TWI624265B - Use of dipsacus asper wall extract for manufacturing compositions to inhibit oxidation for anti aging skin - Google Patents
Use of dipsacus asper wall extract for manufacturing compositions to inhibit oxidation for anti aging skin Download PDFInfo
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Abstract
本發明係有關於一種川續斷萃取物用於製備抑制氧化作用達到皮膚抗老化之組成物的用途，係將一有效劑量之一川續斷萃取物投予至一所需個體，以清除自由基以及螯合亞鐵離子，其中川續斷萃取物係以至少一步驟之有機溶劑萃取製得；藉此，川續斷萃取物可用於製備抗氧化之化妝材料組成物或醫藥組成物，以保護皮膚細胞免於紫外線傷害。 The present invention relates to a use of a continuous extract for preparing a composition for inhibiting oxidation to achieve anti-aging of the skin, and administering an effective dose of one of the extracts to a desired individual to eliminate freedom And chelating ferrous ions, wherein the extract is obtained by extracting at least one step of an organic solvent; thereby, the extract can be used for preparing an antioxidant cosmetic composition or a pharmaceutical composition, Protect skin cells from UV damage.
Description
本發明係有關於一種川續斷萃取物用於製備抑制氧化作用達到皮膚抗老化之組成物的用途，尤其係指一具有清除自由基及螯合亞鐵離子等能力之川續斷萃取物，將其用於製備化妝材料組成物或醫藥組成物，可達到抗氧化、抗老化之功效。 The present invention relates to a use of a continuous extract for the preparation of a composition for inhibiting oxidation to achieve anti-aging of the skin, in particular to a squid extract having the ability to scavenge free radicals and chelate ferrous ions. It can be used to prepare a cosmetic material composition or a pharmaceutical composition to achieve anti-oxidation and anti-aging effects.
按，皮膚係人體內部生理活動的自然反映器，亦是與外界環境接觸的第一層防護。人體內常受到有害化學物質、光照或體內產生的自由基攻擊，例如生物體正常有氧代謝過程中會自然形成許多活性氧物質(reactive oxygen species，ROS)，包括超氧陰離子(superoxide anion radical，O2．)、過氧化氫(hydrogen peroxide，H2O2)、氫氧自由基(hydroxyl radical，OH．)、單重態氧(single oxygen，O2)等，尤其高活躍的自由基係帶有未配對電子，因此非常危險而不穩定，容易攻擊皮膚組織造成皮膚組織老化。因此，如何有效降低皮膚氧化作用以減緩皮膚老化速度，便成為相關領域發明人所思及之方向。 According to the skin, the natural reflector of the internal physiological activities of the human body is also the first layer of protection against the external environment. The human body is often attacked by harmful chemicals, light or free radicals generated in the body. For example, during the normal aerobic metabolism of organisms, many reactive oxygen species (ROS), including superoxide anion radicals, are formed. O 2 .), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH.), single oxygen (O 2 ), etc., especially highly active free radicals There are unpaired electrons, so it is very dangerous and unstable, and it is easy to attack skin tissue and cause skin tissue to age. Therefore, how to effectively reduce the skin oxidation to slow down the skin aging has become the direction that the inventors of the related art have thought.
傳統中草藥針對美容保養，多為一些補氣活血、調整體質的溫補藥物，亦有少許單、複方直接使用於皮膚美白、除痘、消炎等用途。 但是對於中草藥萃取物中之有效成分之機制分析與其於化粧品中之配方設計應用，仍是國內化粧保養品產業需積極開發的重點。如能開發傳統中草藥成為低劑量、高效能之抗氧化之化粧保養產品，將有助於提升化粧品科技產業。 Traditional Chinese herbal medicines are used for beauty care. They are mostly warming and tonifying blood and regulating body warming medicines. There are also a few single and compound directly used for skin whitening, acne removal, anti-inflammatory and other purposes. However, the mechanism analysis of the active ingredients in Chinese herbal extracts and its formulation and application in cosmetics are still the focus of the domestic cosmetics and skin care industry. If you can develop traditional Chinese herbal medicine into a low-dose, high-performance antioxidant cosmetic maintenance product, it will help to enhance the cosmetics technology industry.
川續斷，學名Dipsacus asper Wall，屬多年生草本植物，又稱川斷、續斷、接骨草、南草、鼓捶草、山蘿蔔根、山蘿蔔、屬折、龍豆、和尚頭、滋油菜、六汗等，目前已知川續斷可用以補肝腎、續筋骨、調血脈、益氣力，可治腰背酸痛、足膝無力、胎漏、崩漏、帶下、破癥結瘀血、遺精、婦人乳難，外用可治跌打損傷、刀傷、金瘡、痔漏、癰疽瘡腫。然，目前卻沒有具體研究證實川續斷萃取物於皮膚抗氧化之功效為何。 Chuan Xuan , the scientific name Dipsacus asper Wall , is a perennial herb, also known as Chuanchuan, Dianza, Sambucus, South Grass, Drumstick Grass, Mountain Radish Root, Mountain Radish, Folding, Longdou, Heshangtou, Nourishing Oil, Six sweats, etc., it is known that Sichuan can be used to replenish liver and kidney, continue bones and muscles, regulate blood circulation, benefit Qi, can cure low back pain, foot and knee weakness, fetal leakage, uterine bleeding, vaginal discharge, stagnation, blood stasis, nocturnal emission, Women's milk is difficult, external use can cure bruises, knife wounds, gold sores, leaks, hemorrhoids. However, there is no specific research to confirm the effect of the extract of Chuanxiong on the skin.
今，發明人即是鑑於上述現有川續斷萃取物尚缺乏抗氧化作用之研發，於是乃一本孜孜不倦之精神，並藉由其豐富專業知識及多年之實務經驗所輔佐，而加以改善，並據此研創出本發明。 Nowadays, the inventor is in the light of the lack of anti-oxidation effect of the above-mentioned existing extracts, which is an indefatigable spirit and is improved by its rich professional knowledge and years of practical experience. The present invention has been developed based on this.
本發明主要目的為提供一種川續斷萃取物用於製備抑制氧化作用達到皮膚抗老化之組成物的用途，其係指一具有清除自由基以及螯合亞鐵離子能力之川續斷萃取物，將其用於製備化妝材料組成物或醫藥組成物，可達到抑制皮膚氧化作用之功效。 The main object of the present invention is to provide a use of a continuous extract for the preparation of a composition for inhibiting oxidation to achieve anti-aging of the skin, which is a squid extract having the ability to scavenge free radicals and chelate ferrous ions. It can be used to prepare a cosmetic material composition or a pharmaceutical composition to achieve the effect of inhibiting skin oxidation.
為了達到上述實施目的，本發明一種川續斷萃取物用於製備抑制氧化作用達到皮膚抗老化之組成物的用途，其係將一有效劑量(可例如為50~1000μg/mL，最佳係50~500μg/mL)之一川續斷萃取物投予至一所需個體，以清除DPPH自由基及ABTS自由基及螯合亞鐵離子，其中，川續斷萃 取物係以至少一步驟之有機溶劑萃取製得，再者，有機溶劑係選自酯類、醇類、烷類及鹵烷所構成之族群。 In order to achieve the above-mentioned object, the use of the extract of the present invention for preparing a composition for inhibiting oxidation to achieve anti-aging of the skin is an effective dose (for example, 50 to 1000 μg/mL, preferably 50). ~500μg/mL) One of the continuous extracts is administered to a desired individual to scavenge DPPH free radicals and ABTS free radicals and chelate ferrous ions. The extract is obtained by extracting at least one step of an organic solvent. Further, the organic solvent is selected from the group consisting of esters, alcohols, alkanes, and halothanes.
於本發明之一實施例中，川續斷萃取物之萃取步驟，係包含：步驟一：利用第一有機溶劑萃取一川續斷材料，以獲得一第一萃取物；步驟二：利用第二有機溶劑萃取第一萃取物，以劃分出一第一有機相以及一第二有機相，其中第一有機相具有一第二萃取物，且第二有機相具有一第一剩餘物；步驟三：利用一第三有機溶劑萃取第一剩餘物，以劃分出一第三有機相以及第一水相，其中第三有機相具有一第三萃取物，且第一水相具有一第二剩餘物；以及步驟四：利用一第四有機溶劑萃取第二剩餘物，以劃分出一第四有機相以及一第二水相，其中第四有機相具有一第四萃取物，且第二水相具有一第三剩餘物。 In an embodiment of the present invention, the extracting step of the extract of the extract comprises: step 1: extracting the continuous material by using the first organic solvent to obtain a first extract; and step 2: using the second Extracting the first extract with an organic solvent to divide a first organic phase and a second organic phase, wherein the first organic phase has a second extract and the second organic phase has a first residue; Step 3: Extracting a first residue by using a third organic solvent to divide a third organic phase and a first aqueous phase, wherein the third organic phase has a third extract, and the first aqueous phase has a second residue; And step 4: extracting the second residue by using a fourth organic solvent to divide a fourth organic phase and a second aqueous phase, wherein the fourth organic phase has a fourth extract, and the second aqueous phase has a The third residue.
於本發明之一實施例中，第一有機溶劑為乙醇溶液、第二有機溶劑為正己烷/甲醇溶液、第三有機溶劑為乙酸乙酯/水溶液，而第四有機溶劑為正丁醇/水溶液。 In one embodiment of the present invention, the first organic solvent is an ethanol solution, the second organic solvent is a n-hexane/methanol solution, the third organic solvent is an ethyl acetate/water solution, and the fourth organic solvent is an n-butanol/water solution. .
於本發明之一實施例中，第一有機溶劑為體積百分比95%的乙醇溶液，第二有機溶劑為體積比1：1的正己烷/95%甲醇溶液，第三有機溶劑為體積比2：1的乙酸乙酯/水溶液，且第四有機溶劑為體積比1：1的正丁醇/水溶液。 In one embodiment of the present invention, the first organic solvent is a 95% by volume ethanol solution, the second organic solvent is a 1:1 volume ratio of n-hexane/95% methanol solution, and the third organic solvent is a volume ratio of 2: An ethyl acetate/water solution of 1 and a fourth organic solvent is a n-butanol/water solution in a volume ratio of 1:1.
於本發明之一實施例中，第一有機相為正己烷相，第二有機相為甲醇相，第三有機相為乙酸乙酯相，以及第四有機相為正丁醇相。 In one embodiment of the invention, the first organic phase is a n-hexane phase, the second organic phase is a methanol phase, the third organic phase is an ethyl acetate phase, and the fourth organic phase is a n-butanol phase.
藉此，川續斷萃取物可進一步作為抗氧化之化妝材料組成物或醫藥組成物，提供一較佳抗氧化組合物之選擇。 Thereby, the extract can further serve as an antioxidant composition or a pharmaceutical composition, providing a preferred antioxidant composition.
第一圖：本發明較佳實施例之川續斷萃取步驟流程圖 First Figure: Flowchart of the continuous extraction step of the preferred embodiment of the present invention
第二圖：細胞內活性氧生成之測定 Figure 2: Determination of intracellular reactive oxygen species production
本發明之目的及其結構功能上的優點，將依據以下圖面所示之結構，配合具體實施例予以說明，俾使審查委員能對本發明有更深入且具體之瞭解。 The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.
首先，本發明一種川續斷萃取物用於製備抗氧化之組成物的用途，其係將一有效劑量(可例如為50~1000μg/mL，最佳係50~500μg/mL)之一川續斷萃取物投予至一所需個體，以清除DPPH自由基及ABTS自由基及螯合亞鐵離子；其中，川續斷萃取物係以至少一步驟之有機溶劑萃取製得；有機溶劑係選自酯類、醇類、烷類及鹵烷所構成之族群。 First, the use of the extract of the present invention for preparing an antioxidant composition is an effective dose (for example, 50 to 1000 μg/mL, preferably 50 to 500 μg/mL). The extract is administered to a desired individual to remove DPPH free radicals and ABTS free radicals and chelate ferrous ions; wherein the extract is prepared by extracting at least one step of an organic solvent; A group consisting of esters, alcohols, alkanes, and halos.
再者，根據上述之用途，請參閱第一圖，為本發明較佳實施例之川續斷萃取步驟流程圖，可包含：步驟一(S1)：利用第一有機溶劑(可例如為體積百分比95%乙醇溶液)萃取一川續斷材料，以獲得一第一萃取物；步驟二(S2)：利用第二有機溶劑(可例如為體積比1：1的正己烷/95%甲醇溶液)萃取第一萃取物，以劃分出一第一有機相(可例如為正己烷相)以及一第二有機相(可例如為甲醇相)，其中第一有機相具有一第二萃取物，且第二有機相具有一第一剩餘物；步驟三(S3)：利用一第三有機溶劑(可例如為體積比2：1的乙酸乙酯/水溶液)萃取第一剩餘物，以劃分出一第三有機相(可例如為乙酸乙酯 相)以及第一水相，其中第三有機相具有一第三萃取物，且第一水相具有一第二剩餘物；以及步驟四(S4)：利用一第四有機溶劑(可例如為體積比1：1的正丁醇/水溶液)萃取第二剩餘物，以劃分出一第四有機相(可例如為正丁醇相)以及一第二水相，其中第四有機相具有一第四萃取物，且第二水相具有一第三剩餘物。藉此，由上述方法萃取而得之川續斷萃取物可作為抗氧化之化妝材料組成物或醫藥組成物。 In addition, according to the above application, please refer to the first figure, which is a flow chart of the continuous extraction step of the preferred embodiment of the present invention, which may include: Step 1 (S1): using the first organic solvent (for example, volume percentage) 95% ethanol solution) extracting a continuous material to obtain a first extract; step 2 (S2): extracting with a second organic solvent (for example, a 1:1 volume ratio of n-hexane/95% methanol) a first extract to separate a first organic phase (which may be, for example, a n-hexane phase) and a second organic phase (which may be, for example, a methanol phase), wherein the first organic phase has a second extract, and the second The organic phase has a first residue; Step 3 (S3): extracting the first residue with a third organic solvent (for example, ethyl acetate/water solution in a volume ratio of 2:1) to define a third organic Phase (can be, for example, ethyl acetate And a first aqueous phase, wherein the third organic phase has a third extract and the first aqueous phase has a second residue; and the fourth step (S4): utilizes a fourth organic solvent (which may be, for example, a volume) Extracting a second residue from a ratio of 1:1 n-butanol/water solution to define a fourth organic phase (which may be, for example, a n-butanol phase) and a second aqueous phase, wherein the fourth organic phase has a fourth The extract, and the second aqueous phase has a third residue. Thereby, the extract obtained by the above method can be used as an anti-oxidation cosmetic material composition or a pharmaceutical composition.
此外，藉由下述具體實施例，可進一步證明本發明可實際應用之範圍，但不意欲以任何形式限制本發明之範圍。 In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.
首先，關於川續斷(Dipsacus asper Wall)萃取，係先取得生長於地上部分(aerial part)之川續斷加以乾燥，再藉由如上述萃取步驟流程製得不同階段之川續斷萃取物(第一萃取物~第四萃取物)，進一步配置成50、100、500及1000μg/mL不同濃度，進行以下試驗。 First of all, regarding the Dipsacus asper Wall extraction, the first part is obtained by drying on the aerial part and dried, and the extracts of different stages are obtained by the above extraction step process ( The first to fourth extracts were further divided into different concentrations of 50, 100, 500 and 1000 μg/mL, and the following tests were carried out.
〈細胞存活度試驗〉 Cell viability test
採用MTT assay檢測。將細胞(1×104/well)培養在96-well盤，並在37℃及5% CO2培養箱中培養至少24小時。加入不同濃度的川續斷萃取物以及於指定的時間作用，達反應時間，移除舊的培養液，以PBS清洗一次，並換上新的培養液，加入10μL的MTT(3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)溶液反應，於37℃、5% CO2反應4小時後移除培養液，加入100μl的DMSO溶解formazan沉澱物，最後於波長570nm下測定吸光值(BioTek,SynergyTM2,USA)。 Detection by MTT assay. The cells (1 x 10 4 /well) were cultured in 96-well plates and cultured for at least 24 hours at 37 ° C in a 5% CO 2 incubator. Add different concentrations of Chuanxuan extract and react at the specified time to reach the reaction time, remove the old culture solution, wash once with PBS, and replace with new medium, add 10 μL of MTT (3-(4, 5-cimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution reaction, the culture solution was removed after reacting at 37 ° C, 5% CO 2 for 4 hours, 100 μl of DMSO was added to dissolve the formazan precipitate, and finally at a wavelength of 570 nm. Determination of absorbance (BioTek, Synergy TM 2, USA ).
結果如表一所示，於HaCaT人類皮膚角質細胞株或B16小鼠 黑色素瘤細胞株處理50~500μg/mL的川續斷萃取物，細胞具有較高的存活率，當川續斷萃取物之濃度為1000μg/mL，細胞存活率大量下降，表示較佳川續斷萃取物之濃度為50~500μg/mL。 The results are shown in Table 1, in HaCaT human skin keratinocyte strain or B16 mouse. The melanoma cell line was treated with 50~500μg/mL of Chuanzhuo extract, and the cell had a high survival rate. When the concentration of the extract was 1000μg/mL, the cell survival rate decreased greatly, indicating that the cell survival rate was better. The concentration of the extract is 50-500 μg/mL.
實驗一：川續斷萃取物於清除自由基能力之試驗 Experiment 1: Experiment on the ability of extracts from Sichuan to remove free radicals
〈清除DPPH．自由基試驗〉 <Clear DPPH. Free radical test
1,1-二苯基-2-苦醯肼基(1,1-Diphenyl-2-picrylhydrazyl，DPPH．)自由基係一種穩定的自由基，在波長517nm有最大吸光值。藉由加入樣品乙醇溶液後測量其517nm吸光值變化，即可換算出濃度下之自由基抑制率。當顏色由藍紫轉為淡黃色，吸光值也隨之降低，因此可藉由517nm下吸光值的變化，來評估抗氧化物清除自由基的能力。將配製成不同濃度(50、100、500及1000μg/mL)之川續斷萃取物，依序加入90mL新鮮配置的100μM DPPH乙醇溶液，以分光光度計檢測517nm之吸光值，其中，維生素C(ascorbic acid)係作為對照標準品。當吸光值越低表示樣品清除DPPH．自由基的能力越強，以[1-(樣品於517nm之吸光值/未添加樣品之控制組於517nm之吸光值)]×100%得到清除效應百分率(scavenging effects %)。 The 1,1-diphenyl-2-picrylhydrazyl (DPPH.) radical is a stable free radical having a maximum absorbance at a wavelength of 517 nm. The free radical inhibition rate at the concentration can be converted by measuring the change in absorbance at 517 nm after adding the sample ethanol solution. When the color changes from blue-violet to pale yellow, the absorbance decreases, so the ability of the antioxidant to scavenge free radicals can be evaluated by the change in absorbance at 517 nm. The extracts were prepared at different concentrations (50, 100, 500, and 1000 μg/mL), and 90 mL of freshly prepared 100 μM DPPH ethanol solution was added in sequence, and the absorbance at 517 nm was measured by a spectrophotometer. (ascorbic acid) was used as a control standard. When the absorbance value is lower, the sample clears DPPH. The stronger the ability of the radical, the scavenging effect % was obtained by [1-(the sample absorbance at 517 nm / the absorbance at 517 nm of the control group without added sample)] x 100%.
結果如表二所示，川續斷萃取物具有清除DPPH．自由基的能力，尤其係川續斷第一萃取物及第三萃取物在濃度500μg/mL以下，就具有高達九成的DPPH．自由基清除能力。 The results are shown in Table 2. The extract of Chuanxuan has DPPH clearance. The ability of free radicals, especially the first extract and the third extract of the genus, have a DPPH of up to 90% at a concentration of less than 500 μg/mL. Free radical scavenging ability.
〈清除ABTS陽離子自由基試驗〉 <Cleaning ABTS cation free radical test>
2,2’-次偶氮基-雙(3-乙基苯並噻唑啉-6-磺酸)(2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid，ABTS)在過氧化酶(peroxidase)的催化下，會產生ABTS．+自由基，而呈現穩定的藍綠色。因此藉由加入抗氧化物參與反應，ABTS．+得以還原成ABTS而抑制藍綠色生成，並經由測定734nm吸光值的變化，可評估抗氧化物的抗氧化能力。將2.45mM K2S2O8和7mM ABTS溶液混合後，避光靜置16-18小時後，反應產生自由基，接續取1.5μL不同濃度之萃取物混合3.5μL ddH2O及145μL ABTS與K2S2O8混合溶液，靜置20分鐘，以分光光度計檢測734nm之吸光值。當吸光值越低表示樣品清除ABTS自由基的能力越強。再以不同濃度的水溶性維生素E(trolox)抗氧化劑清除ABTS陽離子自由基的能力作為標準曲線。以[1-(樣品於734nm之吸光值/未添加樣品之控制組於734nm之吸光值)]×100%得到清除效應百分率(scavenging effects %)。 2,2'-Azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid, ABTS) Under the catalysis of oxidase, ABTS. + radicals are produced, and a stable blue-green color is formed. Therefore, ABTS. + can be reduced to ABTS to inhibit blue-green formation by adding antioxidants. The change in absorbance at 734 nm allows evaluation of the antioxidant capacity of the antioxidant. After mixing 2.45 mM K 2 S 2 O 8 and 7 mM ABTS solution, the reaction was allowed to stand for 16-18 hours, and the reaction produced free radicals, followed by 1.5. The μL different concentrations of the extract were mixed with 3.5 μL of ddH 2 O and 145 μL of ABTS and K 2 S 2 O 8 mixed solution, and allowed to stand for 20 minutes to detect the absorbance at 734 nm by spectrophotometer. The lower the absorbance value, the sample clear ABTS free. The stronger the ability of the base, the ability to remove ABTS cationic free radicals with different concentrations of water-soluble vitamin E (trolox) antioxidant as a standard curve. [1-(Sample at 734nm absorbance / no added sample control group) The absorbance at 734 nm)] × 100% gives the scavenging effects %.
結果如表三所示，川續斷萃取物具有清除ABTS自由基的能力，尤其係川續斷第一萃取物、第三萃取物及第四萃取物在濃度500μg/mL以下，就具有高達九成的ABTS自由基清除能力。 The results are shown in Table 3. The extract of Chuanxuan has the ability to scavenge ABTS free radicals. In particular, the first extract, the third extract and the fourth extract are at a concentration of 500 μg/mL or less. The ability to remove ABTS free radicals.
實驗二：川續斷萃取物於螯合亞鐵離子能力之試驗 Experiment 2: Test of the ability of succulent extracts to chelate ferrous ions
在多種金屬離子中，亞鐵離子(Fe2+)是最具影響力的促氧化劑，會促進脂質氧化作用的進行。利用亞鐵離子與亞鐵嗪(4,4'-[3-(2-吡啶基)-1,2,4-三嗪-5,6-二基]-二苯磺酸二鈉鹽；4,4'-(3-(2-pyridinyl)-1,2,4-triazine-5,6-diy1)bisbenzenesulfonic acid,disodium salt；ferrozine)的複合物在波長562nm之下會有呈色反應，可以測得樣品對亞鐵離子的螯合能力。當樣品可螯合亞鐵離子時，會造成562nm之吸光值的降低，因此吸光值越低表示樣品的螯合能力越強。取10μL之萃取物並加入930μL二次去離子水及20μL FeCl2靜置30秒後，再加入40μL、5mM ferrozine，反應10分鐘後，以分光光度計檢測562nm之吸光值；其中，EDTA係作為對照標準品。 Among various metal ions, ferrous ions (Fe 2+ ) are the most influential oxidants, which promote the oxidation of lipids. Using ferrous ions and ferrous iron (4,4'-[3-(2-pyridyl)-1,2,4-triazin-5,6-diyl]-diphenylsulfonic acid disodium salt; 4 , 4'-(3-(2-pyridinyl)-1,2,4-triazine-5,6-diy1)bisbenzenesulfonic acid,disodium salt;ferrozine) has a color reaction at a wavelength of 562 nm, The chelating ability of the sample to ferrous ions was measured. When the sample can chelate ferrous ions, it will cause a decrease in the absorbance at 562 nm, so the lower the absorbance value, the stronger the chelating ability of the sample. After taking 10 μL of the extract and adding 930 μL of secondary deionized water and 20 μL of FeCl 2 for 30 seconds, 40 μL of 5 mM ferrozine was added, and after 10 minutes of reaction, the absorbance at 562 nm was measured by a spectrophotometer; Control standard.
結果請參閱表四，不同濃度(50、100、500及1000μg/mL)之川續斷萃取物具有螯合亞鐵離子能力，尤其係以第二萃取物及第三萃取物，在濃度500μg/mL以下，就具有高達六成的螯合亞鐵離子能力。 The results are shown in Table 4. The different concentrations (50, 100, 500, and 1000 μg/mL) of the Chuan continuous extract have the ability to chelate ferrous ions, especially with the second extract and the third extract at a concentration of 500 μg / Below 5.5, it has up to 60% of the ability to chelate ferrous ions.
實驗三：川續斷萃取物於還原能力之試驗 Experiment 3: Experiment on the reducing ability of the extract
將4.8μL不同濃度的川續斷萃取物加入50μL的磷酸鹽緩衝液，其中磷酸鹽緩衝液含有1%赤血鹽(K3Fe(CN)6)，50℃水浴20分鐘後，加入50μL 10% TCA，混合均勻之後，離心10,000轉10分鐘，取25μL上清液和9.1μL 0.1%的三氯化鐵(FeCl3)，最後加入65.9μL ddH2O，於700nm測其 吸光值；其中，維生素C係作為對照標準品。 4.8 μL of different concentrations of Chuanzhiu extract were added to 50 μL of phosphate buffer containing 1% red blood salt (K 3 Fe(CN) 6 ), and after 50 minutes at 50 ° C, 50 μL of 10 was added. % TCA, after mixing uniformly, centrifugation for 10,000 rpm for 10 minutes, taking 25 μL of the supernatant and 9.1 μL of 0.1% ferric chloride (FeCl 3 ), and finally adding 65.9 μL of ddH 2 O, and measuring the absorbance at 700 nm; Vitamin C was used as a control standard.
結果如表五所示，濃度500~1000μg/mL之川續斷萃取物(尤其係第三萃取物及第四萃取物)具有極佳的還原力。 As a result, as shown in Table 5, the continuous extracts (especially the third extract and the fourth extract) having a concentration of 500 to 1000 μg/mL have excellent reducing power.
實驗四：細胞內活性氧(ROS)生成測定之試驗 Experiment 4: Test for determination of intracellular reactive oxygen species (ROS) production
將1×105cell/mL的細胞數培養在96-well plate中，並在37℃、5% CO2培養箱中生長24小時以上，再加入1μL的川續斷萃取物反應。達反應時間後，移除上清液，以PBS清洗，加入含有0.1mM H2O2之新鮮培養液作用一小時，接著移除培養液，加入含有10μM 2’,7’-dichlorofluorescin diacetate(DCFH2DA)之新鮮培養液避光作用一小時後，移除培養液，加入定量PBS，於激發光502nm/散色光524nm下測其吸光值(BioTek,SynergyTM2,USA)。 The cell number of 1 × 10 5 cells/mL was cultured in a 96-well plate, and grown in a 37 ° C, 5% CO 2 incubator for 24 hours or more, and then 1 μL of the extract was added. After the reaction time, the supernatant was removed, washed with PBS, and fresh culture medium containing 0.1 mM H 2 O 2 was added for one hour, then the culture solution was removed, and 10 μM 2',7'-dichlorofluorescin diacetate (DCFH) was added. 2 DA) Fresh culture solution was protected from light for one hour, the culture solution was removed, and quantitative PBS was added thereto, and the absorbance was measured at 502 nm/scatter light 524 nm (BioTek, Synergy TM 2, USA).
結果請參閱第二圖，加入0.1mM H2O2會增加細胞內活性氧含量(cellular ROS content)；處理500μg/mL之川續斷萃取物則能降低H2O2引起的ROS含量，尤其以第一萃取物及第二萃取物具有較佳抑制活性氧生成之能力。 For the results, please refer to the second figure. Adding 0.1 mM H 2 O 2 will increase the cellular ROS content; treatment of 500 μg/mL of the continuous extract will reduce the ROS content caused by H 2 O 2 , especially The first extract and the second extract have the ability to preferably inhibit the formation of active oxygen.
藉由上述實驗證實，川續斷萃取物確實具有清除自由基、螯合亞鐵離子、抗氧化之能力；藉此，川續斷萃取物可進一步應用於作為皮膚抗氧化之化妝材料組成物、保養品或醫藥組成物，並且此醫藥組成物可與一皮膚外用劑合併使用。上述“皮膚外用劑”意指一通常在化妝品或醫藥品中被使用的外用成份，包括，但不限於：其他的美白劑、保濕劑、抗 氧化劑、紫外線吸收劑、介面活性劑、增稠劑、色料以及皮膚營養劑等等，可例如為濃縮精華液之成分或添加於面膜中，提供使用者以一適當量施予皮膚，進而達到減緩皮膚氧化或老化之功效。 It has been confirmed by the above experiments that the extract of Chuanxuan does have the ability to scavenge free radicals, chelate ferrous ions, and resist oxidation; thereby, the extract can be further applied as a cosmetic material composition for skin anti-oxidation, A skin care product or a pharmaceutical composition, and the pharmaceutical composition can be used in combination with a skin external preparation. The above-mentioned "skin external preparation" means a topical ingredient which is usually used in cosmetics or pharmaceuticals, including, but not limited to, other whitening agents, moisturizers, and anti-aging agents. The oxidizing agent, the ultraviolet absorbing agent, the surfactant, the thickening agent, the coloring material, the skin nutrient, and the like can be, for example, a component of the concentrated essence or added to the mask, and the user can be applied to the skin in an appropriate amount to achieve Reduces the effects of skin oxidation or aging.
由上述之實施說明可知，本發明與現有技術相較之下，本發明具有以下優點： It can be seen from the above description that the present invention has the following advantages compared with the prior art:
1.本發明證實天然中草藥川續斷萃取物具有低細胞毒性、可清除自由基、具有還原力、以及螯合亞鐵離子之能力，其可用於作為塗抹皮膚之化妝材料組成物、保養品或醫藥組成物，達到抗皮膚細胞老化之功效。 1. The invention proves that the natural Chinese herbal medicine Chuanzhi extract has low cytotoxicity, can scavenge free radicals, has reducing power, and has the ability to chelate ferrous ions, and can be used as a makeup material composition, skin care product for applying skin or A pharmaceutical composition that achieves anti-aging effects on skin cells.
2.目前坊間鮮少有川續斷保養品，本發明拓展川續斷於抗氧化之應用，提供消費者一種新的天然保養品選擇。 2. At present, there are few products in the market. The invention expands the application of anti-oxidation and provides consumers with a new choice of natural skin care products.
綜上所述，本發明之川續斷萃取物用於製備抑制氧化作用達到皮膚抗老化之組成物的用途，的確能藉由上述所揭露之實施例，達到所預期之使用功效，且本發明亦未曾公開於申請前，誠已完全符合專利法之規定與要求。爰依法提出發明專利之申請，懇請惠予審查，並賜准專利，則實感德便。 In summary, the use of the continuous extract of the present invention for preparing a composition for inhibiting oxidation to achieve anti-aging of the skin can indeed achieve the intended use efficiency by the above-disclosed examples, and the present invention It has not been disclosed before the application, and Cheng has fully complied with the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.
惟，上述所揭之圖示及說明，僅為本發明之較佳實施例，非為限定本發明之保護範圍；大凡熟悉該項技藝之人士，其所依本發明之特徵範疇，所作之其它等效變化或修飾，皆應視為不脫離本發明之設計範疇。 The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.
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| TW201212948A (en) * | 2010-09-30 | 2012-04-01 | Univ Chia Nan Pharm & Sciency | Extracts from Peony's root cortex for application of antioxidation and method of preparing the same |
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