TW202241482A - Annona montana macf leaves extract for anti-oxidant, anti-inflammation, whitening, repairing and anti-wrinkle - Google Patents

Abstract

The present invention relates to a use of an annona montana macf leaves extract, ant the use is for manufacturing a composition for anti-oxidant, anti-inflammation, whitening, repairing and anti-wrinkle. The composition is used as a raw material of medicine, cosmetics, care products, fragrance, or body cleaning products. The present invention provides another use of an annona montana macf leaves extract, and the use is for manufacturing a composition for killing cancer cells. The composition is used as a raw material of medicine, anti-cancer drugs, nutritional supplements, or health food.

Description

可抗氧化、抗發炎、美白、修復及抗皺的山刺番荔枝葉萃取物Soursop leaf extract for anti-oxidation, anti-inflammation, whitening, repairing and anti-wrinkle

本發明有關一種植物萃取物，特別是一種山刺番荔枝葉萃取物，由該萃取物所製成的組合物可作為醫藥品、化妝品、保養品、香氛或人體清潔用品等產品的原料者。The present invention relates to a plant extract, especially a soursop leaf extract. The composition made from the extract can be used as a raw material for products such as pharmaceuticals, cosmetics, skin care products, fragrances or body cleaning products. .

山刺果番荔枝（Annona montan）包括大約2300種，屬於木瓜木材部門（Annonacea），分佈在美國、非洲、亞洲以及巴西地區。其樹高6〜7m，葉為卵形或長橢圓形且長度為7.5〜15㎝，其葉尖尖、葉片光滑且有光澤，葉柄部分是角膜且其葉柄短；花瓣在六片複葉上，其形狀為蛋形，具有一定厚度，擁有皮革的質感和寬度，但毛髮尖而柔軟；它的漿果重量可以達到2 kg至6 kg，表面可見淡淡的綠色，其形狀為橢圓形且具有香氣。The soursop (Annona montan) includes about 2300 species belonging to the papaya wood division (Annonacea), distributed in the United States, Africa, Asia and Brazil. The height of the tree is 6-7m, the leaves are ovate or oblong and 7.5-15cm in length, the tip of the leaf is pointed, the blade is smooth and shiny, the petiole part is cornea and its petiole is short; the petals are on six compound leaves, the The shape is egg-shaped, has a certain thickness, has the texture and width of leather, but the hair is pointed and soft; its berry weight can reach 2 kg to 6 kg, and a light green color can be seen on the surface. It is oval in shape and has a fragrance.

PCT專利申請號WO2018-178955 A1提出一種組合物，該組合物包含來自番荔枝葉或番荔枝花天然提取物的天然活性成分以及賦形劑和水的特定混合物，該組合物在癌症損傷的治療中達到了已證實的效果。該基於天然化合物的組合物，其特徵在於包含0.5％至5％的番荔枝提取物，至多20％的油、潤膚劑、乳化劑、防腐劑和維生素組成的賦形劑混合物和至多80％的水 。PCT Patent Application No. WO2018-178955 A1 proposes a composition comprising a specific mixture of natural active ingredients from cherimoya leaves or cherimoya flower natural extracts and excipients and water, which is useful in the treatment of cancer lesions have achieved proven results. The composition based on natural compounds, characterized in that it contains 0.5% to 5% of cherimoya extract, up to 20% of excipient mixture consisting of oils, emollients, emulsifiers, preservatives and vitamins and up to 80% of water.

日本特許專利公告號JP 5477731 B2提出一種黑色素生成抑制劑、增白劑和膠原酶抑制劑，其可顯示出對人體安全性極優異的效果，其中以番荔枝科提取物做為有效成分。Japanese Patent Publication No. JP 5477731 B2 proposes a melanin production inhibitor, whitening agent and collagenase inhibitor, which can show excellent effects on human body safety, and the extract of Anemoneaceae is used as the active ingredient.

美國專利公開號US 2004-0101584 A1提出一種來源於番荔枝科植物的提取物組合物及其製備方法，其中該提取物的藥用價值包括腫瘤消退和腫瘤抗原水平降低，其中所述提取物包含至少一種非丙酮類產乙酸原素，此外，所述提取物由非食用類Asimina、Annona、Goniothalamus、Uvaria、Disepalum、Xylopia和Rollinia中的至少一種製備。U.S. Patent Publication No. US 2004-0101584 A1 proposes an extract composition derived from plants of the Annonaceae family and a preparation method thereof, wherein the medicinal value of the extract includes tumor regression and reduction of tumor antigen levels, wherein the extract contains At least one non-acetone acetogen, and furthermore, the extract is prepared from at least one of the non-edible species Asimina, Annona, Goniothalamus, Uvaria, Disepalum, Xylopia and Rollinia.

至少有40種以上的荔枝科乙醯生合成物可從其樹莖、葉片和種子分離出來。許多種類的番荔枝科植物萃取物被應用於民間藥療與殺蟲劑，在這些萃取物的結構成分中，已知具有抗癌作用的番荔枝科乙醯生合成物，被視為主要活性成份構造並且只能從Annonaceae中取得；一般而言，其化學結構為碳數35-37的脂肪酸衍生物連結零到數個的四氫呋喃(Tetrahydrofuran，四氫呋喃)或四氫呋喃(Tetrahydropyran)環與一終端內酯分子團(terminal lactone moiety)。到目前為止已有三百多種化合物被發現並發表，其中大多數為立體異構物。其所含之生物活性如細胞毒素、抗寄生性、殺蟲性與免疫系統抑制性也已被證實。At least 40 kinds of acetylene biosynthetic compounds of Litchiaceae can be isolated from their stems, leaves and seeds. Many kinds of plant extracts of the Annonaceae family are used in folk medicine and insecticides. Among the structural components of these extracts, the acetyl biosynthetic compounds of the Annonaceae family, which are known to have anticancer effects, are considered to be the main active Component structure and can only be obtained from Annonaceae; in general, its chemical structure is a fatty acid derivative with a carbon number of 35-37 linked to zero to several tetrahydrofuran (Tetrahydrofuran, tetrahydrofuran) or tetrahydrofuran (Tetrahydropyran) rings and a terminal lactone Molecular group (terminal lactone moiety). More than 300 compounds have been discovered and published so far, most of which are stereoisomers. Its biological activities such as cytotoxicity, antiparasitic, insecticidal and immune system suppressive properties have also been confirmed.

先前技術指出一種用於預防或治療認知功能障礙的藥物組合物，其包含番荔枝葉提取物作為有效成分，係用於預防或治療認知功能障礙的方法，以及用於預防或治療認知功能障礙的健康食品組合物。治療認知功能障礙的藥物，其包括番荔枝葉提取物或其部分。該發明的組合物抑制澱粉樣β的聚集，並且顯示出對神經細胞的無毒作用和保護神經細胞免受外部刺激的作用。The prior art indicates a pharmaceutical composition for the prevention or treatment of cognitive dysfunction, which comprises an annus apple leaf extract as an active ingredient, a method for preventing or treating cognitive dysfunction, and a method for preventing or treating cognitive dysfunction Healthy food composition. A medicament for the treatment of cognitive dysfunction, which comprises an annus apple leaf extract or a part thereof. The composition of the invention inhibits the aggregation of amyloid β, and exhibits a non-toxic effect on nerve cells and an effect of protecting nerve cells from external stimuli.

本發明之一目的在於提供一種山刺番荔枝葉萃取物的用途，係用於製備抗氧化、抗發炎、美白、修復及抗皺的組合物。One object of the present invention is to provide a use of soursop leaf extract, which is used for preparing anti-oxidation, anti-inflammation, whitening, repairing and anti-wrinkle compositions.

更佳者，該山刺番荔枝葉萃取物的製備方法包含：將山刺番荔枝葉浸泡於水中以取得一山刺番荔枝葉水萃取物。More preferably, the preparation method of the soursop leaf extract comprises: soaking the soursop leaf in water to obtain a soursop leaf water extract.

更佳者，該山刺番荔枝葉水萃取物的製備方法包含：秤取重量比為1：8至1：10之山刺番荔枝葉以及去離子水，放入超音波萃取設備於低溫下連續式萃取， 經高速離心收集上清液，再經抽真空篩檢程式過濾，以獲得澄清過濾液。More preferably, the preparation method of the water extract of soursop leaves comprises: weighing soursop leaves and deionized water with a weight ratio of 1:8 to 1:10, putting them into ultrasonic extraction equipment at low temperature Continuous extraction, the supernatant is collected by high-speed centrifugation, and then filtered through a vacuum screening program to obtain a clarified filtrate.

更佳者，該山刺番荔枝葉水萃取物的製備方法更包含：將該澄清過濾液進行濃縮。More preferably, the preparation method of the soursop leaf water extract further comprises: concentrating the clarified filtrate.

更佳者，每公克該山刺番荔枝葉水萃取物中總多酚之含量為86 mg至 93.8mg，每公克該山刺番荔枝葉水萃取物中總類黃酮之含量為 33.1mg至  39.5mg。More preferably, the content of total polyphenols per gram of the water extract of soursop leaves is 86 mg to 93.8 mg, and the content of total flavonoids per gram of the water extract of soursop leaves is 33.1 mg to 39.5 mg mg.

此外，本發明所揭露上述製成萃取物的用途為用於製備具有活化能量、抗皺、抗發炎、美白、抗氧化、抗老化及保護修復功效的組合物，而此組合物可為醫藥品、化妝品、保養品、香氛或人體清潔用品。In addition, the use of the above-mentioned extracts disclosed in the present invention is used to prepare a composition with activating energy, anti-wrinkle, anti-inflammation, whitening, anti-oxidation, anti-aging and protection repair effects, and this composition can be medicine, Cosmetics, skin care products, fragrances or body cleansing products.

本發明之再一目的在於提供一種山刺番荔枝葉萃取物的用途，係用於製備毒殺癌細胞之組合物。Another object of the present invention is to provide a use of the extract of soursop leaves, which is used to prepare a composition for poisoning and killing cancer cells.

更佳者，該山刺番荔枝葉萃取物的製備方法包含：將山刺番荔枝葉浸泡於乙醇中以取得一山刺番荔枝葉乙醇萃取物。More preferably, the preparation method of the soursop leaf extract comprises: soaking the soursop leaves in ethanol to obtain a soursop leaf ethanol extract.

更佳者，該山刺番荔枝葉之乙醇萃取物的製備方法包含：將該山刺番荔枝葉秤取100公克，於低溫下浸泡於五倍體積之乙醇中，取出後放入超音波萃取設備，於低溫下連續式萃取，經高速離心收集上清液，再經抽真空篩檢程式過濾，以獲得澄清過濾液。More preferably, the preparation method of the ethanol extract of soursop leaves comprises: weighing 100 grams of the soursop leaves, soaking them in five times the volume of ethanol at low temperature, taking them out and putting them into ultrasonic extraction Equipment, continuous extraction at low temperature, the supernatant is collected by high-speed centrifugation, and then filtered through a vacuum screening program to obtain a clarified filtrate.

更佳者，該山刺番荔枝葉乙醇萃取物的製備方法更包含：將該澄清過濾液進行濃縮。More preferably, the preparation method of the ethanol extract of soursop leaves further comprises: concentrating the clarified filtrate.

更佳者，每公克該山刺番荔枝葉乙醇萃取物中總多酚之含量為79 mg至82.4 mg，每公克該山刺番荔枝葉乙醇萃取物中總類黃酮之含量為7.3 mg至15.1 mg。More preferably, the content of total polyphenols per gram of the ethanol extract of soursop leaves is 79 mg to 82.4 mg, and the content of total flavonoids per gram of the ethanol extract of soursop leaves is 7.3 mg to 15.1 mg.

此外，本發明所揭露上述製成萃取物的用途為用於製備具毒殺細胞的組合物，而此組合物可為醫藥品、抗癌藥物、保健食品或健康食品。In addition, the use of the above-mentioned extracts disclosed in the present invention is used to prepare a composition with toxicity to kill cells, and the composition can be medicine, anticancer drug, health food or health food.

為讓本發明上述及/或其他目的、功效、特徵更明顯易懂，下文特舉較佳實施方式，作詳細說明於下：In order to make the above and/or other purposes, effects, and features of the present invention more obvious and understandable, the preferred implementation modes are specifically cited below, which are described in detail below:

本發明之一目的為提出一種山刺番荔枝萃取物，其中該山刺番荔枝萃取物為一山刺番荔枝葉萃取物，但不以此為限。於另一較佳實施例中，該山刺番荔枝葉萃取物包含山刺番荔枝葉水萃取物或山刺番荔枝葉乙醇萃取物，但不以此為限。One purpose of the present invention is to provide a soursop extract, wherein the soursop extract is a soursop leaf extract, but not limited thereto. In another preferred embodiment, the soursop leaf extract comprises soursop leaf water extract or soursop leaf ethanol extract, but not limited thereto.

本發明之另一目的為提出一種上述山刺番荔枝葉萃取物之用途，其中該山刺番荔枝葉萃取物之用途包含：用於製備抗氧化、抗發炎、美白、修復、抗皺或毒殺細胞之組合物，且不以此為限。於一較佳實施例中，所指毒殺之細胞包含癌細胞，但不以此為限。於另一較佳實施例中，該癌細胞代表之癌症類型，包含肺癌、乳癌、結腸直腸癌、***癌、鼻咽癌、胃癌、睾丸癌、皮膚癌、影響神經系統之癌症、骨癌、卵巢癌、肝癌、血液科惡性疾病、胰臟癌、子宮內膜癌、腎癌、子宮頸癌或膀胱癌，但不以此為限。於又一較佳實施例中，該皮膚癌包含：基底細胞癌（BCC）、鱗狀細胞癌（SCC）或黑色素細胞癌（melanoma），但不以此為限。於又一較佳實施例中，該組合物可為醫藥品、化妝品、保養品、香氛、人體清潔用品、抗癌藥物、保健食品或健康食品之原料，但不以此為限。Another object of the present invention is to propose a use of the above-mentioned soursop leaf extract, wherein the use of the soursop leaf extract includes: for preparing anti-oxidation, anti-inflammation, whitening, repairing, anti-wrinkle or killing cells Compositions, and are not limited thereto. In a preferred embodiment, the poisoned cells include cancer cells, but not limited thereto. In another preferred embodiment, the cancer types represented by the cancer cells include lung cancer, breast cancer, colorectal cancer, prostate cancer, nasopharyngeal cancer, gastric cancer, testicular cancer, skin cancer, cancer affecting the nervous system, bone cancer, Ovarian cancer, liver cancer, hematological malignancies, pancreatic cancer, endometrial cancer, kidney cancer, cervical cancer or bladder cancer, but not limited to. In yet another preferred embodiment, the skin cancer includes: basal cell carcinoma (BCC), squamous cell carcinoma (SCC) or melanoma, but not limited thereto. In yet another preferred embodiment, the composition can be medicines, cosmetics, skin care products, fragrances, body cleansing products, anticancer drugs, health food or raw materials of health food, but not limited thereto.

本發明之又一目的為提出一種上述山刺番荔枝葉萃取物之萃取方法。Another object of the present invention is to propose a method for extracting the above-mentioned soursop leaf extract.

首先，將山刺番荔枝葉子洗淨，陰乾約3至5天，再將山刺番荔枝葉子剪成小碎片約0.5cm²大小以進行萃取。於一較佳實施例中，本發明之萃取率公式為：(萃取物重量/樣品原總重)×100%。First, wash the soursop leaves and dry them in the shade for about 3 to 5 days, then cut the soursop leaves into small pieces about 0.5cm² in size for extraction. In a preferred embodiment, the extraction rate formula of the present invention is: (extract weight/total original sample weight)×100%.

再來，將剪碎之山刺番荔枝葉進行一浸泡步驟，於此步驟中包含兩種實施態樣，其中，第一實施態樣為「超音波低溫水萃法」，具體而言，其實施方式為：將山刺番荔枝葉子秤取約100 g於4˚C的環境下浸泡於900 ml的去離子水中，並以超音波振盪萃取設備，於低溫下以總能量300~600 w的能量連續式萃取。於一較佳實施例中，該連續式萃取以震盪3分鐘，休息2分鐘之方式一共震盪60分鐘。於另一較佳實施例中，於連續式萃取完成後，在4°C溫度下以15,000 rpm高速離心30分鐘，收集上清液，再經抽真空篩檢程式過濾，獲得澄清過濾液，利用減壓濃縮機及冷凍乾燥機將半成品萃取液進行濃縮，製成山刺番荔枝葉水萃取物。於又一較佳實施例中，山刺番荔枝葉水萃取物之凍乾物儲存於-80˚C；第二實施態樣為「超音波低溫乙醇萃法」，具體而言，其實施方式為：將山刺番荔枝葉子秤取約100 g浸泡於五倍體積的95%乙醇，於4˚C的環境中避光三天，取出後於低溫下以總能量300~600 w的能量連續式萃取。於一較佳實施例中，該連續式萃取以震盪3分鐘，休息2分鐘之方式一共震盪60分鐘。於另一較佳實施例中，於連續式萃取完成後，在4°C溫度下以15,000 rpm高速離心30分鐘，收集上清液，再經抽真空篩檢程式過濾，獲得澄清過濾液，利用減壓濃縮機及冷凍乾燥機將半成品萃取液進行去除溶劑作業，製成山刺番荔枝葉乙醇萃取物。於又一較佳實施例中，該山刺番荔枝葉乙醇萃取物之凍乾物儲存於-80˚C。Next, the shredded soursop leaves are subjected to a soaking step, which includes two implementations, wherein the first implementation is "ultrasonic low-temperature water extraction method", specifically, its The implementation method is as follows: weigh about 100 g of soursop leaves, soak them in 900 ml of deionized water at 4°C, and use ultrasonic vibration extraction equipment to extract them with a total energy of 300~600 w at low temperature. Energy continuous extraction. In a preferred embodiment, the continuous extraction is shaken for 3 minutes and rested for 2 minutes for a total of 60 minutes. In another preferred embodiment, after the continuous extraction is completed, centrifuge at a high speed of 15,000 rpm for 30 minutes at a temperature of 4° C. to collect the supernatant, and then filter through a vacuum screening program to obtain a clarified filtrate. The decompression concentrator and freeze dryer concentrate the semi-finished extract to make the soursop leaf water extract. In yet another preferred embodiment, the freeze-dried product of the water extract of soursop leaves is stored at -80°C; the second embodiment is "ultrasonic low-temperature ethanol extraction method", specifically, the embodiment is : About 100 g of soursop leaves were weighed and soaked in five times the volume of 95% ethanol, and kept in the dark at 4°C for three days. extraction. In a preferred embodiment, the continuous extraction is shaken for 3 minutes and rested for 2 minutes for a total of 60 minutes. In another preferred embodiment, after the continuous extraction is completed, centrifuge at a high speed of 15,000 rpm for 30 minutes at a temperature of 4° C. to collect the supernatant, and then filter through a vacuum screening program to obtain a clarified filtrate. The decompression concentrator and freeze dryer remove the solvent from the semi-finished extract to produce the ethanol extract of soursop leaves. In yet another preferred embodiment, the lyophilized product of the ethanol extract of soursop leaves is stored at -80°C.

為更進一步瞭解本發明之技術特徵、運用技術手段及所預期達成之功效，茲將本發明相關實施例加以說明敘述如下：In order to further understand the technical characteristics, technical means and expected effects of the present invention, the relevant embodiments of the present invention are described as follows:

實施例1：「AML _S之DPPH·和ABTS· ⁺體外抗氧化試驗」 Example 1: "DPPH· and ABTS· ⁺ in vitro antioxidant test of AML _S "

AML _S具有清除自由基之能力，其中，AML _S對於清除DPPH·自由基之實驗結果如下所示，設定一未與其他物質反應之控制組，其對DPPH·自由基之抑制率為0%，於清除DPPH·自由基之試驗中添加一AMLW或AMLE，其中AMLW與AMLE於清除DPPH·自由基之能力隨其濃度增加而提升，當AMLW於1000 μg/ml時，其對DPPH·自由基之抑制率為64.8%；當AMLE於1000 μg/ml時，其對DPPH·自由基之抑制率48.9%，其結果顯示，AMLW對於清除DPPH·自由基之能力較佳。AML _S對於清除ABTS· ⁺自由基之實驗結果如下所示，設定一未與其他物質反應之控制組，其對ABTS· ⁺自由基之抑制率為0%， 於清除ABTS· ⁺自由基之試驗中添加一AMLW或AMLE，其中AMLW與AMLE於清除ABTS· ⁺自由基之能力隨其濃度增加而提升，AMLW於500 μg/ml時，其對ABTS· ⁺自由基之抑制率即達到最高(99.9%)；當AMLE於1000 μg/ml時，其對ABTS· ⁺自由基之抑制率為98.2%，從結果可得知，AMLW對於清除ABTS· ⁺自由基之能力較佳。半最大效益濃度(concentration for 50% of maximal effect；EC50)為清除50%自由基所需之萃取物濃度，其結果為：在清除DPPH·自由基之實驗中，AMLW之EC ₅₀為670.4 μg/ml，而AMLE之EC ₅₀為1073.5 μg/ml；在清除ABTS· ⁺自由基之實驗中，AMLW之EC ₅₀為180.5 μg/ml，而AMLE之EC ₅₀為231.3 μg/ml，AMLW清除DPPH·或ABTS· ⁺之能力皆優於AMLE，其表示 AMLW的抗氧化效果較AMLE佳。 AML _S has the ability to scavenge free radicals. Among them, the experimental results of AML _S for scavenging DPPH free radicals are shown below. A control group that does not react with other substances is set, and its inhibition rate on DPPH free radicals is 0%. In the test of scavenging DPPH·free radicals, AMLW or AMLE was added, wherein the ability of AMLW and AMLE to scavenge DPPH·free radicals increased with the increase of its concentration. When AMLW was at 1000 μg/ml, its effect on DPPH·free radicals The inhibition rate was 64.8%. When AMLE was at 1000 μg/ml, its inhibition rate on DPPH·free radicals was 48.9%. The results showed that AMLW had a better ability to scavenge DPPH·free radicals. The experimental results of AML _S for scavenging ABTS· ⁺ free radicals are shown below. Set a control group that does not react with other substances, and its inhibition rate for ABTS· ⁺ free radicals is 0%. In the test of scavenging ABTS· ⁺ free radicals AMLW or AMLE was added to AMLW or AMLE, wherein the ability of AMLW and AMLE to scavenge ABTS· ⁺ free radicals increased with the increase of its concentration, and when AMLW was at 500 μg/ml, its inhibitory rate to ABTS· ⁺ free radicals reached the highest (99.9 %); when AMLE is at 1000 μg/ml, its inhibitory rate to ABTS· ⁺ free radicals is 98.2%. From the results, it can be seen that AMLW has a better ability to scavenge ABTS· ⁺ free radicals. The half-maximal effect concentration (concentration for 50% of maximal effect; EC50) is the concentration of the extract required to scavenge 50% of free radicals. The result is: in the experiment of scavenging DPPH free radicals, the EC ₅₀ of AMLW is 670.4 μg/ ml, while the EC ₅₀ of AMLE was 1073.5 μg/ml; in the experiment of scavenging ABTS· ⁺ free radicals, the EC ₅₀ of AMLW was 180.5 μg/ml, while the EC ₅₀ of AMLE was 231.3 μg/ml, AMLW scavenged DPPH· or The ability of ABTS· ⁺ is better than that of AMLE, which means that the antioxidant effect of AMLW is better than that of AMLE.

「AML _S之總類黃酮含量(total flavonoid content ；TFC)和總多酚含量(Total phenolic content；TPC)之含量檢驗」，其檢驗結果如下： "AML _S total flavonoid content (total flavonoid content; TFC) and total polyphenol content (Total phenolic content; TPC) content test", the test results are as follows:

多酚類被認為是具有抗氧化能力之指標。AMLs之TPC及TFC之含量如表1所示，其中，AMLW的TFC之標準含量為89.9±3.9 mg，而TPC之標準含量為36.3±3.2 mg；AMLE之TFC之標準含量為80.7±1.7 mg，而TPC之標準含量為11.2±3.9 mg，由上述結果說明，AMLW的TFC和TPC含量皆高於AMLE，顯示AMLW的抗氧化效果較AMLE佳，與清除DPPH·自由基及清除ABTS· ⁺自由基之試驗結果相同。 表1 樣本 總類黃酮含量 (mg of rutin/g) 總多酚含量 (mg of rutin/g) AMLW 89.9±3.9 36.3±3.2 AMLE 80.7±1.7 11.2±3.9 Polyphenols are considered to be indicators of antioxidant capacity. The contents of TPC and TFC in AMLs are shown in Table 1. Among them, the standard content of TFC in AMLW is 89.9±3.9 mg, and the standard content of TPC is 36.3±3.2 mg; the standard content of TFC in AMLE is 80.7±1.7 mg, The standard content of TPC is 11.2±3.9 mg. According to the above results, the content of TFC and TPC in AMLW is higher than that of AMLE, which shows that the antioxidant effect of AMLW is better than that of ^AMLE . The test results are the same. Table 1 sample Total flavonoid content (mg of rutin/g) Total polyphenol content (mg of rutin/g) AMLW 89.9±3.9 36.3±3.2 AMLE 80.7±1.7 11.2±3.9

實施例2：「AML _S作用於不同細胞之存活度試驗」 Example 2: "Experiment on the viability of AML _S acting on different cells"

依據文獻說明，AMLE萃取物具有抑制癌細胞生成作用，且對正常細胞無毒。本試驗以MTT溶液測試AML _S於不同細胞之存活度，其結果如圖1A至圖1F所示。圖1A說明AMLW 於1~500 μg/ml時之HaCaT細胞存活度，其中，各濃度之AMLW於HaCaT細胞作用24小時後，HaCaT之細胞存活度未降低，其表示AMLW對HaCaT細胞無毒性；圖1B 說明AMLE於1~500 μg/ml時之HaCaT細胞存活度，其中，在AMLE大於100 μg/ml時對HaCaT細胞具有顯著毒性。圖1C說明AMLW 於50~1000 μg/ml時之B16F10細胞存活度，其中，AMLW與黑色素瘤細胞(B16F10)作用72小時後，發現AMLW於50~250 μg/ml時，B16F10細胞仍有80%以上之存活度；圖1D 說明AMLE於50~1000 μg/ml時之B16F10細胞存活度，其中，AMLE於50 μg/ml時，即對B16F10細胞具有毒性，其細胞存活度只剩下30%；圖1E說明，AMLW 於100~1000 μg/ml時之3T3L1細胞存活度，其中，將AMLW於3T3L1細胞作用48小時後，3T3L1細胞之存活度從103.2%增加至121.5%，可推測AMLW具有促進纖維母細胞生長的作用；圖1F說明AMLE 於1~100 μg/ml時之3T3L1細胞存活度，其中，在AMLE100 μg/ml時，3T3L1細胞之存活度降至67%。經過本細胞存活度試驗可發現：AMLW對三株細胞皆無毒殺作用，而濃度100 μg/ml以上之AMLE則對細胞有顯著毒性。根據前述細胞外之抗氧化試驗，結果顯示AMLW之抗氧化能力優於AMLE，故本部分將AMLW用於探討有效性生物活性評估之相關試驗；AMLE則用於探討關於癌細胞毒殺之機制。 According to the literature, AMLE extract has the effect of inhibiting the generation of cancer cells and is non-toxic to normal cells. In this experiment, MTT solution was used to test the viability of _AMLS in different cells, and the results are shown in Figure 1A to Figure 1F. Figure 1A illustrates the HaCaT cell viability of AMLW at 1-500 μg/ml, wherein the HaCaT cell viability did not decrease after AMLW of various concentrations acted on HaCaT cells for 24 hours, which indicated that AMLW had no toxicity to HaCaT cells; 1B shows the viability of HaCaT cells when AMLE is in the range of 1-500 μg/ml, and AMLE is significantly toxic to HaCaT cells when it is greater than 100 μg/ml. Figure 1C shows the viability of B16F10 cells when AMLW was in the range of 50-1000 μg/ml. After 72 hours of action of AMLW on melanoma cells (B16F10), it was found that when AMLW was in the range of 50-250 μg/ml, 80% of the B16F10 cells remained The above viability; Figure 1D shows the viability of B16F10 cells when AMLE is at 50-1000 μg/ml, among them, when AMLE is at 50 μg/ml, it is toxic to B16F10 cells, and the cell viability is only 30%; Figure 1E shows the viability of 3T3L1 cells when AMLW was in the range of 100-1000 μg/ml. After AMLW was applied to 3T3L1 cells for 48 hours, the viability of 3T3L1 cells increased from 103.2% to 121.5%. It can be inferred that AMLW has the ability to promote fiber The role of blast cell growth; Figure 1F shows the viability of 3T3L1 cells when AMLE was 1-100 μg/ml, and the viability of 3T3L1 cells decreased to 67% when AMLE was 100 μg/ml. Through the cell viability test, it can be found that: AMLW has no toxic effect on the three cell lines, while AMLE with a concentration above 100 μg/ml has significant toxicity on the cells. According to the aforementioned extracellular antioxidant test, the results show that the antioxidant capacity of AMLW is superior to that of AMLE. Therefore, in this part, AMLW is used to explore the relevant tests for evaluating the effectiveness and biological activity; AMLE is used to explore the mechanism of cancer cell killing.

實施例3：「AMLW於活性氧(Reactive oxygen species；ROS）與穀胱甘肽(Glutathione；GSH)之細胞內活性試驗」Example 3: "Intracellular Activity Test of AMLW on Reactive Oxygen Species (ROS) and Glutathione (GSH)"

活性氧(ROS)為生物進行有氧代謝過程中的一種副產品，過多的氧化壓力會造成活性氧(ROS)的生成，使細胞受到損傷及誘導發炎反應。穀胱甘肽(GSH)則可從人體細胞中自行合成，是體內新陳代謝重要的營養成分之一，具有使生理功能代謝正常等作用。氧化壓力會造成ROS生成及GSH降低，使得到心血管疾病及老化等風險大增。將AMLW於HaCaT細胞以H ₂O ₂誘發後，以DCFH ₂DA 測定細胞內ROS的含量變化及以OPT測定細胞內GSH含量的變化，其試驗結果為：設定一未與其他物質反應之控制組，其細胞內ROS之活性為100%，經H ₂O ₂反應後其ROS之誘發率提升至126.2%，而再經AMLW (500 μg/ml)作用之組別，其ROS之誘發率下降至92.3%，降低細胞內ROS活性約30%；而在GSH試驗中，其試驗結果為：設定一未與其他物質反應之控制組，其細胞內GSH之活性為100%，經H ₂O ₂反應後其GSH生成率下降至96.4%，而再經AMLW (500 μg/ml)作用後，GSH的含量提升至105.6%，提升了9.2%之生成率，試驗結果證明經AMLW作用的組別能減少ROS之生成，降低經H ₂O ₂誘發之氧化傷害，並可以提升GSH含量來保護細胞免受於氧化壓力之傷害。 Reactive oxygen species (ROS) are a by-product of the aerobic metabolism of organisms. Excessive oxidative stress will cause the generation of reactive oxygen species (ROS), damage cells and induce inflammation. Glutathione (GSH) can be self-synthesized from human cells. It is one of the important nutrients for metabolism in the body, and it can make the metabolism of physiological functions normal. Oxidative stress will cause ROS generation and GSH reduction, which will greatly increase the risk of cardiovascular disease and aging. After AMLW was induced in HaCaT cells with H ₂ O ₂ , DCFH ₂ DA was used to measure the change of intracellular ROS content and OPT was used to measure the change of intracellular GSH content. The test results were as follows: set a control group that did not react with other substances , the activity of ROS in the cells was 100%, and the induction rate of ROS increased to 126.2% after H ₂ O ₂ reaction, and the induction rate of ROS decreased to 92.3%, reduce intracellular ROS activity by about 30%; and in the GSH test, the test result is: set a control group that does not react with other substances, and its intracellular GSH activity is 100%, which is reacted by H ₂ O ₂ Afterwards, the GSH production rate decreased to 96.4%, and after being treated with AMLW (500 μg/ml), the GSH content increased to 105.6%, which increased the production rate by 9.2%. The test results proved that the group treated with AMLW could reduce The generation of ROS reduces the oxidative damage induced by H ₂ O ₂ , and can increase the content of GSH to protect cells from oxidative stress.

實施例4：「AMLW於超氧陰離子O ₂· ^-與抗氧化酵素基因表現量之試驗」 Example 4: "Test of AMLW in Superoxide Anion O ₂ · ^- and Antioxidant Enzyme Gene Expression"

為了避免過多活性氧的生成，如O ₂· ^-出現時，身體會啟動抗氧化機制，其中，抗氧化酵素SOD、GPx及CAT可將O ₂· ^-轉為H ₂O ₂，最後再將其催化成水及氧氣。試驗將AMLW與HaCaT作用後以H ₂O ₂誘發一小時，並透過DHE測量細胞內O ₂· ^-之活性。設定一未與其他物質反應之控制組，其細胞內超氧陰離子(O ₂· ^-)之活性為100%，，細胞經H ₂O ₂誘發之後，O ₂· ^-誘發率上升至111.3%，而經AMLW (500 μg/ml)作用之組別，O ₂· ^-誘發率則下降至99.6%，降低了11.7%之含量。並以qPCR探討抗氧化酵素SOD、GPx及CAT之基因表現時，AMLW於250 μg/ml即具有提升抗氧化酵素SOD、GPx及CAT之表現量而降解O ₂· ^-達到抗氧化之效果。 In order to avoid excessive generation of reactive oxygen species, for example, when O ₂ · ^- appears, the body will start the antioxidant mechanism, among which, the antioxidant enzymes SOD, GPx and CAT can convert O ₂ · ^- into H ₂ O ₂ , and finally Catalyzed into water and oxygen. In the experiment, AMLW was induced with H ₂ O ₂ for one hour after interacting with HaCaT, and the activity of intracellular O ₂ · ^- was measured by DHE. Set a control group that does not react with other substances, and the activity of superoxide anion (O ₂ · ^-) in the cells is 100%. After the cells are induced by H ₂ O ₂ , the O ₂ · ^- induction rate rises to 111.3%. In the group treated with AMLW (500 μg/ml), the induction rate of O ₂ · ^- decreased to 99.6%, and the content decreased by 11.7%. When using qPCR to investigate the gene expression of antioxidant enzymes SOD, GPx and CAT, AMLW at 250 μg/ml can increase the expression of antioxidant enzymes SOD, GPx and CAT and degrade O ₂ · ^- to achieve the effect of antioxidant.

實施例5：「AMLW於抗氧化酵素之蛋白質表現量」Example 5: "Protein expression of AMLW in antioxidant enzymes"

西方墨點法為利用特定抗體具有專一性，可以結合抗原蛋白質的原理觀察其蛋白質表現量，試驗利用HaCaT細胞與AMLW反應後，以H ₂O ₂誘發一小時後萃取細胞內蛋白質。試驗結果如表2所示，設定一對照組之數值為1.0以方便比對，經AMLW (250及500 μg/ml)作用之HaCaT細胞在受到H ₂O ₂誘發後，具有藉由增加抗氧化酵素SOD及CAT含量，以達到清除自由基之作用，其中以增加SOD之表現最為明顯。 表2   控制組 經H ₂O ₂誘發 H ₂O ₂ AMLW (250 μg/ml) AMLW (500 μg/ml) SOD-1 1.0 0.9 3.4 3.8 GPX 1/2 1.0 0.7 0.4 0.9 Catalase 1.0 0.9 1.3 1.5 β-actin 1.0 1.0 1.0 1.0 The western blotting method uses the specificity of specific antibodies and can combine the principle of antigen protein to observe its protein expression. The test uses HaCaT cells to react with AMLW, and then induces with H ₂ O ₂ for one hour to extract intracellular proteins. The test results are shown in Table 2. The value of a control group was set as 1.0 for easy comparison. After being induced by H2O2, HaCaT cells treated with _AMLW ( ₂₅₀ and 500 μg/ml) had the ability to increase antioxidant capacity Enzyme SOD and CAT content to achieve the effect of scavenging free radicals, among which the performance of increasing SOD is the most obvious. Table 2 control group Induced by H ₂ O _{2 H2O2 _} AMLW (250 μg/ml) AMLW (500 μg/ml) SOD-1 1.0 0.9 3.4 3.8 GPX 1/2 1.0 0.7 0.4 0.9 Catalase 1.0 0.9 1.3 1.5 β-actin 1.0 1.0 1.0 1.0

實施例6：「AMLW抑制一氧化氮(NO)體外活性試驗」Example 6: "In vitro Activity Test of AMLW Inhibiting Nitric Oxide (NO)"

氧化反應與發炎反應間有著密不可分的關係，發炎反應是身體抵禦外來物的防禦機制，但過度的發炎反應將導致多種疾病的發生。一氧化氮NO即為其中一種發炎物質，設定一未添加其他物質之控制組，其NO自由基之抑制率為0%，，與未加藥物之控制組別相比，AMLW在100 μg/ml、250 μg/ml、500 μg/ml及1000 μg/ml時，其NO自由基之抑制率分別為3.8%、9.2%、22.4%及27.4%，隨著濃度提高其抑制率有上升趨勢。There is an inseparable relationship between oxidation and inflammation. Inflammation is the body's defense mechanism against foreign substances, but excessive inflammation will lead to the occurrence of various diseases. Nitric oxide NO is one of the inflammatory substances. A control group without adding other substances is set, and the inhibition rate of NO free radicals is 0%. Compared with the control group without drugs, AMLW is at 100 μg/ml , 250 μg/ml, 500 μg/ml and 1000 μg/ml, the inhibitory rates of NO free radicals were 3.8%, 9.2%, 22.4% and 27.4% respectively, and the inhibitory rates tended to increase as the concentration increased.

實施例7：「AMLW經LPS誘導於HaCaT細胞中發炎因子TNF-α、IL-6及IL-1β試驗」Example 7: "TNF-α, IL-6 and IL-1β test of AMLW induced by LPS in HaCaT cells"

透過LPS誘導評估藥物是否能抑制促發炎細胞激素TNF-α、IL-6及IL-1β，設定一未添加其他物質之控制組，其TNF-α、IL-6及IL-1β之誘發率皆為100%，經LPS誘發後，LPS在TNF-α、IL-6及IL-1β之誘發率分別為118.1%、110.5%及143%，而經AMLW (250 μg/ml)作用後，其在TNF-α、IL-6及IL-1β之誘發率分別降為101.1%、88%及130%，說明AMLW在250 μg/ml可以降低發炎因子TNF-α、IL-6及IL-1β之發炎反應。Evaluate whether the drug can inhibit the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β through LPS induction, set a control group without adding other substances, the induction rates of TNF-α, IL-6 and IL-1β are all After being induced by LPS, the induction rates of LPS in TNF-α, IL-6 and IL-1β were 118.1%, 110.5% and 143% respectively, and after being treated with AMLW (250 μg/ml), it was in The induction rates of TNF-α, IL-6 and IL-1β were reduced to 101.1%, 88% and 130% respectively, indicating that AMLW at 250 μg/ml can reduce the inflammation of inflammatory factors TNF-α, IL-6 and IL-1β reaction.

實施例8：「AMLW經脂多醣(LPS)誘導後於人類皮膚角質細胞(HaCaT細胞)中發炎相關路徑之蛋白質表現量」Example 8: "Protein Expression of Inflammation-Related Pathways in Human Skin Keratinocytes (HaCaT Cells) After AMLW Induced by Lipopolysaccharide (LPS)"

受到LPS誘導時，除了促發炎細胞激素被活化外，絲裂原活化蛋白激酶(mitogen-activated protein kinases；MAPKs)家族(ERK1/2、JNK和p38)會使轉錄因子NFκB被活化而生成發炎因子(TNF-α、IL-6及IL-1β)，並活化iNOS與COX-2去生成***素(prostaglandin E2；PGE ₂)與一氧化氮(NO)引起發炎反應。將AMLW作用於HaCaT細胞經LPS (1 μg/ml)誘發24小時後，並萃取其細胞內蛋白質，探討相關發炎因子之蛋白表現量。試驗結果如表3與表4所示，設定一對照組之數值為1.0以方便比較，細胞以LPS (1 μg/ml)誘發，經AMLW (250及500 μg/ml)處理後可以降低促發炎因子TNF-α、IL-6及IL-1β之蛋白質表現，藉由抑制核因子活化B細胞κ輕鏈增強子(nuclear factor kappa-light-chain-enhancer oNFκBactivated B cells；NFκB)的表現量而抑制iNOS與COX-2去生成NO及PGE ₂，亦可調控MAPKs家族中ERK、p-ERK、p38及p-p38發炎相關路徑之蛋白質降低其表現量，而達到抗發炎之效能。 表3   控制組 LPS AMLW (250 μg/ml) AMLW (500 μg/ml) JNK 1.0 1.4 2.2 1.5 p-JNK 1.0 2.0 3.2 4.1 ERK 1.0 1.3 1.0 1.1 p-ERK 1.0 1.7 1.6 0.9 p38 1.0 1.6 1.9 1.3 p-p38 1.0 3.3 2.4 0.6 β-actin 1.0 1.0 1.0 1.0 表4   控制組 LPS AMLW (250 μg/ml) AMLW (500 μg/ml) NFκB 1.0 1.0 0.8 0.9 LaminA 0.0 0.0 0.0 0.0 iNOS 1.0 1.5 1.2 0.7 COX-2 1.0 1.7 1.4 1.1 TNF-α 1.0 1.3 1.0 0.9 1L-1β 1.0 1.6 1.1 0.1 1L-6 1.0 1.4 2.6 1.1 β-actin 1.0 1.0 1.0 1.0 When induced by LPS, in addition to the activation of pro-inflammatory cytokines, the mitogen-activated protein kinases (MAPKs) family (ERK1/2, JNK and p38) will activate the transcription factor NFκB to generate inflammatory factors (TNF-α, IL-6 and IL-1β), and activate iNOS and COX-2 to generate prostaglandin (prostaglandin E2; PGE ₂ ) and nitric oxide (NO) to cause inflammation. AMLW was applied to HaCaT cells induced by LPS (1 μg/ml) for 24 hours, and the intracellular proteins were extracted to investigate the protein expression of related inflammatory factors. The test results are shown in Table 3 and Table 4. The value of a control group was set as 1.0 to facilitate comparison. The cells were induced with LPS (1 μg/ml), and treated with AMLW (250 and 500 μg/ml) can reduce the pro-inflammatory Protein expression of factors TNF-α, IL-6 and IL-1β, inhibited by inhibiting the expression of nuclear factor kappa-light-chain-enhancer oNFκB activated B cells (NFκB) iNOS and COX-2 generate NO and PGE ₂ , and can also regulate the expression of proteins in the inflammatory pathways of ERK, p-ERK, p38 and p-p38 in the MAPKs family, thereby achieving anti-inflammatory effects. table 3 control group LPS AMLW (250 μg/ml) AMLW (500 μg/ml) JNK 1.0 1.4 2.2 1.5 p-JNK 1.0 2.0 3.2 4.1 ERK 1.0 1.3 1.0 1.1 p-ERK 1.0 1.7 1.6 0.9 p38 1.0 1.6 1.9 1.3 p-p38 1.0 3.3 2.4 0.6 β-actin 1.0 1.0 1.0 1.0 Table 4 control group LPS AMLW (250 μg/ml) AMLW (500 μg/ml) NFκB 1.0 1.0 0.8 0.9 Lamin A 0.0 0.0 0.0 0.0 iNOS 1.0 1.5 1.2 0.7 COX-2 1.0 1.7 1.4 1.1 TNF-α 1.0 1.3 1.0 0.9 1L-1β 1.0 1.6 1.1 0.1 1L-6 1.0 1.4 2.6 1.1 β-actin 1.0 1.0 1.0 1.0

實施例9：「AMLW於SKH-2小鼠之發炎相關因子之免疫螢光組織染色試驗」Example 9: "Immunofluorescent Histological Staining Test of AMLW on Inflammation-Related Factors in SKH-2 Mice"

IHC為利用抗原和抗體之間的專一性，於抗體上結合螢光，檢測組織中是否含有目標抗原的存在，此方法不只可以偵測抗原的表現量也可以觀察抗原表現的位置。試驗選用SKH-2小鼠之皮膚，試驗組別分別為未照光之控制組、UVB (180 mJ/cm ²)照射組、HEC gel空白背景組、含AMLW (25 mg/ml)作用之組別與含AMLE (2.5 mg/ml)作用之組別。結果如圖2A與2B所示，與控制組相比，HEC gel之組別受到UVB的照射後，其TNF-α、IL-6及IL-1β、iNOS、COX-2、p-JNK、p-ERK及p-p38螢光表現量皆有明顯表現，而與UVB組相比，AMLW與AMLE組別之TNF-α、IL-6及IL-1β、iNOS、COX-2、p-JNK、p-ERK及p-p38螢光表現皆有降低趨勢，驗證AML _S可以調控發炎相關轉錄因子而達到降低發炎反應之效能。 IHC uses the specificity between antigen and antibody to combine fluorescent light on the antibody to detect the presence of the target antigen in the tissue. This method can not only detect the amount of antigen expression but also observe the position of antigen expression. The skin of SKH-2 mice was selected for the test, and the test groups were the control group without light exposure, the UVB (180 mJ/cm ² ) irradiation group, the HEC gel blank background group, and the group containing AMLW (25 mg/ml) The group with the effect of AMLE (2.5 mg/ml). The results are shown in Figure 2A and 2B. Compared with the control group, after the HEC gel group was irradiated with UVB, the TNF-α, IL-6 and IL-1β, iNOS, COX-2, p-JNK, p -ERK and p-p38 fluorescence expression were significantly expressed, and compared with UVB group, TNF-α, IL-6 and IL-1β, iNOS, COX-2, p-JNK, The fluorescent expression of p-ERK and p-p38 both showed a decreasing tendency, which verifies that AML _S can regulate inflammation-related transcription factors to achieve the effect of reducing the inflammatory response.

實施例10：「AMLW於黑色素瘤細胞內抑制黑色素及酪胺酸酶等試驗」Example 10: "AMLW inhibits melanin and tyrosinase in melanoma cells"

黑色素生成是一個複雜的生理過程，有助於保護皮膚免受陽光及化學物質的有害影響，但過度的黑色素生成會導致色素沉澱。黑色素生成路徑相關酵素包含tyrosinase(酪胺酸酶)、TRP-1及TRP-2，在UVB照射的刺激下，會影響促黑激素α-MSH產生，在黑色素生成的過程中，涉及tyrosinase、TRP-1及TRP-2之活性，藉由酪胺酸酶酵素催化酪胺酸，並形成L-DOPA及DOPA quinone，最後促使黑色素生成。試驗以黑色素瘤細胞(B16F10)將含有α-MSH之培養液與AMLW (50~250 μg/ml)反應72小時後，進行細胞內的酪胺酸酶、黑色素及多巴氧化酶之含量生成測試。設定一未添加其他物質之控制組，其細胞內之黑色素含量為100%，結果如圖3A所示，在經α-MSH (100 nM)刺激下，單獨之α-MSH誘發組別中，其細胞內黑色素含量為157.6%，而再經AMLW (250 μg/ml)作用後，其細胞內黑色素含量降至140.3%，其降低了細胞內黑色素分泌17.3%之活性，以及設定一未添加其他物質之控制組，其細胞酪胺酸酶活性為100%，經α-MSH (100 nM)刺激下，單獨之α-MSH誘發組別其細胞酪胺酸酶活性為131.6%，經AMLW (250 μg/ml)作用後之細胞酪胺酸酶活性為111.1%，以及設定一未添加其他物質之控制組，其細胞多巴氧化酶含量為100%經α-MSH (100 nM)刺激下，單獨之α-MSH誘發組別其多巴氧化酶含量為110.3%，經AMLW (250 μg/ml)作用後之多巴氧化酶含量為87.8%，與單獨之α-MSH誘發組別相比，皆降低了20%以上之生成率。說明AMLW可降低黑色素生成酵素的活性，藉此達到降低黑色素的生成。Melanin production is a complex physiological process that helps protect the skin from the harmful effects of the sun and chemicals, but excessive melanin production can lead to pigmentation. The enzymes related to melanin production pathway include tyrosinase (tyrosinase), TRP-1 and TRP-2. Under the stimulation of UVB irradiation, it will affect the production of melanotropic hormone α-MSH. In the process of melanin production, tyrosinase, TRP are involved The activities of -1 and TRP-2 catalyze tyrosine through tyrosinase enzymes, and form L-DOPA and DOPA quinone, and finally promote melanin production. The test used melanoma cells (B16F10) to react the culture medium containing α-MSH with AMLW (50~250 μg/ml) for 72 hours, and then tested the content of tyrosinase, melanin and dopa oxidase in the cells. . A control group without adding other substances was set, and the melanin content in the cells was 100%. The results are shown in Figure 3A. The intracellular melanin content is 157.6%, and after being treated with AMLW (250 μg/ml), the intracellular melanin content drops to 140.3%, which reduces the activity of intracellular melanin secretion by 17.3%, and setting 1 does not add other substances In the control group, the cell tyrosinase activity was 100%, stimulated by α-MSH (100 nM), the cell tyrosinase activity of the separate α-MSH induced group was 131.6%, after AMLW (250 μg /ml) after the action of the cell tyrosinase activity was 111.1%, and set a control group without adding other substances, the content of dopa oxidase in the cells was 100% stimulated by α-MSH (100 nM), alone The content of dopa oxidase in the α-MSH-induced group was 110.3%, and the content of dopa oxidase after AMLW (250 μg/ml) was 87.8%, which was lower than that of the α-MSH-induced group alone A production rate of more than 20%. It shows that AMLW can reduce the activity of melanin-producing enzymes, so as to reduce the production of melanin.

實施例11：「AMLW於黑色素瘤細胞中與黑色素生成相關因子之蛋白質表現量」Example 11: "AMLW Protein Expression of Factors Related to Melanoma Production in Melanoma Cells"

當紫外線照射時會活化p53進而影響α-MSH的產生，並與受體MC1R結合，活化環腺苷酸cAMP並使cAMP反應元件結合蛋白CREB磷酸化，導致MITF轉錄因子上調而調控tyrosinase、TRP-1及TRP-2促使黑色素生成。將AMLW (250 μg/ml)經α-MSH誘發作用與於黑色素瘤細胞中72小時，以西方墨點法測定AMLW之黑色素生成相關因子MC1R、MITF、tyrosinase、TRP-1及TRP-2之蛋白質表現。試驗結果如表5所示，AMLW (250 μg/ml)可以藉由調控MC1R而降低CREB磷酸化之表現，導致MITF轉錄因子的表現被抑制而降低黑色素生成相關之蛋白Tyrosinase、TRP-1及TRP-2表現，達到抑制黑色素生成而具有美白之效用。 表5   控制組 經α-MSH(100 nM誘發) α-MSH AMLW (250 μg/ml) MC1R 1.0 1.7 0.9 CREB 1.0 1.1 0.9 MITF 1.0 1.2 0.9 Tyrosinase 1.0 1.7 1.5 Trp-1 1.0 1.1 0.3 Trp-2 1.0 1.2 0.9 β-actin 1.0 1.0 1.0 When irradiated by ultraviolet light, p53 will be activated to affect the production of α-MSH, and bind to the receptor MC1R, activate cyclic AMP and phosphorylate the cAMP response element binding protein CREB, leading to the up-regulation of MITF transcription factors and the regulation of tyrosinase, TRP- 1 and TRP-2 promote melanin production. AMLW (250 μg/ml) was induced by α-MSH in melanoma cells for 72 hours, and the proteins of AMLW's melanin production-related factors MC1R, MITF, tyrosinase, TRP-1 and TRP-2 were measured by Western blot method which performed. The test results are shown in Table 5. AMLW (250 μg/ml) can reduce the expression of CREB phosphorylation by regulating MC1R, resulting in the inhibition of the expression of MITF transcription factors and the reduction of melanin production-related proteins Tyrosinase, TRP-1 and TRP -2 performance, to inhibit the production of melanin and have the effect of whitening. table 5 control group Induced by α-MSH (100 nM) α-MSH AMLW (250 μg/ml) MC1R 1.0 1.7 0.9 CREB 1.0 1.1 0.9 MITF 1.0 1.2 0.9 Tyrosinase 1.0 1.7 1.5 Trp-1 1.0 1.1 0.3 Trp-2 1.0 1.2 0.9 β-actin 1.0 1.0 1.0

實施例12：「AMLW於老鼠成纖維母細胞3T3L1中膠原蛋白生成試驗」Example 12: "AMLW in collagen production test in mouse fibroblast 3T3L1"

膠原蛋白存在於結締組織，是真皮層中不可或缺的蛋白質，因具有高度的彈性張力，並負責維持細胞間質正常的功能，而膠原蛋白會隨著年齡增長或紫外線照射而流失，當膠原蛋白流失將會使皮膚的彈力下降，形成皺紋的產生。試驗利用3T3L1與AMLW作用48小時，並利用Sircol collagen kit測定上清液與細胞內蛋白質中膠原蛋白之含量，設定一未添加其他物質之控制組，其細胞內膠原蛋白含量為100%，其中添加AMLW， AMLW於100 μg/ml、250 μg/ml及500 μg/ml時可以增加細胞內膠原蛋白之含量，其中，各濃度之細胞內膠原蛋白含量分別為115.3%、121.3%及137.4%，與控制組相比，經AMLW500 μg/ml作用後之細胞內膠原蛋白含量增加了37.4%。Collagen exists in connective tissue and is an indispensable protein in the dermis. Because of its high elastic tension, it is responsible for maintaining the normal function of the intercellular matrix. Collagen will be lost with age or ultraviolet radiation. When collagen The loss of protein will reduce the elasticity of the skin and form wrinkles. The experiment used 3T3L1 and AMLW to act for 48 hours, and used the Sircol collagen kit to measure the collagen content in the supernatant and intracellular protein. A control group without adding other substances was set up, and the intracellular collagen content was 100%. AMLW, AMLW can increase the content of intracellular collagen at 100 μg/ml, 250 μg/ml and 500 μg/ml, among which, the intracellular collagen content of each concentration is 115.3%, 121.3% and 137.4%, respectively, and Compared with the control group, the intracellular collagen content increased by 37.4% after being treated with AMLW500 μg/ml.

實施例13：「AMLW抑制基質金屬蛋白酶之活性評估試驗」Example 13: "Assessment test for the activity of AMLW inhibiting matrix metalloproteinases"

紫外線的刺激會引起細胞膜產生脂質過氧化的反應，並促進基質金屬蛋白酶(MMPs)之生成，透過此種膠原蛋白分解酵素之作用，會引起皮膚彈力下降，使皺紋生成。MMP-9與MMP-2皆屬於明膠蛋白酶，其中，MMP-2大量存在於纖維母細胞、內皮細胞及上皮細胞中；MMP-9存在於白血球與轉性細胞。試驗將AMLW 於100~500 μg/ml時作用於3T3L1細胞48小時，並利用含有明膠gelatin之膠片進行SDS-PAGE電泳分析，經過不同的buffer染色後，會於對應分子量位置呈現，具有抑制效果之組別會將gelatin分解，並以Image J定量。設定一未添加其他物質之控制組，其MMP-9與MMP-2之抑制率為0%，其中添加AMLW，AMLW於500 μg/ml時抑制MMP-9與MMP-2之抑制率分別為8.1%與38.4%，其結果說明AMLW對於抑制MMP-2的效果較佳，可以藉由抑制MMP-2而達到預防光老化之效果。The stimulation of ultraviolet rays will cause the lipid peroxidation reaction of the cell membrane and promote the production of matrix metalloproteinases (MMPs). Through the action of this collagen decomposing enzyme, it will cause the decrease of skin elasticity and cause wrinkles. Both MMP-9 and MMP-2 belong to gelatin proteases, among which, MMP-2 exists in fibroblasts, endothelial cells and epithelial cells in large quantities; MMP-9 exists in leukocytes and transformed cells. In the experiment, AMLW was applied to 3T3L1 cells at 100-500 μg/ml for 48 hours, and the film containing gelatin was used for SDS-PAGE electrophoresis analysis. After staining with different buffers, it will appear at the corresponding molecular weight position, which has an inhibitory effect The group will decompose gelatin and quantify it with Image J. Set a control group without adding other substances, the inhibition rate of MMP-9 and MMP-2 is 0%, and AMLW is added, and the inhibition rate of AMLW at 500 μg/ml inhibits MMP-9 and MMP-2 is 8.1% % and 38.4%, the results show that AMLW has a better effect on inhibiting MMP-2, and can achieve the effect of preventing photoaging by inhibiting MMP-2.

實施例14：「AMLW於SKH-2光老化相關因子之蛋白質表現量」Example 14: "Protein expression of AMLW in SKH-2 photoaging-related factors"

MMPs與Elastase-3A為膠原蛋白分解之酵素，當UVB曝曬時會大量表現，進而降解細胞外基質(ECM)使真皮層中的膠原蛋白降解，其中COL1A與COL3A1分別為第一型(type I collagen)膠原蛋白與第三型膠原蛋白(type III collagen)膠原蛋白。在細胞外基質被破壞的同時，生物體內的細胞黏附分子濃度也會下降，使細胞間的鍵結變弱，而導致角質的排列變得鬆散。E-cadherin與β-catenin存在與表皮細胞中，當兩者的表現異常時，會使細胞失去黏附的特性，進而造成細胞的異常增生。試驗組別分別有未照光之組別、UVB組別、HEC gel空白背景組、含AMLW之gel組別與AMLE之gel組別，連續照光並投藥三天後，取其小鼠之皮膚，進行組織蛋白質之萃取，並以西方墨點法探討其組織之蛋白質表現量變化。結果如表6與表7所示，將一控制組之數值設定為1.0以方便對照，僅受UVB與HEC背景組之組別，與降解膠原蛋白之基質金屬蛋白酶MMP-1、MMP-2、MMP-7、MMP-9及Elastase -3A表現量明顯地提升，而代表膠原蛋白的指標COL1A、COL3A1、β-catenin及E-cadherin則下降，顯示經AMLW (25 mg/ml)與AMLE (2.5 mg/ml)作用之組別，則為降解膠原蛋白之基質金屬蛋白酶表現量降低，而膠原蛋白指標之表現量呈現上升趨勢，結果證實AML _S具有抑制基質金屬蛋白酶的活性進而避免膠原蛋白分解、並促進膠原蛋白生長與增加細胞間質黏合力之效能。 表6   控制組 經UVB(180 mJ/cm ²)照射 UVB HEC AMLW (25 mg/ml) AMLE (2.5 mg/ml) MMP1 1.0 1.5 2.5 1.0 1.2 MMP2 1.0 1.6 1.9 1.2 0.9 MMP7 1.0 1.7 2.0 1.6 1.2 MMP9 1.0 1.4 1.7 1.3 1.2 MMP13 1.0 1.1 0.8 0.7 0.6 β-actin 1.0 1.0 1.0 1.0 1.0 表7   控制組 經UVB(180 mJ/cm ²)照射 UVB HEC AMLW (25 mg/ml) AMLE (2.5 mg/ml) Elastase-3A 1.0 2.2 3.1 1.7 1.6 COL1A 1.0 0.7 0.6 0.8 0.9 COL3A1 1.0 0.9 1.2 1.3 1.4 β-catenin 1.0 0.9 1.4 1.7 2.2 E-cadherin 1.0 0.9 1.5 1.8 2.4 β-actin 1.0 1.0 1.0 1.0 1.0 MMPs and Elastase-3A are collagen-decomposing enzymes, which will be expressed in large quantities when exposed to UVB, and then degrade the extracellular matrix (ECM) to degrade the collagen in the dermis. Among them, COL1A and COL3A1 are type I collagen ) collagen and type III collagen (type III collagen) collagen. When the extracellular matrix is destroyed, the concentration of cell adhesion molecules in the organism will also decrease, which will weaken the bond between cells and cause the arrangement of cutin to become loose. E-cadherin and β-catenin exist in epidermal cells, and when the two behave abnormally, the cells will lose their adhesion properties, resulting in abnormal proliferation of cells. The test groups include non-irradiated group, UVB group, HEC gel blank background group, AMLW-containing gel group and AMLE gel group. After three days of continuous irradiation and drug administration, the skin of the mice was taken and tested. Tissue protein extraction, and Western blot method to explore the changes in protein expression in the tissue. The results are shown in Table 6 and Table 7. The value of a control group is set as 1.0 to facilitate the comparison. The groups that are only subjected to UVB and HEC background groups, and matrix metalloproteinases MMP-1, MMP-2, The expression levels of MMP-7, MMP-9 and Elastase -3A were significantly increased, while the indicators representing collagen COL1A, COL3A1, β-catenin and E-cadherin were decreased, showing that after AMLW (25 mg/ml) and AMLE (2.5 mg/ml) group, the expression of matrix metalloproteinases that degrade collagen decreased, while the expression of collagen indicators showed an upward trend. The results confirmed that AML _S has the ability to inhibit the activity of matrix metalloproteinases, thereby preventing collagen decomposition, And promote the growth of collagen and increase the performance of intercellular matrix adhesion. Table 6 control group Irradiated by UVB (180 mJ/cm ² ) UVB HEC AMLW (25mg/ml) AMLE (2.5mg/ml) MMP1 1.0 1.5 2.5 1.0 1.2 MMP2 1.0 1.6 1.9 1.2 0.9 MMP7 1.0 1.7 2.0 1.6 1.2 MMP9 1.0 1.4 1.7 1.3 1.2 MMP13 1.0 1.1 0.8 0.7 0.6 β-actin 1.0 1.0 1.0 1.0 1.0 Table 7 control group Irradiated by UVB (180 mJ/cm ² ) UVB HEC AMLW (25mg/ml) AMLE (2.5mg/ml) Elastase-3A 1.0 2.2 3.1 1.7 1.6 COL1A 1.0 0.7 0.6 0.8 0.9 COL3A1 1.0 0.9 1.2 1.3 1.4 β-catenin 1.0 0.9 1.4 1.7 2.2 E-cadherin 1.0 0.9 1.5 1.8 2.4 β-actin 1.0 1.0 1.0 1.0 1.0

實施例15：「AMLW於SKH-2小鼠中，抑制預防光老化相關因子之免疫螢光活性」Example 15: "AMLW inhibits the immunofluorescence activity of photoaging-related factors in SKH-2 mice"

試驗以SKH-2小鼠經UVB照光，並塗抹含藥物之HEC gel，組別分別為未照光之組別、UVB組別、不含藥物HEC gel空白背景組、含AMLW之gel組別與AMLE之gel組別，連續照光並投藥三天後，取其小鼠之皮膚試驗利用SKH-2小鼠之皮膚，探討AMLW (25 mg/ml)與AMLE (2.5 mg/ml)於光老化因子之免疫螢光染色表現量，結果如圖4A與4B所示，UVB組別，與未照光之控制組相比，其降解膠原蛋白之相關因子MMP-1、2、7、9及Elastase -3A表現量之FITC綠色螢光表現量於皆上升，而含AMLW與AMLE之藥物組別照光之後，其螢光表現與UVB組別相比，於降解膠原蛋白相關因子MMP-1、2、7、9及Elastase -3A中皆有降低之趨勢，並且在膠原蛋白指標之相關因子中，亦提升了螢光表現量，驗證AML _S可以調控光老化相關因子，降低降解膠原蛋白相關轉錄因子而達到提升膠原蛋白之生成，達到降低光老化之效能。 In the experiment, SKH-2 mice were irradiated with UVB and smeared with drug-containing HEC gel. The groups were the non-irradiated group, the UVB group, the HEC gel blank background group without drug, the gel group containing AMLW, and the AMLE group. In the gel group, after three days of continuous light irradiation and drug administration, the skin test of the mice was performed using the skin of SKH-2 mice to investigate the effect of AMLW (25 mg/ml) and AMLE (2.5 mg/ml) on photoaging factors Immunofluorescence staining expression, the results are shown in Figure 4A and 4B, UVB group, compared with the control group without light, the expression of MMP-1, 2, 7, 9 and Elastase-3A related factors for collagen degradation The amount of FITC green fluorescence expression increased, and after the drug group containing AMLW and AMLE was illuminated, its fluorescence expression was better than that of the UVB group in degrading collagen-related factors MMP-1, 2, 7, and 9 and Elastase -3A have a decreasing trend, and among the factors related to collagen indicators, the fluorescence expression level has also been increased. It is verified that AML _S can regulate the factors related to photoaging and reduce the degradation of collagen-related transcription factors to achieve the enhancement of collagen The production of protein achieves the effect of reducing photoaging.

實施例16：「AMLW於pUC119之DNA保護試驗」Example 16: "DNA Protection Test of AMLW in pUC119"

經過UVB照射之DNA pUC119試驗，當受到破壞時，DNA會從環形環繞結構(supercoiled-form, S-form)斷裂成直線結構(linear-form, L-form)，利用不同分子量的核酸在膠體孔徑中移動速度的差異特性，將pUC19 DNA以H ₂O ₂(1 mM)誘導及UVB (20 mJ/cm ²)照射，若DNA能受到保護作用時，則能呈現至S-form之位置，而當受到破壞時，DNA便會呈現在L-form之位置。試驗結果如圖5A所示，控制組未受到傷害可以保持完整的DNA型態並呈現環繞結構，未加藥物之UVB傷害組別，DNA結構受到損傷而呈現直線形結構，而AML _S(50及100 μg/ml)在經UVB照射後，AMLW在50及100 μg/ml具有完整之DNA環繞結構，具有DNA保護作用，而AMLE在100 μg/ml時才有較完整的片段，經圖5B定量結果表示，AMLW之保護能力較勝於AMLE。 In the DNA pUC119 test irradiated by UVB, when damaged, the DNA will break from the circular surrounding structure (supercoiled-form, S-form) to a linear structure (linear-form, L-form). The differential characteristics of moving speed in the middle, pUC19 DNA induced by H ₂ O ₂ (1 mM) and irradiated with UVB (20 mJ/cm ² ), if the DNA can be protected, it can appear in the position of S-form, while When damaged, the DNA will appear in the L-form position. The test results are shown in Figure 5A. The control group was not injured and could maintain a complete DNA pattern and present a circular structure. In the UVB injury group without drugs, the DNA structure was damaged and presented a linear structure, while the AML _S (50 and 100 μg/ml) after UVB irradiation, AMLW has a complete DNA surround structure at 50 and 100 μg/ml, which has a DNA protective effect, while AMLE has a relatively complete fragment at 100 μg/ml, which is quantified in Figure 5B The results showed that the protection ability of AMLW was better than that of AMLE.

實施例17：「AMLW經UVB照射後於HaCaT細胞之保護修復存活度試驗」Example 17: "Experiment on the protection and repair survival of AMLW in HaCaT cells after UVB irradiation"

將無添加血清之培養液於HaCaT細胞中與AMLW (100~500 μg/ml)反應4小時，並抽乾照射UVB (20 mJ/cm ²)，再加入無添加血清之培養液與AMLW反應4小時，當存活度增加時，說明具有保護及修復細胞使細胞增生之能力。設定一未添加其他物質之控制組，其細胞活性為100%，結果如圖6表示，經UVB照射處理後之組別，其細胞存活度降至75.6%，而AMLW (100~500 μg/ml)經照射UVB後，存活度分別提升至96.4%、106.6%及109.6%，與UVB組別相比，AMLW於500 μg/ml時提升了34%之存活度，說明具有保護及修復細胞之能力。 React the culture solution without added serum with AMLW (100~500 μg/ml) in HaCaT cells for 4 hours, drain and irradiate with UVB (20 mJ/cm ² ), then add the culture solution without added serum to react with AMLW for 4 hours Hours, when the viability increases, it shows that it has the ability to protect and repair cells and make cells proliferate. Set a control group without adding other substances, and its cell viability is 100%. The results are shown in Figure 6. After UVB irradiation treatment, the cell viability dropped to 75.6%, while AMLW (100~500 μg/ml ) after UVB irradiation, the survival rate increased to 96.4%, 106.6% and 109.6% respectively. Compared with the UVB group, AMLW increased the survival rate by 34% at 500 μg/ml, indicating that it has the ability to protect and repair cells .

實施例18：「AMLW經UVB照射之後光化產物(CPD及6-4 PPs)之含量變化」Example 18: "Changes in content of photochemical products (CPD and 6-4 PPs) in AMLW after UVB irradiation"

由前述提到，紫外線的輻射會造成DNA的損傷，尤其是環丁烷嘧啶二聚體(CPD)和6-4光化產物(6-4 PPs)，此種光化產物是使突變產生的原因之一，其會藉由改變DNA原有結構，使聚合酶無法正常運作及複製，當損傷無法被修復時，最終會引發突變。試驗將AMLW (250及500 μg/ml)經UVB誘導後，利用可以辨識CPD及6-4 PPs之抗體進行免疫螢光染色，觀察其螢光表現變化及含量變化。結果如圖7A與7B所示，僅受到UVB作用組別之CPD與6-4 PPs有較強之螢光訊號，CPD表現上升至135.7%，而6-4 PPs表現上升至107.5%，表示細胞核受到較嚴重之損傷，而經AMLW 250 μg/ml作用後，使CPD表現量降低至94.9%，以及使6-4 PPs表現量降低至104.7%；經AMLW 於500 μg/ml作用後使CPD表現量降低至87.8%，以及使6-4 PPs表現量降低至91.6%，說明AMLW可以修復UVB所引起的細胞光化產物CPD及6-4 PPs之損傷。As mentioned above, ultraviolet radiation can cause DNA damage, especially cyclobutane pyrimidine dimer (CPD) and 6-4 photochemical products (6-4 PPs), which cause mutations One of the reasons is that by changing the original structure of DNA, the polymerase cannot function and replicate normally, and when the damage cannot be repaired, it will eventually cause mutations. In the experiment, after AMLW (250 and 500 μg/ml) was induced by UVB, immunofluorescent staining was performed with antibodies that can recognize CPD and 6-4 PPs, and the changes in fluorescence expression and content were observed. The results are shown in Figures 7A and 7B. Only CPD and 6-4 PPs in the UVB group had strong fluorescent signals, and the expression of CPD increased to 135.7%, while the expression of 6-4 PPs increased to 107.5%, indicating that the nucleus After more severe damage, the expression of CPD was reduced to 94.9% and the expression of 6-4 PPs was reduced to 104.7% after being treated with AMLW at 250 μg/ml; after being treated with 500 μg/ml of AMLW, the expression of CPD was The expression level of 6-4 PPs was reduced to 87.8%, and the expression level of 6-4 PPs was reduced to 91.6%, indicating that AMLW can repair the damage of cell photochemical product CPD and 6-4 PPs caused by UVB.

實施例19：「AMLW經UVB照射後之傷口癒合(wound healing)試驗」Example 19: "Wound healing test of AMLW after UVB irradiation"

試驗將含有AMLW (50~250 μg/ml)之無血清培養液作用於HaCaT細胞中4小時後，抽乾培養液再將細胞照射UVB (20 mJ/cm ²)，再以AMLW培養另一個4小時，利用顯微鏡觀察細胞移動之變化，藉此評估細胞修復之能力。其中，控制組與AMLW (50~250 μg/ml)於12、24及48小時其細胞間距即有向中間生長之趨勢，至48小時已幾乎癒合，而單受UVB照射之組別，其細胞間距變化緩慢，並透過量化結果如圖8所示，AMLW(250 μg/ml)於48小時時，其細胞間距相比於12小時時減少了42.5%，說明AMLW確實具有促進細胞修復並使傷口癒合之能力。 In the experiment, the serum-free culture solution containing AMLW (50~250 μg/ml) was applied to HaCaT cells for 4 hours, the culture solution was drained, and the cells were irradiated with UVB (20 mJ/cm ² ), and then another 4 hours were cultured with AMLW. After hours, use a microscope to observe the changes in cell movement to evaluate the ability of cell repair. Among them, the intercellular space between the control group and AMLW (50-250 μg/ml) tended to grow towards the middle at 12, 24 and 48 hours, and almost healed at 48 hours, while the cells of the group exposed to UVB alone The spacing changes slowly, and the quantification results are shown in Figure 8. At 48 hours, the cell spacing of AMLW (250 μg/ml) was reduced by 42.5% compared with that at 12 hours, indicating that AMLW does have the ability to promote cell repair and make wounds The ability to heal.

實施例20：「AMLW於SKH-2小鼠皮膚組織中HE-stain染色」Example 20: "HE-stain staining of AMLW in SKH-2 mouse skin tissue"

蘇木紫-伊紅染色(Hematoxylin & Eosin stain)是組織學最常用的組織化學染色(Histochemistry stain)方法之一，常用於呈現組織中的特殊成分或病原，藉由組織結構對不同染料酸鹼的結合程度不同，造成染色差異。蘇木紫透過氧化作用將細胞核染成藍色，伊紅則將組織纖維和細胞質染成不等程度的粉紅色。經染色後的正常的皮膚切片，皮膚結構緊密且排列整齊，當受到損傷時，會依受損程度有不同之表現，如角質層增厚和排列鬆散等等。試驗結果如圖9所示，與控制組相比，受到UVB (180 mJ/cm ²)照射的組別，傷害太強導致表皮剝落而無法觀察到增厚的完整表皮且真皮層排列鬆散，HEC gel背景組，因有膠體保護損傷較小，維持較完整之表皮但角質層增厚，而受到AMLW (25 mg/ml)與AMLE (2.5 mg/ml)處理之組別改善了角質增厚現象，並且角質的排列緊密，具有保護效果。 Hematoxylin & Eosin stain is one of the most commonly used histochemical staining methods in histology. It is often used to display special components or pathogens in tissues, and the pH of different dyes can be determined by tissue structure. The degree of binding is different, resulting in differences in staining. Hematoxylin stains cell nuclei blue through oxidation, while eosin stains tissue fibers and cytoplasm pink to varying degrees. The normal skin section after dyeing, the skin structure is tight and neatly arranged, when it is damaged, it will have different manifestations according to the degree of damage, such as thickening of the stratum corneum and loose arrangement. The test results are shown in Figure 9. Compared with the control group, the group irradiated by UVB (180 mJ/cm ² ) was too damaged to cause the epidermis to peel off so that the thickened intact epidermis could not be observed and the dermis was loosely arranged. HEC In the gel background group, because of the colloid protection, the damage was small, the epidermis was maintained relatively intact, but the stratum corneum was thickened, and the group treated with AMLW (25 mg/ml) and AMLE (2.5 mg/ml) improved the phenomenon of horny thickening , and the horny arrangement is tight, which has a protective effect.

實施例21：「AMLW於SKH-2小鼠皮膚組織中修復相關因子之蛋白質表現量」Example 21: "AMLW protein expression of repair-related factors in SKH-2 mouse skin tissue"

核苷酸切除修復(NER)為哺乳動物中主要的修復系統，主要為消除紫外線所導致之損傷，PARP會參與損傷位置的識別與修復蛋白XPA會確保損傷位置正確，並啟動NER修復系統，XPC會辨識光化產物CPD及6-4PP，並且透過酵素打開雙股螺旋DNA，XPA及RPA會穩定受損蛋白，而ERCC-XPF及XPG將損傷位置進行切除，並以PCNA填補新的DNA，並由接合酶黏合完成修復。故利用西方墨點法觀察SKH-2小鼠皮膚中，觀察控制組、照射UVB (180 mJ/cm ²)組別、HEC gel空白背景組、含AMLW (25 mg/ml)組與含AMLE (2.5 mg/ml)組之於修復路徑蛋白質之表現量。結果如表8所示，設定一控制組，其蛋白質表現為1.0，與未照射UVB之控制組相比，經UVB照射組別，降低了修復路徑相關蛋白XPC、XPG、RPA、XPA、XPF、ERCC、PARP及PCNA等蛋白質之表現，而經UVB照射之HEC空白背景組，因為有膠的保護亦具有些微修復相關蛋白質之能力，以及經UVB照射後，含AMLW (25 mg/ml)與AMLE (2.5 mg/ml)之藥物組別，於修復相關因子之蛋白質XPC、XPG、RPA、XPA、XPF、ERCC、PARP及PCNA具有更佳上升趨勢之表現。說明AML _S具有增加NER修復系統之蛋白質表現量，而達到修復紫外線引起之DNA損傷之能力。 表8   控制組 以UVB照射 UVB HEC AMLW (25 mg/ml) AMLE (2.5 mg/ml) XPC 1.0 0.9 1.1 1.5 1.5 XPE 1.0 0.4 0.4 0.7 0.7 RPA 1.0 0.9 1.1 1.0 1.4 XPA 1.0 0.7 1.5 3.3 3.8 ERCC1 1.0 0.7 0.9 1.4 1.6 XPF 1.0 0.6 1.2 1.4 1.7 XPG 1.0 0.9 1.2 1.3 1.1 PCNA 1.0 0.9 1.4 1.7 2.2 β-actin 1.0 1.0 1.0 1.0 1.0 Nucleotide excision repair (NER) is the main repair system in mammals, mainly to eliminate the damage caused by ultraviolet rays. PARP will participate in the identification and repair of the damage site. The protein XPA will ensure the correct position of the damage and start the NER repair system, XPC It will recognize the photochemical products CPD and 6-4PP, and open the double-stranded DNA through enzymes, XPA and RPA will stabilize the damaged protein, while ERCC-XPF and XPG will excise the damaged position, and fill the new DNA with PCNA, and Repair is accomplished by ligase bonding. Therefore, the Western Blot method was used to observe the skin of SKH-2 mice, and observed the control group, the UVB (180 mJ/cm ² ) group, the HEC gel blank background group, the AMLW (25 mg/ml) group and the AMLE ( 2.5 mg/ml) group expressed in the repair pathway proteins. The results are shown in Table 8. A control group is set, and its protein performance is 1.0. Compared with the control group not irradiated with UVB, the group irradiated with UVB has reduced the repair pathway-related proteins XPC, XPG, RPA, XPA, XPF, The expression of proteins such as ERCC, PARP, and PCNA, and the HEC blank background group irradiated by UVB, because of the protection of glue, also has the ability to slightly repair related proteins, and after UVB irradiation, AMLW (25 mg/ml) and AMLE (2.5 mg/ml) drug group has a better upward trend in repair-related factors proteins XPC, XPG, RPA, XPA, XPF, ERCC, PARP and PCNA. This shows that AML _S has the ability to increase the protein expression of the NER repair system, thereby achieving the ability to repair DNA damage caused by ultraviolet rays. Table 8 control group Irradiate with UVB UVB HEC AMLW (25mg/ml) AMLE (2.5mg/ml) XPC 1.0 0.9 1.1 1.5 1.5 XPE 1.0 0.4 0.4 0.7 0.7 RPA 1.0 0.9 1.1 1.0 1.4 XPA 1.0 0.7 1.5 3.3 3.8 ERCC1 1.0 0.7 0.9 1.4 1.6 XPF 1.0 0.6 1.2 1.4 1.7 XPG 1.0 0.9 1.2 1.3 1.1 PCNA 1.0 0.9 1.4 1.7 2.2 β-actin 1.0 1.0 1.0 1.0 1.0

實施例22：「AML _S於SKH-2小鼠皮膚組織中修復相關因子之免疫螢光表現量」 Example 22: "Immunofluorescence expression of repair-related factors of AML _S in SKH-2 mouse skin tissue"

如前述所示，免疫螢光染色法為利用抗原與特異性抗體結合的原理，並利用螢光顯色劑顯色對組織細胞內抗原進行定性、定位及定量。試驗利用SKH-2小鼠，並分為控制組、照UVB (180 mJ/cm ²)組、HEC gel組、含AMLW (25 mg/ml)與AMLE (2.5 mg/ml)組別進行，並以皮膚之組織切片，以免疫組織螢光染色法觀察經藥物作用後其NER修復相關路徑之免疫螢光表現量。試驗結果如圖10A與10B所示，與控制組相比，受到UVB照射與HEC之組別，其修復因子之螢光表現量XPC、XPG、RPA、XPA、XPF、ERCC及PCNA等皆為下降趨勢，而在含AMLW (25 mg/ml)與AMLE (2.5 mg/ml)之組別中，XPC、XPG、RPA、XPA、XPF、ERCC、PARP及PCNA等修復因子之螢光表現量為提升趨勢。 As mentioned above, the immunofluorescent staining method utilizes the principle of combining an antigen with a specific antibody, and uses a fluorescent chromogenic reagent to develop the color to identify, locate and quantify the antigen in the tissue cells. The experiment used SKH-2 mice and divided them into control group, UVB (180 mJ/cm ² ) group, HEC gel group, AMLW (25 mg/ml) and AMLE (2.5 mg/ml) group, and Using immunohistofluorescence staining method to observe the immunofluorescence expression of NER repair-related pathways after drug action on skin tissue sections. The test results are shown in Figures 10A and 10B. Compared with the control group, the fluorescent expression levels of repair factors XPC, XPG, RPA, XPA, XPF, ERCC and PCNA were all decreased in the group exposed to UVB irradiation and HEC In the group containing AMLW (25 mg/ml) and AMLE (2.5 mg/ml), the fluorescence expression levels of repair factors such as XPC, XPG, RPA, XPA, XPF, ERCC, PARP and PCNA were increased trend.

無none

以下本發明之「山刺番荔枝葉萃取物」簡稱為「AML _S」；本發明之「山刺番荔枝葉水萃取物」簡稱為「AMLW」；本發明之「山刺番荔枝葉乙醇萃取物」簡稱為「AMLE」； 圖1A為一長條圖，說明「AMLW(1~500 μg/ml)於人類皮膚角質細胞(HaCaT)作用24小時後之HaCaT細胞存活度」； 圖1B為一長條圖，說明「AMLE(1~500 μg/ml)於人類皮膚角質細胞(HaCaT)作用24小時後之HaCaT細胞存活度」； 圖1C為一長條圖，說明「AMLW(50~1000 μg/ml)於老鼠黑色毒瘤細胞(B16F10)作用72小時後之B16F10細胞存活度」； 圖1D為一長條圖，說明「AMLE(50~1000 μg/ml)於老鼠黑色毒瘤細胞(B16F10)作用72小時後之B16F10細胞存活度」； 圖1E為一長條圖，說明「AMLW(100~1000 μg/ml)於老鼠成纖維母細胞(3T3L1)作用48小時後之3T3L1細胞存活度」； 圖1F為一長條圖，說明「AMLE(1~100 μg/ml)於老鼠成纖維母細胞(3T3L1)作用48小時後之3T3L1細胞存活度」； 圖2A至2B為一照片圖，說明「AMLW於SKH-2小鼠之發炎相關因子之免疫螢光組織染色試驗」； 圖3A為一長條圖，說明「AMLW與Arbutin經α-MSH於黑色素瘤細胞中作用72小時之細胞內黑色素含量」； 圖3B為一長條圖，說明「AMLW與Arbutin經α-MSH於黑色素瘤細胞中作用72小時之細胞外黑色素分泌之含量」； 圖4A至4B為一照片圖，說明「AMLW於SKH-2小鼠中，抑制預防光老化相關因子之免疫螢光活性」； 圖5A為一plasmid圖像，說明「AML _S於pUC119之DNA保護試驗，透過冷光照相系統之plasmid圖像」； 圖5B為一長條圖，說明「AML _S於pUC119DNA之保護試驗，以Image J定量AML _S於超螺旋DNA形式之位置」； 圖6為一長條圖，說明「AMLW經UVB照射後於HaCaT細胞促進細胞增生之能力」； 圖7A為一照片圖，說明「以免疫螢光染色法測定，經UVB照射後，與AMLW作用後光化產物CPD之含量變化」； 圖7B為一照片圖，說明「以免疫螢光染色法測定，經UVB照射後，與AMLW作用後6-4PPs之含量變化」； 圖8為一長條圖，說明「AMLW經UVB照射後於12小時、24小時及48小時之傷口癒合結果，其使用Image J定量之結果」； 圖9為一照片圖，說明「AMLW於SKH-2小鼠皮膚組織中HE-stain染色之結果」； 圖10A至10B為一照片圖，說明「AML _S於SKH-2小鼠皮膚組織中修復相關因子之免疫螢光表現量」。 The following "Soursop Leaf Extract" of the present invention is referred to as "AML _S "; the "Soursop Leaf Water Extract" of the present invention is referred to as "AMLW"; “AMLE” is abbreviated as “AMLE”; Figure 1A is a bar graph illustrating “the survival rate of HaCaT cells after AMLW (1~500 μg/ml) was applied to human skin keratinocytes (HaCaT) for 24 hours”; Figure 1B is a A bar graph illustrating "the viability of HaCaT cells after AMLE (1-500 μg/ml) was applied to human skin keratinocytes (HaCaT) for 24 hours"; Figure 1C is a bar graph illustrating "AMLW (50-1000 μg /ml) on mouse melanoma cells (B16F10) for 72 hours after B16F10 cell viability”; Figure 1D is a bar chart, illustrating “AMLE (50~1000 μg/ml) on mouse melanoma cells (B16F10) B16F10 cell viability after 72 hours"; Fig. 1E is a bar chart, illustrating "3T3L1 cell viability after AMLW (100~1000 μg/ml) was applied to mouse fibroblasts (3T3L1) for 48 hours"; Fig. 1F is a bar graph, illustrating "3T3L1 cell viability after AMLE (1~100 μg/ml) was applied to mouse fibroblasts (3T3L1) for 48 hours"; Figure 2A to 2B is a photo graph, illustrating "AMLW Immunofluorescent tissue staining test of inflammatory-related factors in SKH-2 mice"; Fig. 3A is a long bar graph illustrating "the intracellular melanin content of AMLW and Arbutin treated with α-MSH in melanoma cells for 72 hours"; Figure 3B is a long bar graph illustrating "the content of extracellular melanin secreted by AMLW and Arbutin in melanoma cells through α-MSH for 72 hours"; Figures 4A to 4B are a photo graph illustrating "AMLW in SKH- 2 In mice, inhibit the immunofluorescence activity of photoaging-related factors"; Figure 5A is a plasma image, illustrating "AML _S in the DNA protection test of pUC119, a plasma image through a luminescent camera system"; Figure 5B is A bar graph illustrating "AML _S in the pUC119 DNA protection test, using Image J to quantify the position of AML _S in the supercoiled DNA form"; The ability to proliferate”; Figure 7A is a photo graph illustrating “the change in the content of the photochemical product CPD after UVB irradiation and AMLW as determined by immunofluorescence staining”; Figure 7B is a photo graph illustrating “the Measured by immunofluorescence staining, after UVB irradiation, the content change of 6-4PPs after interacting with AMLW"; Hour, 24 hour and 48 hour wound healing results, which were quantified using Image J"; Figure 9 is a photo graph illustrating "AMLW HE-stain staining results in SKH-2 mouse skin tissue"; Figure 10A 10B to 10B is a photo graph illustrating "the immunofluorescent expression of repair-related factors of AML _S in the skin tissue of SKH-2 mice".

Claims (10)

一種山刺番荔枝葉萃取物的用途，係用於製備抗氧化、抗發炎、美白、修復及抗皺的組合物。The use of the soursop leaf extract is used for preparing anti-oxidation, anti-inflammation, whitening, repairing and anti-wrinkle compositions. 如請求項第1項所述之用途，其中該山刺番荔枝葉萃取物的製備方法包含：將山刺番荔枝葉浸泡於水中以取得一山刺番荔枝葉水萃取物。The use as described in item 1 of the claim, wherein the preparation method of the soursop leaf extract comprises: soaking the soursop leaves in water to obtain a soursop leaf water extract. 如請求項2所述之用途，其中該山刺番荔枝葉水萃取物的製備方法包含：秤取重量比為1：8至1：10之山刺番荔枝葉以及去離子水，放入超音波萃取設備於低溫下連續式萃取， 經高速離心收集上清液，再經抽真空篩檢程式過濾，以獲得澄清過濾液。The use as described in claim 2, wherein the preparation method of the soursop leaf water extract comprises: weighing the soursop leaves and deionized water with a weight ratio of 1:8 to 1:10, putting them into a super The sonic extraction equipment extracts continuously at low temperature. The supernatant is collected by high-speed centrifugation, and then filtered through a vacuum screening program to obtain a clear filtrate. 如請求項3所述之用途，其中，該山刺番荔枝葉水萃取物的製備方法更包含：將該澄清過濾液進行濃縮。The use as described in claim 3, wherein the preparation method of the water extract of soursop leaves further comprises: concentrating the clarified filtrate. 如請求項第4項所述之山刺番荔枝葉水萃取物，其中，每公克該山刺番荔枝葉水萃取物中總多酚之含量為86 mg至93.8 mg，每公克該山刺番荔枝葉水萃取物中總類黃酮之含量為33.1 mg至39.5 mg。The soursop leaf water extract as described in item 4 of the request, wherein the content of total polyphenols per gram of the soursop leaf water extract is 86 mg to 93.8 mg, and each gram of the soursop leaf water extract The content of total flavonoids in the water extract of litchi leaves ranged from 33.1 mg to 39.5 mg. 一種山刺番荔枝葉萃取物的用途，係用於製備毒殺癌細胞之組合物。The use of the soursop leaf extract is for preparing a composition for poisoning and killing cancer cells. 如請求項第6項所述之用途，其中該山刺番荔枝葉萃取物的製備方法包含：將山刺番荔枝葉浸泡於乙醇中以取得一山刺番荔枝葉乙醇萃取物。The use as described in item 6 of the claim, wherein the preparation method of the soursop leaf extract comprises: soaking the soursop leaves in ethanol to obtain an ethanol extract of soursop leaves. 如請求項7所述之用途，其中該山刺番荔枝葉之乙醇萃取物的製備方法包含：將該山刺番荔枝葉秤取100公克，於低溫下浸泡於五倍體積之乙醇中，取出後放入超音波萃取設備，於低溫下連續式萃取， 經高速離心收集上清液，再經抽真空篩檢程式過濾，以獲得澄清過濾液。The use as described in claim 7, wherein the preparation method of the ethanol extract of soursop leaves comprises: weighing 100 grams of the soursop leaves, soaking them in five times the volume of ethanol at low temperature, taking out Then put it into the ultrasonic extraction equipment, extract continuously at low temperature, collect the supernatant by high-speed centrifugation, and then filter through the vacuum screening program to obtain the clarified filtrate. 如請求項8所述之用途，其中，該山刺番荔枝葉乙醇萃取物的製備方法更包含：將該澄清過濾液進行濃縮。The use as described in Claim 8, wherein the preparation method of the ethanol extract of soursop leaves further comprises: concentrating the clarified filtrate. 如請求項第9項所述之山刺番荔枝葉乙醇萃取物，其中，每公克該山刺番荔枝葉乙醇萃取物中總多酚之含量為79 mg至82.4 mg，每公克該山刺番荔枝葉乙醇萃取物中總類黃酮之含量為7.3 mg至15.1 mg。The ethanol extract of soursop leaves as described in item 9 of the claim, wherein the content of total polyphenols in the ethanol extract of soursop leaves per gram is 79 mg to 82.4 mg, and each gram of soursop leaves The content of total flavonoids in the ethanol extract of litchi leaves ranged from 7.3 mg to 15.1 mg.

Images