the present invention.
實施例Example
實施例1:桂花萃取物之製備及其處理Example 1: Preparation and treatment of Osmanthus fragrans extract
取1.0 kg桂花(Osmanthus fragrans )加入10-20倍體積之純水,加熱萃取並過濾,接著將萃取液濃縮後,再以乙醇溶液進行萃取,過濾且濃縮後,可得桂花萃取物(約250.4 g)。Take 1.0 kg of osmanthus ( Osmanthus fragrans ) into 10-20 times the volume of pure water, heat to extract and filter, then concentrate the extract, then extract with ethanol solution, filter and concentrate, you can get osmanthus extract (about 250.4 g).
實施例2:毛髮生長模式評估Example 2: Evaluation of hair growth patterns
本發明之實驗動物係購自國家實驗動物中心之C57BL/6小鼠,年齡為6週。該實驗動物飼養於國防醫學院動物中心,其動物房溫度維持18~26°C,相對溼度為30%~70%,光照週期為12小時光照,12小時黑夜,並且提供充足飼料和飲水。The experimental animal of the present invention is C57BL/6 mice purchased from the National Laboratory Animal Center, and the age is 6 weeks. The experimental animal was raised in the Animal Center of the National Defense Medical College. The temperature of the animal room was maintained at 18~26°C, the relative humidity was 30%~70%, the photoperiod was 12 hours of light and 12 hours of night, and sufficient feed and water were provided.
為確認桂花萃取物之促進毛髮生長的活性進行分組處理,其中,正對照組為以習知的生髮成分敏諾西迪(Minoxidil)處理。將小鼠隨機分為三組如下:空白對照組(BC;10%乙醇做為載劑)、正對照組(PC;3% 敏諾西迪)及桂花萃取物(CE;1%加入10%乙醇中)。每一組各5隻小鼠予以測試。桂花萃取物之促建毛髮生長的活性係使用部分修改Choi等人的方法(Choi et al., Biol Pharm Bull 37:44-53, 2014)。簡而言之,小鼠藉由電剪小心剃除背側部一2 cm×3 cm 面積的毛髮。待測物(100 μL)係局部施加於小鼠背側部的皮膚,每天1次連續4週。該待測物的毛髮生長增進活性係以背側部皮膚變黑作為評估標準,其表示該毛髮之毛囊係於生長期。In order to confirm the hair growth-promoting activity of the Osmanthus fragrans extract, group treatments were carried out. Among them, the positive control group was treated with the known hair growth ingredient Minoxidil. The mice were randomly divided into three groups as follows: blank control group (BC; 10% ethanol as a carrier), positive control group (PC; 3% minosidic) and osmanthus extract (CE; 1% with 10% ethanol) in). Five mice in each group were tested. The hair growth-promoting activity of Osmanthus fragrans extract was partially modified by Choi et al. (Choi et al., Biol Pharm Bull 37:44-53, 2014). In short, the mouse carefully shaved a 2 cm×3 cm area of hair on the dorsal side with electric scissors. The test substance (100 μL) was locally applied to the skin of the back of the mouse once a day for 4 weeks. The hair growth enhancing activity of the test substance is based on the darkening of the dorsal skin as an evaluation standard, which means that the hair follicle of the hair is in the growth phase.
毛髮生長評分係由兩位不知悉實施方案的獨立實驗室成員執行。毛髮生長係每週測量一次,連續4週,毛髮生長分數定義如下:0分=沒有觀察到生長;1分=達到20%生長;2分=達到20-40%生長;3分=達到40-60%生長;4分=達到60-80%生長;及5分=達到80-100%生長。4週之毛髮總生長數位影像係使用Nikon Cool Pix P100(Tokyo, Japan)攝得。將該二個別評分的平均作為評估毛髮生長之毛髮生長指數。The hair growth scoring system was performed by two independent laboratory members who were not aware of the implementation plan. The hair growth line is measured once a week for 4 consecutive weeks. The hair growth score is defined as follows: 0 points = no growth is observed; 1 point = 20% growth; 2 points = 20-40% growth; 3 points = 40- 60% growth; 4 points = achieve 60-80% growth; and 5 points = achieve 80-100% growth. The digital image of total hair growth for 4 weeks was taken with Nikon Cool Pix P100 (Tokyo, Japan). The average of the two individual scores is used as the hair growth index for evaluating hair growth.
觀察小鼠處理後結果,於第16天時,發現桂花萃取物組較其他組其傷口外觀較完整、傷口組織表皮層已完全復原、真皮層結構與正常小鼠較為相似。各組小鼠自處理後第0-28天之傷口照片影像如圖1A所示,相較於空白對照組及正對照組,桂花萃取物組在第14日起小鼠背部皮膚已長出毛髮,其毛囊發育、毛髮生成面積與毛髮量在第16日起至28日止均顯著高於其他組別。其量化結果以毛髮生長指數呈現,如圖1B顯示,以桂花萃取物處理之毛髮生長相較於其他組有顯著差異(詳見圖1B)。各組小鼠傷口處理後之皮膚組織染色照片影像如圖1C所示,由皮膚組織染色可知相較於空白對照組及正對照組,桂花萃取物組其毛囊發育明顯且有毛髮生長。由以上結果顯示,桂花萃取物具有促進毛髮生長之作用。Observing the results after treatment of the mice, on the 16th day, it was found that the Osmanthus fragrans extract group had a more complete wound appearance, the epidermal layer of the wound tissue was completely restored, and the dermal layer structure was more similar to that of normal mice. The wound photos of mice in each group from 0-28 days after treatment are shown in Figure 1A. Compared with the blank control group and the positive control group, the osmanthus extract group has grown hair on the back skin of the mice on the 14th day , Its hair follicle development, hair growth area and hair volume were significantly higher than other groups from
由實施例結果可知,本發明所提供之桂花萃取物能明顯促進毛髮生長之作用。It can be seen from the results of the examples that the Osmanthus fragrans extract provided by the present invention can significantly promote hair growth.
實施例3:傷口癒合實驗動物模式評估Example 3: Evaluation of experimental animal model for wound healing
1. 實驗動物及模式1. Experimental animals and models
本實驗之實驗動物係購自國家實驗動物中心之C57BL/6J Narl品系小鼠,體重40~45 g,年齡為8週。該實驗動物飼養於國防醫學院動物中心,其動物房溫度維持18~26°C,相對溼度為30%~70%,光照週期為12小時光照,12小時黑夜,並且提供充足飼料和飲水。The experimental animals used in this experiment were purchased from the C57BL/6J Narl strain mice of the National Laboratory Animal Center, weighing 40-45 g, and aged 8 weeks. The experimental animal was raised in the Animal Center of the National Defense Medical College. The temperature of the animal room was maintained at 18~26°C, the relative humidity was 30%~70%, the photoperiod was 12 hours of light and 12 hours of night, and sufficient feed and drinking water were provided.
各實驗模式之組別如下表所示: 其中,正對照組為敏諾西迪(Minoxidil)。The groups of each experimental mode are shown in the table below: Among them, the positive control group is Minoxidil.
2. 傷口處理2. Wound treatment
手術前將小鼠以腹腔注射方式給予0.6 g/kg Avertin,其中該Avertin 係以0.6 g 2,2,2-三溴乙醇(2,2,2-Tribromoethanol)及1.5 ml 2-甲基2丁醇(2- Methyl-2-butanol)混合後添加生理食鹽水至50 ml,以0.2μm 濾紙過濾,並以4°C保存。待小鼠麻醉後,將其背部毛髮剃除。將紙卡固定於小鼠背部確定傷口位置,以麥克筆描繪出1.2 x 1.2 cm2的面積。使用手術剪刀將此面積內的皮膚剪掉,形成傷口。於感染性創傷模式中,將金黃色葡萄球菌稀釋至105
CFU/150 μl,並於每隻小鼠加入150 μl菌液,使菌液完全滲入皮膚內約5分鐘(非感染性創傷模式不需進行此步驟)。使用棉花棒沾取適量待測試藥物後,將其均勻塗抹在傷口上。使用防水透氣敷料(Tegaderm,3M)覆蓋傷口,以透氣膠帶將防水透氣敷料予以固定,避免防水透氣敷料脫落與其他傷口感染。Before the operation, mice were given 0.6 g/kg Avertin by intraperitoneal injection, where the Avertin was 0.6
3. 金黃色葡萄球菌培養與計算3. Cultivation and calculation of Staphylococcus aureus
以接種環挑取單一金黃色葡萄球菌菌落,以分區畫線法接種於LB瓊脂碟(LB agar plate)上,並於37°C培養箱中培養約16小時。以接種環挑取單一金黃色葡萄球菌菌落,將其放入含有LB培養基之細菌培養試管培養約16小時。將試管中菌液取出,於4°C、7,000 rpm 下離心20分鐘。移除上清液,並將離心所得金黃色葡萄球菌菌體以LB培養基稀釋至1 x 107 CFU/ml備用。Pick a single colony of Staphylococcus aureus with an inoculating loop, and inoculate it on an LB agar plate by dividing and drawing lines, and incubate it in an incubator at 37°C for about 16 hours. Pick a single colony of Staphylococcus aureus with an inoculating loop, and place it in a bacterial culture test tube containing LB medium for about 16 hours. Take out the bacterial solution from the test tube and centrifuge at 4°C and 7,000 rpm for 20 minutes. Remove the supernatant, and dilute the Staphylococcus aureus cells obtained by centrifugation with LB medium to 1 x 107 CFU/ml for later use.
將菌液以序列稀釋至10-9。從不同稀釋倍數試管中取出100 μl菌液並加入LB瓊脂碟並均勻塗抹。在37°C培養箱培養約16小時並紀錄菌落數。原始菌液中每毫升所含菌數(CFU/ml)=菌落數*稀釋倍數*10。Dilute the bacterial solution to 10-9 in sequence. Take out 100 μl of bacterial solution from the test tube of different dilution multiples and add it to the LB agar dish and spread evenly. Incubate in a 37°C incubator for about 16 hours and record the number of colonies. The number of bacteria per milliliter in the original bacterial solution (CFU/ml) = the number of colonies * the dilution factor * 10.
4. 抑菌環試驗4. Antibacterial ring test
從不同稀釋倍數試管中取出100 μl金黃色葡萄球菌液,分別滴於瓊脂碟並均勻塗抹,待菌液被瓊脂吸入後,以鑷子將濾紙平貼於瓊脂上,將每張濾紙滴入50 μl 桂花萃取物。在37°C培養箱中培養約16小時後,測量液菌直徑抑菌環範圍 = 抑菌環直徑(mm)-濾紙直徑(mm)。Take out 100 μl of Staphylococcus aureus liquid from the test tubes of different dilution multiples, drop them on the agar plate and apply evenly. After the bacterial liquid is sucked into the agar, use tweezers to flatten the filter paper on the agar, and drop each filter paper into 50 μl Osmanthus extract. After culturing in a 37°C incubator for about 16 hours, measure the diameter of the liquid bacteria and the range of inhibition ring = diameter of inhibition ring (mm)-diameter of filter paper (mm).
5. 傷口組織細菌含量試驗5. Bacteria content test in wound tissue
在感染性創傷模式小鼠實驗第七天後,收集創傷小鼠背部傷口的化膿組織。將組織剪成碎片,並以超音波細胞粉碎機將組織打碎。於4°C下以1,500 rpm離心8分鐘後,收集上清液。將該上清液進行序列稀釋,菌液均勻塗抹於瓊脂上後,於37°C培養箱中培養約16小時後,計算菌落數。每克組織所含菌數(CFU/g)=(菌落數*稀釋倍數*10)/ 組織克數。After the seventh day of the infectious wound model mouse experiment, the purulent tissue from the back wound of the wounded mouse was collected. Cut the tissue into pieces and break the tissue with an ultrasonic cell crusher. After centrifugation at 1,500 rpm for 8 minutes at 4°C, the supernatant was collected. The supernatant was serially diluted, the bacterial solution was evenly smeared on the agar, and then incubated in a 37°C incubator for about 16 hours, and the number of colonies was counted. The number of bacteria per gram of tissue (CFU/g) = (number of colonies * dilution factor * 10) / grams of tissue.
6. 皮膚組織切片及蘇木精及伊紅(Hematoxylin & Eosin, H&E)染色6. Skin tissue section and Hematoxylin & Eosin (H&E) staining
將適量OCT(optimum cutting temperature)包埋劑加入鋁箔組織包埋盒內,把新鮮取下之皮膚組織放入包埋盒內,再加入OCT包埋劑將整個組織予以覆蓋,並放在液態氮上快速冷凍後,至於-70°C保存。在鋁合金組織標本台上加入適量OCT包埋劑,把預處理後之組織至於標本台上,接著將標本台放在切片機冷凍台上冷凍,並把標本台放在切片機上且鎖緊。接著,以粗切模式(20 μm)使組織塊形成平面,再利用細切模式(5~7 μm)開始切片。將載玻片貼附於組織薄片,並將玻片浸泡在丙酮中,置於-20°C,30分鐘後將玻片放在-70°C保存。Add an appropriate amount of OCT (optimum cutting temperature) embedding agent into the aluminum foil tissue embedding box, put the freshly removed skin tissue into the embedding box, and then add OCT embedding agent to cover the entire tissue and place it in liquid nitrogen After being quickly frozen, store at -70°C. Add an appropriate amount of OCT embedding agent to the aluminum alloy tissue specimen table, put the pretreated tissue on the specimen table, then place the specimen table on the microtome freezing table for freezing, and place the specimen table on the microtome and lock it tightly . Next, use the rough cut mode (20 μm) to flatten the tissue block, and then use the fine cut mode (5-7 μm) to start sectioning. Attach the glass slide to the tissue slice, and soak the glass slide in acetone at -20°C. After 30 minutes, store the glass slide at -70°C.
將組織玻片浸泡在PBS緩衝液約3~4分鐘後,將玻片以蘇木精染色10分鐘。將玻片以純水清洗三次後,再浸泡於純水,分別為5、5、5、10分鐘。將著將玻片以伊紅染色15秒。將玻片分別浸泡於95%,95%,100%,100%酒精,各1分鐘。再浸泡於二甲苯(xylene)1分鐘。以70%酒精將玻片背面擦拭乾淨,等待樣本乾燥。最後以蓋玻片封片(封片劑:二甲苯=2:1)。After immersing the tissue slides in PBS buffer for about 3 to 4 minutes, stain the slides with hematoxylin for 10 minutes. After washing the slides with pure water three times, soak them in pure water for 5, 5, 5, and 10 minutes respectively. Stain the slide with eosin for 15 seconds. Soak the slides in 95%, 95%, 100%, and 100% alcohol, respectively, for 1 minute. Soak in xylene for 1 minute. Wipe the back of the slide with 70% alcohol and wait for the sample to dry. Finally, mount the slide with a cover glass (mounting tablet: xylene=2:1).
7. 結果7. Results
在非感染性創傷模式中,對小鼠傷口外觀變化進行評估。首先,在C57BL/6小鼠背部皮膚開創傷口後,每二天清理傷口並更換藥物。如圖2A所示,相較於其他組別,非感染性傷口以桂花萃取物進行處理後,其傷口具有較佳的癒合效率。如圖2B所示,於第4至8天時,空白對照組及凡士林組其傷口癒合並不明顯。相較之下,桂花萃取物組其傷口外觀已明顯開始癒合。由結果顯示,在給予桂花萃取物治療後,非感染性傷口可提前約4至8天癒合。In the non-infectious wound model, changes in the appearance of wounds in mice were evaluated. First, after creating a wound on the back skin of C57BL/6 mice, the wound was cleaned and the medicine was changed every two days. As shown in Figure 2A, compared with other groups, non-infectious wounds treated with Osmanthus fragrans extract have better wound healing efficiency. As shown in Fig. 2B, on the 4th to 8th day, the wound healing of the blank control group and the petrolatum group was not obvious. In contrast, the appearance of the wound in the Osmanthus fragrans extract group has obviously begun to heal. The results show that after treatment with Osmanthus fragrans extract, non-infectious wounds can heal about 4 to 8 days earlier.
而在感染性創傷模式中,以金黃色葡萄球菌感染傷口,於感染第七天後,每兩天清理傷口並更換藥物。如圖3A所示,相較於其他組別,感染性傷口以桂花萃取物進行處理後,其傷口有顯著縮小的趨勢。在第6天時,以桂花萃取物處理之傷口,已達到60%以上的傷口癒合。如圖3B所示,於第6天時,空白對照組及凡士林組其傷口尚未開始癒合。相較之下,桂花萃取物組其傷口外觀已顯著開始癒合。由結果顯示,於感染性創傷模式中給予桂花萃取物治療後,傷口可提前約8至12天癒合。In the infectious wound mode, the wound is infected with Staphylococcus aureus. After the seventh day of infection, the wound is cleaned and the medicine is changed every two days. As shown in Figure 3A, compared with other groups, the infected wounds have a significant shrinking trend after being treated with Osmanthus fragrans extract. On the 6th day, the wounds treated with Osmanthus fragrans extract had reached 60% wound healing. As shown in Figure 3B, on the 6th day, the wounds of the blank control group and the petrolatum group had not yet begun to heal. In contrast, the osmanthus extract group had a significant appearance of wounds and began to heal. The results show that after treatment with Osmanthus fragrans extract in the infectious wound mode, the wound can heal about 8 to 12 days earlier.
無論在非感染性創傷模式(圖4A)或感染性創傷模式(圖4B)中,其傷口皮膚外觀可發現,相較於空白對照組和凡士林組,桂花萃取物組其具有顯著促進血管面積、長度和分支發展的情形,顯示桂花萃取物顯著具有促進血管新生之功效。No matter in the non-infectious trauma model (Figure 4A) or infectious trauma model (Figure 4B), the appearance of the wound skin can be found. Compared with the blank control group and the petroleum jelly group, the Osmanthus fragrans extract group has a significant promotion of blood vessel area, The length and branch development show that the extract of Osmanthus fragrans has a significant effect of promoting angiogenesis.
因膠原蛋白的合成有助於傷口癒合,進一步檢視傷口癒合時,桂花萃取物醋促進膠原蛋白合成的效果。對於非感染性創傷模式,在小鼠創傷第21天,而對於感染性創傷模式,則在第27天,取傷口周圍皮膚組織,以Masson’s trichrom染色法觀察皮膚組織內膠原蛋白合成的情形,膠原蛋白被染成藍色呈現。觀察實驗結果,發現無論在非感染性或感染創傷模式,與正常小鼠比較下,在空白對照組和凡士林組中,組織內部膠原蛋白合成數量少且結構鬆散;而在以桂花萃取物處理的組別中,組織內部膠原蛋白合成數量和結構緊密程度都與正常小鼠相似。由以上結果顯示,桂花萃取物具有促進膠原蛋白合成的功效。As the synthesis of collagen helps wound healing, further examine the effect of osmanthus extract vinegar in promoting collagen synthesis during wound healing. For the non-infectious trauma model, on the 21st day of the mouse trauma, and for the infectious trauma model, on the 27th day, take the skin tissue around the wound and observe the synthesis of collagen in the skin tissue by Masson’s trichrom staining method. The protein is dyed blue to appear. Observing the experimental results, it was found that compared with normal mice in non-infectious or infected trauma mode, in the blank control group and petrolatum group, the amount of collagen synthesis in the tissue was less and the structure was loose; while in the osmanthus extract treated In the group, the amount of collagen synthesis and the tightness of the structure within the tissue are similar to those of normal mice. The above results show that Osmanthus fragrans extract has the effect of promoting collagen synthesis.
傷口感染為造成傷口無法有效癒合的原因之一,因此以抑菌環試驗測試桂花萃取物是否具有抑菌的活性。試驗結果顯示金黃色葡萄球菌以藥物處理後,與對照組比較後並無發現抑菌現象,顯示桂花萃取物無直接抑菌的作用。Wound infection is one of the reasons why the wound cannot heal effectively. Therefore, the antibacterial circle test is used to test whether the Osmanthus fragrans extract has antibacterial activity. The test results showed that after Staphylococcus aureus was treated with drugs, there was no antibacterial phenomenon after comparison with the control group, indicating that the Osmanthus fragrans extract had no direct antibacterial effect.
進一步將小鼠背部皮膚開創傷口後,以金黃色葡萄球菌感染傷口並以藥物處理,感染7天後,收集傷口周圍化膿組織,分析含菌量。如圖5所示,相較於空白對照組及凡士林組,以桂花萃取物處理後其傷口表面膿菌數明顯減少。結果得知,桂花萃取物具有間接抑菌活性。After the wound was created on the back skin of the mouse, the wound was infected with Staphylococcus aureus and treated with drugs. Seven days after the infection, the purulent tissue around the wound was collected and the bacterial content was analyzed. As shown in Figure 5, compared with the blank control group and the petrolatum group, the number of pus on the wound surface was significantly reduced after treatment with Osmanthus fragrans extract. As a result, it was found that Osmanthus fragrans extract has indirect antibacterial activity.
由以上結果可知,本發明桂花萃取物能促進傷口癒合,具有促進傷口血管新生及促進膠原蛋白生成之功效。From the above results, it can be seen that the Osmanthus fragrans extract of the present invention can promote wound healing, has the effects of promoting wound angiogenesis and promoting collagen production.
無no
在本發明所呈現的較佳具體實施例圖示係以闡述本發明為目的。應被理解的是,本發明並不侷限於所示之較佳具體實施例。The illustrations of preferred specific embodiments presented in the present invention are for the purpose of illustrating the present invention. It should be understood that the present invention is not limited to the preferred embodiments shown.
圖1顯示本發明桂花萃取物其促進毛髮生長的結果。圖1A顯示各組別第0至28天毛髮生長情形。圖1B顯示第0至28天的毛髮生長分數(誤差槓,SE;*,P
<0.05)。圖1C顯示各組別的皮膚組織染色結果。Figure 1 shows the results of the Osmanthus fragrans extract of the present invention in promoting hair growth. Figure 1A shows the hair growth of each group from
圖2顯示本發明桂花萃取物於非感染性創傷模式中傷口癒合之結果。圖2A顯示第0至18天的傷口癒合效率(誤差槓,SE;*,P
<0.05)。圖2B顯示第0至18天該傷口的癒合情形。Figure 2 shows the results of wound healing in the non-infectious wound mode of the Osmanthus fragrans extract of the present invention. Figure 2A shows the wound healing efficiency from
圖3顯示本發明桂花萃取物於感染性創傷模式中該傷口的癒合結果。圖3A顯示第0至18天的傷口癒合效率(誤差槓,SE;*,P
<0.05)。圖3B顯示第0至18天該傷口的癒合情形。Figure 3 shows the healing results of the Osmanthus fragrans extract of the present invention in the infectious wound mode. Figure 3A shows the wound healing efficiency from
圖4顯示本發明桂花萃取物於非感染性創傷模式或感染性創傷模式中促進血管新生的效果。圖4A顯示在非感染性創傷模式中各組別對於血管新生的影響。圖4B顯示在感染性創傷模式中各組別對於血管新生的影響。Figure 4 shows the effect of the Osmanthus fragrans extract of the present invention in promoting angiogenesis in a non-infectious wound mode or an infectious wound mode. Figure 4A shows the effect of each group on angiogenesis in the non-infectious trauma model. Figure 4B shows the influence of each group on angiogenesis in the infectious trauma model.
圖5顯示本發明桂花萃取物之間接抑菌效果;係於非感染性創傷模式或感染性創傷模式中,以金黃色葡萄球菌感染傷口,經各組別處理後之傷口周圍化膿組織之含菌數。Figure 5 shows the bacteriostatic effect of the Osmanthus fragrans extract of the present invention; in the non-infectious wound mode or the infectious wound mode, the wound is infected with Staphylococcus aureus, and the purulent tissue around the wound after treatment of each group contains bacteria number.
無no
Claims (4)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW107123164A TWI702960B (en) | 2015-09-10 | 2015-09-10 | Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW107123164A TWI702960B (en) | 2015-09-10 | 2015-09-10 | Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing |
Publications (2)
Publication Number | Publication Date |
---|---|
TW201838644A TW201838644A (en) | 2018-11-01 |
TWI702960B true TWI702960B (en) | 2020-09-01 |
Family
ID=65033770
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW107123164A TWI702960B (en) | 2015-09-10 | 2015-09-10 | Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing |
Country Status (1)
Country | Link |
---|---|
TW (1) | TWI702960B (en) |
-
2015
- 2015-09-10 TW TW107123164A patent/TWI702960B/en active
Non-Patent Citations (3)
Title |
---|
Chem Phys Lipids. 2010 Feb;163(2):190-9. doi: 10.1016 |
Chem Phys Lipids. 2010 Feb;163(2):190-9. doi: 10.1016 Chem. Pharm. Bull. 47(9) 1341—1343 (1999) * |
Chem. Pharm. Bull. 47(9) 1341—1343 (1999) |
Also Published As
Publication number | Publication date |
---|---|
TW201838644A (en) | 2018-11-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Fikru et al. | Evaluation of in vivo wound healing activity of methanol extract of Achyranthes aspera L. | |
Abd Elgadir et al. | Carica papaya as a source of natural medicine and its utilization in selected pharmacetical applications | |
CN110538198A (en) | application of Sipunculus nudus water extract in wound repair | |
Sharil et al. | Systematic review of flaxseed (Linum usitatissimum L.) extract and formulation in wound healing | |
Charde et al. | Evaluation of ethanolic extract of Moringa oleifera for wound healing, anti-inflammatory and antioxidant activities on rats | |
Muhammed et al. | Effect of application of Fenugreek (Trigonella foenum-graecum) on skin wound healing in rabbits | |
Nema et al. | In vivo topical wound healing activity of punica granatum peel extract on rats | |
KR102026409B1 (en) | Pharmaceutical composition comprising root bark or cortex powder of elm tree for wound healing and skin regeneration | |
Getahun et al. | In vivo evaluation of 80% methanolic leaves crude extract and solvent fractions of buddleja polystachya fresen (buddlejaceae) for wound healing activity in normal and diabetic mice | |
Kumari et al. | Spider web ointment: A traditional based approach in cutaneous wound healing | |
CN109820945B (en) | Composite medicine for promoting skin wound healing and hair growth and preparation method thereof | |
Betul et al. | Wound healing effects of Nigella sativa L. essential oil in streptozotocin induced in diabetic rats | |
US20080102106A1 (en) | Wound Healing Composition Comprising Substances From Diptera Larvae | |
Ghodrati Azadi et al. | Macroscopic evaluation of wound healing activity of the Persian shallot, Allium hirtifolium in rat | |
TWI702960B (en) | Uses of osmanthus fragrans extracts for manufacturing a composition for wound healing | |
CN110638999A (en) | Application of Sipunculus nudus collagen peptide in wound repair | |
Nafiu et al. | Histomorphological evaluation of wound healing-comparison between use of honey and vernonia amygdalina leaf juice | |
CN107693771B (en) | Composition containing catfish body surface antibacterial peptide and active polysaccharide for promoting wound healing | |
Salmah et al. | Synergistic effects of alcoholic extract of sweet basil (Ocimum basilicum L.) leaves and honey on cutaneous wound healing in rats | |
TWI634896B (en) | Uses of osmanthus fragrans extracts for manufacturing a composition for enhancing hair growth and a composition for wound healing | |
CN106511480A (en) | Application of sweet osmanthus flower extract in preparation of composition for promoting hair growth and wound healing | |
CN104887661B (en) | Medical component for promoting wound healing and application thereof | |
KR20220128539A (en) | Composition for wound healing containing Stellera chamaejasme extract using supercritical extraction | |
CN111514119A (en) | Adhesive bandage containing industrial hemp root extract and preparation method thereof | |
CN112206281A (en) | Anti-infection wound repair pharmaceutical composition, preparation method and application thereof |