TWI436771B - Flos lonicerae extract, manufacturing method and use thereof - Google Patents
Flos lonicerae extract, manufacturing method and use thereof Download PDFInfo
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本發明係關於一金銀花萃取物,尤其是關於一種具有抗痤瘡桿菌(Propionibacterium acnes) 功效的金銀花萃取物、以及其製備方法及應用。The present invention relates to a honeysuckle extract, and more particularly to a honeysuckle extract having the efficacy against Propionibacterium acnes , and a preparation method and application thereof.
台灣東部地區由於地理環境因素影響,且具有豐富天然資源可供利用,天然物年產值約計12億元,尤其東部保有許多台灣特有原生植物,其中以台東的金銀花及花蓮的丹蔘為東部地區推廣栽種面積最廣且具特色。如何衍生東部特用作物其產品應用,有助於帶動東部經濟價值。而其中一項特用作物-金銀花,於《本草綱目》記載其善於化毒,故治癰疽、腫毒、瘡癬等”。自古以來,金銀花常用於清熱解毒,治療溫病發熱,熱毒血痢,癰瘍等癥,亦用於風熱感冒,支氣管炎等病癥。而現代藥理研究表明,金銀花具有抑菌、抗病毒、抗炎、解熱、調節免疫等作用。近年來文獻報導指出,金銀花含環己六醇、黃酮類、皂苷類等,具有廣泛抗菌作用,文獻指出對金黃色葡萄球菌、痢疾桿菌、雙球菌、蠟樣芽胞桿菌等均有較強的抑制作用,且對鉤端螺旋體、流感病毒等多種病原微生物亦有抑制作用。Due to geographical and environmental factors, and the abundant natural resources are available in the eastern part of Taiwan, the annual output value of natural products is about 1.2 billion yuan. In particular, there are many Taiwanese native plants in the east, including the honeysuckle in Taitung and the Tanjung in Hualien. The area is the most widely spread and unique. How to derive the use of Eastern special products for their products helps to drive the economic value of the East. One of the special uses - honeysuckle, in the "Compendium of Materia Medica" records that it is good at poisoning, so cure phlegm, swollen poison, sore sputum, etc.. Since ancient times, honeysuckle is often used for detoxification, treatment of fever, fever Toxic blood stasis, phlegm and ulcers are also used in wind-heat flu, bronchitis and other diseases. Modern pharmacological research shows that honeysuckle has antibacterial, antiviral, anti-inflammatory, antipyretic, immune regulation and other effects. It is pointed out that honeysuckle contains cyclohexanol, flavonoids, saponins, etc., which has a wide range of antibacterial effects. The literature indicates that it has strong inhibitory effects on Staphylococcus aureus, dysentery bacilli, diplococcus, and Bacillus cereus, and It also inhibits various pathogenic microorganisms such as Leptospira and influenza virus.
痤瘡是由痤瘡桿菌(Propionibacterium acnes )所引起的一種慢性皮膚毛囊炎症,目前治療青春痘大多使用過氧化苯(Benzoyl Peroxide)、硫磺(Sulfur)、三氯生(Triclosan)、口服抗生素-四環黴素(Tetracycline),外用抗生素-紅黴素(Erythromycin)、克林達黴素(Clindamycin)等。而造成青春痘的病原菌為痤瘡桿菌(Propionibacterium acnes) ,也包含其他共生菌如金黃色葡萄球菌(Staphylococcus aureus ),因此若能找到一種中草藥種同時具備抗這類菌種的能力,將有助於應用於青春痘照護相關產品開發。Acne is a chronic skin follicle inflammation caused by Propionibacterium acnes . Most of the current treatments for acne use Benzoyl Peroxide, Sulfur, Triclosan, Oral Antibiotics - Tetrahymena Tetracycline, topical antibiotics - erythromycin (Erythromycin), clindamycin (Clindamycin) and the like. The pathogen causing acne is Propionibacterium acnes , which also contains other commensal bacteria such as Staphylococcus aureus . Therefore, if a Chinese herbal medicine can be found and has the ability to resist such species, it will help. Used in the development of acne care related products.
中華人民共和國專利申請號CN200710003451.0,”含慶大黴素的藥物(Medicine containing gentamycin)”專利,雖然此前案專利提到採用與金銀花同屬的銀花,約占中藥材8.6%,然而此萃取法是以水萃取,主要目的在於應用中藥萃取物進行氣血運行的調理,並未探討金銀花萃取物對於抗痤瘡桿菌的功效。The People's Republic of China patent application number CN200710003451.0, "Medicine containing gentamycin" patent, although the previous patent mentioned the use of silver flowers of the same family as honeysuckle, accounting for 8.6% of Chinese herbal medicines, however This extraction method is based on water extraction. The main purpose is to apply the Chinese medicine extract for the conditioning of blood and blood. The effect of the honeysuckle extract on the anti-acne bacillus is not discussed.
美國專利號6083921“Pharmaceutical compositions and method of using same”,揭示一種藥物的組成,由黃芩(Radix scutellariae)、金銀花(Flos Lonicera)、以及連翹(Fructus forsythiae)萃取活性物質而來,不同的成分組成其抗菌效果有異,但亦無抗痤瘡桿菌的功效探討,且此前案的金銀花萃取物為水溶性的綠原酸,而非萃取油溶性的活性物質。U.S. Patent No. 6,083,921, "Pharmaceutical Composition and method of using same", discloses a composition of a drug which is obtained by extracting active substances from Radix scutellariae, Flos Lonicera, and Fructus forsythiae. The antibacterial effect is different, but there is no anti-acne bacillus effect, and the previous honeysuckle extract is water-soluble chlorogenic acid, rather than extracting oil-soluble active substances.
中華民國專利公開第200932205號”痤瘡肌膚用皮膚外用劑”,此前案之痤瘡肌膚用皮膚外用劑的特徵在於杜鵑醇及其衍生物中之至少1種以上,再結合炎症抑制效果的植物萃取物,例如:由山黃梔(Gardenia Florida)、芍藥(Paeonia Albiflora root)、金銀花(Lonicera japonica)之萃取物。該案並未提供金銀花萃取物之萃取方法,且未探討金銀花具抗痤瘡桿菌之功效。The Republic of China Patent Publication No. 200932205 "External Agent for Skin for Acne Skin", the skin external preparation for acne skin of the previous case is characterized by at least one of rhododendrol and its derivatives, and a plant extract which combines an inhibitory effect on inflammation. For example, an extract from Gardenia Florida, Paeonia Albiflora root, and Lonicera japonica. The case did not provide an extraction method for honeysuckle extract, and did not investigate the efficacy of honeysuckle against acne bacteria.
美國專利號5,834,000“Antiviral and antimicrobial herbal complex”,揭示以95%乙醇(ethanol)以及5%異丙醇(isopropyl alcohol)於82℃下萃取金銀花,其萃取物成分以黃酮類為主,例如木犀草素(Luteolin)、忍冬苷(Lonicerin)、木犀草素葡萄糖苷(Luteolin glucoside)、肌醇(inositol)等,具有抗菌及減緩喉嚨痛功效,然而此前案並未揭示金銀花萃取物具有抑制痤瘡桿菌或治療青春痘的功效。U.S. Patent No. 5,834,000 "Antiviral and antimicrobial herbal complex" discloses the extraction of honeysuckle with 95% ethanol and 5% isopropyl alcohol at 82 ° C. The extract is mainly composed of flavonoids such as wood rhinoceros. Luteolin, Lonicerin, Luteolin glucoside, inositol, etc., have antibacterial and slowing sore throat effects, but the previous case did not reveal that honeysuckle extract has anti-acne Bacillus or the efficacy of treating acne.
美國專利號6063383“Pharmaceutical suppository composites for fever and influenza and method of producing the composites”,揭示一種治療發燒與流行性之感冒的栓劑合成物,其成分中包含金銀花(flos lonicerae japonicae)。此前案係以高溫煎煮的方式進行活性物質的萃取,其具有抗菌功效,除了可抑制發燒、排毒外,還提到可治療青春痘。U.S. Patent No. 6,063,383, "Pharmaceutical suppository composites for fever and influenza and method of producing the composites", discloses a suppository composition for the treatment of fever and epidemic cold, which contains honeysuckle (flos lonicerae japonicae). Previously, the case was extracted by high-temperature decoction, which has antibacterial effect. In addition to inhibiting fever and detoxification, it also mentioned that it can treat acne.
由於目前講求天然的皮膚照護,以避免多餘的化學產品造成皮膚負擔,因此尋求其他天然抗痤瘡桿菌的中草藥,進行青春痘照護相關材料開發,具市場需求。Since natural skin care is currently being sought to avoid the burden of skin caused by excess chemical products, it is necessary to seek other natural Chinese herbal medicines against acne bacteria for the development of acne care related materials.
本發明提供一種金銀花萃取物,其組成包括:20-50重量份的綠原酸(chlorogenic acid);1-5重量份的齊墩果酸(oleanolic acid);以及0.001-5重量份的熊果酸(ursolic acid)。The present invention provides a honeysuckle extract comprising: 20-50 parts by weight of chlorogenic acid; 1-5 parts by weight of oleanolic acid; and 0.001-5 parts by weight of bear Ursolic acid.
本發明亦提供一種金銀花萃取物的製備方法,包括:利用一包含乙醇的極性溶劑,對金銀花進行一超音波萃取,以得到一金銀花粗萃物,其中上述超音波萃取溫度介於約15-45℃;以及以一分子拓印高分子對上述金銀花粗萃物進行純化以得到一金銀花萃取物。The invention also provides a method for preparing a honeysuckle extract, comprising: performing ultrasonic extraction on a honeysuckle by using a polar solvent containing ethanol to obtain a crude extract of honeysuckle, wherein the ultrasonic extraction temperature is about 15-45 ° C; and purification of the above crude honeysuckle extract by a molecularly imprinted polymer to obtain a honeysuckle extract.
本發明尚提供一種皮膚用藥,包括上述之金銀花萃取物。The present invention also provides a dermatological drug comprising the above-described honeysuckle extract.
本發明提供一種金銀花的萃取及純化製程,所得之金銀花脆取物具抑制痤瘡桿菌的活性成分。上述金銀花的萃取及純化製程係將中草藥-金銀花先以適合溶劑進行低溫超音波製程以萃取金銀花中的活性成分,其具有抗痤瘡桿菌的功效。本發明另透過分子拓印高分子(Molecular Imprinted Polymer;MIP)技術,其可運用於短管柱層析或是浸泡過濾的方式進行萃取物的純化,同時提高金銀花萃取物多項活性成分,以應用於青春痘照護相關皮膚用藥。此外,本發明亦可採用高速細胞破碎輔助萃取製程,以加快活性成分的萃取效果。The invention provides a process for extracting and purifying honeysuckle, and the obtained honeysuckle brittle material has the active ingredient inhibiting the acne bacillus. The above-mentioned extraction and purification process of honeysuckle is to extract the active ingredient in the honeysuckle by the Chinese herbal medicine-honeysuckle in a low temperature ultrasonic process suitable for the solvent, which has the anti-acne bacteria effect. The invention further utilizes Molecular Imprinted Polymer (MIP) technology, which can be applied to the purification of the extract by short tube column chromatography or immersion filtration, and simultaneously improves the multiple active ingredients of the honeysuckle extract, Used in acne care related skin medications. In addition, the invention can also adopt a high-speed cell disruption-assisted extraction process to accelerate the extraction effect of the active ingredients.
在一實施例中,上述金銀花萃取物的組成可包括:20-50重量份,較佳20-30重量份的綠原酸(chlorogenic acid);1-5重量份,較佳3-5重量份的齊墩果酸(oleanolic acid);以及0.001-5重量份,較佳0.002-5重量份的熊果酸(ursolic acid)。在另一實施例中,本發明之金銀花萃取物基本上由上述綠原酸(chlorogenic acid)、齊墩果酸(oleanolic acid)、以及熊果酸(ursolic acid)所組成(consisting essentially of)。在又一實施例中,本發明之金銀花萃取物僅由上述綠原酸(chlorogenic acid)、齊墩果酸(oleanolic acid)、以及熊果酸(ursolic acid)所組成(consisting of)。In one embodiment, the composition of the above honeysuckle extract may include: 20-50 parts by weight, preferably 20-30 parts by weight of chlorogenic acid; 1-5 parts by weight, preferably 3-5 weight A portion of oleanolic acid; and 0.001 to 5 parts by weight, preferably 0.002 to 5 parts by weight, of ursolic acid. In another embodiment, the honeysuckle extract of the present invention consists essentially of the above-described chlorogenic acid, oleanolic acid, and ursolic acid (consisting essentially of) . In still another embodiment, the honeysuckle extract of the present invention consists solely of the above-described chlorogenic acid, oleanolic acid, and ursolic acid.
上述金銀花粗萃物的製備方法可包括:選擇性地(optionally)利用一均質機對金銀花進行一細胞破碎製程,以使上述金銀花微小化;接著利用一包含乙醇的極性溶劑,對金銀花進行一超音波萃取,而後進行過濾以去除固體渣,以得到一金銀花粗萃物。此處之金銀花可使用金銀花的市售乾貨。上述細胞破碎製程為一選擇性的步驟,用以提高萃取效率,此技藝人士亦可使用均質機以外之其他慣用於破碎的工具或手段。上述包含乙醇的極性溶劑中乙醇濃度可介於約50-95 vol%,較佳介於約70-95 vol%,若乙醇濃度低於50 vol%,則具有抑制痤瘡桿菌的功效性成分,被提取出來的量不足。上述超音波萃取時間介於約1-5小時,較佳2-3小時,上述超音波萃取溫度介於約15-45℃,較佳15-35℃。The method for preparing the above-mentioned crude extract of honeysuckle may include: selectively performing a cell disruption process on the honeysuckle by using a homogenizer to miniaturize the honeysuckle; and then using a polar solvent containing ethanol to treat gold and silver The flower is subjected to an ultrasonic extraction, followed by filtration to remove the solid residue to obtain a crude extract of honeysuckle. The honeysuckle here can use the commercially available dry goods of honeysuckle. The cell disruption process described above is an optional step to increase extraction efficiency, and the skilled artisan may also use other tools or means than conventional homogenizers for disruption. The above polar solvent containing ethanol may have an ethanol concentration of about 50-95 vol%, preferably about 70-95 vol%, and if the ethanol concentration is less than 50 vol%, it has an efficacy component for inhibiting acne bacillus, and is extracted. The amount coming out is insufficient. The ultrasonic extraction time is about 1-5 hours, preferably 2-3 hours, and the ultrasonic extraction temperature is about 15-45 ° C, preferably 15-35 ° C.
接著,以一分子拓印高分子對所得之金銀花粗萃物進行純化以得到一金銀花萃取物。本發明採用分子拓印高分子(Molecular Imprinted Polymer;MIP)技術來萃取並純化金銀花中的有效成分。分子拓印高分子乃目前常用來辨識分子的合成高分子,主要是利用官能基分子(Function Monomer)與模版分子(Template)之間的相互作用力,達到分離的效果,是一種具特異選擇性辨識方法,通常當層析管柱的充填物可加強純化的效果。Next, the obtained crude extract of honeysuckle is purified by a molecularly imprinted polymer to obtain a honeysuckle extract. The invention adopts Molecular Imprinted Polymer (MIP) technology to extract and purify the active ingredients in honeysuckle. Molecularly-printed polymer is a synthetic polymer commonly used to identify molecules. It mainly uses the interaction force between a functional molecule (Function Monomer) and a template to achieve separation. It is a specific selectivity. Identification method, usually when the column column filling can enhance the purification effect.
在一實施例中,利用上述分子拓印高分子對所得之金銀花粗萃物進行純化的方法包括:準備欲萃取之一有效成分以作為一模板分子,接著利用一官能單體(Functional monomer)捕捉上述模板分子,再將上述官能單體(Functional monomer)與一基材(Matrix)單體進行一聚合反應,然後去除上述有效成分以得到一分子拓印材料。在一實施例中,上述有效成分可包括:綠原酸(chlorogenic acid)、齊墩果酸(oleanolic acid)、熊果酸(ursolic acid)、或上述之組合。比起基材單體,官能單體具有羧酸基,可與有效成分形成氫鍵以利於捕捉有效成分,上述官能單體可具有羧酸、磺酸、或雜環基(例如含氮、氧的環類),或可包括但不限於:甲基丙烯酸(Methacrylic Acid,MAA)、丙烯酸(Acrylic acid,AA)、衣康酸(Itaconic acid)、乙烯基苯甲酸(4-vinylbenzoic acid)、丙烯氨基-甲基丙烷磺酸(2-Acrylamido-2-methyl-1-propanesulfonic acid)、2-乙烯基吡啶(2-Vinylpyridine)、4-乙烯基吡啶(4-Vinylpyridine)、或上述之組合。上述基材單體可包括但不限於:甲基丙烯酸甲酯(methylmethacrylate,MMA)、4-二乙烯基苯(4-divinylbenzene)、乙二醇二甲基丙烯酸酯(ethylene glycol dimethacrylate,EGDMA)、或季戊四醇三丙烯酸酯(Pentaerythritol triacrylate,PETA)。In one embodiment, the method for purifying the obtained crude extract of honeysuckle by using the above molecular printing polymer comprises: preparing an active ingredient to be extracted as a template molecule, and then using a functional monomer (Functional monomer) The template molecule is captured, and the functional monomer is polymerized with a matrix monomer, and then the active ingredient is removed to obtain a molecularly imprinted material. In one embodiment, the above active ingredient may include: chlorogenic acid, oleanolic acid, ursolic acid, or a combination thereof. The functional monomer has a carboxylic acid group than the substrate monomer, and can form a hydrogen bond with the active ingredient to facilitate capturing the active ingredient. The above functional monomer may have a carboxylic acid, a sulfonic acid, or a heterocyclic group (for example, nitrogen and oxygen). Rings), or may include, but are not limited to, Methacrylic Acid (MAA), Acrylic Acid (AA), Itaconic Acid, 4-vinylbenzoic acid, Propylene 2-Acrylamido-2-methyl-1-propanesulfonic acid, 2-Vinylpyridine, 4-Vinylpyridine, or a combination thereof. The above substrate monomers may include, but are not limited to, methylmethacrylate (MMA), 4-divinylbenzene, ethylene glycol dimethacrylate (EGDMA), Or Pentaerythritol triacrylate (PETA).
然後,再將上述分子拓印材料裝入一管柱或一濾袋中;並且將上述金銀花粗萃物選擇性地(optionally)經過一濃縮及/或冷凍乾燥步驟後,再溶於乙醇/或甲醇中以得到一金銀花粗萃物乙醇/或甲醇溶液,並使其通過上述管柱或上述濾袋,以利用上述管柱或上述濾袋中的分子拓印材料捉取金銀花粗萃物中的有效成分。上述乙醇或甲醇濃度介於約30-95 vol%,較佳介於約60-95 vol%,若乙醇濃度低於30 vol%或高於95 vol%,則金銀花粗萃物的溶解度降低,進而降低純化步驟的回收率。在另一實施例中,上述利用分子拓印高分子進行純化的步驟,亦可直接使用前述未經濃縮之金銀花粗萃物,使其通過上述管柱或上述濾袋,以利用上述管柱或上述濾袋中的分子拓印材料捉取金銀花粗萃物中的有效成分。最後利用例如40-60℃的溫水,流洗出上述有效成分並收集以得到一金銀花萃取物。Then, the above molecular rubbing material is further loaded into a column or a filter bag; and the above-mentioned crude honeysuckle extract is selectively subjected to a concentration and/or freeze-drying step, and then dissolved in ethanol/ Or methanol to obtain a honeysuckle crude extract ethanol / or methanol solution, and pass it through the above column or the above filter bag, to capture the honeysuckle rough extract by using the molecular column material in the above-mentioned column or the above filter bag The active ingredient in the product. The above ethanol or methanol concentration is about 30-95 vol%, preferably about 60-95 vol%, and if the ethanol concentration is less than 30 vol% or more than 95 vol%, the solubility of the crude extract of the honeysuckle is lowered, and further Reduce the recovery of the purification step. In another embodiment, the step of purifying the molecularly imprinted polymer may also directly use the unconcentrated honeysuckle crude extract to pass through the column or the filter bag to utilize the column. Or the molecular rubbing material in the above filter bag captures the active ingredient in the crude extract of honeysuckle. Finally, the above active ingredient is washed off with warm water of, for example, 40-60 ° C and collected to obtain a honeysuckle extract.
本發明所使用的分子拓印高分子(Molecular Imprinted Polymer;MIP)技術乃運用短管柱層析(<10 cm)或是浸泡過濾方式使得分離的效果較為模糊,以同時獲得尺寸相當且含同樣官能基的數種活性成分,如綠原酸(chlorogenic acid)、齊墩果酸(oleanolic acid)、以及熊果酸(ursolic acid)等,對青春痘照護相關產品而言,本發明之金銀花萃取物可同時具備抗病原菌痤瘡桿菌及其他共生菌如金黃色葡萄球菌的效果,更具應用於青春痘照護相關材料的機會。The Molecular Imprinted Polymer (MIP) technique used in the present invention uses a short column chromatography (<10 cm) or a immersion filtration method to make the separation effect relatively fuzzy, so that the size is equivalent and the same Several active ingredients of a functional group, such as chlorogenic acid, oleanolic acid, and ursolic acid, etc., for the acne care related products, the honeysuckle of the present invention The extract can also have the effect of resisting the pathogenic bacteria Acne and other commensal bacteria such as Staphylococcus aureus, and is more suitable for the application of acne care related materials.
在一實施例中,本發明之銀花萃取物的製備方法更可包括經高分子拓印純化得到一金銀花萃取物之後,對此金銀花萃取物進行一濃縮與冷凍乾燥步驟以得到一固態的金銀花萃取物。In one embodiment, the method for preparing the silver flower extract of the present invention may further comprise: after obtaining a honeysuckle extract by polymer blotting, performing a concentration and freeze drying step on the honeysuckle extract to obtain a solid state. Honeysuckle extract.
在一實施例中,由上述金銀花萃取物的製備方法所得之金銀花萃取物可作為一皮膚用藥,其中上述皮膚用藥中之未經高分子拓印純化過程,直接進行過濾、濃縮與冷凍乾燥步驟的金銀花粗萃取物濃度大於約10 mg/mL,若為經高分子拓印純化過程、濃縮與冷凍乾燥步驟之固態的金銀花萃取物,則其濃度大於約0.1 mg/mL,較佳介於約0.5-10 mg/mL。上述藥物的型態可包括但不限於:乳液型、貼片型、或膠體型,提供青春痘照護一種新的中草藥選擇標的。In one embodiment, the honeysuckle extract obtained by the method for preparing the honeysuckle extract described above can be used as a dermatological drug, wherein the dermatological drug is directly filtered, concentrated and freeze-dried without a polymer printing and purification process. The concentration of the crude extract of the honeysuckle in the step is greater than about 10 mg/mL. If the solid honeysuckle extract is subjected to the polymer purification process, concentration and freeze-drying steps, the concentration is greater than about 0.1 mg/mL, preferably It is about 0.5-10 mg/mL. The types of the above drugs may include, but are not limited to, an emulsion type, a patch type, or a colloid type, and provide a new Chinese herbal medicine selection target for acne care.
綜上所述,本發明所提供之金銀花萃取物,除了具備金銀花之抗金黃色葡萄球菌(Staphy1ococcus aureus )的功效外,更發現其具備抗痤瘡桿菌的能力,因此本發明之金銀花萃取物能同時抗造成青春痘(痤瘡)病原的痤瘡桿菌(Propionibacterium acnes ),與共生菌金黃色葡萄球菌(Staphylococcus aureus ),可應用於皮膚用藥並具有青春痘照護的條件。In summary, the honeysuckle extract provided by the present invention, in addition to the efficacy of honeysuckle against Staphy1ococcus aureus , is found to have anti-Acne bacillus ability, so the honeysuckle extract of the present invention Propionibacterium acnes , which is simultaneously resistant to the pathogen of acne (acne), and the commensal Staphylococcus aureus , can be applied to dermatological conditions and have conditions for acne care.
為讓本發明之上述和其他目的、特徵、和優點能更明顯易懂,下文特舉出實施例,作詳細說明如下:The above and other objects, features, and advantages of the present invention will become more apparent and understood.
實施例1. 製備金銀花粗萃物Example 1. Preparation of crude extract of honeysuckle
將200g乾燥後金銀花運用均質機,於大於10,000 rpm高速轉速下,進行原料料微小化及細胞壁的破碎,預期透過高速細胞破碎輔助萃取製程,提高活性物質萃取率。200g of dried honeysuckle is applied to the homogenizer, and the raw material is miniaturized and the cell wall is broken at a high speed of more than 10,000 rpm. It is expected that the high-speed cell disruption assists the extraction process to increase the extraction rate of the active material.
接著將微小化後的金銀花粉末,添加1.8升95%高濃度酒精為溶劑,攪拌混合均勻後,室溫下(25℃)以超音波設備進行萃取2小時。Next, the miniaturized honeysuckle powder was added with 1.8 liters of 95% high-concentration alcohol as a solvent, stirred and uniformly mixed, and then extracted by ultrasonic equipment at room temperature (25 ° C) for 2 hours.
再進行一過濾步驟:經超音波萃取後的萃取液,以TOYO定性濾紙NO1(孔徑6 μm/厚度0.2 mm/直徑110 mm/濾紙材質:棉纖維)進行過濾以去除固體渣,每張濾紙過濾使用時間約45秒即更換濾紙,避免濾紙阻塞。Further, a filtration step is carried out: the extract after ultrasonic extraction is filtered with TOYO qualitative filter paper NO1 (pore size 6 μm/thickness 0.2 mm/diameter 110 mm/filter paper material: cotton fiber) to remove solid residue, and each filter paper is filtered. Replace the filter paper in about 45 seconds to avoid clogging the filter paper.
接著進行一濃縮步驟:過濾後的濾液,利用旋轉減壓濃縮機去除溶劑,水浴溫度:40℃,真空度650-720 mmHg;將初始體積約2000 ml,濃縮約至300 ml,再加入2倍二次去離子水,再濃縮至200 mL,分裝成兩袋,每袋約100 ml,放置-20℃冰箱一天。Then, a concentration step is carried out: the filtrate after filtration is removed by a rotary vacuum concentrator, the temperature of the water bath is 40 ° C, and the degree of vacuum is 650-720 mmHg; the initial volume is about 2000 ml, and the concentration is about 300 ml, and then added twice. Double deionized water, concentrated to 200 mL, divided into two bags, about 100 ml per bag, placed in a refrigerator at -20 ° C for one day.
最後進行一冷凍乾燥步驟:利用真空冷凍乾燥機(溫度:-86.9℃、真空度:4.9Pa),乾燥時間約兩天,去除多於水分及溶劑,得到乾燥後的粗萃物以利後續成分使用。要進行MIP純化的樣品則省略此冷凍乾燥步驟。Finally, a freeze-drying step is carried out: using a vacuum freeze dryer (temperature: -86.9 ° C, vacuum: 4.9 Pa), the drying time is about two days, more than water and solvent are removed, and the dried crude extract is obtained to facilitate subsequent components. use. This freeze-drying step is omitted for samples to be MIP purified.
實施例2. 金銀花粗萃物成分分析及抗痤瘡桿菌、金黃色葡萄球菌的效果Example 2. Analysis of the components of the crude extract of honeysuckle and the effect of anti-acne bacillus and Staphylococcus aureus
測試菌種以培養液於厭氧狀態下37℃培養24~48小時,於2天後以培養液進行系列10倍稀釋調整細菌濃度。測試細菌濃度為106 -107 CFU/mL。試驗物質與測試細菌以適當體積比例混合,並使各別最終濃度為50、40、30、20、10、0.5、0.3 mg/mL等所需濃度與(1~5)×106 CFU/mL(上述濃度實驗時的測試細菌分別為2.7×106 、2.5×106 、2.5×106 、2.5×106 、3.5×106 、6.5×106 、9.2×106 CFU/mL)。於設計之時間點取出0.1 mL混合培養液塗抹於洋菜培養基上,放置於37℃培養箱於厭氧狀態下培養24~48小時,於菌落出現後進行計數。The test strain was cultured in an anaerobic state at 37 ° C for 24 to 48 hours, and after 2 days, the bacterial concentration was adjusted by serial 10-fold dilution with the culture solution. The test bacteria concentration was 10 6 -10 7 CFU/mL. The test substance and the test bacteria are mixed in an appropriate volume ratio, and the respective final concentrations are 50, 40, 30, 20, 10, 0.5, 0.3 mg/mL and the like, and (1~5)×10 6 CFU/mL. (The test bacteria at the above concentration experiments were 2.7 × 10 6 , 2.5 × 10 6 , 2.5 × 10 6 , 2.5 × 10 6 , 3.5 × 10 6 , 6.5 × 10 6 , 9.2 × 10 6 CFU / mL, respectively). At the time of design, 0.1 mL of the mixed culture solution was taken and applied to the vegetable medium, and placed in an incubator at 37 ° C for 24 to 48 hours under an anaerobic state, and counted after the colony appeared.
抑菌率=(A-B)/A×100%Inhibition rate = (A-B) / A × 100%
因長青春痘時,除痤瘡桿菌外常伴隨金黃色葡萄球菌等共生菌,而粗萃取也含綠原酸,表1顯示金銀花95%酒精粗萃物20 mg/mL與對照組處理金黃色葡萄球菌(Staphylococcus aureus ,ATCC 6538P)18小時後之抑菌效果,其數據顯示本發明之金銀花酒精粗萃物具抑金黃色葡萄球菌的效果,因而可加強治療青春痘的效果。Due to the long acne, acne bacteria such as Staphylococcus aureus are often accompanied by acne bacteria, and the crude extract also contains chlorogenic acid. Table 1 shows that the crude extract of 95% alcohol of honeysuckle 20 mg/mL and the control group are treated with golden yellow. The antibacterial effect of Staphylococcus aureus (ATCC 6538P) after 18 hours, the data shows that the crude extract of honeysuckle alcohol of the present invention has the effect of inhibiting Staphylococcus aureus, thereby enhancing the effect of treating acne.
參見表2,係探討以不同酒精濃度萃取之金銀花粗萃物的抗菌效果。其中以95%酒精粗萃物的抗痤瘡桿菌效果較佳,由粗萃物的成分分析(HPLC)推斷,應是齊墩果酸與熊果酸的貢獻,令95%的酒精粗萃物具抗痤瘡桿菌的能力。See Table 2 for the antibacterial effect of the crude extract of honeysuckle extracted at different alcohol concentrations. Among them, the anti-acne bacillus with 95% alcohol extract is better, and it is inferred from the component analysis (HPLC) of the crude extract that it should be the contribution of oleanolic acid and ursolic acid, so that 95% of the alcohol extracts are The ability to fight acne bacteria.
為驗證抗痤瘡桿菌的活性物質種類,選用標準品評估其抗痤瘡桿菌的效果,表3與表4顯示不同標準品定量菌種培養計數法結果,其中齊墩果酸(Oleanolic acid)、熊果酸(Ursolic acid)具抗痤瘡桿菌的效果、而綠原酸(Chlorogenic acid)反而會令痤瘡桿菌孳生。In order to verify the anti-acne bacillus active substance type, the standard was used to evaluate the anti-acne bacillus effect. Table 3 and Table 4 show the results of different standard quantitative strain culture counting methods, among which Oleanolic acid and bear fruit Ursolic acid has an anti-acne bacillus effect, while chlorogenic acid (Chlorogenic acid) will cause acne bacteria to germinate.
表5係根據本發明實施例2,顯示不同濃度粗萃物定量菌種培養計數法結果。由表5可知以95%酒精萃取金銀花所得之金銀花粗萃物,其濃度大於20 mg/ml時的抗痤瘡桿菌(ATCC 6919)效果可達98%以上。Table 5 shows the results of the culture count method for quantitative strains of different concentrations of crude extracts according to Example 2 of the present invention. Table 5 shows that the crude extract of honeysuckle obtained by extracting honeysuckle with 95% alcohol can achieve an effect of more than 98% against acne bacteria (ATCC 6919) at a concentration greater than 20 mg/ml.
第1a-1d圖係根據本發明實施例1,顯示金銀花粗萃物經過HPLC成分分析扣除層析液背景值所得之結果,其中第1d圖顯示以95%酒精萃取金銀花所得之金銀花粗萃物的成分,可明顯看出其具有綠原酸(chlorogenic acid)、齊墩果酸(oleanolic acid)、以及熊果酸(ursolic acid)等活性成分(齊墩果酸與熊果酸的延遲時間之峰值幾乎重疊)。1a-1d shows the results obtained by HPLC analysis of the crude extract of the honeysuckle by subtracting the background value of the chromatographic solution according to Example 1 of the present invention, wherein the first graph shows the crude honeysuckle obtained by extracting the honeysuckle with 95% alcohol. The composition of the extract is clearly found to have chlorogenic acid, oleanolic acid, and ursolic acid active ingredients (latitude of oleanolic acid and ursolic acid) The peak of time almost overlaps).
實施例3. 金銀花粗萃物進行分子拓印方式純化得到金銀花萃取物Example 3. The crude extract of honeysuckle was purified by molecular rubbing to obtain honeysuckle extract
分子拓印高分子製備方式:將模板分子(Oleanolic acid)60mg,與1.5 ml甲基丙烯酸(Methacrylic Acid,MAA)攪拌均勻後,加入10.5ml甲基丙烯酸甲酯(methylmethacrylate,MMA)溶液中再混合攪拌均勻,並一邊吹入氮氣除氧。接著將上述溶液在惰性氣體下加熱至70℃,再加入10 mg的偶氮二異丁腈(2,2-azobisisobutyronitrile,AIBN,Initiator),持續約2~3小時至形成高分子聚合物。再將上述聚合物以機械力粉碎,研磨成細緻粉體,並以100網目金屬網過篩。然後將過篩的白色粉末狀MIP材,加入60℃溫水中以洗去模板分子,清洗數次後乾燥,以得乾燥後的MIP材(分子拓印高分子)。Molecular printing polymer preparation method: 60mg of template molecule (Oleanolic acid), and 1.5 ml of methacrylic acid (MAA), after mixing, add 10.5ml methyl methacrylate (MMA) solution and then mix Stir well and deaerate by blowing nitrogen gas. Next, the solution was heated to 70 ° C under an inert gas, and then 10 mg of azobisisobutyronitrile (AIBN, Initiator) was added for about 2 to 3 hours to form a high molecular polymer. The above polymer was then pulverized by mechanical force, ground into a fine powder, and sieved through a 100 mesh metal mesh. Then, the sieved white powdery MIP material is added to warm water of 60 ° C to wash away the template molecules, washed several times, and then dried to obtain a dried MIP material (molecular blotting polymer).
分子拓印方式純化方式:乾燥後的金銀花粗萃物先溶於30%或70%酒精中。取上述乾燥後MIP材,裝入短管柱中,或裝入濾袋中,然後將上述金銀花粗萃物乙醇溶液通過上述管柱或上述濾袋,以利用上述管柱或上述濾袋中的上述分子拓印材料捉取上述金銀花粗萃物中與齊墩果酸(Oleanolic acid)相似官能基的熊果酸(Ursolic acid)及綠原酸(Chlorogenic acid)等活性成分,最後再將已捉取活性成分的MIP,加入60℃溫水中以沖提出活性成分並收集,透過冷凍乾燥方式,乾燥純化後得到一固態的金銀花萃取物。Molecular rubbing purification method: The dried honeysuckle crude extract is first dissolved in 30% or 70% alcohol. Taking the dried MIP material, loading into a short pipe column, or loading into a filter bag, and then passing the above-mentioned honeysuckle crude extract ethanol solution through the above-mentioned pipe string or the above-mentioned filter bag to utilize the above-mentioned pipe string or the above filter bag The above-mentioned molecular rubbing material captures active ingredients such as Ursolic acid and Chlorogenic acid, which are similar to Oleanolic acid in the above-mentioned crude extract of honeysuckle, and finally The MIP of the active ingredient has been taken, added to warm water of 60 ° C to extract the active ingredient and collected, and dried and purified by freeze-drying to obtain a solid honeysuckle extract.
第2a-2b圖係根據本發明實施例,顯示以HPLC分析純化後的金銀花萃取物之成分。第2a圖顯示未經過分子拓印純化之金銀花粗萃物的HPLC成分分析結果(已扣除層析液背景值)。第2b圖顯示經過分子拓印純化之金銀花萃取物的HPLC成分分析結果。在第2b圖之經過分子拓印純化之金銀花萃取物中,以延遲時間(Retention time)3.49峰(綠原酸)及63.15峰(oleanolic acid)為例,各比第2a圖提高>10%及2倍的純度,第2a-2b圖之HPLC分析結果所計算出之組成成分比例係整理於表6。2a-2b is a diagram showing the composition of the purified honeysuckle extract by HPLC analysis according to an embodiment of the present invention. Figure 2a shows the HPLC component analysis of the crude extract of honeysuckle without molecular blotting (the background value of the chromatographic solution has been subtracted). Figure 2b shows the results of HPLC analysis of the honeysuckle extract purified by molecular blotting. In the molecularly purified and purified honeysuckle extract of Fig. 2b, the retardation time 3.49 peak (chlorogenic acid) and 63.15 peak (oleanolic acid) are taken as examples, and the ratio is increased by >10% compared with the second graph. And the purity of 2 times, the composition ratio calculated by the HPLC analysis result of the 2a-2b figure is compiled in Table 6.
實施例4. 比較金銀花粗萃物與純化後之固態的金銀花萃取物的抗痤瘡桿菌效果Example 4. Comparison of the anti-acne bacillus effect of the crude extract of honeysuckle and the purified solid honeysuckle extract
分析含20 mg/ml乾燥之金銀花粗萃取物的抗痤瘡桿菌效果為98%。表7係根據本發明實施例3,分子拓印純化後的金銀花萃取物定量菌種培養計數法結果。而純化後萃取物之中齊墩果酸(Oleanolic acid)提高2倍,其僅需含10 mg/ml乾燥之固態的金銀花萃取物即可達到>99.99%抗菌效果。The anti-acne bacillus effect containing 20 mg/ml of dried crude honeysuckle extract was analyzed to be 98%. Table 7 shows the results of quantitative counting of cultures of honeysuckle extract after molecular rubbing purification according to Example 3 of the present invention. The purified extract has a 2-fold increase in Oleanolic acid, which requires only 10 mg/ml of dried solid honeysuckle extract to achieve >99.99% antibacterial effect.
實施例5. 金銀花粗萃取物的DPPH(α,α-diphenyl-β-picrylhydrazyl)抗氧化分析Example 5. Antioxidant analysis of DPPH (α,α-diphenyl-β-picrylhydrazyl) of crude extract of honeysuckle
配製1000 ppm的粗萃取液5 ml,再取0.0197 g之DPPH(MW=394.32),配製成50 ml之甲醇溶液(濃度為1 mM),接著取萃取液5ml,加入1 ml新配製之DPPH(1 mM),混和靜置30分鐘,於517 nm測其吸光值。以萃取液之溶劑取代樣品做為控制組。Prepare 5 ml of 1000 ppm crude extract, then take 0.0197 g of DPPH (MW=394.32), prepare 50 ml of methanol solution (concentration of 1 mM), then take 5 ml of extract and add 1 ml of freshly prepared DPPH. (1 mM), mix and let stand for 30 minutes, and measure the absorbance at 517 nm. The sample was replaced with a solvent of the extract as a control group.
DPPH自由基清除能力百分率=[1-(樣品吸光值/未加樣品之控制組吸光值)]×100DPPH free radical scavenging percentage = [1 - (sample absorbance / unabsorbed control group absorbance)] × 100
第3圖係根據本發明之實施例5,顯示以不同濃度的乙醇對金銀花進行粗萃,所得之金銀花粗萃物之DPPH自由基清除能力,其中以維他命C與維他命E作為控制組,當今銀花粗萃物濃度為250 ppm時即具備超過80%的DPPH去除能力,且接近約95%之維他命C及維他命E的DPPH去除能力水準。表8係顯示根據第3圖之DPPH測試結果所得之抗氧化能力。Figure 3 is a diagram showing the DPPH free radical scavenging ability of the crude extract of honeysuckle obtained by crude extraction of honeysuckle with different concentrations of ethanol according to Example 5 of the present invention, wherein vitamin C and vitamin E are used as control groups. Today's silver flower extract has a DPPH removal capacity of more than 80% at a concentration of 250 ppm, and is close to about 95% of the DPPH removal capacity of vitamin C and vitamin E. Table 8 shows the antioxidant capacity obtained according to the DPPH test results of Fig. 3.
透過標準品驗證抗痤瘡桿菌的效果,數據顯示Oleanolic acid(齊墩果酸)、Ursolic acid(熊果酸)具抗痤瘡桿菌的效果,而因青春痘照護相關產品因最好具備抗病原菌痤瘡桿菌及其他共生菌如金黃色葡萄球菌效果,因此最好萃取純化過程能包含幾項活性物質,如綠原酸(chlorogenic acid)、齊墩果酸(oleanolic acid)、以及熊果酸(ursolic acid)等,因此的分子拓印高分子運用於短管柱(<10cm)層析或是浸泡過濾的純化,有機會同時提高金銀花萃取物多項活性物質,更具應用於青春痘照護相關材料的機會。The anti-acne bacillus effect was verified by standard data. The data showed that Oleanolic acid and ursolic acid have anti-acne bacillus effect, and the acne care related products have the best anti-pathogenic bacteria acne And other commensal bacteria such as Staphylococcus aureus, so the best extraction and purification process can contain several active substances, such as chlorogenic acid, oleanolic acid, and ursolic acid Etc. Therefore, the molecularly-printed polymer is applied to the purification of short column (<10cm) chromatography or immersion filtration, which has the opportunity to simultaneously improve the active substances of honeysuckle extract and more opportunities for acne care related materials. .
雖然本發明已以數個實施例揭露如上,然其並非用以限定本發明,任何所屬技術領域中具有通常知識者,在不脫離本發明之精神和範圍內,當可作任意之更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。The present invention has been disclosed in several embodiments, and is not intended to limit the invention, and any one of ordinary skill in the art can make any changes and modifications without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims.
第1a-1d圖係根據本發明實施例,顯示金銀花粗萃物經過HPLC成分分析所得之結果(已扣除層析液背景值),其中第1a-1d圖分別顯示以標準品、與30%、60%、以及95%酒精萃取金銀花所得之金銀花粗萃物的成分。1a-1d shows the results obtained by HPLC component analysis of the crude extract of honeysuckle (the background value of the chromatographic solution has been subtracted) according to an embodiment of the present invention, wherein the 1a-1d chart shows the standard, and 30% respectively. 60%, and 95% alcohol extracts of honeysuckle obtained from the honeysuckle crude extract.
第2a-2b圖係根據本發明實施例,顯示以HPLC分析純化後的金銀花萃取物之成分(已扣除層析液背景值),其中第2a圖顯示未經過分子拓印純化之金銀花粗萃物的HPLC成分分析結果,第2b圖顯示經過分子拓印純化之金銀花萃取物的HPLC成分分析結果。2a-2b is a diagram showing the composition of the purified honeysuckle extract by HPLC analysis (after subtracting the background value of the chromatographic solution) according to an embodiment of the present invention, wherein the 2a image shows that the honeysuckle is not subjected to molecular rubbing and purification. The HPLC component analysis results of the extract, and the 2b chart shows the HPLC component analysis results of the molecularly purified honeysuckle extract.
第3圖係根據本發明之實施例,顯示以不同濃度的乙醇對金銀花進行粗萃,所得之金銀花粗萃物之DPPH自由基清除能力。Figure 3 is a graph showing the DPPH free radical scavenging ability of the crude extract of honeysuckle obtained by crude extraction of honeysuckle with different concentrations of ethanol according to an embodiment of the present invention.
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