CN100574744C - Extract of cercis chinensis having anti-oxidant activity and anti-aging activity, and cosmetic composition containing the extract for anti-oxidation, skin-aging protection and wrinkle improvement - Google Patents

Extract of cercis chinensis having anti-oxidant activity and anti-aging activity, and cosmetic composition containing the extract for anti-oxidation, skin-aging protection and wrinkle improvement Download PDF

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CN100574744C
CN100574744C CN 200380107721 CN200380107721A CN100574744C CN 100574744 C CN100574744 C CN 100574744C CN 200380107721 CN200380107721 CN 200380107721 CN 200380107721 A CN200380107721 A CN 200380107721A CN 100574744 C CN100574744 C CN 100574744C
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任坤爀
刘载国
李灿馥
田永旻
罗敏均
金镇杓
闵东日
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株式会社韩国新药;株式会社韩生化妆品
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Abstract

The present invention relates to extract of Cercis chinensis having anti-oxidant activity and antiaging activity containing compound of chemical formula 1 to chemical formula 20, and cosmetical composition for anti-oxidation, skin-aging protection and wrinkle improvement containing the extract as effective ingredient. The extract of present invention having protective effect on oxidative damage and skin damage, and inhibitory effect on age-dependent telomere shortening, so it can effectively used as skin-aging protection comestic.

Description

具有抗氧化活性和抗衰老活性的中国紫荆的提取物,以及含有该提取物并用于抗氧化、皮肤衰老防护和改善皱纹的化妆品组合物 Having antioxidant activity and anti-aging activity of China Bauhinia extract, and the extracts were used containing antioxidant, skin aging and wrinkle protection cosmetic composition

技术领域 FIELD

本发明涉及具有抗衰老活性的植物提取物和含有该提取物作为有效成分的化妆品组合物。 The present invention relates to plant extracts having anti-aging activity and a cosmetic composition containing the extract as an active ingredient. 更具体而言,本发明涉及具有抗氧化活性和抗衰老活性的中国紫荆(Cwc^ A/^mz》的提取物,以及涉及含有作为有效成分的该提取物的化妆品组合物,该组合物可用于抗氧化、皮肤衰老防护和改善皱 More particularly, the present invention relates to Chinese Bauhinia (Cwc ^ A / ^ mz having antioxidant activity and anti-aging active "extract, and relates to cosmetic compositions containing the extract as an active ingredient, the composition can be in oxidation, protection and improvement of skin aging wrinkles

背景技术 Background technique

衰老表示随着时间流逝而出现的身体的所有生理变化,而衰老状况和速度因个体情况而不同,并且受到多种原因的影响。 Aging represents all the physiological changes that occur over time in the body, and the status and speed of aging due to individual circumstances vary, and is influenced by a variety of reasons. 即使在一个个体中, 衰老对于每个器官也表现出不同的状况,因此面向个体的衰老研究具有局限性。 Even in one individual, aging also performed for every organ in different conditions, the study of aging individuals facing limitations. 更具体而言,每个器官和组织的功能都随时间而改变,这种情况是由于细胞功能的改变引起的。 More specifically, the function of every organ and tissue are changing over time, this situation is due to changes in cell function caused. 例如,脑神经细胞的损伤导致认知下降,皮下脂肪细胞的损伤会导致皮肤弹性的损失,毛根黑素细胞生产黑色素能力的损失会导致白发,等等此类的情况。 For example, damage to brain cells leads to cognitive decline, damage to the subcutaneous fat cells can lead to loss of elasticity of the skin, loss of hair root melanin melanocyte production capacity will lead to gray hair, and so such a situation. 因此,个体细胞的衰老会导致个体的衰老。 Thus, the aging of individual cells can lead to aging individuals. 于是,最近对于衰老的研究都集中在以细胞为基础的研究上。 Thus, a recent study for aging research has focused on cell-based. 在很多科学家把所有精力都致力于彻底解释衰老之后,衰老的确切机理依然没有被揭开,这是因为衰老有多种方面并且非常复杂的缘故。 In many scientists we are committed to a thorough explanation of all the energy After aging, the exact mechanism of aging still not been lifted, because there are many aspects of aging and very complex reasons. 通过现象研究只是提出了一些关于衰老的理论。 By Phenomenon just made a number of theories about aging. 其中,氧自由基理论和端粒理论给出了一些结果。 Wherein the oxygen free radical theory telomere theory gives some results. 前者认为由在通常新陈代谢过程中产生的氧自由基导致的累积氧化应激(oxidative stress)是衰老的主要原因,而后者认为反复细胞分裂之后,位于染色体末端的端粒逐渐消失,因而导致细胞分裂停止以及最终细胞死亡。 The former accumulated that oxidative stress by the oxygen radicals generated in the metabolic process typically results in the (oxidative stress) is the main cause of aging, and that the latter after repeated cell division, the telomeres on chromosome ends gradually disappear, resulting in cell division stop and ultimately cell death. 其它理论也共同有助于对衰老进行完善的解释。 Other common theory of aging contribute to perfect interpretation. 更精确地,对于氧自由基理论,在通常的新陈代谢过程中产生的氧自由基随机破坏了诸如脂质、蛋白质、糖或DNA之类的细胞成分,引起细胞或组织的氧化应激,由此不仅会导致各种诸如癌症之类的疾病、诸如脑溢血和动脉硬化之类的心血管疾病、诸如风湿病之类的慢性炎症、自身免疫性疾病、等 More precisely, for a theoretical oxygen radicals, oxygen radicals generated during normal metabolism such as random damage lipids, proteins, sugars or cell components like DNA, cells or tissues caused by oxidative stress, thereby not only cause various diseases such as cancer, cardiovascular disease, chronic inflammation and arteriosclerosis such as cerebral hemorrhage and the like, such as rheumatism and the like, autoimmune diseases, etc.

(Halliwell, B和Gutteridge, JM C, Biochem. J., 1984, 219,1-14; Freeman, BA和Grapo, JD , Lab Invest, 1982,47, 412-426; Ames, BN, Sc&"ce, 1983, 221, 1256-1264; Fridovich, I., Arch. Biochem. Biophys. , 1986, 247,1-11 ; Vishwanath, MS , Nutrition in Clinical Practice, 1995,10, 19-25),而且由于该氧化性损伤长时间累积会衰老至死亡。1956年Harman首次提出了关于衰老的氧自由基理论(Harman, D. , Free radical theory of aging, Alan R Liss, New York, 1986,3-49),从那时起,很多实验都获得了支持该理论的结果。 举例来说,通过控制基础代谢率(BMR),即氧消耗、通过限制食物或通过限制运动可以延长寿命(Medvedev, ZA,所o/. i?ev., 1990, 65, 375-398; Loe, J" Northrop, JH , J.所o/. C7je附.,1971,32, 103-121; Sohal, RS, Jraecf a—g, 5^77'wgar-旨/"g, T7ezV/e/6e;^, 1986, 23-44 ; Sohal, RS,々•, 1982, 5, 21-24)。 (Halliwell, B and Gutteridge, JM C, Biochem J., 1984, 219,1-14;. Freeman, BA and Grapo, JD, Lab Invest, 1982,47, 412-426; Ames, BN, Sc & "ce, 1983, 221, 1256-1264;.. Fridovich, I., Arch Biochem Biophys, 1986, 247,1-11;. Vishwanath, MS, Nutrition in Clinical Practice, 1995,10, 19-25), and since the oxidation injury for a long time accumulation of aging to death. in 1956 Harman first proposed the free radical theory of aging (Harman, D., Free radical theory of aging, Alan R Liss, New York, 1986,3-49), from since then, many experiments were obtained results support the theory. for example, by controlling the basal metabolic rate (the BMR), i.e. oxygen consumption, the life (Medvedev, ZA movement by limiting or by restricting food, the o / .? i ev, 1990, 65, 375-398;. Loe, J "Northrop, JH, J. of the o / C7je attached, 1971,32, 103-121;.. Sohal, RS, Jraecf a-g, 5 ^ 77'wgar- purpose / "g, T7ezV / e / 6e; ^, 1986, 23-44; Sohal, RS, 々 •, 1982, 5, 21-24).

活体为了保护其免受氧化性损伤,它具有抗氧化物质和抗氧化酶,如过氧化物歧化酶(SOD)、过氧化氢酶或过氧化物酶。 In order to protect the living body from oxidative damage, which has antioxidant and antioxidant enzymes such as superoxide dismutase (SOD), catalase or peroxidase. 但是,随着年龄增长, 这些酶对于氧自由基的防御力变差(Orr,WC和Sohal, RS , &/e"ce, 1994, 263,1128-1130; Sohal, RS等,J!说0/.C/2e附.,1995, 270, 15671-15674)。例如,从老年小鼠中分离出的SOD活性比从年轻小鼠中分离出的SOD活性低。尤其是,增加抗氧化酶、SOD和过氧化氢酶的活性,可以使果蝇的寿命延长30%,这说明氧自由基是与衰老紧密相连的。因此,能够除去氧自由基或抑制脂质过氧化作用的抗氧化剂可以有效用于治疗由氧自由基引起的疾病,以及可以有效用于防止衰老。 However, with age, these enzymes for the degradation of oxygen radical defense (Orr, WC and Sohal, RS, & / e "ce, 1994, 263,1128-1130; Sohal, RS et al, J say 0! /.C/2e attachment., 1995, 270, 15671-15674). For example, isolated from aged mice SOD activity lower than the activity of SOD isolated from young mice in particular, increases antioxidant enzymes, activities of SOD and catalase, and can extend the life of flies 30%, indicating that oxygen free radicals are closely linked with aging. Therefore, the oxygen radicals or suppress lipid peroxidation antioxidants can be removed utility in the treatment of diseases caused by oxygen free radicals, and can be effectively used to prevent aging.

将活体持续置于由诸如空气污染、UV、应激(stress)或疾病之类的有害环境导致的氧化应激中,会增加活体中的自由基,会破坏透明质酸、弹性蛋白、胶原蛋白和真皮的结缔组织而导致皱纹,甚至会由于氧化细胞膜中的脂质而破坏了细胞,从而导致如皮炎、皮疹或皮肤癌之类的疾病。 The living body placed in a continuously such as air pollution, UV, stress, oxidative stress (Stress) such a disease or environmentally harmful lead, a living body increases free radicals will destroy hyaluronic acid, elastin, collagen dermis and connective tissues and causes wrinkles, or even due to lipid oxidation in cell membranes and damage the cells, leading to diseases such as dermatitis, rash or skin cancer and the like. 自由基与黑色素的产生有关,这种关系被认为是变色、雀斑和皱纹的原因。 For the production of melanin and free radicals, this relationship is thought to be discoloration, freckles, and wrinkles. 迄今为止,抗坏血酸、OC-生育酚或SOD用于制备保护皮肤的化妆品或作为自由基消除剂的医疗用品。 To date, ascorbic acid, tocopherol or SOD OC- protection of the skin for cosmetic preparation as radical scavengers or medical supplies. 但是,其价格高昂,并且由于化学混合物的不稳定性,而使其效果不确定。 However, its high price, due to the instability and chemical mixtures, but results uncertain. 因此,在医疗用品、食品和化妆品工业中的共同目标是开发出即安全又能满意去除自由基效果的物质。 Therefore, medical supplies, food and cosmetic industries common goal is to develop a safe substance that is the effect of free radicals can be removed with satisfaction.

端粒理论是解释衰老的另一种主要理论。 Telomere theory is another major theories to explain aging. 人类的正常细胞在体外只经历确定数量的细胞分裂。 Normal human cells in vitro undergo only a certain number of cell divisions. 即,在完成预定分裂之后,细胞分裂终止,这种 That is, after completion of a predetermined division, termination of cell division, this

情况称作复制型老化。 It referred to the case of replicative aging. 端粒理论解释了为什么发生复制型老化(Kim, SH, 等,(9"coge"e 21: 503-511 (2002); Harley, CB,等,iVa加re 345: 458-460 (1990); Olovnikov, AM J T7zeo〜f.历o/. 41: 181-190 (1973); Harley, CB, 五x;?. Gero"to/. 27: 375-382 (1992); Allsopp, RC, Weissman,IL, (9wcog幼e, 21:3270-3273(2002))。端粒是真核细胞线性染色体的末端部分,具有非常独特的结构,在该结构中'TTAGGG'序列是重复的。尤其是,鸟嘌呤(G)通过氢键形成了非常稳定的G-四连体结构(G-quartet structure),因此它可稳定并保护染色体(Moyzis, RK,等,户rac.Mw/. icW. 85: 6622-6626 (1988))。然而,已知在人类体细胞中每当细胞分裂发生时,端粒就逐渐縮短(Harley, CB , Futcher, AB, Greider, CW, Atowe 345: 458-460 (1990); Harley, CBet al.,五x;?.GWw7to/. 27: 375-382 (1992); Allsopp, RC, Weissman, IL, 21 : 3270-3273 (2002))。这是因为"末端复制问 Telomere theory explains why replicating aging (Kim, SH occurs, and the like, (9 "coge" e 21: 503-511 (2002); Harley, CB, and the like, IVA plus re 345: 458-460 (1990); Olovnikov, AM J T7zeo~f calendar o / 41: 181-190 (1973); Harley, CB, five x; Gero "to / 27:..?. 375-382 (1992); Allsopp, RC, Weissman,. IL, (9wcog immature e, 21: 3270-3273 (2002)).. telomeres are the end portions of linear eukaryotic chromosomes, has a very unique structure, in the structure 'TTAGGG' sequences are repeated in particular, guanine (G) by a very stable hydrogen bonds are formed four-piece structure G- (G-quartet structure), it can be protected stably and chromosome (Moyzis, RK, et al., households rac.Mw/ icW 85..: . 6622-6626 (1988)), however, is known in human somatic cells when cell division occurs each time, gradually shortened telomeres (Harley, CB, Futcher, AB, Greider, CW, Atowe 345: 458-460 (1990 ); Harley, CBet al, five x; GWw7to / 27:.?.. 375-382 (1992); Allsopp, RC, Weissman, IL, 21:. 3270-3273 (2002)) this is because the "end replication asked

题"的缘故,所述"末端复制问题"表示当DNA复制时,3'-末端的引物区域是不复制的(01ovnikov, AMJ T7犯w饥祝o/. 41: 181- 190 (1973))。因此, 每次发生细胞分裂,已复制DNA都縮短引物的该部分,在染色体中的端粒也缩短引物的该部分。当端粒继续縮短超过临界点时,DNA的单链和双链被切断,导致细胞分裂在Gl阶段被细胞周期蛋白依赖激酶抑制剂(cyclin dependent kinase inhibitors)阻石寻(Harley, CB , et al.,(7erawto/, 27:375-382(1992))。根据最近研究,端粒长度随氧化应激而变化。也就是说,氧化应激促使端粒变短,这是因为与染色体的其它部分相比,在端粒DNA部分中的氧化性损伤较少被修复的缘故(Saretzki, G. , von Zglinicki, T. , j朋.7Vew K?A Jo^. 959: 24-29 (2002); von Zglinicki, T. , Ann. 7Vew 'Sake, the "title end replication problem" indicates when DNA replication, the 3'-end region of the primer is not replicated (01ovnikov, AMJ T7 hunger wish to commit w o / 41:. 181- 190 (1973)) Thus, every occurrence of cell division, DNA replication have been shortened primer portion, also in the chromosome telomeres shorten the portion of the primer. when telomere shortening continues over the critical point, and double-stranded DNA is single stranded cutting, leading to cell division at the Gl phase of the cyclin-dependent kinase inhibitors (cyclin dependent kinase inhibitors) find stone barrier (Harley, CB, et al, (7erawto /, 27:. 375-382 (1992)) of recent. Research, telomere length varies with oxidative stress. that is, oxidative stress cause telomere shortening, because compared with other portions of the chromosome, telomeric DNA oxidative damage is repaired portion is less the reason (Saretzki, G., von Zglinicki, T., j friends .7Vew K a Jo ^ 959: 24-29 (2002); von Zglinicki, T., Ann 7Vew?..

」c"d 908: 99-110 (2000); von Zglinicki, T" ri^A^5"所0c/2em. 27: 339-344 (2002) ; Lorenz, M.,等,F〜ei?^/zc历o/. 31: 824-831(2001》。 "C" d 908: 99-110 (2000); von Zglinicki, T "ri ^ A ^ 5" The 0c / 2em 27:.? 339-344 (2002); Lorenz, M., et, F~ei ^ . / zc calendar o / 31: 824-831 (2001 ".

本发明人以那些关于衰老的理论为基础,致力于从植物中找出抑制皮肤衰老的新物质。 The inventors on the theory that aging is based, is dedicated to finding new substances to inhibit skin aging from plants. 植物具有建立完善的自我防御体系,这种自我防御体系保护它们自身避免遭受由包括过氧化物自由基(光合作用的残留产物)的许多氧自由基引起的氧化应激。 Plants establish perfect self-defense system, this self-defense system to avoid being themselves protected from the oxidative stress radical include peroxides (photosynthesis residual products) a number of oxygen free radicals. 因此,植物自身是抗氧化剂的重要来源。 Therefore, the plant itself is an important source of antioxidants. 因此,本发明人对于350种植物研究了清除自由基的活性和抑制脂质过氧化作用的活性,并且选择了几种具有抗氧化活性的候选物。 Accordingly, the present inventors investigated the activity of free radical scavenging activity and lipid peroxidation inhibition for 350 kinds of plants, and selects the candidate having several antioxidant activity. 从易于确保来源以及其成分和活性没有被公开考虑,本发明人选择了中国紫荆作为用于抗氧化剂的最终候选物。 From various sources and easily secured and the active ingredient is not considered to be disclosed, the present invention is the Chinese Bauhinia selected as the final candidate for the antioxidant.

中国紫荆, 一种落叶灌木,它属于豆科族并在中国生长。 China Bauhinia, a deciduous shrub, which belongs to the legume family and grow in China. 中国紫荆高3~5 m,其末梢没有绒毛,但有很多皮孔。 Chinese high Bauhinia 3 ~ 5 m, its distal lint, but there are many lenticels. 它具有简单的互生叶(altemate leaves),叶子形成直径为6-llcm的圆形心,没有绒毛、无花纹,边缘光滑。 It has a simple alternate leaves (altemate leaves), leaves a circular core having a diameter of 6-llcm, lint, no pattern, smooth edges. 叶子上端为墨绿色并有光泽,但叶子背部为淡绿色。 The upper end of the leaf is dark green and shiny, but the leaves back light green. 托叶为四边形并且早期脱落。 Stipules quadrilateral and early fall. 花为l-2cm长,叶腋具有多个花。 Flowers for the l-2cm long, leaf axils have more to spend. 花没有花轴但有花梗。 Rachis but no flowers pedicellate. 花萼为伞膜状,其上面边缘具有5个钝锯齿。 Calyx umbrella a film thereon having 5 crenellated edge. 花冠为蝶形和絳红色,它具有5个不规则的花瓣。 Corolla butterfly and magenta, which has five petals irregular. 它具有10个全部分离的雄蕊。 It has all of the ten separate stamens. 雄蕊底部附着在花萼上, 雄蕊的花丝薄并且很长。 Attached to the bottom of stamens calyx, stamens and Silk flowers long. 它具有单个雌蕊。 It has a single pistil. 子房平滑且没有绒毛。 Ovary smooth and free of lint. 子房也具有囊(bag)。 Ovary also has a pouch (bag). 上部形状弯曲,花的柱头小、短且无花纹。 An upper curved shape, small flower stigma, short and no pattern. 开花时间约为4 月,开花后长出叶子。 Flowering time is about 4 months after flowering grow leaves. 作为豆科植物,果实具有扁平带状,其末端部分收縮形成短喙(short bill)。 As legumes, fruit has a flat strip, which end portion is formed short beak shrinkage (short bill). 豆荚长7〜12 cm,在八月或九月成熟。 Pod length 7~12 cm, mature in August or September. 种子为圆形、扁平并接近黑色(Lee, YN , Flora of Korea, Kyo-Hak Publishing Co., Ltd., Seoul, 1996,362-363)。 Seeds are circular, flat and close to black (Lee, YN, Flora of Korea, Kyo-Hak Publishing Co., Ltd., Seoul, 1996,362-363). 中国紫荆的茎皮、根皮和茎用于改善血液循环、 痛经、水肿、碰伤以及各种伤害(Bae, KH, The medicinal plants of Korea, Kyo-Hak Publishing, Seoul, Korea, 2000)。 China Bauhinia bark, root bark and stems for improving blood circulation, dysmenorrhea, swelling, bruising and a variety of injuries (Bae, KH, The medicinal plants of Korea, Kyo-Hak Publishing, Seoul, Korea, 2000).

本发明人证实中国紫荆的提取物与其它合成的抗氧化剂不同,它对人类没有伤害,它对氧化应激具有优异的保护细胞活性,它甚至可以通过降. 低端粒縮短速度而延长细胞寿命,因此该提取物可以有效用作用于抗衰老、保护皮肤弹性和皱纹护理的化妆品组合物,因而本发明人完成了本发明。 The present inventors confirmed China Bauhinia extract with other synthetic antioxidants different, it does not harm humans, it is excellent in oxidative stress protection cell activity, it can even drop. Shortening velocity particles extend the low end of cell life , so that the extract can be effectively used as anti-aging, skin elasticity and wrinkles protection care cosmetic composition, and thus the present invention completed the present invention. 发明概述 SUMMARY OF THE INVENTION

本发明目的是提供中国紫荆的提取物,它具有抗氧化、抗皮肤衰老、 保护皮肤弹性和防止皱纹的活性,并且该提取物是使用水或醇作为提取剂进行提取的。 Object of the present invention is to provide a Chinese Bauhinia extract, which has antioxidant, anti-aging skin, protect the skin elasticity and preventing wrinkles activity, and the extract is extracted using water or alcohol as an extraction agent.

本发明的另一目的是提供化妆品组合物,它用于抗氧化、改善皮肤弹性或皱纹护理,它含有作为有效成分的从由上述提取物或从其中分离的成分组成的组中选取的化合物,该化合物由化学式1〜20表示。 Another object of the present invention is to provide a cosmetic composition for anti-oxidation, improvement of skin elasticity or wrinkle treatment, which contains as an active ingredient a compound selected from the group consisting of the above-described extract or from the separated component consisting of wherein, the compound represented by chemical formula 1~20.

本发明的再一个目的是提供药物组合物,它含有作为有效成分的上述 A further object of the present invention is to provide a pharmaceutical composition comprising as an active ingredient the

S本发明的还一个目的是提供本发明上述提取物的制备方法。 S is also an object of the present invention is to provide a preparation method of the extract of the present invention. 优选实施方案的详细描述 Detailed Description of the Preferred Embodiments

为了实现本发明的上述目的,本发明提供了中国紫荆的提取物,它具有抗氧化、抗皮肤衰老、保护皮肤弹性和防止皱纹的活性,并且该提取物是使用水或醇作为提取剂进行提取的。 To achieve the above object of the present invention, the present invention provides a Chinese Bauhinia extract, which has antioxidant, anti-aging skin, protect the skin elasticity and preventing the activity of wrinkles, and the extract is the use of water or alcohol as an extraction agent extraction of.

本发明也提供了化妆品组合物,它用于抗氧化、改善皮肤弹性或皱纹护理,它含有作为有效成分的从由上述提取物或从其中分离的成分组成的 The present invention also provides a cosmetic composition for anti-oxidation, improvement of skin elasticity or wrinkle treatment, which contains as an active ingredient from the above extract or isolated from a composition wherein component

组中选取的化合物,该化合物由化学式1〜20表示。 Group selected compound, the compound represented by Chemical Formula 1~20.

本发明还提供了药物组合物,它含有作为有效成分的上述提取物。 The present invention also provides a pharmaceutical composition which contains the above extract as an active ingredient. 本发明也提供了本发明上述提取物的制备方法。 The present invention also provides a method for preparing the extract of the present invention. 下面,详细描述本发明。 The present invention will be described in detail.

本发明提供了中国紫荆的提取物,它具有抗氧化、抗皮肤衰老、保护皮肤弹性和防止皱纹的活性,并且该提取物是使用水或醇作为提取剂进行提取的。 The present invention provides a Chinese Bauhinia extract, which has antioxidant, anti-aging skin, protect the skin elasticity and preventing wrinkles activity, and the extract is extracted using water or alcohol as an extraction agent.

基于氧自由基理论,本发明人收集了超过140种草本药和210种植物以研究抗氧化活性,然后选择了有用的候选物。 Oxygen-based free radical theory, the present invention is a collection of more than 140 and 210, the drug grass plants to investigate antioxidant activity, and then select a useful candidate. 其中,因为中国紫荆易于确保来源并且还没有被充分研究,所以它被选作最终的候选物。 Among them, because the Chinese redbud easy to ensure that the source and has not been fully studied, so it was chosen as the final candidate. 而且中国紫荆最终被发明人确认为具有抗氧化活性。 And finally Bauhinia Chinese inventor identified as having antioxidant activity. 在本发明中使用的中国紫荆是由Daeduk Science Town (Daejeon, Korea)禾d Chungnam National University (Daejeon, Korea)在2001年9月收集的,并且由KiHwan Bae教授(College ofPharmacy, Chimgnam National University)确定的。 China Bauhinia used in the present invention is collected in September 2001 by the Daeduk Science Town (Daejeon, Korea) Wo d Chungnam National University (Daejeon, Korea), and is determined by KiHwan Professor Bae (College ofPharmacy, Chimgnam National University) of. 凭证标本(HK U22)陈列于Hansaeng化妆品公司的Jakwang石开究所中(Jakwang Research Institute of the Hansaeng Cosmetics Co., Ltd.)。 Voucher specimens (HK U22) Hansaeng cosmetic companies to display Jakwang stone in the open study (Jakwang Research Institute of the Hansaeng Cosmetics Co., Ltd.).

为了制备本发明的中国紫荆提取物,使用醇的水溶液作为溶剂,该醇的水溶液优选从由甲醇水溶液、乙醇水溶液、丙醇水溶液和丁醇水溶液组成的组中选择。 For the preparation of the present invention Bauhinia Chinese extract used as an aqueous solution of an alcohol solvent, preferably an aqueous solution of the alcohol is selected from the group consisting of aqueous methanol, aqueous ethanol, propanol, butanol and aqueous solution thereof. 其中,更优选乙醇水溶液,尤其是优选50〜80%的乙醇水溶液,更优选60%的乙醇水溶液。 Wherein, more preferably aqueous ethanol, particularly preferably 50~80% ethanol, more preferably 60% aqueous ethanol.

在本发明中,中国紫荆的提取物通过如下步骤制备:通过使用乙酸乙酯(EtOAc)和丁醇(BuOH)提取中国紫荆的醇粗提物,更优选地乙醇(EtOH) In the present invention, Bauhinia Chinese extract prepared by the steps of: extracting Chinese Bauhinia alcohol using ethyl acetate (EtOAc) and butanol (BuOH) crude extract, more preferably ethanol (EtOH)

粗提物;从以上获得各种级分;分离具有抗氧化活性的乙酸乙酯级分和丁醇级分;以及进行色谱分析。 The crude extract; various fractions obtained from above; separated with ethyl acetate and butanol fractions fraction antioxidant activity; and chromatographed. 最终,从乙酸乙酯级分和丁醇级分中获得包括化学式1〜20表示的化合物的提取物(参见图4和图5)。 Finally, obtaining an extract comprising the compound of formula 1~20 represented (see FIG. 4 and FIG. 5) from the fraction of ethyl acetate and butanol fractions. 而且在本发明中获得的化学式15表示的化合物(丁香亭_3-0(2"-0-没食子酰基)-芸香糖苷) 被证实是一种新的化合物。 Further compounds (Ding Xiangting _3-0 (2 '- O galloyl) - rutinoside) represented 15 obtained in the present invention, the formula has proven to be a new compound.

<化学式1><formula>formula see original document page 9</formula><化学式5> <Chemical Formula 1> <formula> formula see original document page 9 </ formula> <Formula 5>

云杉鞣酸醇(Piceatarmol) Spruce tannin alcohol (Piceatarmol)

<化学式6> <Formula 6>

没食子! Gallic!

<化学式7> <Formula 7>

没食子酸甲酯<化学式8> Methyl gallate <Chemical Formula 8>

O O

杨梅黄酮<化学式10> Myricetin <Formula 10>

OH OH OH OH

阿福豆碱(afzelin) <化学式11><formula>formula see original document page 12</formula><化学式14> Fu bean base (afzelin) <Chemical Formula 11> <formula> formula see original document page 12 </ formula> <Chemical Formula 14>

丁香亭-3-0-芸香糖苷 Ding Xiangting -3-0- rutinoside

<化学式15> <Chemical Formula 15>

丁香亭-3-0-(2"-0-没食子酰基)-芸香糖苷 Ding Xiangting -3-0- (2 '- O galloyl) - rutinoside

<化学式16> <Chemical Formula 16>

(+)-儿茶素<化学式17> (+) - catechin <Chemical Formula 17>

<formula>formula see original document page 14</formula> <Formula> formula see original document page 14 </ formula>

(-)-表JL茶素(Epicatechin)-3-0-没食子酸酯 (-) - JL tea table hormone (Epicatechin) -3-0- gallate

<化学式18> <Chemical Formula 18>

<formula>formula see original document page 14</formula> <Formula> formula see original document page 14 </ formula>

(-)-表倍儿茶素(Epigallocatechin)-3-O-没食子酸酯 (-) - catechin fold table (Epigallocatechin) -3-O- gallate

<化学式19> <Chemical Formula 19>

(-)-lyoniresi加l 3a-0-PD-吡喃木糖苷<化学式20> (-) - lyoniresi plus l 3a-0-PD- xylopyranoside <Chemical Formula 20>

(+)-lyoniresinol-3a-0-PD-妣喃葡糖苷 (+) - lyoniresinol-3a-0-PD- deceased mother thiopyran-glucosidase

在化学式1至20表示的化合物中,对于本发明的中国紫荆提取物,以提取物的总重量计,优选包括0.01〜1.00重量%的化学式6表示的化合物、 0.01〜1.00重量%的化学式12表示的化合物和0.01〜0.5重量%的化学式5表示的化合物。 In the compound represented by Formula 1 to 20, for the Chinese extract Bauhinia present invention to the total weight of the extract, preferably 6 comprising a compound represented by formula 0.01~1.00 wt%, 0.01~1.00 wt% represented by Chemical Formula 12 and a compound of chemical formula 0.01~0.5 5% by weight expressed.

化学式1至20表示的本发明化合物具有抗氧化活性,如清除1,1-二苯基-2-Pycryl-偕腙肼(Hydrazyi)自由基的活性(见表3)、脂质过氧化作用抑制活性(见表4)、清除羟基自由基活性、清除一氧化氮的活性(见表6)以及清除过氧化物自由基的活性(见表5)。 A compound represented by Chemical Formula 1-20 of the present invention having antioxidant activity, such as scavenging activity of 1,1-diphenyl -2-Pycryl- hydrazyl (Hydrazyi) radicals (Table 3), inhibition of lipid peroxidation activity (see Table 4), remove the active hydroxyl radical, nitric oxide scavenging activity (see Table 6), and peroxide radical scavenging activity (see Table 5).

在好氧生物中氧对于能量代谢是必需的,但是一旦给予物理、化学和生物应激,氧就变成有害的活性氧种类如超氧化物阴离子自由基、H202 和羟自由基等,由此导致严重的生理障碍。 The superoxide anion radicals in oxygen for aerobic energy metabolism is essential, but once given the physical, chemical and biological stress, oxygen becomes harmful reactive oxygen species, hydroxyl radicals, and H202, whereby cause serious physiological disorders. 该活性氧种类攻击不饱和脂肪酸,导致过氧化作用,而不饱和脂肪酸是细胞膜的成分之一。 The reactive oxygen species attack unsaturated fatty acids, resulting in peroxidation, while unsaturated fatty acid is a component of the cell membrane. 而累积的脂质过氧化物可以是包括衰老在内的各种疾病的原因。 The accumulation of lipid peroxides may be the cause of various diseases, including aging, including. 在本发明中,中国紫荆提取物的抗氧化活性是通过研究提取物的清除活性氧种类活性以及脂质过氧化作用抑制活性进行测定的。 In the present invention, the antioxidant activity of the extract is China Bauhinia inhibitory activity of peroxidation by scavenging reactive oxygen species and lipid research activity was measured in extracts. 结果,中国紫荆提取物的抗氧化活性与维生素E、常规的抗氧化剂、或BHA(叔丁基-4-羟基苯甲醚)、合成抗氧 As a result, the antioxidant activity of extract of Chinese Bauhinia and vitamin E, conventional antioxidants or BHA (tert-butyl-4-hydroxyanisole), synthetic antioxidant

化剂的抗氧化活性相当或比它们更好。 Agents antioxidant activity comparable to or better than them. 因此,本发明的中国紫荆提取物被证实具有优异的抗氧化活性。 Thus, China Bauhinia extract of the invention were confirmed to have excellent antioxidant activity.

化学式1〜20表示的本发明化合物也具有如下的作用:保护细胞免于被叔丁基氢过氧化物(叔-BuOOH)引起的氧化性损伤(见表7)、保护细胞抵抗UV辐射(见图6a和6b),保护裸鼠抵抗UV辐射(见图7)、抑制由UV 辐射引起的脂质过氧化作用的活性(见表8)、延长细胞寿命(见图8)以及延长端粒长度(见图9和图10)。 1~20 represented by the compounds of formula the present invention also has the following functions: to protect cells from oxidative damage induced by tert-butyl hydroperoxide (t -BuOOH) (see Table 7), protects cells against the UV radiation (see FIG. 6a and 6B), the protection against the UV radiation in nude mice (see FIG. 7), the inhibitory activity of UV radiation-induced lipid peroxidation (see Table 8), to extend the life of the cell (see FIG. 8) and the lengthening of telomeres length (see FIGS. 9 and 10). 因此,证实了本发明的中国紫荆提取物以及由此分离的活性成分不仅具有优异的抗氧化活性,而且具有令人满意的细胞衰老抑制作用。 Thus, it was confirmed Bauhinia Chinese extract thus isolated the active ingredient and the present invention has not only excellent antioxidant activity, but also a satisfactory inhibition of cell senescence.

本发明也提供了化妆品组合物,它用于抗氧化作用、皮肤衰老抑制、 促进皮肤弹性或改善皱纹,它含有从由中国紫荆提取物组成的组中或由上述提取物中分离出的化学式1〜20表示的化合物中选择的一种化合物,所选择的化合物作为组合物的有效成分。 The present invention also provides a cosmetic composition for anti-oxidation effect, skin aging inhibition, promoting skin elasticity or improving wrinkles, comprising or isolated from the group consisting of Chinese Bauhinia extract consisting of the above extracts in Chemical formula 1 one compound represented by the ~ 20 selected, the selected compound as an active ingredient of the composition.

本发明的化妆品组合物包含从中国紫荆提取物中分离的活性组分,该活性组分选自由以下化学物组成的组:化学式l(异甘草素)、化学式2(2',4'-二羟基-4-甲氧基查耳酮)、化学式3(甘草素)、化学式4(白藜芦醇)、化学式5(云杉鞣酸醇)、化学式6(没食子酸)、化学式7(没食子酸甲酯)、化学式8(没食子酸乙酯)、化学式9(杨梅黄酮)、化学式10(阿福豆碱)、化学式11 (栎素)、化学式12 (杨梅苷)、化学式13(杨梅黄酮-3-0-(2"-0-没食子酰基)-aL-鼠李吡喃糖苷)、化学式14 (丁香亭-3-0-芸香糖苷)、化学式15 (丁香亭_3—O-(2"-0-没食子酰基)-芸香糖苷)、化学式16( (+)-儿茶素)、化学式17((-)-表儿茶素(Epicatechin)-3-0-没食子酸酯)、化学式18 ((-)-表倍儿茶素(Epigallocatechin)-3-0-没食子酸酯)、化学式19((-)4yoniresinol 3a-0-卩-D-吡喃木糖苷)和化学式20((+)-lyoniresinol-3a-0-PD-吡喃葡糖苷)表示的化合物。 The cosmetic composition of the present invention comprise an active ingredient isolated from Chinese Bauhinia extract, the active ingredient is selected from the group consisting of the following chemical composition: Chemical Formula L (isoliquiritigenin), Chemical Formula 2 (2 ', 4'- hydroxy-4-methoxy chalcone), chemical formula 3 (liquiritigenin), chemical formula 4 (resveratrol), chemical formula 5 (spruce tannin alcohol), chemical formula 6 (gallic acid), chemical formula 7 (gallic acid methyl ester), chemical formula 8 (ethyl gallate), chemical formula 9 (myricetin), formula 10 (Fu bean base), chemical formula 11 (quercitrin), chemical formula 12 (myricitrin), chemical formula 13 (myricetin -3- O- (2 '- O-galloyl) -Al- rhamnose pyranoside), chemical formula 14 (Ding Xiangting -3-0- rutinoside), chemical formula 15 (Ding Xiangting _3-O- (2 "-0 - galloyl) - rutinoside), of the formula 16 ((+) - catechin), of the formula 17 ((-) - epicatechin (epicatechin) -3-0- gallate), the formula 18 ((- ) - catechin fold table (Epigallocatechin) -3-0- gallate), the formula 19 ((-) 4yoniresinol 3a-0- Jie -D- xylopyranoside) and chemical formula 20 ((+) - lyoniresinol- 3a-0-PD- glucopyranoside) a compound represented by.

中国紫荆的提取物或从该提取物中分离的活性成分具有优异的诸如过氧化作用抑制活性和清除自由基活性之类的抗氧化活性,因此它可以有效用作化妆品组合物,该组合物具有抑制皮肤衰老、改善皮肤弹性或皱纹护理作用,这是因为该组合物能够保护皮肤并延长细胞寿命的缘故。 China Bauhinia extracts or isolated from the extract of the active ingredient have excellent inhibiting activity such as over oxidation and removal radical active antioxidant activity and the like, so it can be effectively used as a cosmetic composition, the composition having inhibition of skin aging, improving skin elasticity or wrinkle treatment, and this is because the composition can protect the skin and because of prolonged cell life.

本发明的化妆品组合物可以用作基础皮肤护理的原材料,如软性洗剂、 营养洗剂、营养乳膏剂、香精、面膜(pack)或浴粉,或者可以用作皮肤的外用制剂。 The cosmetic composition of the present invention raw materials may be used as a basis for skin care, such as soft lotion, nutrition lotion, nutrition cream, essence, pack (Pack) or bath powder, or may be used as an external preparation for skin.

制备化妆品组合物时,在考虑乳化作用和经济效率之后就要确定油成分的含量。 When the cosmetic composition is prepared, it is necessary to determine the content of the oil component after emulsification and considering economic efficiency. 作为油成分,可以使用从由植物油、矿物油、硅油和合成油组 As the oil component, use may be made from vegetable oils, mineral oils, synthetic oils and silicone groups

16成的组中选择的一种或多种油。 16 into a selected group of one or more oils. 此外,为了提高乳化作用,可以加入0.1-5 重量%的表面活性剂和高级醇。 In order to improve the emulsification action and to be 0.1 to 5% by weight of a surfactant and a higher alcohol. 可利用普通的表面活性剂如非离子表面活性剂,而至于高级醇,可以单独使用具有12〜20个碳数的醇,或者将该醇与其它种类的醇混合使用。 May utilize conventional surfactants such as nonionic surfactants, as for the higher alcohols may be used alone alcohols having 12~20 carbon number, or the alcohol mixed with other types of alcohol.

为制备化妆品组合物,可以以0.001〜5重量%将诸如卡波姆(carbomer)、膨润土、黄原胶等之类的增稠剂中的至少一种添加到水性组分中以调节粘度或凝固。 To prepare the cosmetic composition, it may be 0.001~5 wt% of at least one additive such as a carbomer thickener (Carbomer), bentonite, xanthan gum and the like in the aqueous component to adjust viscosity or solidification .

为制备化本发明的妆品组合物,也可以增添诸如高级脂肪酸、维生素等之类的药物性质以及诸如UV保护剂、抗氧化剂、防腐剂、香料、着色剂、pH值调节剂等之类的用于化妆品的其它常用添加剂。 To prepare the cosmetic composition of the present invention, it may be added to the pharmaceutical properties such as higher fatty acids, vitamins and the like, such as a UV protective agent or the like, antioxidants, preservatives, fragrances, coloring agents, pH adjusting agent other common additives for cosmetics.

在本发明的优选实施方案中,通过使用本发明的中国紫荆提取物,可以制备软性洗剂、粘稠溶液、乳状洗剂和乳膏剂(见表9〜表11)。 In a preferred embodiment of the present invention, by using the present invention, Chinese Bauhinia extract, a lotion may be prepared soft, viscous solution, milky lotions and creams (Table 9~ Table 11).

为了制备含有中国紫荆提取物的化妆品组合物,向普通组合物中,另外加入1-15重量%的提取物,更优选加入2-10重量%。 For the preparation of a cosmetic composition containing an extract of Chinese Bauhinia, the conventional composition, the further addition of 15% by weight of the extract, more preferably 2-10 wt% was added.

本发明还提供用于抗氧化作用和皮肤抗衰老的药物组合物,该组合物含有本发明的中国紫荆提取物,该提取物作为有效成分。 The present invention further provides for the antioxidant and anti-aging skin pharmaceutical compositions, China Bauhinia composition comprising the extract of the present invention, the extract as an active ingredient.

用于抗氧化作用和皮肤抗衰老的药物组合物含有作为有效成分的本发明中国紫荆提取物,该药物组合物对于治疗或预防由氧自由基对细胞成分氧化而引起的各种疾病是非常有用的。 For the antioxidant and anti-aging skin pharmaceutical composition comprising Bauhinia Chinese extract of the invention as an active ingredient, various diseases of the pharmaceutical composition for the treatment or prevention of oxygen free radicals caused by oxidation of cell components is very useful of. 目标疾病是癌症、衰老、冠心病、 高血脂症、动脉硬化症、多发性硬化、自身免疫脑脊髓炎、脑溢血、早老性痴呆症(Alzheimer's disease)和肠炎,但是目标疾病未必总是限制于这些疾病。 Target disease is cancer, aging, coronary heart disease, hyperlipidemia, atherosclerosis, multiple sclerosis, autoimmune encephalomyelitis, stroke, Alzheimer's disease (Alzheimer's disease) and inflammatory bowel disease, but the goal is not always limited to these diseases disease.

含有中国紫荆提取物的药物组合物可以另外包含稀释剂、崩解剂、甜味剂、润滑剂、香料等,通常可以制备成片剂、胶囊、粉末、粒剂、混悬剂、乳剂、糖浆剂的形式以及其它液体形式。 China Bauhinia extract containing pharmaceutical composition may additionally contain diluents, disintegrating agents, sweetening agents, lubricants, perfumes, etc., can generally be prepared as tablets, capsules, powders, granules, suspensions, emulsions, syrups, in the form of agent, and other liquid forms.

尤其是,含有本发明中国紫荆提取物作为有效成分的药物组合物可以制备成片剂、锭剂、糖锭、水溶或油性混悬剂、粉末或颗粒、乳剂、硬或软胶囊、糖浆剂或酏剂的形式,以用于口服。 In particular, the present invention comprises Bauhinia Chinese extract as an active ingredient of the pharmaceutical composition can be prepared into tablets, troches, lozenges, aqueous or oily suspensions, powders or granules, emulsions, hard or soft capsules, or syrups elixirs, for oral administration. 为了制备片剂或胶囊形式, 可以包含诸如乳糖、蔗糖、山梨(糖)醇、甘露糖醇(manitol)、淀粉、支链淀粉、纤维素或明胶之类的粘合剂;诸如磷酸二钙之类的稀释剂、诸如玉 For the preparation of tablets or capsules, may contain information such as lactose, sucrose, sorbitol (a sugar) alcohol, starch, amylopectin, cellulose adhesive mannitol (manitol) or gelatin and the like; such as dicalcium phosphate of thinners, such as jade

17米淀粉或甘薯淀粉之类的崩解剂;诸如硬脂酸镁、硬脂酸钙、硬脂酰富马酸镁或聚乙二醇蜡之类的润滑剂。 17 meters starch or sweet potato starch disintegrants like; a lubricant such as magnesium stearate, calcium stearate, magnesium stearyl fumarate, polyethylene glycol waxes or the like. 为了制备胶囊形式的制剂,还^T向上述物质中加入液体载体如脂肪油。 To prepare a capsule form preparations ^ T also added to the liquid carrier material, such as a fatty oil.

包含本发明的中国紫荆提取物的药物组合物可以是非肠道施用的。 Comprising a pharmaceutical composition of the present invention Bauhinia Chinese extract may be administered parenterally. 非肠道施用的方式有静脉注射、肌内注射或皮下注射。 Parenteral administration methods are intravenous, intramuscular or subcutaneous injection. 为了制备适用于非肠道施用的组合物,本发明的中国紫荆提取物应该与稳定剂或缓冲剂在水中混合,以制备溶液或混悬剂形式,最后以安瓿或小瓶形式配制。 For the preparation of compositions suitable for parenteral administration, China Bauhinia extract of the invention in water should be mixed with a stabilizer or buffer to prepare solutions or suspensions, and finally formulated in the form of ampoules or vials.

本发明的有效剂量是考虑活性成分在活体内的吸收、失活速率、排泄速度、年龄、性别和病人的其它条件、疾病的严重性等而确定的。 Effective dose of the present invention is to consider the active ingredient, inactivation rate, excretion rate, age, sex and other conditions of the patient, the severity of the disease in vivo absorption determined. 通常, usually,

如果是口服,则每lkg重量建议使用2〜200mg的本发明提取物, 一天一次或多次,更优选剂量为10〜100mg。 When administered orally, the recommended weight per lkg extract 2~200mg the present invention, the one or more times, more preferred dose 10~100mg.

本发明也提供了中国紫荆提取物的制备方法。 The present invention also provides a method for preparing an extract of Chinese Bauhinia.

本发明中国紫荆的制备方法由下列步骤构成: China Bauhinia preparation of the present invention consists of the following steps:

1) 用醇对中国紫荆的粉碎粉末进行粗提; 1) to be pulverized powder Bauhinia Chinese crude extract with an alcohol;

2) 按顺序使用己烷、乙酸乙酯和丁醇,对上述步骤l)的醇粗提物进行提取; 2) sequentially using hexane, ethyl acetate and butanol, the alcohol of the above-described step l) extracting the crude extract;

3) 将上述步骤2)中获得的乙酸乙酯级分或丁醇级分进行甲醇:水的密度梯度柱色谱;和 3) The above step 2) fractions obtained in ethyl acetate or butanol fractions of methanol: water density gradient column chromatography; and

4) 通过将在上述步骤3)中获得的具有抗氧化剂活性的级分进行柱色谱、TLC或HPLC,获得最终的抗氧化剂提取物。 4) For column chromatography, TLC or by HPLC grade having antioxidant activity obtained in) step 3 above, to obtain the final antioxidant extract.

在步骤1)中,醇优选甲醇、乙醇、丙醇或丁醇中的一种,其中,更优选乙醇。 In step 1), an alcohol preferably methanol, ethanol, propanol or butanol, wherein, more preferably ethanol. 如果优选乙醇,则更优选60%的乙醇。 If ethanol, even more preferably 60% ethanol. 在本发明的优选实施方案中,通过研究浓度为0〜100。 In a preferred embodiment of the present invention, from a study of the concentration of 0 to 100. /。 /. 乙醇的中国紫荆粗提物级分清除DPPH自由基的活性,证实60%乙醇的粗提物具有最高的活性(见图2)。 Chinese Bauhinia ethanol crude extract fraction DPPH radical scavenging activity, demonstrated 60% ethanol crude extract had the highest activity (see FIG. 2).

附图简述 BRIEF DESCRIPTION

参考附图,可最好地理解本发明优选实施方案的应用,其中: 图l是示出在从中国紫荆中获得乙醇粗提物后,按顺序进行己烷、乙:乙酯和丁醇的提取工艺的示意图。 Referring to the drawings, the application can be best understood preferred embodiments of the present invention, wherein: Figure l is a diagram illustrating after obtaining crude ethanol extract from Bauhinia in China, in sequence hexane, B: butanol and ethyl extraction process schematic.

图2是示出从中国紫荆获得的乙醇粗提物的清除DPPH自由基活性的图,其中乙醇浓度为0〜100%(每个粗提物之间相差10%)。 FIG 2 is a diagram illustrating Bauhinia Chinese radical from ethanol to obtain crude extract was FIG DPPH radical scavenging activity, wherein the ethanol concentration is 0 to 100% (a difference of 10% between each of the crude extract).

图3是示出己烷、乙酸乙酯、丁醇和水级分中的每一种以及乙醇提取 3 is a diagram illustrating hexane, ethyl acetate, butanol and water fractions, and each of the ethanol extract

物的清除DPPH自由基活性的图。 FIG scavenging DPPH radical activity thereof.

图4所示为从乙酸乙酯级分中分离具有抗氧化活性的化合物的工艺。 Figure 4 shows the separated fraction from ethyl acetate with the process of antioxidant activity of compounds.

图5所示为从丁醇级分中分离具有抗氧化活性的化合物的工艺。 Figure 5 is a process having antioxidant activity of compounds isolated from butanol fraction FIG.

图6a所示为一组DNA损伤的细胞照片,反映出本发明的中国紫荆提 A group of cells is shown in FIG picture DNA damage, reflecting the present invention provide 6a China Bauhinia

取物或从该提取物中分离的化合物对于保护细胞抵抗UV辐射的作用。 Extract or a compound isolated from the extract to protect cells against the action of UV radiation. 图6b所示为表示DNA损伤的相对荧光强度,反映出本发明的中国紫 As shown in FIG. 6b is a diagram showing the relative fluorescence intensity of DNA damage, the present invention reflects purple China

荆提取物或从该提取物中分离的化合物对于保护细胞抵抗UV辐射的作用。 Jing extracts or isolated compounds from the extract to protect cells against the action of UV radiation.

图7所示为一组表示裸鼠皮肤损伤的照片,证实了本发明的中国紫荆提取物或从该提取物中分离的化合物对于保护细胞抵抗UV辐射的作用。 Figure 7 is a photograph showing a group of nude mice skin damage, confirmed China Bauhinia extract of the invention or isolated from the extract a compound to protect cells against UV radiation.

图8所示为用本发明的中国紫荆提取物或从该提取物中分离的化合物延长细胞寿命。 As shown in FIG. 8 extend cell life of the compound of the invention or the extract of Chinese Bauhinia isolated from the extract.

图9所示为DNA印迹分析产生的一组照片,证实了本发明的中国紫荆提取物或从该提取物中分离的化合物降低了端粒的縮短速度。 Analysis As shown in FIG. 9 is a set of photographs generated DNA blots confirmed China Bauhinia compound of the invention or an extract isolated from the extract reduced telomere shortening velocity.

图10所示为端粒的縮短速度,表明本发明的中国紫荆提取物或从该提取物中分离的化合物延长了端粒的长度。 Figure 10 shows the telomere shortening velocity, China showed Bauhinia extract of the invention or isolated from the extract a compound of extended telomere length.

图ll所示为一组示出中国紫荆的花、叶、根皮(rootbark)、茎的照片。 Figure ll is a set of photographs showing China Bauhinia flowers, leaves, root bark (rootbark), stem picture.

实施例 Example

如下面实施例所示解释本发明的实用并当前优选的实施方案。 As shown in the following Examples explain the practical embodiment of the present invention and presently preferred embodiments. 然而,要意识到本领域普通技术人员在考虑本公开的基础上是可以在本发明的精神和范围之内进行修改和改进的。 However, it should be appreciated by those of ordinary skill in the art based on the present disclosure is to be considered can be modified within the spirit and scope of the invention and improved. 实施例l:从中国紫荆中提取有效成分<1-1>抗氧化活性级分的初步分离 Example l: active ingredient extracted from Chinese Bauhinia in <1-1> Preliminary isolated fractions antioxidant activity

为了从中国紫荆中提取具有抗氧化活性的有效成分,按在图1示意图中出现的工艺进行实验。 In order to extract the active ingredient with antioxidant activity from Bauhinia in China, according to the process occurring in the schematic in FIG. 1 experiment. 具体地,使用粉碎机将l Kg阴干的中国紫荆的叶和茎碾碎成粉末,该粉末在室温用乙醇(EtOH)提取两次,两次之间相隔两周时间。 Specifically, l Kg of dried leaves and stems of Chinese Bauhinia pulverizer using crushed into powder, the powder was extracted twice with ethanol (EtOH) at room temperature, separated by two weeks between the two. 对于该提取物,向其中逐歩加入0〜100%的乙醇(每次递增10 For the extract, was added thereto by ho 0 to 100% ethanol (10 per increment

19%)以制备乙醇溶液。 19%) to prepare an ethanol solution. 所制备的乙醇溶液用于提取。 Ethanol was prepared for extraction. 通过使用DPPH (1,1- By using DPPH (1,1-

二苯基-2-Pycryl-偕腙肼)(Taco, T.等,Biosci. Biotech. Biochem. , 1994, 58, 1780-1783; Na, MK等,Nat. Prod. Sci. , 2002, 8, 26-29)的方法对提取物的抗氧化活性进行研究。 .-Diphenyl -2-Pycryl- hydrazyl) (Taco, T., etc., Biosci Biotech Biochem, 1994, 58, 1780-1783;. Na, MK, etc., Nat Prod Sci, 2002, 8,... study of antioxidant activity of extracts 26-29) method. DPPH是一种稳定的自由基,它示出作为自由基在517nm处的最大光密度。 DPPH is a stable free radical, which is shown as a radical maximum optical density at 517nm. 但是,当其被清除时,它就失去了吸光度。 However, when it is cleared, it loses its absorbance. 基于该观点,可以使用DPPH测定抗氧化活性。 From this viewpoint, the antioxidant activity may be measured using DPPH. 更具体而言,根据不同浓度从中国紫荆中获得每种乙醇提取物,再用DMSO(Sigma)分别稀释到3.125、 6.25、 12.25、 25和50 jug/ml。 More specifically, each of the ethanol extract obtained from Chinese Bauhinia according to different concentrations, and then DMSO (Sigma) were diluted to 3.125, 6.25, 12.25, 25 and 50 jug / ml. 然后,每种溶液以每种10 jul分配进入96 孔板中,再向其中加入190 pi DPPH(Sigma, St. Louis, Mo, USA)溶液,该溶液的乙醇浓度为2xl0—4 M/ml。 Then, each solution was dispensed into each of 10 jul 96 well plate, to which was added 190 pi DPPH (Sigma, St. Louis, Mo, USA) solution, ethanol concentration of the solution is 2xl0-4 M / ml. 该板在室温只放置30分钟。 The plate was left at room temperature for 30 minutes only. 最后,在517nm测定OD5n。 Finally, OD5n measured at 517nm. 用于对照,加入DMSO以代替样品,并研究光密度的变化。 For the control, DMSO was added instead of the sample, and to study the change in optical density. 清除DPPH自由基的活性通过下面<数学式1>计算,而能够清除50 %的DPPH的样品浓度定作IC5()。 DPPH free radical scavenging activity by the following <Equation 1> is calculated, it is possible to remove a sample of DPPH concentration of 50% set for IC5 (). <数学式1> <Mathematical Formula 1>

清除DPPH自由基的活性(%) =(A对照一A样品)/A对照x100 A对照:对照组的光密度(没有加入样品), A样品:实验组的光学密度(加入样品)。 DPPH free radical scavenging activity (%) = (A-A control sample) / A Control A Control XlOO: the optical density of control (no sample added), A sample: Optical density of the experimental group (added to the sample).

结果,清除DPPH的活性根据剂量而增加。 As a result, scavenging DPPH activity increased according to the dose. 0%、 10°/。 0%, 10 ° /. 、 20%和90%的乙醇提取物显示出低的清除自由基活性,而30%、 40%、 50%、 60%、 70%、 80%和100%的乙醇提取物总体上表示出高的清除自由基活性。 , 20% and 90% ethanol extract showed a low free radical scavenging, and 30%, 40%, 50%, 60%, 70%, generally shown high 80% and 100% ethanol extract free radical scavenging activity. 尤其是在60%乙醇提取物的情况下,清除自由基活性随着提取物的浓度增加而变得非常高,在所有乙醇提取物中,ICsq是最低的(26.6),这情况反映出该提取物具有最高的抗氧化活性(表1和图2)。 Especially in the case of 60% ethanol extract, free radical scavenging activity with increasing concentration of the extract becomes very high in all the ethanol extract, ICsq is the lowest (26.6), which reflects the case where extraction having the highest anti-oxidative activity (table 1 and FIG. 2).

<表1> <Table 1>

<table>table see original document page 20</column></row> <table><table>table see original document page 21</column></row> <table> <Table> table see original document page 20 </ column> </ row> <table> <table> table see original document page 21 </ column> </ row> <table>

在确认60X乙醇提取物具有最高的清除DPPH自由基的活性之后,用不同提取剂获得各种提取物。 After confirming 60X ethanol extract having the highest DPPH free radical scavenging activity of various extracts obtained by different extraction agents. 具体地,60%的乙醇提取物悬浮在蒸馏水中, 再用己烷提取三次。 Specifically, 60% of the ethanol extract was suspended in distilled water, extracted three times with hexane. 在减压下浓縮该己垸提取物,获得11 g的己烷级分(下面称作'Fr,)。 Concentrated under reduced pressure has the embankment extract to obtain 11 g of hexane fraction (hereinafter referred to as' Fr,). 残余悬浮液用乙酸乙酯(EtOAc)提取三次。 The residue suspension was extracted three times with ethyl acetate (EtOAc). 在减压下将其浓縮,获得25g的乙酸乙酯级分(下面称作'EtOAcFr,)。 Concentrated under reduced pressure to give 25g of ethyl acetate fraction (hereinafter referred to as' EtOAcFr,). 剩余悬浮液再用水-饱和的丁醇(BuOH)提取三次。 The remaining suspension was washed with water - saturated butanol (BuOH) and extracted three times. 将该提取液在减压下浓縮,获得19 g 的丁醇级分(下面称作《BuOH Fr')。 The extract was concentrated under reduced pressure to obtain 19 g of butanol fraction (hereinafter referred to as "BuOH Fr '). 而20 g的残余级分当作是水级分(图1)。 And 20 g of water as the residual fraction fraction (FIG. 1).

为了在上述获得的己烷Fr、 EtOAc Fr和BuOH Fr中确定具有抗氧化活性的级分,使用各个级分的各个提取物用上述方法测定清除DPPH自由基的活性。 To determine the fraction having antioxidant activity obtained above Fr in hexanes, EtOAc in BuOH Fr and Fr, a respective fraction of each extract was measured by the above method DPPH radical scavenging activity. 此时,已知具有高的清除DPPH自由基活性的维生素E用作对比组。 In this case, it is known to have a high DPPH radical scavenging activity of vitamin E used as a control group.

结果,EtOAc Fr和BuOH Fr的IC50分别为24.0和27.0吗/ml,这结果表明具有与对比组维生素E(IC5。: 24.9吗/ml)相类似的活性。 Results, EtOAc Fr and BuOH Fr of IC50 were 24.0 and 27.0 do / ml, which results showed a similar activity of vitamin group with Comparative E (IC5 .: 24.9 yet / ml). 但是,其它级分表示出弱的活性(图3)。 However, other fractions shown weak activity (FIG. 3).

<1-2>抗氧化活性级分的二次分离 Secondary Separation <1-2> Antioxidant active fraction

在上述实施例<1-1>结果的基础上,将EtOAc Fr和BuOHFr进行柱色谱,这两者级分都具有高活性。 Based on the results of the above Example <1-1> on the BuOHFr EtOAc Fr and subjected to column chromatography, both of which are highly active fractions. 将所获得的级分再次进行抗氧化活性测试, 以选择具有高抗氧化活性的级分。 Fractions obtained points for antioxidant activity test again to select the fractions with high antioxidant activity.

首先,使用甲醇:水(h 4—1: O)作为流动相,使EtOAc Fr (25 g)通过YMC柱色谱(柱尺寸:5x30 cm),获得11个亚级分(Fr.l〜Fr.11)。 First, using methanol: water (h 4-1: O) as the mobile phase, so EtOAc Fr (25 g) by YMC column chromatography (column size: 5x30 cm), with 11 sub-fractions (Fr.l~Fr. 11). 在上述的ll个亚级分中将Fr.l (3.6 g)再进行YMC柱色谱(柱尺寸:3x30 cm),获得了5个亚级分(Fi'.ll〜Fr.l-5)。 Then YMC column chromatography (column size: 3x30 cm) above the level in the sub-fraction ll Fr.l (3.6 g) in the obtained five sub-fractions (Fi'.ll~Fr.l-5). 选择Fd-l (450 mg)用于HPLC[柱: fiBondapakTMC18(3.9x300 mm,水),流动相:ACN: 0. 1% TCA (16: 18),流动速度:lml/min, UV: 280腿],分别获得18 mg和21mg的具有保留时间(以下称为"tr")为11.1分钟和6.2分钟的化合物,这两种化合物分别命名为'CCEA111'和'CCEA112'。 Select Fd-l (450 mg) for HPLC [column: fiBondapakTMC18 (3.9x300 mm, water), mobile phase: ACN: 0. 1% TCA (16: 18), flow rate: lml / min, UV: 280 Leg ], 18 mg and 21mg respectively having a retention time (hereinafter referred to as "tr") of 11.1 minutes and 6.2 minutes compounds, the two compounds are named as 'CCEA111' and 'CCEA112'.

其次,对Fr. 1-2 (500 mg)进行HPLC [YMC-Pack ODS-A柱:(20x250 mm),流动相:甲醇:水(3:7),流动速度:6 ml/min, UV: 254 nm]级分收集,获得77mg具有16分钟tR的化合物以及19mg具有24分钟tR的化合物,这两种化合物分别命名为'CCEA1211邻'CCEA1212'。 Next, Fr. 1-2 (500 mg) for HPLC [YMC-Pack ODS-A column: (20x250 mm), mobile phase: methanol: water (3: 7), flow rate: 6 ml / min, UV: 254 nm] to collect fractions, obtained with 16 minutes tR compound 77mg and 19mg of compound having tR 24 minutes, the two compounds are named as' CCEA1211 o 'CCEA1212'.

第三,对Fr. 3 (4.8 g)进行HPLC [YMC-Pack ODS-A柱:(20x250 mm), 流动相:ACN:O. 1%TCA(25:75),流动速度:6 ml/min,检测器:UV(280 nm)]级分收集,获得19mg具有24.5分钟tR的化合物,该化合物命名为'CCEA33'。 Third, Fr. 3 (4.8 g) for HPLC [YMC-Pack ODS-A column: (20x250 mm), mobile phase: ACN:. O 1% TCA (25:75), flow rate: 6 ml / min detector: UV (280 nm)] fractions were collected to give 19mg of compound having tR 24.5 minutes, the compound is named 'CCEA33'.

接着,Fr. 4 (4.0 g)通过硅胶柱色谱(4x25cm, 230-400目,流动相:氯仿:甲醇(85: 15))分成6个亚级分(Fr.4-l〜Fr. 4-6)。 Subsequently, Fr 4 (4.0 g) by silica gel column chromatography (4x25cm, 230-400 mesh, mobile phase: chloroform: methanol (85: 15)). Is divided into six sub-fraction (Fr.4-l~Fr 4-. 6). 其中,Fr. 4-1 (320 mg) 用于HPLC收集[流动相:氯仿:甲醇(35:65),流动速度:6ml/min., UV: 254 nm],获得30mg具有25分钟tR的化合物,并将该化合物命名为'CCEA413'。 Among them, Fr 4-1 (320 mg) was collected for HPLC [eluent: chloroform: methanol (35:65), flow rate:. 6ml / min, UV: 254 nm], to obtain 30mg of compound having tR 25 minutes and the compound is named 'CCEA413'. 同样,Fr. 4-4(1 g)也用于收集HPLC[流动相:甲醇:水(l: 1), 流动速度:6 ml/min., UV: 254 nm],获得57 mg具有15分钟tR的化合物, 并将该化合物命名为'CCEA442'。 Similarly, Fr 4-4 (1 g) is also used to collect HPLC [mobile phase: methanol: water (l: 1), flow rate: 6 ml / min, UV:. 254 nm], with 15 minutes to obtain 57 mg tR compound, and the compound is named 'CCEA442'.

Fr. 6 (2.2 g)通过硅胶柱色谱(3x30cm, 230-400目,流动相:氯仿:甲醇(10: 1))分成3个亚级分(Fr.6-l〜Fr. 6-3)。 Fr. 6 (2.2 g) by silica gel column chromatography (3x30cm, 230-400 mesh, mobile phase: chloroform: methanol (10: 1)) is divided into three sub-fractions (Fr.6-l~Fr 6-3.) . 其中,Fr. 6-2 (200 mg)用于HPLC 收集[流动相:甲醇:水(l:l),流动速度:6 ml/min., UV:254nm],获得25 mg具有20分钟tR的化合物,并将该化合物命名为'CCEA622'。 Among them, Fr 6-2 (200 mg) was collected for HPLC [mobile phase: methanol: water (l: l), flow rate:. 6 ml / min, UV: 254nm], with 20 minutes to obtain 25 mg of tR compound, and the compound is named 'CCEA622'.

接着,将Fr. 8(2.1 g)进行硅胶柱色谱(流动相:氯仿:水(10: 1)),获得20mg化合物,并将该化合物命名为'CCEA82'。 Subsequently, Fr. 8 (2.1 g) silica gel column chromatography (mobile phase: chloroform: water (10: 1)) to give 20mg compound, and the compound is named 'CCEA82'. 将上述CCEA82分离之后,残余级分溶解在甲醇中。 After the above CCEA82 separation, the residue fraction was dissolved in methanol. 进行重结晶,获得10mg化合物,并将其命名为'CCEA83'。 Recrystallized to give compound 10mg, and named it 'CCEA83'. Fr.9(2.8g)通过硅胶柱色谱(流动相:氯仿:甲醇(15: 1))分成2个亚级分。 Fr.9 (2.8g) purified by silica gel column chromatography (mobile phase: chloroform: methanol (15: 1)) is divided into two sub-fractions. 其中,将Fr.9-1 (220mg)用于收集HPLC[流动相:甲醇:水(4: 1), 流动速度:6 ml/min., UY: 254 nm],获得32 mg具有25分钟tR的化合物, 并将该化合物命名为'CCEA913'(图4)。 Wherein the Fr.9-1 (220mg) for collecting HPLC [mobile phase: methanol: water (4: 1), flow rate: 6 ml / min, UY:. 254 nm], with 25 minutes to obtain 32 mg tR compound, and the compound is named 'CCEA913' (FIG. 4).

将BuOHFr(19g)进行YMC柱色谱(柱尺寸:5x30cm,流动相:甲醇: ZR(0:1—1:0),获得8个亚级分(Fr.l-Fr. 8)。 The BuOHFr (19g) for YMC column chromatography (column dimensions: 5x30cm, mobile phase: methanol: ZR (0: 1-1: 0), to obtain 8 sub-fractions (Fr.l-Fr 8)..

Fr.2(3.8g)进行硅胶柱色谱(流动相:氯仿:甲醇:水(70 : 30: 5),柱尺寸: 3x30 cm, 230-400目),获得5个亚级分(Fr.2-l〜Fr.2-5)。 Fr.2 (3.8g) silica gel column chromatography (mobile phase: chloroform: methanol: water (70: 30: 5), Column size: 3x30 cm, 230-400 mesh) to obtain 5 sub-fractions (Fr. 2 -l~Fr.2-5). 在那些级分中, Fr. 2-3 (420 mg)用于收集HPLC[流动相:乙腈:水(18: 82),流动速度:6 ml/min., UV: 254腿],以获得Fr. 2-3-1和Fi'. 2-3-2。 In those fractions, Fr. 2-3 (420 mg) for collecting HPLC [mobile phase: acetonitrile: water (18: 82), flow rate: 6 ml / min, UV:. 254 legs] to obtain Fr . 2-3-1 and Fi '. 2-3-2. Fr. 2-3-1再次用于收集HPLC[流动相:乙腈:水(10:90),流动速度:6 ml/min., UV:254nm], 获得32 mg具有15分钟tR的化合物,并将该化合物命名为'CCBt231'。 Fr. 2-3-1 again for collecting HPLC [mobile phase: acetonitrile: water (10:90), flow rate: 6 ml / min, UV:. 254nm], to obtain 32 mg of the compound having tR 15 minutes, and this compound is named 'CCBt231'.

Fr. 5 (3 g)进行硅胶柱色谱(3x30 cm, 230-400目,流动相:氯仿:甲醇: 水(70:30:5)),获得4个亚级分(Fr.5-l〜Fr.5-4)。 Fr. 5 (3 g) silica gel column chromatography (3x30 cm, 230-400 mesh, mobile phase: chloroform: methanol: water (70: 30: 5)) to obtain four subfractions (Fr.5-l~ Fr.5-4). 其中,Fr. 5-2 (300 mg)用于收集HPLC[流动相:甲醇:水(35: 65),流动速度:6 ml/min., UV: 254 nm], 产生20 mg具有25分钟tR的化合物以及13 mg具有30分钟tR的化合物。 Among them, Fr 5-2 (300 mg) for collecting HPLC [mobile phase: methanol: water (35: 65), flow rate: 6 ml / min, UV:. 254 nm], with 25 minutes to produce 20 mg tR compound and 13 mg of a compound having tR 30 minutes. 这两种化合物命名为'CCBt521'和'CCBt522'。 Both compounds are named as 'CCBt521' and 'CCBt522'. 将Fr. 5- 3 (600 mg)进行YMC 柱色谱(柱尺寸:3x30cm,流动相:30%甲醇),以将其分成4个级分(Fr.5-3-l〜Fr.5-3-4)。 The Fr. 5- 3 (600 mg) for YMC column chromatography (column dimensions: 3x30cm, mobile phase: 30% methanol), so as to be divided into four fractions (Fr.5-3-l~Fr.5-3 -4). 在那些亚级分中,使Fr.5-3-3的沉析物纯化以获得29 mg化合物,并将该化合物命名为'CCBt533'。 In those sub-fractions, The purified Fr.5-3-3 coagulum to obtain 29 mg compound, and the compound is named 'CCBt533'.

Fr. 6(3.2 g)再次进行YMC柱色谱(柱尺寸:3x30cm)以分成4个亚级分(Fr.6-l〜Fr.6-4)。 Fr. 6 (3.2 g) again YMC column chromatography (column size: 3x30cm) to be divided into four sub-fractions (Fr.6-l~Fr.6-4). 其中,Fr. 6-2 (370 mg)用于收集HPLC[流动相:甲醇: 水(3:7),流动速度:6 ml/min., UV:254nm],产生具有26分钟tR的化合物(9 mg)以及具有28分钟tR的化合物(16 mg)。 Among them, Fr 6-2 (370 mg) for collecting HPLC [mobile phase: methanol: water (3: 7), flow rate: 6 ml / min, UV:. 254nm], tR 26 minutes to produce a compound having the ( 9 mg) and the compound (16 mg) having a tR of 28 minutes. 这两种化合物命名为'CCBt622'和'CCBt623'。 Both compounds are named as 'CCBt622' and 'CCBt623'. 将Fr. 6-4 (200 mg)也用于收集HPLC(流动相:甲醇: 水(4:6),流动速度:6ml/min.),产生具有18分钟tR的化合物(5 mg),该化合物命名为'CCBt641'。 The Fr. 6-4 (200 mg) is also used to collect HPLC (mobile phase: methanol: water (4: 6), flow rate: 6ml / min.), To give compound (5 mg) with 18 minutes of tR, which compound named 'CCBt641'.

Fr. 7 (1.9 g)进行硅胶柱色谱(流动相:氯仿:甲醇,柱尺寸:3x30 cm) 分离,产生4个亚级分(Fr.7-l〜Fr.7-4)。 Fr. 7 (1.9 g) silica gel column chromatography (mobile phase: chloroform: methanol, column size: 3x30 cm) to yield the four sub-fractions (Fr.7-l~Fr.7-4). Fr. 7-2 (50 mg)用于Sepadex LH-20 柱色谱(流动相:80%甲醇,柱尺寸:3x30 cm),以分成5个亚级分(Fr.7-2-l〜 Fr. 7-2 (50 mg) for Sepadex LH-20 column chromatography (mobile phase: 80% methanol, column size: 3x30 cm), to be divided into five sub-fractions (Fr.7-2-l~

23Fr.7-2-5)。 23Fr.7-2-5). 其中,Fr.7-2-2溶解在甲醇中以再次重结晶,获得化合物(9mg) 并将该化合物命名为'CCBt722'。 Wherein, Fr.7-2-2 dissolved in methanol again recrystallization to obtain compound (9 mg of) and the compound is named 'CCBt722'. 此外,通过使Fr.7-2-4提取的沉析物纯化获得另外的化合物(10mg),该化合物命名为'CCBt724'(图5)。 Further, by purification of the extracted precipitation Fr.7-2-4 obtain additional compound (10 mg), the compound is named 'CCBt724' (FIG. 5).

<1-3>分离化合物的结构分析 <1-3> Analysis of the structure of the compound isolated

研究在上述实施例<1-2>中分离的21种级分的结构。 In the above embodiment Study <1-2> Structure 21 kinds of separated fractions. 具体地,使用电热熔点仪(Electrothermal Eng. Ltd., AZ 9003)研究熔点,使用DIP- 370数字偏振计(JASCO)研究旋转程度,使用IR记录-IOO分光光度计(JASCO)研究IR光谱,用串连质谱计(Jeol, JMS HX-110/110A)进行质量分析,使用NMR分光光度计(Bmker, NMR AMX-600光谱仪)进行NMR分析,而使用STAR 3400 CX GC (Varian)根据出厂说明进行气相色谱分析。 Specifically, electric melting point apparatus (Electrothermal Eng. Ltd., AZ 9003) m.p. research studies using DIP- 370 degree rotation digital polarimeter (a JASCO), using an IR spectrophotometer recording -IOO (a JASCO) IR SPECTROSCOPY, with tandem mass spectrometer (Jeol, JMS HX-110 / 110A) mass analysis using NMR spectrometer (Bmker, NMR AMX-600 spectrometer) NMR analysis, the use of STAR 3400 CX GC (Varian) according to the factory instructions vapor spectrum analysis. 鉴于上述测试的所有结果(熔点、旋转度、IR光谱、质量和NMR),确定化合物结果。 In view of all the results of the above tests (melting point, degree of rotation, the IR spectrum, NMR and mass), the results of the compound is determined.

结果,化合物根据结构分成如下表2所示的查耳酮、1,2-二苯乙烯、 酚酯(phenolic)、黄酮醇、黄烷醇、木酚素类。 As a result, the compound according to the structure is divided into the following table 2 shown chalcone, stilbene, phenol esters (Phenolic), flavonols, flavanols, lignans. 每一个化合物都被证实为由<化学式1>〜<化学式20>表示的化合物。 Each of the compounds are confirmed compound of <Formula 1> ~ <Chemical Formula 20> represented by. 尤其地, <化学式15 〉表示的化合物(丁香亭-3-0-(2"-0-没食子酰基)-芸香糖苷)具有以下解释的性质, 从这些性质可以确定该化合物为新化合物。 In particular, <Chemical Formula 15> Compound (Ding Xiangting -3-0- (2 '- O galloyl) - rutinoside) has properties explained below, these properties may be determined from the new compounds.

<化学式15的性质> <Properties Chemical Formula 15>

浅黄色粉末, Pale yellow powder,

FeCl3,Mg-HCl,Zn-HCl测试:阳性, 阳性FAB-MS: m/z 823 [M + H]+ [a]D-80 (cO.l,MeOH) FeCl3, Mg-HCl, Zn-HCl test: positive, positive FAB-MS: m / z 823 [M + H] + [a] D-80 (cO.l, MeOH)

IR v丽cm": 3400 (-OH), 1650(00), 1610, 1500, 1455 (芳香OC), 1200,1020 (糖苷CO) Li IR v cm ": 3400 (-OH), 1650 (00), 1610, 1500, 1455 (aromatic OC), 1200,1020 (glycoside CO)

UV Xmax nm (log s): 257 (3.60), 360 (3.82) UV Xmax nm (log s): 257 (3.60), 360 (3.82)

iH-NMR (600 MHz, DMSO-d6): 7.46 (2H, s, H-2, 6), 6.91 (2H, s,没食子酰基-2,6), 6.48(1H, d, J =1. 8 Hz, H-8), 6.21(1H, d, J =1. 8 Hz, H-6), 5.48(1H, d, J=7. 2Hz,.糖苷-l), 4.49(1H, s,鼠李-l), 3.84 (6H, s, OMe-3,5), 3.70(1H, d,J=10. 2 Hz,糖苷-6), 3.38(1H, d,J=10. 2 Hz,糖苷-6), 1.00 (3H, d,J=5. 0 Hz, 鼠李-6) iH-NMR (600 MHz, DMSO-d6): 7.46 (2H, s, H-2, 6), 6.91 (2H, s, galloyl -2,6), 6.48 (1H, d, J = 1 8. Hz, H-8), 6.21 (1H, d, J = 1. 8 Hz, H-6), 5.48 (1H, d, J = 7. 2Hz ,. glycoside -l), 4.49 (1H, s, murine Li -l), 3.84 (6H, s, OMe-3,5), 3.70 (1H, d, J = 10. 2 Hz, glycoside -6), 3.38 (1H, d, J = 10. 2 Hz, glycoside -6), 1.00 (3H, d, J = 5. 0 Hz, rhamnose -6)

24"C-NMR (150 MHz, DMSO-d6): 156.3 (C-2), 133.1 (C-3), 177.3 (C-4), 161.1 (C-5), 98.6(C-6), 164.0(C-7), 93.9(C-8), 156.4(C-9), 104.0(C-10), 119.7(C-1), 106.9(C-2, 6), 147.4 (C-3, 5), 138.6(C-4), 100,8(糖苷-1), 76.4(糖苷-2), 74.9(糖苷-3), 70.1(糖苷-4), 74.3(糖苷-5), 66.7(糖苷-6), lOl.O(鼠李-l), 70.2(鼠李-2), 70.3(鼠李-3), 71.7(鼠李-4), 68.3(鼠李-5), 17.6(鼠李-6), 165.3(C=0), 120.4 (没食子酰基-l), 108.7 (没食子酰基-2,6), 145.3 (没食子酰基-3,5), 137.9 (没食子酰基-4)。 24 "C-NMR (150 MHz, DMSO-d6): 156.3 (C-2), 133.1 (C-3), 177.3 (C-4), 161.1 (C-5), 98.6 (C-6), 164.0 (C-7), 93.9 (C-8), 156.4 (C-9), 104.0 (C-10), 119.7 (C-1), 106.9 (C-2, 6), 147.4 (C-3, 5 ), 138.6 (C-4), 100,8 (glycoside -1), 76.4 (glycoside -2), 74.9 (glycoside -3), 70.1 (glycoside -4), 74.3 (glycoside -5), 66.7 (glycoside - 6), lOl.O (rhamnose -l), 70.2 (rhamnose -2), 70.3 (rhamnose -3), 71.7 (rhamnose -4), 68.3 (rhamnose -5), 17.6 (rhamnose - 6), 165.3 (C = 0), 120.4 (galloyl -l), 108.7 (galloyl 2,6), 145.3 (galloyl-3,5), 137.9 (galloyl -4).

<表2> <Table 2>

<table>table see original document page 25</column></row> <table><table>table see original document page 26</column></row> <table>实施例2:清除DPPH自由基活性的测试 <Table> table see original document page 25 </ column> </ row> <table> <table> table see original document page 26 </ column> </ row> <table> Example 2: DPPH free radical scavenging activity test

为了研究在上述实施例1分离并含有<化学式1>-<化学式20>表示化合物的提取物的抗氧化活性,通过在上述实施例1中所使用的相同方法测定它们的清除DPPH自由基的活性。 In order to study isolated and contained in the above Example 1 <Chemical Formula 1> - <Chemical Formula 20> represents the antioxidant activity of extract compound, measuring the activity thereof DPPH free radical scavenging by the same method as in the above Example 1 was used . 此时,BHA(叔丁基-4-羟基苯甲醚) At this time, BHA (tert-butyl-4-hydroxyanisole)

和a-生育酚、合成抗氧化剂用作阳性对照。 And a- tocopherol, synthetic antioxidant used as a positive control.

结果,酚酸和黄酮类化合物显示出依赖于剂量的强清除自由基活性。 As a result, phenolic acids and flavonoids showed strong free radical scavenging activity is dependent on dose. 而1,2-二苯乙烯化合物显示出较强的清除自由基活性。 The stilbene compound exhibits strong free radical scavenging activity. 具体地,包括没食子酸的没食子酸酯具有强的活性。 In particular, not including the sub-gallic acid, gallate having strong activity. 例如,由以下表示的化合物的ICso值分别为5.1土0.4、 5.3±0.3、 7.0±1.1、 6.8±0.5、 6.7±0.4和8.6±0.7 g/ml: <化学式6>(没食子酸)、 <化学式7>(没食子酸甲酯)、 <化学式8>(没食子酸乙酯)、 <化学式17〉((-)-表儿茶素(epicatechin)-3-O-没食子酸酯)、 <化学式18^(-)-表倍儿茶素(epigallocatechin)-3-0-没食子酸酯)和< 化学式13 >(杨梅黄酮-3-0-(2-0-没食子酰基)-aL-鼠李吡喃糖苷),这表明在活性上这些化合物之间没有大的差异,并且它们中的所有物质与(x-生育酚(IC50 25.4土0.9g/ml)和BHA(IC5o 15.3士0.6g/ml)相比,显示出显著更强的清除自由基活性,而a-生育酚和BHA两者都用于阳性对照(表3)。 For example, the ICso values ​​of the compounds represented below were 5.1 Soil 0.4, 5.3 ± 0.3, 7.0 ± 1.1, 6.8 ± 0.5, 6.7 ± 0.4 and 8.6 ± 0.7 g / ml: <Formula 6> (gallic acid), <Chemical Formula 7> (methyl gallate), <chemical formula 8> (ethyl gallate), <chemical formula 17> ((-) - epicatechin (epicatechin) -3-O- gallate), <chemical formula 18 ^ (-) - catechin fold table (epigallocatechin) -3-0- gallate) and <chemical formula 13> (myricetin -3-0- (2-0- galloyl) -Al- rhamnose pyranoside ), indicating that the activity was not a large difference between these compounds, and all substance (x- tocopherol (IC50 25.4 soil 0.9g / ml) of them and BHA (IC5o 15.3 Disabled 0.6g / ml) compared to , showed significantly greater radical scavenging activity, while both a- tocopherol and BHA are a positive control (table 3).

如果供电子基团放置在苯环的邻位上,则苯氧自由基变得容易稳定, 这情况导致清除自由基活性的增加(Cuvdier, ME, Richard,H., Berset, C., Biosci. Biotechnol. Biochem. 56: 324-325 (1992); Kikuzaki,H"等,J. Agric. Food Chem. 50: 2161-2168 (2002))。因为没食子酰基放置在邻近3个羟基的位置上,因此该基团显示强清除自由基活性,这情况可有效地使酚氧自由基稳定。如表3所示,除没食子酰基外,由以下表示的黄酮类化合物也证实具有强的清除自由基活性: <化学式16>((+)-儿茶素)、 <化学式9> (杨梅黄酮)、 <化学式10 > (阿福豆碱)、 <化学式11> (栎素)和< 化学式12> (杨梅苷),这情况似乎是因为酚取代基使在黄酮类结构中的酚氧自由 If the electron-donating group placed ortho position of the benzene ring, a phenoxy radical is easily stabilized, which has led to increased free radical scavenging activity (Cuvdier, ME, Richard, H., Berset, C., Biosci. Biotechnol Biochem 56:...... 324-325 (1992); Kikuzaki, H "and the like, J Agric food Chem 50: 2161-2168 (2002)) as gallic acid is placed adjacent the 3 hydroxyl position so this group exhibits strong free radical scavenging activity, such circumstances can effectively stabilize phenolic oxygen radical as shown in table 3, in addition to gallic acid, the following compounds represented by the flavonoids have also confirmed a strong free radical scavenging activity: <chemical formula 16> ((+) - catechin), <chemical formula 9> (myricetin), <formula 10> (Fu bean base), <chemical formula 11> (quercitrin) and <chemical formula 12> (myricitrin) this seems to be because the case that the substituted phenol group flavonoid structure consisting of a phenoxy

26基稳定的缘故。 26 sake stable group. 总之,含有供电子能力大的取代基的化合物可以增加清除 In summary, compounds containing an electron-donating substituent large capacity can be increased clearance

自由基活性,这情况与Pekkarinene等的报道一致(Pekkarinen, SS, et al., J. Agric. Food Chem. 47:3036-3043 (1999))。 Consistent radical activity, which like the case of Pekkarinene reported (Pekkarinen, SS, et al, J. Agric Food Chem 47:... 3036-3043 (1999)).

<表3> <Table 3>

<table>table see original document page 27</column></row> <table>实施例3:脂质过氧化作用的抑制活性的测试 <Table> table see original document page 27 </ column> </ row> <table> Example 3: lipid peroxidation inhibitory activity test

为了测定在上述实施例1分离并含有<化学式1> - <化学式20 >表示化合物的抗氧化活性,研究了这些化合物的脂质过氧化作用的抑制活性。 To determine the separated and contained in the above Example 1 <Chemical Formula 1> - <Chemical Formula 20> represents the antioxidant activity of the compounds was studied inhibitory activity of these compounds is lipid peroxidation.

脂质氧化产生诸如脂质氢过氧化物、共轭脂肪酸(HODEs)、环氧脂肪酸、 丙醛(MDA)和4-羟基壬烯醛(4-HNE)之类的氧化物,这样就导致了对构成 Lipid hydroperoxides such as lipid oxidation, conjugated fatty acids (Hodes), epoxidised fatty acid, malonic aldehyde (MDA) and 4-hydroxy-nonenal (4-HNE) oxide or the like, thus leading to to constitute

细胞的生物膜的直接损伤或与其它细胞组分的间接反应。 Damage directly or indirectly to react with other cellular components of the biofilm cells. 因此,脂质的氧化被认为是变性疾病和衰老的主要原因之一(Esterbauer, H.等,i^^i?"Ac. Sz.o/. 1991,11 : 81-128; Esterbauer, H. , Cheeseman, KH , Therefore, oxidation of lipids is considered to be one of the main degenerative diseases and aging (Esterbauer, H. et, i ^^ i "Ac Sz.o / 1991,11:?.. 81-128; Esterbauer, H. , Cheeseman, KH,

五w2^附o/., 1990,186 : 407-421; Jira, W.,等,Cfe肌尸/z".丄—1996,84 : 165-173; Jira,W.等,所oyc/, 1998, 53 : 1061-1071)。通常,在细胞的电子转递系统如线粒体或微粒体中产生的过氧化物自由基在体内通过酶反应转变成H202,还通过诸如过氧化氢酶或谷胱甘肽过氧化物酶之类的酶转变成无害的水。但是,作为其它原因增加的活性氧,H202通过Fenton反应(Fe" + H202—Fe3+ + HO + HO')或金属-催化的Haber-Weiss反应(02> H202 —02 + HO + HO—)产生的羟自由基(HO),这情况就导致了脂质过氧化物的产生。 Five attachment w2 ^ o /, 1990,186: 407-421; Jira, W., et al., Cfe muscle dead / z "Shang -1996,84:... 165-173; Jira, W, etc., the oyc /, 1998, 53: 1061-1071) typically, the peroxide generated in the electronic transmitting system cells such as mitochondria or microsomes radical is converted in vivo by an enzyme reaction to H202, further, such as by catalase or glutathione GSH-Px enzymes and the like into harmless water, however, other causes increased activity as oxygen, H202 by Fenton reaction (Fe "+ H202-Fe3 + + HO + HO ') or a metal - catalyzed Haber -Weiss reaction (02> H202 -02 + HO + HO-) produced hydroxyl radical (HO), this situation results in the production of lipid peroxides. 该脂质过氧化物再次与金属离子在体内反应,从而生成脂质自由基(L,)或脂质过氧自由基(LOO),这导致了脂质过氧化物的链反应(Halliwell, B. , Gutteridge, JMC,尸7狄ra^Ycafo Z?f0/0^譜J me血Z"e, 3rd Edition, Oxford University Press, Oxford, 1999; Halliwell, B. , Gutteridge, JMC,历0c/7置《/• , 1999, 219 :1-14 ; Harman, D. , Free radical theory of aging. Alan R Liss, New York, 1986,3-49 ; Stadtman, ER , Levine, RL, j朋.A^o^. , 2000, 899: 191-208)。因此,具有供电子能力的化合物被认为是具有脂质过氧化作用抑制的活性。 The lipid peroxides in the body again reacted with the metal ions, thereby producing a lipid radical (L,) or lipid peroxy radicals (LOO), which results in a chain reaction of lipid peroxidation (Halliwell, B ., Gutteridge, JMC, 7 dead Di ra ^ Ycafo Z f0 / 0 ^ J me blood spectrum Z "e, 3rd Edition, Oxford University Press, Oxford, 1999;? Halliwell, B., Gutteridge, JMC, calendar 0c / 7 set "/ •, 1999, 219: 1-14; Harman, D., Free radical theory of aging Alan R Liss, New York, 1986,3-49; Stadtman, ER, Levine, RL, j friends .A ^. o ^, 2000, 899:.. 191-208) Accordingly, a compound having an electron-donating ability is considered to be active in the inhibition of lipid peroxidation.

脂质过氧化作用的抑制活性可以通过光谱法进行量化,在该光谱法中由羟自由基导致的脂质氧化作用产生的丙醛与硫代巴比妥酸(下面称作1BA,)反应,所述羟自由基是Fe^抗坏血酸反应体系的最终产物。 Lipid peroxidation inhibitory activity can be quantified by spectroscopy, propionaldehyde thiobarbituric acid in the oxidation of the spectroscopy caused by hydroxyl radicals generated (hereinafter referred 1BA,) reaction, the hydroxyl radical is the final product of the reaction Fe ^ ascorbate system. 具体地,10 pi由<化学式1 > - <化学式20 >表示的各个化合物与50 具有10mg/ml的蛋白浓度的小鼠脑组织均浆和740)^1的50mM磷酸盐缓冲液(pH 7.4)混合。 Specifically, 10 pi of <Chemical Formula 1> - respective compound 50 in the mouse brain protein concentration with 10mg / ml of the expressed <Chemical Formula 20> homogenate and 740) ^ 50mM phosphate buffer solution 1 (pH 7.4) mixing. 然后,向其中加入Ol mMFeS04. 7H202禾B 1 mM抗坏血酸的混合物(200|Lil),随后在37。 Then, the mixture was Ol mMFeS04 7H202 thereto Wo B 1 mM ascorbic acid was added (200 | Lil), followed by 37. C反应30分钟。 C for 30 minutes. 向该反应溶液中加入250)Li1的20%三氯乙酸(下文称作7CA' )( Sigma)以终止反应。 To the reaction solution was added 250) Li1 20% trichloroacetic acid (hereinafter referred 7CA ') (Sigma) to terminate the reaction. 然后,加入250 ^的1 %TBA(Sigma),该混合物在100。 Then, 250 ^ of 1% TBA (Sigma), and the mixture was at 100. C反应10分钟。 C for 10 minutes. 该反应溶液用10,000 rpm离心10分钟,随后测定OD532。 The reaction solution was centrifugation at 10,000 rpm for 10 minutes and then measuring OD532. 根据下面<数学式2>计算每个样品的脂质过氧化作用的抑制活性,而将足以抑制50%的脂质过氧化的浓度确定为IC,BHA和a-生育酚用于比较组,而没有向对照组中加入样品或溶液。 The following <Equation 2> inhibiting activity was calculated for each sample of lipid peroxidation, and 50% will be sufficient to inhibit lipid peroxide concentration was determined for the comparative group IC, BHA and a- tocopherol, and no sample was added to a solution or a control group. <数学式2> <Equation 2>

脂质过氧化作用的抑制活性(%) =(A对照-A样品)/(A对照-A空白)x 100 Lipid peroxidation inhibitory activity (%) = (A sample -A Control) / (A -A blank control) x 100

对照组的光密度(没有加入样品), A船:实验组的光密度(加入样品), A空白:没有加入样品、TCA和TBA的组的光密度。 Optical density of control (no sample added), A boat: the optical density of the experimental group (added to the sample), A Blank: No optical density of the sample was added, TCA and TBA group.

结果,黄酮类、1,2-二苯乙烯和酚酯化合物表现出强的依赖剂量的脂质过氧化作用抑制活性。 As a result, flavonoids, phenolic esters and stilbene compounds exhibit a strong dose-dependent lipid peroxidation inhibitory activity. 具体地,1,2-二苯乙烯的由<化学式5>(云杉鞣酸醇)表示的化合物被证明具有与BHA(IC5Q: 0.11±0.02 g/ml)—样强的活性(IC5Q: 0.09±0.01g/ml), BHA是已知作为强的脂质过氧化作用抑制剂。 Specifically, a compound represented by <Formula 5> (spruce tannin alcohol) represented by stilbene has proved BHA (IC5Q: 0.11 ± 0.02 g / ml) - strong like activity (IC5Q: 0.09 ± 0.01g / ml), BHA is known as a strong inhibitor of lipid peroxidation. and

多数黄酮类化合物都表示出强的脂质过氧化作用抑制活性。 Most flavonoids have shown strong lipid peroxidation inhibitory activity. 具体地,由以下表示的化合物的IC5o值分别为0.95±0.06、2.9±0.06、 1.0±0.08、 6.21±0.40、 5.27±0.32和4.73±0. 41g/ml: <化学式9> (杨梅黄酮)、 <化学式17> ((-)-表儿茶素-3-0-没食子酸酯)、 <化学式18> ((-)-表倍儿茶素-3-0-没食子酸酯)、 <化学式11>(栎素)、 <化学式12>(杨梅苷)和<化学式13 >(杨梅黄酮一3-0—(2-0-没食子酰基)-aL-鼠李吡喃糖苷),这些化合物比a-生育酚的脂质过氧化作用的抑制活性更好(表4),该oc-生育酚用作阳性对照。 Specifically, the IC5o values ​​for the compounds represented by the following were 0.95 ± 0.06,2.9 ± 0.06, 1.0 ± 0.08, 6.21 ± 0.40, 5.27 ± 0.32 and 4.73 ± 0 41g / ml:. <Chemical Formula 9> (myricetin), <chemical formula 17> ((-) - epicatechin gallate -3-0-), <chemical formula 18> ((-) - catechin -3-0- table times gallate), <chemical formula 11 > (quercitrin), <chemical formula 12> (myricitrin) and <chemical formula 13> (myricetin a 3-0- (2-0- galloyl) -Al- rhamnose pyranoside), these compounds than a- tocopherol lipid peroxidation inhibitory activity is better (table 4), which oc- tocopherol as a positive control. 类似上述实施例2的DPPH清除自由基活性,脂质过氧化作用的抑制活性似乎与酚氧自由基的稳定化相关。 Example 2 is similar to the above-described embodiment DPPH radical scavenging activity, lipid peroxidation inhibitory activity appears to be associated with a stabilized phenolic oxygen radicals. 即,在苯环上放置越多的供电子基团,酚氧自由基越易于变得稳定,这种情况会导致活性的增加。 That is, the benzene ring is placed on the more electron donating groups, phenolic oxygen radicals tend to become more stable, this situation leads to an increase in activity. 尤其地,黄酮类化合物的结构不仅有助于酚氧自由基的稳定,而且也有助于金属离子的螯合,这是因为该化合物具有强的脂质过氧化作用抑制活性的缘故。 In particular, the structure of flavonoids not only help to stabilize the phenolic oxygen radicals, but also helps to chelate metal ions, since this compound has a strong inhibition of lipid peroxidation sake activity. <表4> <Table 4>

<table>table see original document page 30</column></row> <table> <Table> table see original document page 30 </ column> </ row> <table>

实施例4:清除羟自由基活性以及清除一氧化氮活性的测定 Example 4: hydroxyl radical scavenging activity and to clear the measured activity of nitric oxide

羟自由基(HO)通过金属离子或过氧化物自由基的反应产生。 Hydroxyl radical (HO) is generated by reaction of metal ions, or peroxide radicals. 由于强的反应活性,羟自由基在体内会导致对DNA、蛋白质和脂质的氧化性损伤。 Due to strong reactivity, hydroxyl radicals in the body can cause oxidative damage to DNA, proteins and lipids. 而一氧化氮(NO)与过氧化物自由基反应,从而产生过氧亚硝酸根(ONOO-),该过氧亚硝酸根也具有强的反应活性。 And nitric oxide (NO) with a peroxide radical reaction, to produce peroxynitrite (of ONOO-), which also have a strong peroxynitrite reactivity. 因此, 一氧化氮在体内 Thus, nitric oxide in vivo

表示出类似于羟自由基的反应(Yan, LJ , Sohal, RS, B说i?^Z/c.历o/. AfW. , 2000, 29 : 1143-1150)。 The reaction is shown similar to hydroxyl radical (Yan, LJ, Sohal, RS, B said i ^ Z / c calendar o / AfW, 2000, 29:?... 1143-1150). 因此,为了研究在上述实施例1分离并由< 化学式1>至<化学式20>表示化合物的抗氧化活性,本发明人根据Halliwell方法(Halliwell, B.等,Anal Biochem., 1987,165, 215- 219)测定了清除羟自由基活性。 Therefore, in order to study the above-described Example 1 separated by <Chemical Formula 1> to <Chemical Formula 20> represents the antioxidant activity of the compounds, the present invention (Halliwell, B. et, Anal Biochem method according Halliwell., 1987,165, 215 --219) determined to scavenge hydroxyl radicals. 具体±也,每个化合物与10 jal溶解在DMSO的样品、 磷酸盐缓冲液(20 mM, pH 7.4)、5.6mM脱氧核糖、0.1 mM FeCl3、 1 mM H202 和0.1 mM抗坏血酸混合,使总体积为lml,然后该混合物在37°C反应60分钟。 Specifically ± also, each compound was dissolved in 10 jal sample in DMSO, phosphate buffer (20 mM, pH 7.4), 5.6mM deoxyribonucleotide 0.1 mM FeCl3 1 mM mixed,, H202 and 0.1 mM ascorbic acid to a total volume of lml, then the mixture was reacted at 37 ° C 60 min. 向该反应溶液中加入250 pi的20%TCA以及250 的1 %TBA 溶液(溶解在50mM的NaOH中),随后在95°C热反应5分钟。 To the reaction solution was added 250 PI of 20% TCA and 250 of 1% TBA solution (dissolved in 50mM NaOH solution), followed by reaction at 95 ° C heat for 5 minutes. 反应完毕后,测定OD^。 After completion of the reaction, the measured OD ^. 通过下面<数学式3>计算清除羟自由基的活性。 Hydroxyl radical scavenging activity by the following <Equation 3> is calculated. BHA和oc-生育酚用作阳性对照。 Oc- BHA and tocopherol as a positive control. <数学式3> <Equation 3>

清除羟自由基的活性(%) = (A对照-A样品)/(A对照-A空白)x 100 Hydroxyl radical scavenging activity (%) = (A sample -A Control) / (A -A blank control) x 100

A对照:对照组孔的光密度(没有加入样品), Control A: The optical density of a control set of holes (no sample added),

A縣:实验组孔的光密度(加入样品), A County: optical density test set of holes (added to the sample),

A空自:没有加入样品、TCA和TBA的孔的光密度。 Air from the A: no addition of the sample, the optical density of the holes of TCA and TBA.

结果,与对照组相比,黄酮类、1,2-二苯乙烯和酚酯的化合物表示出12.7-54.0%的清除羟自由基活性。 As a result, compared with the control group, a flavonoid, a stilbene compound and phenolic esters shows 12.7-54.0% of the hydroxyl radical scavenging activity. 具体地,由<化学式17>((->表儿茶素-3-0-没食子酸酯)、 <化学式18>((-)-表倍儿茶素-3-0-没食子酸酯)、 <化学式7> (没食子酸甲酯)、 <化学式8> (没食子酸乙酯)和<化学式13>(杨梅黄酮-3-0_(2_0—没食子酰基)-a—L-鼠李吡喃糖苷表示的化合物(其中所有的没食子酰基被取代)表示出显著更高的清除氧自由基活性(表5)。 In particular, the <Chemical Formula 17> ((-> -3-0- epigallocatechin gallate), <Chemical Formula 18> ((-) - catechin -3-0- Table times gallate), <formula 7> (methyl gallate), <chemical formula 8> (ethyl gallate) and <chemical formula 13> (myricetin -3-0_ (2_0- galloyl) -a-L- rhamnose represented pyranoside compound (wherein all galloyl substituted) shows activity of scavenging oxygen free radicals significantly higher (table 5).

另一方面,由<化学式18 >((-)-表倍儿茶素-3-0-没食子酯), < 化学式17 >((-)-表儿茶素-3-0-没食子酯), <化学式16>( (+)-儿茶素)和<化学式9> (杨梅黄酮)表示的化合物表现出较高的清除一氧化氮的活性(表5)。 On the other hand, the <Chemical Formula 18> ((-) - catechin -3-0- Table times gallic esters), <Chemical Formula 17> ((-) - epicatechin gallate esters -3-0-), <chemical formula 16> ((+) - catechin) and <chemical formula 9> (myricetin) represented by the compounds exhibit high activity of scavenging nitric oxide (table 5). <表5> <Table 5>

<table>table see original document page 32</column></row> <table> <Table> table see original document page 32 </ column> </ row> <table>

实施例5:清除过氧自由基活性的测定 Example 5: Determination of peroxy radicals Activity Clear

过氧化物自由基(02—)自身具有少量的反应活性,但是它易于改变成H202,而11202可产生具有强反应活性的羟自由基,或者它与一氧化氮(NO-) 反应以产生也具有强反应活性的过氧亚硝酸根(ONOO-)。 Peroxide radicals (02-) itself has a small amount of reactivity, it is easy to change to H202, and 11202 may be generated hydroxyl radical having strong reactivity, or it with nitric oxide (NO-) also reacted to produce peroxynitrite has a strong reactivity (ONOO-). 因此,过氧化物自由基可能是SH-基氧化、蛋白质酪氨酸的硝化、脂质过氧化作用和DNA Thus, peroxide radicals may be the SH- group oxidation, nitration of tyrosine protein, DNA and lipid peroxidation

损伤的原因。 The reason injury. 为了测定在上述实施例1中制备并由< 化学式1>-<化学式 To determine prepared in the above Example 1 by <Chemical Formula 1> - <Chemical formula

20>表示化合物的抗氧化活性,测定这些化合物的清除过氧化自由基(02—) 的活性,因为过氧化物自由基是许多有害活性氧的前体。 20> represents the antioxidant activity of the compounds, peroxide radical scavenging activity was measured (02-) of these compounds, since peroxide radicals is a precursor to many harmful reactive oxygen species. 主要地,通过研究超氧化物岐化酶(SOD)的活性进行清除超氧化物自由基的测定,超氧化物岐化酶是一种通过过氧化物的歧化作用清除过氧化物的酶。 Primarily, the study by the activity of superoxide dismutase (SOD) was measured clearance of superoxide radicals, superoxide dismutase is an enzyme that destroy peroxide by disproportionation of peroxide. 在本发明中,测定样品抑制由黄嘌呤/黄嘌呤氧化酶的酶反应制备的过氧化物产物的活性以及抑制通过硝基蓝四唑的还原产生甲月替的反应(NBT +202—— NBTH2 + 202)的其它活性。 In the present invention, the inhibitory activity measurement sample prepared by the xanthine / xanthine oxidase enzyme reaction products of the peroxide and the reaction for formazan suppressed by reduction of nitro blue tetrazolium (NBT + 202-- NBTH2 + 202) of the other active. 具体地,96-孔平板的孔内填充有50)^1的4 mM 的黄嘌呤(Sigma)、 50lal的250 mMNBT(Sigma)、 50 的50 mM磷酸盐缓冲液(pH值为7.8, lmMEDTA)以及10)^1各个样品,向该混合物中加入40jil黄嘌呤氧化酶以诱发反应。 Specifically, the hole is filled with a 96-well plate 50) ^ 4 mM 1 xanthine (Sigma), 250 mMNBT (Sigma) 50lal is, 50 mM phosphate buffer (pH value of 50 is 7.8, lmMEDTA) and 10) ^ 1 for each sample, added to the mixture to induce 40jil xanthine oxidase reaction. 反应完成后,按预定收集各个反应溶液, 并用ELISA读数器测定OD55。 After completion of the reaction, each reaction solution was collected in a predetermined and measured with an ELISA reader OD55. . 如下面的<数学式4>所示,通过比较NBT 还原与对照组还原的降低计算各个样品的清除过氧化物自由基的活性,而IC5o确定为能够清除50%过氧化物自由基的样品浓度。 The <Equation 4> shown below, the activity of scavenging peroxide radicals each sample was calculated by comparing the reduction of NBT reducing reduction of the control group, and can be determined as IC5o remove a sample concentration of 50% peroxide radicals . a-生育酚和咖啡酸用于阳性对照以与其它物质比较抗氧化活性。 a- tocopherol and caffeic acid in a positive control for comparison with other substances antioxidant activity.

<数学式4> <Equation 4>

清除过氧化物自由基的活性(%) = (A对照-A样品)/(A对照-A空自)x 100 Clear peroxide radicals activity (%) = (A sample -A Control) / (A -A blank from control) x 100

A,:对照组孔的光密度(没有加入样品), A ,: optical density of a control set of holes (no sample added),

A样。 A sample. . "'实验组孔的光密度(加入样品), 'Optical density' experimental set of apertures (added to the sample),

A空白:没有加入样品、TCA和TBA的孔的光密度。 A Blank: No added optical density of sample well, TCA and the TBA.

结果,黄酮类、1,2-二苯乙烯和酚酯的化合物表现出优异的清除自由基活性,其与剂量有关。 As a result, flavonoids, stilbene compounds and phenolic esters exhibits excellent radical scavenging activity, which is dose-related. 具体地,由下面所表示的黄烷-3-醇化合物的IC50 值分别为11.9±2.1、 24.0土3.5禾卩13.2±2.5 g/ml: <化学式17>((-)-表儿茶素-3-0-没食子酸酯)、 <化学式18>((-)-表倍儿茶酸-3-0-没食子酸酯)和<化学式15>(杨梅黄酮-3-0-(2-0-没食子酰基)-aL-鼠李吡喃糖苷),这反映出类似与咖啡酸的强活性,所述咖啡酸是有效的过氧化物自由基清除剂(IQo 11.0±1.8g/ml)。 Specifically, flavan -3- IC50 value of an alcohol compound represented by the following were 11.9 ± 2.1, 24.0 3.5 Soil Wo Jie 13.2 ± 2.5 g / ml: <Chemical Formula 17> ((-) - epicatechin - 3-0- gallate), <chemical formula 18> ((-) - catechin -3-0- table times gallate) and <chemical formula 15> (myricetin -3-0- (2-0- galloyl) -Al- rhamnose pyranoside), reflecting similar strong activity caffeic acid, caffeic acid is effective in the peroxide radical scavenger (IQo 11.0 ± 1.8g / ml). 而且,黄酮类和酚酯酚酯化合物表现出优异的清除过氧化物自由基的活性。 Moreover, flavonoids and phenolic esters phenolic ester compounds exhibit excellent activity scavenging peroxide radicals. 具体地,由<化学式9>(杨梅黄酮)、< 化学式16 >((+)-儿茶素)、 <化学式7> (没食子酸甲酯)和<化学式8> (没食子酸乙酯)表示的 In particular, the <Chemical Formula 9> (myricetin), <Chemical Formula 16> ((+) - catechin), <Formula 7> (methyl gallate) and represents <Chemical formula 8> (ethyl gallate) of

化合物的IC5o值分别为12.1±1.1、 16.5±2.0、 16.5±1.4和15.肚1.6g/ml。 IC5o values ​​for compounds were 12.1 ± 1.1, 16.5 ± 2.0, 16.5 ± 1.4 and 15. The belly 1.6g / ml. another

一方面,查耳酮化合物具有较弱的清除活性。 In one aspect, chalcone compounds having a weak scavenging activity. 具有使得酚氧自由基稳定的结构的化合物被证实具有优异的清除过氧化物自由基的活性,所述酚氧自由基是在与过氧化物反应的期间产生。 A phenolic compound such that oxygen radicals having a stable structure were confirmed to have excellent activity scavenging peroxide radicals, the phenoxy radical and peroxide is produced during the reaction. 尤其是没食子酰基被取代的化合物显示出强的活性(表6)。 In particular galloyl substituted compounds showed strong activity (Table 6).

<表6> <Table 6>

<table>table see original document page 34</column></row> <table><table>table see original document page 35</column></row> <table>实施例6:保护细胞免受由叔丁基氢过氧化物诱发的氧化性损伤的活 <Table> table see original document page 34 </ column> </ row> <table> <table> table see original document page 35 </ column> </ row> <table> Example 6: Protection of cells from t live-butyl hydroperoxide induced oxidative damage

Sex

当叔丁基氢过氧化物进入细胞时,其新陈代谢成为自由基中间体,因此它引起脂质过氧化物作用,从而导致细胞损伤。 When t-butyl hydroperoxide into the cells, which are metabolized to free radical intermediates, it role of lipid peroxides, resulting in cell damage. 这种现象与由累积在细胞和组织中的氧化应激所引起的现象相似。 This phenomenon is similar to the phenomenon caused by accumulation in cells and tissues caused by oxidative stress. 因此,事实上,该现象对于评 So, in fact, this phenomenon for comment

价抑制氧化应激引起的衰老的作用是非常有效的。 Monovalent inhibiting oxidative stress induced aging is very effective. HEK-N/F是新生儿的一种表皮细胞系,它用1.5mM的叔丁基氢过氧化物处理3小时。 HEK-N / F is a neonatal epidermal cell line, it was treated with tert-butyl hydroperoxide 1.5mM for 3 hours. 结果,细胞的存活率显著降低11.2±1.2%。 As a result, a significant reduction in cell viability was 11.2 ± 1.2%. 但是,当上述分离的化合物在其中另外由50.0g/ml浓度处理,检测到强的细胞保护活性。 However, when the above compound in which the separation process by the addition 50.0g / ml concentration detected strong cytoprotective activity. 尤其是当由< 化学式5 >(云杉鞣酸醇)表示的化合物一起处理时,细胞存活率达到84.7±6.9%,这反映出有非常强的细胞保护活性。 Especially when dealing with a compound represented by <Formula 5> (spruce tannin alcohol), the cell survival rate of 84.7 ± 6.9%, reflecting a very strong cytoprotective activity. 此外,由<化学式9>(杨梅黄酮)和<化学式11> (栎精)表示的黄酮-3-醇化合物表现出61.0±4.5%和48.1±5.7%的细胞存活率。 In addition, the flavone-3-ol compound represented by <Chemical Formula 9> (myricetin) and <Chemical Formula 11> (quercetin) showed 61.0 ± 4.5% and 48.1 ± 5.7% cell viability. 而由<化学式6>(没食子酸)、 <化学式7>(没食子酸甲酯)和<化学式8 > (没食子酸乙酯)所表示化合物的细胞保护活性分别为41.5±3.1%、 43.0 ±5.6%和43.1±4.1%。 And <Formula 6> (gallic acid), <Formula 7> (methyl gallate) a and <Chemical Formula 8> (ethyl gallate) represented cytoprotective activity compound was 41.5 ± 3.1%, 43.0 ± 5.6%, respectively and 43.1 ± 4.1%. 从该结果看,本发明的来自中国紫 From the results, the present invention is derived from China violet

荆的化合物提取物被证实能够通过保护细胞抵抗氧化应激来抑制由氧化应激引起的衰老以及抑制由过氧化作用诱发的细胞死亡。 Jing compounds extract was confirmed due to aging can be suppressed by oxidative stress by protecting cells against oxidative stress and to suppress the peroxidation induced cell death.

35<表7> 35 <Table 7>

<table>table see original document page 36</column></row> <table>实施例7:保护细胞抵抗由UV辐照引发的氧化应激的活性 <Table> table see original document page 36 </ column> </ row> <table> Example 7: Protection of cells against oxidative stress caused by UV irradiation activity

<7-1 >保护细胞抵抗UV辐照的作用(体内) <7-1> protect cells against UV irradiation (in vivo)

UV辐照由于增加了细胞内的活性氧而诱发DNA损伤和DNA蛋白结合。 UV radiation due to the increase of intracellular reactive oxygen species induced DNA damage and DNA binding proteins. 当HEK-N/F细胞被35mJ/cm2的UVB辐照时,细胞核内的DNA链被切割。 When HEK-N / F cells were irradiated UVB 35mJ / cm2 to, DNA strand is cleaved in the nucleus. 断裂链与乙啡啶同型二聚体(Et2) (DNA链插入荧光染料)联合, 测定荧光范围以确定DNA损伤的数量。 Fragmentation chain with ethidium homodimer (Et2) (insert DNA strand fluorescent dye) jointly, fluorescence was measured to determine the range of the amount of DNA damage. 具体地,HEK-N/F细胞在无血清的KGM培养基(Clonetics)中培养,将该细胞接种在24-孔板的孔中,接种量为0.5-4xl0"细胞/2 cm2。然后,该细胞培养18小时,随后样品在其中处理2小时。该细胞用PBS洗涤之后,使用UV反射照射仪(transilluminator) (Spectronics UV反射照射仪EBF-260,最大波长312 nm;半峰强度范围,297-328 nm)对该细胞进行辐照。暴露于UV的细胞再培养l-7小时,然后,加入5M的乙啡啶同型二聚体(Et2, Millipore)。 30分钟之后,用Millipore微型板荧光计Cytofluor 2350(激发:485 nm,发射:645 證)领!定荧光。 Specifically, HEK-N / F cells in serum-free KGM medium (Clonetics) were cultured, the cells are seeded in the wells of 24-well plates in an amount of inoculum 0.5-4xl0 "cells / 2 cm2. Then, the cells were cultured for 18 h before sample processing in which the cells 2 hours after washed with PBS and irradiated using a UV reflecting instrument (transilluminator) (Spectronics UV irradiator reflection EBF-260, the maximum wavelength of 312 nm;. half peak intensity range, 297- 328 nm) irradiation of the cells. the cells were exposed to UV culture l-7 hours, then, after the addition of 5M ethidium homodimer (Et2, Millipore). 30 minutes with Millipore microplate fluorometer Cytofluor 2350 (excitation: 485 nm, emission: 645 certificate) collar set fluorescent!.

结果,如图6a和图6b所示,在用中国紫荆提取物以及由<化学式9> (杨梅黄酮)和<化学式5> (云杉鞣酸醇)表示的化合物处理的组中所看到的荧光各自为54.7°/。 As a result, as shown in FIG. 6a and 6b, in the group treated with the extract of Chinese Bauhinia and a compound represented by <Chemical Formula 9> (myricetin) and <Chemical Formula 5> (spruce tannin alcohol) as seen fluorescence each 54.7 ° /. 和66.7%,该荧光比对照的荧光低。 And 66.7%, lower than the control of the fluorescent phosphor. 因此,中国紫荆提取物和由该提取物分离出的活性成分被证实可通过抑制由UV辐照引发的氧化应激而显著降低DNA损伤。 Thus, China Bauhinia extracts and isolated from the extract the active ingredient can be demonstrated significantly reduced DNA damage by inhibition of oxidative stress caused by UV irradiation.

< 7-2 >保护皮肤组织抵抗UV辐照(体内)的活性 <7-2> active protection against the UV irradiated skin tissue (in vivo)

5-6周龄的雌性无毛小鼠(SKH-hr-1)在24土2。 5-6 week-old female hairless mice (SKH-hr-1) at 24 ± 2. C、 50±10%的相对湿度和12小时的白天/黑夜循环下饲养14天。 C, 50 ± 10% relative humidity and 12 hours day / night cycle fed for 14 days. 在实验之前30-60分钟,在小鼠的5个位置上皮下注射样品,并且用UVB辐照该小鼠背部。 30-60 minutes, the sample injected subcutaneously at 5 positions prior to the experiment on mice, and the mice were irradiated with UVB back. 每组5只小鼠, 将其放进笼子中(20xl5x5 cm),并用UV灯(HP-15M, 280-400 nm,最大波长312 nm; Atto公司,日本)在15cm的距离处以15kJ/m2的量对小鼠进行UVB辐照。 5 mice per group, which was put in a cage (20xl5x5 cm), and with a UV lamp (HP-15M, 280-400 nm, a maximum wavelength of 312 nm; Atto Corporation, Japan) impose 15kJ / m2 at a distance of 15cm mice were UVB irradiated amount. 在UV辐照24小时之后,切下小鼠的背部皮肤,并保存在-70。 24 hours after UV irradiation, excised mouse dorsal skin, and stored at -70. C, 直到测定过氧化作用时为止。 C, until such time as measured peroxidation.

UVB辐照在皮肤组织中导致了严重的脂质过氧化作用,因此,过氧化脂质含量的测定就是皮肤损伤的测定。 UVB radiation causes the skin tissue in a serious lipid peroxidation, therefore, by measuring lipid peroxide content is measured skin damage. 具体而言,UVB辐照在SKH-1无毛小鼠的背部皮肤上。 Specifically, UVB irradiated on back skin of SKH-1 hairless mice. 48小时之后,切下其背部皮肤组织,通过硫代巴比妥酸(TBA)方法测定脂质过氧化作用。 After 48 hours, the dorsal skin was cut assay (TBA) by the thiobarbituric acid method of lipid peroxidation. 为匀浆,将该小鼠的背部皮肤置于10x50 mM KP缓冲溶液中。 Is homogenized, placed in the dorsal skin of mice 10x50 mM KP buffer solution.

同样,对氢过氧化作用进行研究。 Likewise, the role of hydrogen peroxide was studied. 冷冻背部皮肤切片放置在0.1 MTris-HCl缓冲溶液(pH 7.5)中,该缓冲液含有lmg/ml的葡萄糖和lmg/ml 二氨基联苯胺(DAB),该冷冻背部皮肤切片在37。 Frozen sections were placed on the dorsal skin of 0.1 MTris-HCl buffer solution (pH 7.5) in buffer containing lmg / ml glucose and lmg / ml diaminobenzidine (DAB), the dorsal skin sections were frozen at 37. C培养5-6小时。 C culture for 5-6 hours. 皮肤切片用蒸馏水洗涤后,溶液用2%甲基绿染色60分钟。 Skin sections were washed with distilled water, for 60 minutes a solution of 2% methyl green. 细胞核染色成蓝色, 并且在显微镜下观察到了DAB-过氧化物酶(褐色)。 Nuclei were stained blue and observed DAB- peroxidase (brown) under a microscope.

UV辐照在皮肤组织中诱发活性氧种类的产生,从而导致DNA损伤、 蛋白质氧化和脂质过氧化,这些就是诸如炎症、癌症、衰老等之类皮肤损伤的原因。 UV irradiation induced skin tissue in the generated reactive oxygen species, leading to DNA damage, lipid peroxidation and protein oxidation, which is the reason such as inflammation, cancer, aging and other skin damage and the like. 如图7所示,用90mJ/cn^的UVB辐照引发严重的皮肤损伤。 As shown in FIG 7, cause serious skin damage with 90mJ / UVB irradiation of cn ^. 但是,当中国紫荆提取物和由<化学式5> (云杉鞣酸醇)表示的化合物各自以50mg/kg的量进行预处理时,SKH-1无毛小鼠的皮肤损伤可显著减少。 However, when the Chinese Bauhinia extract and the compound represented by <Formula 5> (spruce tannin alcohol) are each in an amount of pretreatment 50mg / kg of skin damage SKH-1 hairless mice can be significantly reduced. 因此,中国紫荆提取物及其活性成分即由<化学式5> (云杉鞣酸醇)表示的化合物必须具有保护皮肤细胞免于氧化应激的活性、抑制脂质过氧化作用的活性以及清除自由基的活性。 Thus, China Bauhinia extract and its active ingredient, i.e. a compound represented by <Formula 5> (spruce tannin alcohol) must protect the skin cells from oxidative stress activity, inhibitory activity of lipid peroxidation and free Clear active group. 而且,保护皮肤免于UVB辐照的活性似乎是由该抗氧化活性产生的。 Also, protect the skin from UVB radiation activity appears to be produced by the anti-oxidation activity. 二氨基联苯胺(DAB)通过在组织中与过氧化物酶反应产生茶褐色的DAB-过氧化物酶。 Diaminobenzidine (DAB) peroxidase to produce a dark brown DAB- by reaction with a peroxidase in the tissue. 因此,可观察到由UVB辐照产生的H202。 Thus, the observed H202 produced by UVB irradiation. 如图7所示,在90mJ/cm2的UVB辐照下,可观察到更多的茶褐色DAB-过氧化物酶,但是当SKH-1无毛小鼠的皮肤组织用中国紫荆的提取物以及由<化学式5> (云杉鞣酸醇)表示的化合物以各自50 mg/kg的量进行预处理,则在H202产生在SKH-1无毛小鼠的皮肤组织中得到显著抑制。 7, UVB irradiation at 90mJ / cm2, the brownish-red can be observed more DAB- peroxidase, but when the SKH-1 hairless mouse skin using extract of Chinese and the Bauhinia <chemical formula 5> compound (spruce tannin alcohol) represented by the amount of each 50 mg / kg was pretreated, H202 generated at the skin tissue SKH-1 hairless mice significantly suppressed.

从脂质过氧化作用的研究可证实,在由UVB辐照的组中平均TBARS(硫代巴比妥酸反应性物质)含量约为0.68nmol/mg蛋白质,该含量是没有被UVB辐照的对照组过氧化作用(0.32土0.05 nmol/mg蛋白质)的2 倍(表8)。 From lipid peroxidation studies confirmed, by UVB irradiation from the group average of TBARS (thiobarbituric acid reactive substances) content of approximately 0.68nmol / mg protein, which is not the content of UVB irradiation peroxidation control group (0.32 soil 0.05 nmol / mg protein) of 2-fold (table 8). 在用中国紫荆提取物以及由<化学式5>(云杉鞣酸醇)表示的化合物处理的组中,在皮肤中的脂质过氧化作用依赖处理剂量减小。 In the group treated with the extract of Chinese Bauhinia and a compound represented by <Formula 5> (spruce tannin alcohol), the skin lipid peroxidation in a dose-dependent decrease process. 具体而言,在相同浓度下,由<化学式5>表示的化合物比用于阳性对照的MAP (镁-L-抗坏血酸基-2-磷酸盐)表现出更高的活性。 Specifically, at the same concentration, compound of <Formula 5> represents a ratio MAP (magnesium ascorbyl-2-phosphate -L-) for positive controls exhibit higher activity. <表8> <Table 8>

<table>table see original document page 39</column></row> <table> <Table> table see original document page 39 </ column> </ row> <table>

实施例8^细胞寿命的延长 ^ Prolong cell life Example 8

在用溴化尿苷处理其成纤维细胞(皮肤的另一种主要成分)以后,从新生婴儿包皮中获得HEK-N/F细胞。 After the treatment with uridine bromide which fibroblasts (the other major component of the skin) to obtain HEK-N / F cells from the newborn baby foreskin. 然后,该细胞在提供有10%FBS的DMEM培养基中培养。 Then, the cell is provided with a DMEM medium with 10% FBS in culture. 而HEK-N/F细胞在培养过程中以1:4的比例连续稀释。 And HEK-N / F cells during culture 1: 4 dilution ratio continuously. 在细胞用样品处理之前,该细胞生长达到3倍PDL(群体倍增水平)。 Before cells were treated with sample, the cells were grown up to 3 times the PDL (population doubling level). 在培养过程中,每个样品施用3g/ml。 During the culture, the administration of each sample 3g / ml. 培养基每隔三天用新鲜培养基替换, 该新鲜培养基补充有包含等量浓度样品的培养液。 Medium was replaced with fresh medium every three days, fresh medium supplemented with the culture liquid containing an equal amount of sample concentration. 培养之后,本发明的中国紫荆提取物、<化学式5>(云杉鞣酸醇)表示的化合物以及<化学式9> (杨梅黄酮)表示的化合物进行处理,随后进行细胞分裂研究。 After compound incubation, China Bauhinia extract of the present invention, the compound (spruce tannin alcohol) represented by <Formula 5> and <Chemical Formula 9> (myricetin) is processed, followed by the study of cell division.

结果,证实了所有的中国紫荆提取物以及它的活性成分都具有延长细胞寿命的效果。 As a result, it confirmed that all the Chinese Bauhinia extract and its active ingredient has the effect of prolonged cell life. 没有处理对照的细胞平均寿命为约35天,而用3 pg/ml 的中国紫荆提取物处理组的平均寿命为约42天,后者寿命约为前者的1.2 倍。 Cells not treated control the average life of about 35 days, but with 3 pg / average life expectancy of China Bauhinia ml of the extract-treated group was about 42 days, the latter life of about 1.2 times the former. 而且,用其它活性成分、 <化学式9>(杨梅黄酮)表示的化合物以及由<化学式5> (云杉鞣酸醇)表示的化合物处理的组用于培养的平均寿命分别为56天和76天,这两者寿命分别是对照组寿命的1.6倍和2.1倍(图8)。 Moreover, the average life expectancy compound with other active ingredients, <Chemical Formula 9> (myricetin) and a group of compounds represented by the process represented by <Formula 5> (spruce tannin alcohol) were cultured for 56 days and 76 days , both of life were 1.6 and 2.1 times the life of the control group (FIG. 8). 因此,中国紫荆的提取物以及从该提取物中分离的活性成分都被证实具有延长细胞寿命的作用。 Thus, China and the Bauhinia extract isolated from the extract the active ingredients have been shown to have the effect of prolonged cell life. '实施例9:细胞寿命的延长以及端粒的延长 'Example 9: prolong cell life and extend telomeres

在上述实施例8中,中国紫荆的提取物以及从该提取物中分离的活性成分都被证实具有延长细胞寿命的作用。 Example 8 In the above embodiments, and Bauhinia Chinese extract isolated from the extract the active ingredients have been shown to have the effect of prolonged cell life. 因此,在本实施例中,研究寿命的延长作用与端粒长度之间的关系。 Accordingly, in the present embodiment, the study of the relationship between the lifetime and prolong the effect of telomere length. 具体而言,使用核酸提取试剂盒 Specifically, nucleic acid extraction kit

(IsoQuick核酸提取试剂盒,ORCA研究公司),从不同年龄的各个细胞中提取基因组DNA,所提取DNA溶解在Tris-EDTA溶液(lO mM Tris-HCl, lmMEDTA, pH 8.0)之后,在4°C储存。 (IsoQuick nucleic acid extraction kit, ORCA Research Inc.), genomic DNA was extracted from individual cells of different ages, after which DNA was dissolved in Tris-EDTA solution (lO mM Tris-HCl, lmMEDTA, pH 8.0) extracted at 4 ° C store. 将2jal的10xH缓冲溶液(TaKaRa)、 2pl所提取的染色体组DNA溶液和蒸馏水在1.5 ml的试管中混合,并使最终体积为19^1。 The buffer solution 2jal of 10xH (TaKaRa), 2pl extracted genomic DNA solution and distilled water were mixed in a 1.5 ml test tube, to a final volume of 19 ^ 1. 然后,向其中加入1 )nl的限制酶i^/I(6U/1, TaKaRa)。 Then, thereto was added 1) nl restriction enzyme i ^ / I (6U / 1, TaKaRa). 该混合物在37°C反应3-4小时,随后进行电泳。 The mixture was reacted for 3-4 hours at 37 ° C, followed by electrophoresis. 对于电泳,电桥区域的琼脂糖(type I, Sigma)凝胶浓度被调节为1 % ,而底座区域(bed region)的琼脂糖凝胶浓度被调节为0.8%以制备凝胶板(Marisol KS-8405, 20X14 cm), 并且使用lx Boyer's缓冲溶液(50 mM Tris-HCl, 20 mM乙酸钠,2 mM EDTA, 18 mM NaCl, pH 8.0)。 For agarose (type I, Sigma) electrophoresis, gel bridge region is adjusted to a concentration of 1%, while the base region (bed region) agarose gel concentration is adjusted to 0.8% to prepare a gel plate (Marisol KS -8405, 20X14 cm), and using lx Boyer's buffer solution (50 mM Tris-HCl, 20 mM sodium acetate, 2 mM EDTA, 18 mM NaCl, pH 8.0). 装入0.5昭的1 Kb DNA梯(ladder)作为标记物。 Akira charged with 0.5 1 Kb DNA ladder (Relay Ladder Logic) as a marker. 向所有的样品中加入3fil的上样缓冲液,随后用35V/cm进行电泳20小时。 3fil added the loading buffer to all samples, followed by 35V / cm electrophoresis for 20 hours. 在电泳完成之后,该凝胶用溴化乙锭(2 )Lig/ml)染色15分钟,染色可以使用UV确定。 After completion of the electrophoresis, the gel was stained with ethidium bromide (2) Lig / ml) for 15 min, staining can be determined using UV. 然后,该凝胶浸泡在0.25NHC1中,再振荡15分钟。 Then, the gel was soaked in 0.25NHC1, and then shaken for 15 minutes. 该凝胶用蒸馏水洗涤两次。 The gel was washed twice with distilled water. 凝胶进入变性溶液中(0.2 N NaOH, 0.6 M NaCl),随后在室温摇动25分钟。 Into the gel in denaturing solution (0.2 N NaOH, 0.6 M NaCl), followed by shaking at room temperature for 25 minutes. 然后,该凝胶用蒸馏水洗涤三次。 Then, the gel was washed with distilled water three times. 在填充有6xSSC的印迹装置中,按顺序装填硝化纤维素膜(Optitran BA-S 85, Schleicher&Schuel)、 3mm滤纸、纸巾、玻璃板和重物(2kg),然后印迹过夜。 6xSSC filled with imprinting means, the nitrocellulose membrane sequentially loaded (Optitran BA-S 85, Schleicher & Schuel), 3mm filter paper, paper towels, a glass plate, and the weight (2kg), and blotted overnight. 印迹完成之后,膜滤纸吸收3xSSC,然后将水均匀排去。 After completion of blotting, membrane filter absorption 3xSSC, water is drained and then uniformly. 标记膜上的孔位置。 Mark hole locations membrane. 将该膜放置在滤纸之间,随后在80。 The film was placed between filter paper, followed by 80. C烘焙过夜。 Baking C overnight. 然后,在65。 Then, at 65. C 与变性鲑精DNA (Wako)进行预混合,之后在50°C通过将杂交缓冲液(lxDenhan溶液,1 M NaCl、 50 mM Tris-HCl, 10 mM EDTA, 0.1 。/。 SDS, 50 g/ml变性鲑精DNA)与5-末端,P]-标记的(TTAGGG)4进行杂交。 C and denatured salmon sperm DNA (Wako) were premixed, then by 50 ° C and hybridization buffer (lxDenhan solution, 1 M NaCl, 50 mM Tris-HCl, 10 mM EDTA, 0.1 ./. SDS, 50 g / ml denatured salmon sperm DNA) with 5-end, P] - labeled (TTAGGG) 4 hybridization. 该杂交膜浸泡在洗涤溶液(4xSSC/0. 1% SDS)中,然后在55°C振荡15分钟。 The membrane was immersed in a hybridization wash solution (4xSSC / 0. 1% SDS), followed by shaking at 55 ° C 15 min. 干燥之后,该膜覆盖上覆盖物,并放置在附装有X-射线胶片(Scientific Imaging Film,Kodak)和光增强屏的盒子中。 After drying, the cover film covering and placed in X- ray film with attachment (Scientific Imaging Film, Kodak) with an intensifying screen and a light box. 放射自显影在-80。 Autoradiography at -80. C进行过夜。 C overnight. 基于显影胶片和膜的位置,孔位置用魔术笔进行标记。 Hole position is labeled with a magic pen based on the position of the developed film and the film. TRFs的密度峰用激光密度计(UltroScan XL, Pharmacia)检测,并计算其迁移率。 TRFs peak density with a laser densitometer (UltroScan XL, Pharmacia) is detected, and calculates its mobility.

结果,端粒随细胞分裂的进行而逐渐变得更短。 As a result, telomeres with cell division progresses gradually become shorter. 如图9所示,对照组中的端粒在第14.8次分裂之后縮短了8.0 kbp,此后没有更多分裂。 9, the control group after the first telomere shortening division 14.8 8.0 kbp, after which no more divisions. 相反, 在使用中国紫荆的提取物或从该提取物中分离的活性成分处理的组中,端粒长度在分裂进行比对照组中更多次之后达到临界点(在本发明中约为8.0 kbp)。 In contrast, in the group using the Chinese Bauhinia extracts or isolated from the extract the active ingredient in the process, telomere length more times than the control group after reaching a critical point in the division (about 8.0 kbp in the present invention, ). 只要端粒DNA的长度小于临界点(对于人类皮肤角化细胞,其临界点推测为7.9-8.4 kb),细胞分裂就将继续进行。 As long as the length of telomeric DNA is less than a critical point (for human skin keratinocytes, its critical point estimated to be 7.9-8.4 kb), cell division will continue. 因此,已证实通过延缓端粒縮短速度(意味着延迟到达临界点)就可以延长细胞循环。 Therefore, it has been confirmed by slowing telomere shortening rate (mean delay to reach the critical point) can prolong cell cycle. 对照组的最大PDL为14.1,而使用中国紫荆提取物、 <化学式9> (杨梅黄酮)表示的化合物以及<化学式5> (云杉鞣酸醇)表示的化合物处理的组的PDL分别为17.2、 23.1和29.9,这说明最大PDL被显著延长。 The maximum PDL in the control group is 14.1, and the use of Chinese Bauhinia extract, a compound represented by <Chemical Formula 9> (myricetin) and PDL group treated with the compound <Formula 5> (spruce tannin alcohol) represented respectively 17.2, 23.1 and 29.9, indicating that the maximum PDL is significantly extended.

基于细胞分裂频率和端粒长度之间的关系,可计算端粒縮短速度。 The relationship between cell division and frequency based on telomere length, telomere shortening rate may be calculated. 结果,与对照组相比,使用中国紫荆提取物、 <化学式9> (杨梅黄酮)所表示化合物以及<化学式5> (云杉鞣酸醇)所表示化合物处理的组的速度各自延迟1.2倍、1.6倍和2.1倍(图10)。 As a result, compared with the control group, China Bauhinia extract, <Chemical Formula 9> (myricetin) and the compound of <Formula 5> (spruce tannin alcohol) represents a compound represented by the processing speed of each group of delay times 1.2, 1.6-fold and 2.1-fold (FIG. 10). 因此,中国紫荆提取物及其活性成分对于皮肤细胞寿命延长的作用被证实是由延迟端粒縮短速度而产生的。 Thus, China Bauhinia extract and its active ingredients to the skin effect of prolonged cell life proved to be a delay telomere shortening velocity generated.

制备实施例1:含有中国紫荆提取物的化妆品组合物的制备本发明人制备了含有中国紫荆提取物作为有效成分的化妆品组合物(制备1〜制备6),该化妆品组合物如下面的表9和表11所示。 Preparation Example 1: Chinese Bauhinia cosmetic composition comprising an extract as an active ingredient of the present invention comprises the preparation of Chinese people Bauhinia extract cosmetic compositions were prepared (Preparation 6 Preparation of 1 ~), the cosmetic composition is as following Table 9 and shown in table 11. 除中国紫荆提取物之外,可以另外包括通常用于制备化妆品的其它提取物。 In addition to China Bauhinia extract, the extract may additionally comprise other commonly used for preparing cosmetics. 其它提取物的实例有桑树皮、褐藻类、独活、薏苡属、牡丹、马齿苋(Portulaca oleraceaLinne)、柿子叶、榛子提取物和积雪草提取物。 Examples of other extracts have mulberry bark, brown algae, independent living, Coix, peony, purslane (Portulaca oleraceaLinne), persimmon leaves, hazelnut extract and Centella asiatica extract.

首先,本发明人通过下列常用方法制备了含有本发明的中国紫荆提取物的软性洗剂以及粘性溶液,该软性洗剂以及粘性溶液以表9中所例举的下列成分为基础。 First, the present invention is by following the conventional method and lotions soft viscous solution containing the extract of Chinese Bauhinia present invention, the preparation of the flexible lotions and viscous solution the following ingredients in Table 9 based exemplified. <表9> <Table 9>

软性洗剂以及粘性溶液的制备<table>table see original document page 42</column></row> <table> Preparation of soft and lotion viscosity of the solution <table> table see original document page 42 </ column> </ row> <table>

其次,本发明人通过下面的常用方法制备了含有中国紫荆提取物的乳 Secondly, the present invention is the conventional method by the following Chinese milk containing extract prepared Bauhinia

状洗剂和其它洗剂,该洗剂的构成由下表io所列出。 Like lotions and other lotions, lotion constituted of the listed in the following table io. <table>table see original document page 43</column></row> <table><table>table see original document page 44</column></row> <table> <Table> table see original document page 43 </ column> </ row> <table> <table> table see original document page 44 </ column> </ row> <table>

接着,本发明人通过下面的常用方法制备了含有中国紫荆提取物的乳 Next, the present invention is the conventional method by the following Chinese milk containing extract prepared Bauhinia

膏剂,该乳膏剂的构成由下表ll所列出。 Ointments, creams constituting the listed in the following table ll. <表11> <Table 11>

乳膏剂的制备 Preparation of creams

<table>table see original document page 44</column></row> <table><table>table see original document page 45</column></row> <table> <Table> table see original document page 44 </ column> </ row> <table> <table> table see original document page 45 </ column> </ row> <table>

制备实施例2:制备具有抗氧化活性和抗衰老活性的药物组合物本发明人制备了一种具有抗氧化和抗衰老活性的药物组合物,它含有作为有效成分的中国紫荆提取物。 Preparation Example 2: Preparation of having antioxidant activity and anti-aging activity of the pharmaceutical compositions of the present invention was prepared having an antioxidant and anti-aging activity of the pharmaceutical composition, which comprises Bauhinia Chinese extract as an active ingredient. <2-1>糖浆剂的制备 Preparation <2-1> Syrup

按如下制备含有2%(重量/体积)的本发明中国紫荆提取物作为有效成分的糖浆剂。 This contains 2% (w / v) of the present invention is prepared as follows Chinese Bauhinia syrup extract as an active ingredient.

中国紫荆提取物、糖精和糖溶解在80 g的温水中。 China Bauhinia extract, saccharin and sugar were dissolved in 80 g of warm water. 然后,该溶液冷却。 Then, the solution was cooled. 甘油、糖精、调味剂、乙醇、山梨酸和蒸馏水一起混合制备溶液,然后向其中加入上述冷却的溶液。 Mixing glycerin, saccharin, flavor, ethanol, sorbic acid solution prepared with distilled water, and then the cooling was added thereto. 向混合物中加入水使其总体积为IOOiliK表12)。 To the mixture was added water so that the total volume of IOOiliK Table 12).

<表12> <Table 12>

<table>table see original document page 45</column></row> <table><2-2>片剂的制备 Preparation <table> table see original document page 45 </ column> </ row> <table> <2-2> tablet

按如下制备每个含有15mg的提取物作为有效成分的片剂。 Each extract was prepared as follows as a tablet containing 15mg of the active ingredient. 250g的中国紫荆提取物与175.9g乳糖、180g马铃薯淀粉以及32g胶体状硅酸混合。 250g of China Bauhinia extract with 175.9g of lactose, 180g of potato starch, and 32g of colloidal silicic acid mixture. 向该混合物中加入10%明胶溶液,然后充分粉碎以通过14目的筛子。 To this mixture was added a 10% gelatin solution, and then sufficiently pulverized to pass 14 mesh sieve. 将粉碎混合物干燥,再向其中加入160g的马铃薯淀粉、50g 滑石粉和5g硬脂酸镁以制备片剂(表13)。 The milled mixture was dried, and thereto was added 160g of potato starch, talc and 50g 5g of magnesium stearate to prepare tablets (Table 13).

<表13> <Table 13>

<table>table see original document page 46</column></row> <table> <Table> table see original document page 46 </ column> </ row> <table>

如上所述,由于本发明中国紫荆提取物的强抗氧化活性,因此含有该提取物作为有效成分的药物组合物可以用于预防和治疗与过氧化作用相关的疾病。 The pharmaceutical composition described above, due to the strong antioxidant activity Bauhinia Chinese extract of the present invention, thus containing the extract as an active ingredient may be associated with for preventing and treating diseases peroxidation.

下面,解释能够应用含本发明中国紫荆提取物的药物组合物的具体疾病。 Next, an explanation can be applied to the particular disease present invention is a pharmaceutical composition containing an extract of Chinese Bauhinia.

然而,要意识到本领域普通技术人员在考虑本公开内容之上是可以在本发明的精神和范围之内作出修改和改进的。 However, it should be appreciated by those of ordinary skill in the art modifications may be made within the spirit and scope of the present invention on consideration of this disclosure and improved.

可应用实施例l:癌症 Example l may be applied: Cancer

癌症是由很多原因导致的,但其最主要原因被认为是活性氧。 Cancer is caused by a number of reasons, but the main reason is considered to be the most active oxygen. 即,活性氧破坏细胞并且使被破坏的细胞不能修复,由此导致细胞机能失常,因 That is, active oxygen disrupting the cell and the cells are destroyed not be repaired, thereby leading to cell malfunction, because

而产生癌症(Ames, BN , Science, 1983, 221,1256-1264)。 Produce cancer (Ames, BN, Science, 1983, 221,1256-1264). 顺便提及,包含在水果中的苯酚酸对于肝癌是有益的(Sun, J等,J Agric Food Chem, 2002,4 ; 50 (25), 7449-7454),包含在西红柿制备中的番茄素对于乳癌是有益的(Hadley CW等,Exp Biol Med, 2002,227 (10), 869-80), 而isoverbascoside具有对胃癌有益的作用(Chen RC等,Acta Pharmacol Sin, 2002,23 (11), 997-1001),同样该抗氧化剂也被认为是对其它癌症有效的。 Incidentally, the phenol contained in the fruit of the acid is beneficial for liver cancer (Sun, J, etc., J Agric Food Chem, 2002,4; 50 (25), 7449-7454), contained in the preparation for lycopene in tomatoes breast cancer is beneficial (Hadley CW like, Exp Biol Med, 2002,227 (10), 869-80), while isoverbascoside having a beneficial effect on gastric cancer (Chen RC, etc., Acta Pharmacol Sin, 2002,23 (11), 997 -1001), the same antioxidants also considered to be effective against other cancers. 因此,该抗氧化剂可以有效用于各种癌症的防止和治疗,具有优异抗氧化活性的本发明药物组合物对于癌症的防止和治疗也是有效的。 Thus, the antioxidant is effective for preventing and treating various cancers, having excellent antioxidant activity of the pharmaceutical compositions of the invention for the prevention and treatment of cancer, is also effective.

可应用实施例2:衰老 Example 2 may be applied: Aging

在通常新陈代谢过程中产生的活性氧随机并不可逆地破坏了诸如脂质、蛋白质和糖或DNA之类的细胞成分,因而细胞受到氧化性损伤。 Generated in the normal metabolic processes of active oxygen does not irreversibly destroy random such as lipids, proteins and sugars or cellular components like DNA, cells are thus oxidative damage. 该损伤的长期累积就导致衰老,甚至导致死亡(Harm叫D, Free radical theory of aging, 1986,3-49)。 The long-term cumulative damage leads to aging and even death (Harm called D, Free radical theory of aging, 1986,3-49). 相反,通过不同条件的各种方法,例如限制饮食、 限制运动等来研究减少氧消耗而延长寿命,即意味着减少基础新陈代谢率(Medvedev, ZA, Biol Rev., 1990,65, 375-398; Loe, J.等,J. Biol. Chem., 1971,32, 103-121; Sohal, RS, Aging, 1982,5,21-24)。 Instead, different conditions by various methods, such as restricted diet, exercise and other restrictions to reduce the oxygen consumption study prolong life, which means reduced basal metabolic rate (Medvedev, ZA, Biol Rev., 1990,65, 375-398; .. Loe, J. like, J Biol Chem, 1971,32, 103-121;. Sohal, RS, Aging, 1982,5,21-24). 也就是说,清除活性氧是延迟衰老的一种途径,因此迄今为止一直在发展消除活性氧的抗氧化剂。 In other words, oxygen scavenging activity is a way to delay aging, so far has been the elimination of anti-oxidants in the development of reactive oxygen species. 因此具有优异抗氧化活性的本发明药物组合物可用于延迟衰老。 Thus the pharmaceutical compositions of the invention have excellent antioxidant activity may be used to delay senescence.

可应用实施例3:冠心病、高胆甾醇血、动脉硬化当胆固醇合酶抑制剂用于治疗有冠心病和有高胆甾醇血的病人时,在低密度脂质中胆固醇含量降低,但是因为泛醌Q10的合成被抑制,因而低密度脂蛋白仍然被保护,这表明该试剂对于防止活性氧的过氧化作用并不是非常有效,而且对于治疗上述疾病也不是非常有效。 Example 3 is applicable: coronary heart disease, hypercholesterolemia, arteriosclerosis when the cholesterol synthase inhibitors for the treatment of patients with coronary heart disease and high blood cholesterol, reduce cholesterol levels in the low density lipid, but because synthetic ubiquinone Q10 is suppressed, and thus is still protected LDL, indicating that the agent is not very effective in preventing peroxidation active oxygen, and is not very effective for treatment of these diseases. 相反,当抗氧化剂如西立伐他汀或普罗布考施用于那些病人时,病人的低密度脂蛋白迅速降低(Lankin VZ等,Bull Exp Biol Med, 2002, 134(1), 39-42)。 On the contrary, when the anti-oxidants such as West cerivastatin or probucol administered to those patients, the patient rapidly reduce low-density lipoprotein (Lankin VZ, etc., Bull Exp Biol Med, 2002, 134 (1), 39-42). 在另外的临床试 In another clinical trial

验中,将去氢吡啶钙拮抗剂拉西地平(也是一种抗氧化剂)施用于具有动脉粥样硬化的患者。 Test, the calcium antagonist lacidipine pyridine dehydroepiandrosterone (also an antioxidant) administered to a patient having atherosclerosis a. 结果,血压降低,在血管壁中的胆固醇减少,动脉粥样硬化的损伤尺寸减小(HallerH等,Drugs RD, 2002,3 (5),311-23)。 As a result, lower blood pressure, reduce cholesterol in the vessel wall, atherosclerotic lesion size reduction (HallerH the like, Drugs RD, 2002,3 ​​(5), 311-23). 因此,抗 Therefore, the anti

氧化活性被证明对于预防和治疗与胆固醇相关的血管疾病如冠心病,高胆甾醇血和动脉硬化是非常有效的。 Oxidation activity was demonstrated for the prevention and treatment of cholesterol-related vascular diseases such as coronary heart disease, high blood cholesterol and atherosclerosis is very effective. 因此,具有优异抗氧化活性的本发明药物组合物对于预防和治疗与胆固醇相关的血管疾病如冠心病,高胆甾醇血和动脉硬化是非常有效的。 Accordingly, the pharmaceutical compositions of the invention have excellent antioxidant activity of prevention and treatment of cholesterol-related vascular diseases such as coronary heart disease, high blood cholesterol and atherosclerosis is a very effective.

可应用实施例4:多发性硬化和自身免疫性脑脊髓炎ALA(a硫辛酸)是一种抗氧化剂,它施用于具有多发性硬化和自身免疫性脑脊髓炎的模型小鼠,其中施药后,这两种病状减轻。 Example 4 may be applied: multiple sclerosis and autoimmune encephalomyelitis ALA (a lipoic acid) is an antioxidant, it is administered to the model mice with autoimmune encephalomyelitis and multiple sclerosis, wherein the administration after the two relieve symptoms. 表明氧化应激是多发性硬化和自身免疫性脑脊髓炎的主要原因,因此抗氧化剂能够有效用于治疗所述与神经过分紧张有关的疾病(Marracci GH等,J Neuroimmunol, 2002,131 (1-2),104-14)。 Indicates that oxidative stress is the main cause autoimmune encephalomyelitis and multiple sclerosis, and thus can be an effective antioxidant for treating the nerve disorders excessive stress related (Marracci GH like, J Neuroimmunol, 2002,131 (1- 2), 104-14). 因此,具有优异抗氧化活性的本发明药物组合物对于预防和治疗与神经有关的疾病如多发性硬化和自身免疫性脑脊髓炎是非常有用的。 Accordingly, the pharmaceutical compositions of the invention have excellent antioxidant activity for the prevention and treatment of diseases related to such neurological autoimmune encephalomyelitis and multiple sclerosis is useful.

可应用实施例5:脑溢血和早老性痴呆症 Stroke and Alzheimer's disease: 5 embodiments may be applied

由活性氧引起的氧化应激会氧化细胞成分,由此导致那些细胞机能失常。 Active oxygen caused by oxidative stress oxide cell components, thereby causing malfunction of those cells. 这些不正常的功能会引起神经细胞的功能性紊乱、伴随中风、创伤等。 These abnormal function causes of functional disorders of nerve cells, along with stroke, trauma and so on. 如果该氧化应激长时间累积而没有被合适处理时,就会发展成为严重的脑疾病如脑溢血、早老性痴呆症等。 If the accumulation of oxidative stress for a long time without being properly prepared, it will develop into a serious brain diseases such as stroke, Alzheimer's disease psychosis. 通过使用能够消除活性氧的抗氧化试剂可以有效治疗诸如脑溢血和早老性痴呆症之类的脑疾病(Perry G et al., Comp BiochemPhysiol C ToxicolPhaarmacol, 2002, 133 (4),507-13 ; Cecchi C 等,Free Radic Biol Med,2002, 15: 33(10), 1372-9; Smith MA等,Free Radic BiolMed,2002,1 : 33 (9),1194-9)。 Can be effectively treated by eliminating active oxygen capable of oxidizing agents such as anti-stroke and Alzheimer's disease brain disease such (Perry G et al, Comp BiochemPhysiol C ToxicolPhaarmacol, 2002, 133 (4), 507-13;. Cecchi C etc., Free Radic Biol Med, 2002, 15: 33 (10), 1372-9; Smith MA et, Free Radic BiolMed, 2002,1: 33 (9), 1194-9). 因此,具有优异抗氧化活性的本发明药物组合物对于预防和治疗诸如脑溢血和早老性痴呆症等之类的脑疾病是非常有用的。 Accordingly, the pharmaceutical compositions of the invention have excellent antioxidant activity of brain diseases such as stroke and Alzheimer's dementia and the like are useful for the prevention and treatment.

可应用实施例6:肠炎 Example 6 may be applied: enteritis

过量的过氧化副产物会累积在肠炎病人的白细胞中。 Excess amount of oxidation byproducts accumulate leukocytes enteritis patient. 由肠炎病人中累积的过氧化副产物导致的细胞损伤作为肠感染的首要和次要的病理机理。 Primary and secondary pathological mechanisms accumulated by patients had colitis oxidation byproducts resulting from cell damage as intestinal infections. 即,氧化应激导致肠炎症,发展成为发炎性肠炎(KmidenierL等,Aliment Pharmacol Ther, 2002, 16 (12), 1997-2015)。 I.e., intestinal inflammation oxidative stress, the development of inflammatory bowel disease (KmidenierL the like, Aliment Pharmacol Ther, 2002, 16 (12), 1997-2015). 因此,具有优异抗氧化活性的本发明药物组合物可以有效地用于预防和治疗诸如发炎性肠炎等之类的与炎症相关的疾病。 Accordingly, the pharmaceutical compositions of the invention have excellent antioxidant activity may be effective for preventing and treating diseases associated with inflammation such as inflammatory bowel disease and the like.

工业适用性 Industrial Applicability

如以上所解释的那样,与其它合成抗氧化剂不同,与其它天然抗氧化剂相比,本发明的中国紫荆提取物以及从该提取物分离并由<化学式1>至<化学式20>表示的化合物不会损伤人类,而且具有优异的抗氧化活性, 因此它们可以有效抑制细胞内的氧化应激并防止与细胞衰老有关的端粒縮短,由此导致细胞寿命延长。 As explained above, other synthetic antioxidants is different compared with other natural antioxidants, China Bauhinia extract of the invention is isolated by <Chemical Formula 1> and from the extract the compound to <Chemical Formula 20> means not human damage, but also excellent antioxidant activity, so they can effectively inhibit the oxidation in the cell stress and prevent cell senescence associated with telomere shortening, thus causing prolonged cell life. 因此,含有上述提取物或化合物作为有效成分的本发明化妆品组合物可以非常有效地用于开发抗皮肤衰老、支撑皮肤弹性和铍纹护理的化妆品。 Therefore, the cosmetic composition of the invention comprises the above-described extracts or compounds as an active ingredient can be very effectively used for the development of anti skin aging, skin elasticity and beryllium support groove care cosmetics.

本领域普通技术人员将意识到在前面描述中所公开的概念和具体实施方案可以容易地用作用于改进或设计其它实施方案的基础,而该其它实施方案是可实施本发明相同目的的。 Those of ordinary skill in the art will recognize that the foregoing description of the conception and the specific embodiment disclosed may be readily utilized as a program for modifying or designing other embodiments, while other embodiments are the same purposes of the present invention.

本领域普通技术人员也将意识到该等效实施方案是不会背离如所附权利要求所述的本发明精神和范围的。 Those of ordinary skill in the art will also appreciate that this embodiment is equivalent to the appended claims without departing from the spirit and scope of the present invention described.

Claims (5)

  1. 1.中国紫荆提取物用于制备化妆品组合物的应用,所述化妆品组合物用于抗氧化、抗皮肤衰老、保护皮肤弹性或防止皱纹,其中所述提取物是通过使用水或醇的水溶液作为提取剂提取的。 1. China Bauhinia extract for the preparation of a cosmetic composition, the cosmetic composition for anti-oxidation, anti-aging skin, protect the skin elasticity or prevent wrinkles, wherein said extract is an aqueous solution by using water or alcohol as extraction agent extraction.
  2. 2. 权利要求1所述的应用,其中所述化妆品组合物含有选自由中国紫荆提取物分离的异甘草素、2', 4'-二羟基-4-甲氧基查耳酮、甘草素、白藜芦醇、云杉鞣酸醇、没食子酸、没食子酸甲酯、没食子酸乙酯、杨梅黄酮、阿福豆碱、栎素、杨梅苷、杨梅黄酮-3-O(2"-0-没食子酰基)-aL-鼠李吡喃糖苷、丁香亭-3-0-芸香糖苷、丁香亭-3-0-(2"-0-没食子酰基)-芸香糖苷、(+)-儿茶素、(-)-表儿茶素-3-0-没食子酸酯、 (-)-表倍儿茶素-3-0-没食子酸酯、(-)-lyoniresinol 3a-0-(3-D-吡喃木糖苷和(+)-lyoniresio1 3a-0-PD-吡喃葡糖苷表示的化合物组成的组中的一种或多种化合物。 Use according to claim 1, wherein said cosmetic composition comprising an extract selected from the group consisting of Chinese Bauhinia isolated isoliquiritigenin, 2 ', 4'-dihydroxy-4-methoxy chalcone, glycyrrhizin, resveratrol, alcohol spruce tannin, gallic acid, methyl gallate, ethyl gallate, myricetin, Fu bean base, oak, myricetin glycoside, myricetin -3-O (2 "-0- gallate acyl) -Al- rhamnose pyranoside, Ding Xiangting -3-0- rutinoside, Ding Xiangting -3-0- (2 '- O galloyl) - rutinoside, (+) - catechin, ( -) - -3-0- epicatechin gallate, (-) - catechin -3-0- table times gallate, (-) - lyoniresinol 3a-0- (3-D- pyran one or more compounds of the group consisting of compounds lyoniresio1 3a-0-PD- glucopyranoside represented by - xylosidase and (+).
  3. 3. 如权利要求1或权利要求2所述的应用,其中所述化妆品组合物选自由柔性洗剂、营养洗剂、营养乳膏剂、香精、面膜或浴粉组成的组。 Group 1 or claim 3. The use as claimed in claim 2, wherein the cosmetic composition is selected from the group consisting of a flexible lotion, nutrition lotion, nutrition cream, essence, pack or bath powder composition.
  4. 4. 中国紫荆提取物用于制备药物组合物的应用,所述药物组合物用于抗氧化和抗衰老,其中所述提取物是通过使用水或醇的水溶液作为提取剂提取的。 4. Chinese Bauhinia extract for the preparation of a pharmaceutical composition, said pharmaceutical composition for the antioxidant and anti-aging, wherein said extract is extracted by the extraction agent used as an aqueous solution of water or an alcohol.
  5. 5. 如权利要求4所述的应用,其中所述药物组合物以注射剂、片剂或糖浆形式制备。 5. The use according to claim 4, wherein the pharmaceutical composition is prepared in an injection, a tablet or syrup form.
CN 200380107721 2002-12-27 2003-12-04 Extract of cercis chinensis having anti-oxidant activity and anti-aging activity, and cosmetic composition containing the extract for anti-oxidation, skin-aging protection and wrinkle improvement CN100574744C (en)

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KR100853377B1 (en) * 2006-10-31 2008-08-21 (주)아모레퍼시픽 Anti-aging composition for promoting the proteasome activity of skin cells
CN101152173A (en) * 2007-10-15 2008-04-02 崔福贵 Use of liquiritigenin in preparing medicament for treating neurodegenerative diseases
CN102329355B (en) * 2011-07-05 2015-09-02 中国医学科学院药用植物研究所 Method for preparing myricitrin and pharmaceutical compositions
CN102258530A (en) * 2011-07-05 2011-11-30 中国医学科学院药用植物研究所 Application of cardiovascular diseases medicament for the treatment in myricitrin
CN103957998B (en) * 2011-12-06 2018-06-12 荷兰联合利华有限公司 Skin anti-aging composition
CN102552102B (en) * 2012-03-28 2013-04-10 秦至臻 Natural lipstick
WO2014034977A1 (en) * 2012-08-30 2014-03-06 Bio Spectrum, Inc. Composition for blocking uv rays or reducing skin wrinkles comprising afzelin as active ingredient
CN104151374B (en) * 2014-07-16 2016-05-11 南京农业大学 One kind of chalcone daisy Kunlun blood preparation and use of a compound
CN104215592B (en) * 2014-09-26 2016-08-24 浙江省萧山棉麻研究所 One kind of pineapple stigma detection method
CN105232350B (en) * 2015-11-05 2018-03-23 浙江中医药大学 Water mask and manufacturing method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110604A1 (en) 2000-08-11 2002-08-15 Ashni Naturaceuticals, Inc. Composition exhibiting synergistic antioxidant activity

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110604A1 (en) 2000-08-11 2002-08-15 Ashni Naturaceuticals, Inc. Composition exhibiting synergistic antioxidant activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
IFSCC CONFERENCE 2003. NA MK et al, Antioxidant activity of Cercis chinensis and itsprotective effect on skin aging,117-138. 2003
The isolation of the inhibitory constituents on melanin polymerformation from the leaves of Cercis chinese. Lee Seung-Ho et al.Saengyak Hakhoechi, Korea,Vol.30 No.4. 1999
紫荆花红色素开发利用德研究. 李尚德等.广东微量元素科学,第1卷第2期. 1994

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