CN103520279B - Application of Cinnamomum plant extract in the manufacture of a medicament for the treatment or prevention of tumor or of health care products - Google Patents

Application of Cinnamomum plant extract in the manufacture of a medicament for the treatment or prevention of tumor or of health care products Download PDF

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CN103520279B
CN103520279B CN 201310486325 CN201310486325A CN103520279B CN 103520279 B CN103520279 B CN 103520279B CN 201310486325 CN201310486325 CN 201310486325 CN 201310486325 A CN201310486325 A CN 201310486325A CN 103520279 B CN103520279 B CN 103520279B
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CN103520279A (en )
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沈涛
李国辉
任冬梅
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山东大学
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Abstract

本发明公开了樟属植物提取物的新用途——樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物在制备治疗或预防肿瘤药物或保健品中的应用。 The present invention discloses a new use of a plant extract of Cinnamomum - camphor plants (including cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract for treating or preventing cancer drugs or health care products application. 本发明还提供了樟属植物提取物的制备方法。 The present invention further provides a method for preparing an extract of plants of the genus Cinnamomum. 研究表明,樟属植物提取物能够激活Nrf2信号通路,促进抗氧化和II相解毒酶的表达,增加细胞内谷胱甘肽水平,增强细胞内还原能力,并能够抑制致癌物砷和活性氧H2O2引起的细胞毒性。 Research shows that cinnamon plant extract can activate Nrf2 signaling pathway, and promote the expression of anti-oxidation phase II detoxification enzyme, increasing intracellular glutathione levels, enhance the intracellular reducing ability, and to suppress arsenic carcinogens and reactive oxygen species H2O2 induced cytotoxicity. 因此,可将其制备成相应的口服制剂和注射剂,并用于肿瘤的预防。 Thus, it can be prepared into the respective oral preparations and injections, and for the prevention of cancer.

Description

樟属植物提取物在制备治疗或预防肿瘤的药物或保健品中的应用 Application of Cinnamomum plant extract in the manufacture of a medicament for the treatment or prevention of tumor or of health care products

技术领域 FIELD

[0001] 本发明涉及樟属植物提取物的新用途,具体涉及樟属植物提取物在制备治疗或预防肿瘤的药物或保健品中的应用。 Applications [0001] The present invention relates to novel use of a plant extract of Cinnamomum, cinnamon particularly relates to a plant extract in the manufacture of a medicament for treating or preventing tumor or health-care product.

背景技术 Background technique

[0002] 随着社会的发展,人类生活水平和医疗环境的改善,人类寿命得到了显著的延长,但经济高速发展的同时,环境污染严重,导致恶性肿瘤的发病率也在提高,严重威胁人类健康,给国家和社会带来沉重的负担。 [0002] With the development of society, living standards improve human health and the environment, human life has been significantly extended, but rapid economic development, environmental pollution, resulting in the incidence of malignant tumors is also increasing, serious threat to human health, to the country and society a heavy burden. 在尚无有效药物实现对恶性肿瘤有效治疗的前提下,研发具有预防作用的药物和保健品,防止疾病的早期形成和发生成为有效的应对策略。 Under the premise of no effective medication to achieve effective treatment of malignant tumors, research and development have a preventive effect of drugs and health care products, to prevent the formation of early disease and the occurrence of an effective coping strategies.

[0003]核转录因子 Nrf2 (Nuclear factor-erythroid 2-related factor2)是调节细胞氧化还原平衡的重要转录因子,通过与抗氧化酶和II相解毒酶启动子区域抗氧化反应原件(ARE)结合调控细胞氧化还原平衡,其目标基因包括编码GST、UGT、yGCS、NQ01的II相解毒酶,及GCUHO-1等内源性抗氧化蛋白。 [0003] Nuclear transcription factor Nrf2 (Nuclear factor-erythroid 2-related factor2) regulate cellular redox balance is an important transcription factor binding original regulated by an oxidation reaction (the ARE) and antioxidant enzymes and anti phase II detoxification enzyme promoter region cellular redox balance, which comprises a gene encoding a GST, UGT, yGCS, NQ01 phase II detoxification enzyme, and the other endogenous antioxidant GCUHO-1 protein. 当机体暴露于亲电性异物、化学致癌物、内源性氧化剂等对机体有损伤作用的物质下,Nrf2便会被激活并通过上调抗氧化酶和II相解毒酶清除有害物,保护机体免受毒物的损伤(Lau et al.,Pharmacol.Res.,2008,58,262-270)。 When the body is exposed to foreign matter electrophilic substances, chemical carcinogens, oxidants damage endogenous effect on the body, of Nrf2 will be activated and purified by removing harmful and increase antioxidant enzyme phase II detoxification enzyme, protecting the body Free damaged poison (Lau et al., Pharmacol.Res., 2008,58,262-270). 因此,通过激活Nrf2信号通路,增强机体自身清除毒物和致癌物的能力,能够预防多种疾病的发生,例如恶性肿瘤。 Thus, by activation of Nrf2 signaling pathway, enhancing the body's own ability to scavenge poisons and carcinogens, can prevent a variety of diseases, such as cancer.

[0004] 在氧化应激状态下,面对内源性和外源性毒物对机体的侵害,利用外源性Nrf2激动剂上调机体Nrf2水平,能够增强机体自身防御能力,预防内源性活性氧、外源性致癌物等毒物对机体的损伤,对于预防恶性肿瘤的发生具有重要意义(Magesh et al.,Med.Res.Rev.,2012,32,4,687-726)。 [0004] Under oxidative stress, the inner face of endogenous and exogenous toxic damage to the body by exogenous agonists Nrf2 Nrf2 levels up body, can enhance the body's own defense capability, the prevention of endogenous active oxygen exogenous carcinogens and other toxic damage to the body, is of great significance (Magesh et al., Med.Res.Rev., 2012,32,4,687-726) for the prevention of cancer. 激活Nrf2信号通路,能够上调II相解毒酶水平,清除致癌物或降低其毒性,抑制致癌物诱导的DNA损伤、基因突变,通过上调Nrf2表达,能够增加细胞内谷胱甘肽水平,增强抗氧化酶活性,清除细胞内活性氧,抑制细胞脂质过氧化,从而预防恶性肿瘤的发生。 Nrf2 activation of signaling pathways can be upregulated phase II detoxification enzyme level, clearing or reducing the toxicity of carcinogens, inhibiting carcinogen-induced DNA damage, genetic mutation, by upregulating the expression of Nrf2 possible to increase the intracellular glutathione levels, increase the antioxidative activity, clearing of ROS, inhibition of lipid peroxidation in the cell, thereby preventing the occurrence of cancer. 因此,具有Nrf2激动作用的单体化合物、植物提取物等能够有效预防恶性肿瘤的发生率。 Therefore, Nrf2 agonism monomeric compounds and plant extracts can effectively prevent the occurrence of malignancy.

[0005] 樟属(Cinnamomum),为樟科的一属,约250种,分布于热带亚洲、澳大利亚至太平洋岛屿和热带美洲。 [0005] cinnamon (Cinnamomum), is a genus of Lauraceae, about 250 species, distributed in tropical Asia, the Pacific Islands and Australia to tropical America. 我国约有46种,主产南方各省区,种数最多是云南,其次是广东和四川,其中肉桂、柴桂、华南桂、川桂是重要药材。 China has about 46 kinds of main producing southern provinces, the largest number of species in Yunnan, followed by Guangdong and Sichuan, where the cinnamon, laurel wood, Huanan Gui, Gui Chuan is an important medicine. 樟属植物中主要含有挥发油、黄酮、苯丙素等化学成分。 Genus Cinnamomum mainly contains chemical components volatile oil, flavonoids, phenylpropanoids like.

发明内容 SUMMARY

[0006] 针对上述现有技术,本发明公开了樟属植物提取物的新用途一一樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物,在制备治疗或预防肿瘤的药物或保健品中的应用。 [0006] For the above-described prior art, the present invention discloses a plant extract of Cinnamomum new uses eleven genus Cinnamomum (cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract, in the manufacture of a medicament for the treatment or prevention of tumor or health-care product. 本发明还提供了樟属植物提取物的制备方法。 The present invention further provides a method for preparing an extract of plants of the genus Cinnamomum.

[0007] 本发明是通过以下技术方案实现的: [0007] The present invention is achieved by the following technical solutions:

[0008] 本发明的发明人通过研宄发现,樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物,能够激活Nrf2信号通路,并能保护细胞抵抗致癌物砷和活性氧双氧水产生的细胞毒性,因此,可以以上述樟属植物提取物为原料制备预防肿瘤的保健品,也可以制备治疗肿瘤的药物(经查阅文献,迄今为止未见有樟属植物提取物激活Nrf2信号通路的报道,亦未见樟属植物用于肿瘤预防和治疗的报道)。 [0008] The present inventors discovered by study based, cinnamon plants (including cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract, can activate Nrf2 signaling pathway, and can protect cells against the cytotoxicity of arsenic carcinogens and reactive oxygen species generated by the hydrogen peroxide, thus, may be the above-mentioned plant extract of Cinnamomum preparation of starting materials health preventing tumors, tumor therapy may also be prepared (by the literature, to date there is no camphor genus plant extract reported Nrf2 activation signaling pathway, also no cinnamon plants reported for cancer prevention and treatment).

[0009] 具体应用时,可以以樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物为原料,添加医学上可接受的辅料,制成胶囊剂(包括软胶囊)、片剂、散剂、颗粒、口服液、酒剂、丸剂、微丸剂、合剂、酊剂等剂型,优选颗粒剂、胶囊剂、片剂;或者将樟属植物提取物进一步纯化制成注射制剂。 [0009] When the specific application, may be cinnamon plants (including cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract as raw material, medically acceptable materials, made of capsules (including soft capsules), tablets, powders, granules, oral liquid, wine, pills, micro pills, mixture, tinctures and other formulations, preferably granules, capsules, tablets; or Cinnamomum further purified plant extract prepared as an injection preparation. 也可以将樟属植物提取物与其它对肿瘤有治疗或预防作用的药物混合,再添加或不添加医学上可接受的辅料,制成胶囊剂(包括软胶囊)、片剂、散剂、颗粒、口服液、酒剂、丸剂、微丸剂、合剂、酊剂、注射剂等剂型。 Cinnamomum may be mixed with other plant extracts drugs have therapeutic or prophylactic effect on tumor before or without the addition medically acceptable excipients to prepare capsules (including soft capsules), tablets, powders, granules, oral, wine, pills, micro pills, cement, tincture, injections and the like dosage forms.

[0010] 所述樟属植物提取物,是通过以下方法制备得到的: [0010] The Cinnamomum plant extract was prepared by the following method:

[0011] 将樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)地上部分粉碎,用O〜95%乙醇(体积百分数)回流提取I〜8次,每次溶剂用量为药材重量的I〜20倍,每次提取时间为0.5〜10小时;合并提取液,过滤,回收乙醇,进行浓缩、干燥,即得樟属植物提取物。 [0011] The genus Cinnamomum (cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) aboveground pulverized, extracted twice with I~8 O~95% ethanol (percent by volume) was refluxed for each times the amount of solvent I~20 times by weight of herbs, extraction time is 0.5~10 hours; the combined extracts, filtration, recover ethanol, then concentrated and dried to obtain a plant extract cinnamon.

[0012] 优选的,用30〜90%乙醇回流提取2〜4次,每次溶剂的用量为药材重量的3〜7倍,每次提取时间为2〜4小时。 [0012] Preferably, 30~90% ethanol was extracted with 2 ~ 4 times, the amount of each solvent is 3~7 times the weight of the herbs, extraction time is 2 ~ 4 hours.

[0013] 所述任意一种樟属植物提取物,均可以用于制备治疗或预防肿瘤的药物或保健品O [0013] any one of the plants of the genus Cinnamomum extract, treating or preventing tumors can be drugs or health care products for the preparation of O

[0014] 本发明的发明人完成了樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物对Nrf2信号激活作用,及对致癌物砷和活性氧双氧水引起的细胞毒性的保护作用的研宄。 [0014] The present inventors completed the genus Cinnamomum (cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract Nrf2 activation signals, arsenic and carcinogens and reactive oxygen species protective effect study based on hydrogen peroxide cytotoxicity caused. 结果表明,上述樟属植物提取物能够在蛋白水平激活Nrf2,增加Nrf2及其下游基因NQOl和GCS的表达,通过增加细胞内谷胱甘肽的水平增强细胞还原能力。 The results showed that the plant Cinnamomum extract can activate Nrf2 protein level, and increased expression of downstream genes Nrf2 NQOl and GCS enhance cell reducing ability by increasing intracellular glutathione levels. 发明人还发现它们能够抑制致癌物砷和活性氧双氧水诱导的细胞毒性,表现出抑制细胞癌变的能力。 The inventors have also found that they can inhibit carcinogen arsenic hydrogen peroxide and active oxygen induced cytotoxicity, exhibited the ability to inhibit carcinogenesis. 下面以樟属植物,包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟,介绍其对Nrf2信号通路及对致癌物砷和活性氧双氧水引起的细胞毒性的作用: Below to the genus Cinnamomum, cinnamon, camphor leaf Kennedy, hairy leaves camphor, linalyl burmannii, sassafras, describes the effect of Nrf2 signaling and cellular toxicity of arsenic carcinogens and reactive oxygen species induced hydrogen peroxide:

[0015] 采用能够稳定表达含ARE依赖的荧光素酶质粒的MDA-MB-231-Luc细胞系,评价了樟属植物提取物对Nrf2的作用,结果表明樟属植物提取物具有激活Nrf2信号通路的作用(图1)。 [0015] The can stably express luciferase containing ARE dependent plasmid MDA-MB-231-Luc cell line was evaluated cinnamon plant extracts on Nrf2 results show that cinnamon plant extract having activated Nrf2 signaling pathway effect (FIG. 1).

[0016] 选择人乳腺癌MDA-MB-231细胞作为检测细胞,评价樟属植物提取物对Nrf2信号通路的作用。 [0016] The selection of human breast cancer cells MDA-MB-231 cells as detected, evaluate the role of plant Cinnamomum extract Nrf2 signaling pathway. 蛋白质印迹分析结果表明,樟属植物提取物对能够上调Nrf2蛋白的表达,并能促进下游抗氧化和II相解毒酶蛋白NQOl和yGCS的表达(图2)。 Western blot analysis showed that the plant extract of Cinnamomum capable of upregulating the expression of Nrf2 protein, and can promote the expression of the downstream oxidation and phase II detoxification enzyme NQOl and yGCS protein (Figure 2).

[0017] 选择人正常肺上皮HBE细胞,检测了樟属植物提取物对细胞内谷胱甘肽水平的影响。 [0017] HBE selected normal human lung epithelial cells, examined the effect of Cinnamomum plant extract intracellular glutathione levels. 结果显示,樟属植物提取物对能增加细胞内谷胱甘肽水平(图3),增强细胞内还原能力和清除活性氧能力。 The results showed that the plant extract of Cinnamomum increase in intracellular glutathione levels (FIG. 3), and the reducing ability to enhance clearance of intracellular reactive oxygen species.

[0018] 选择致癌物砷诱导的细胞毒性模型,评价樟属植物提取物对对人正常肺上皮HBE细胞的保护作用。 [0018] Cytotoxicity arsenic carcinogen-induced model selection, the protective effect of normal human lung epithelial cells evaluated HBE Cinnamomum plant extract. 经樟属植物提取物对处理后,细胞生存率显著高于未加药处理组,证明樟属植物提取物对能够抑制三价砷诱导的细胞毒性(图4),表明樟属植物提取物对可用于肿瘤的预防和治疗。 Cinnamomum plant extract was after-treatment, cell viability was significantly higher than group treated dosing, cinnamon plant extract proved capable of inhibiting cytotoxicity induced trivalent arsenic (FIG. 4), suggesting that the plant extract of Cinnamomum useful for the prevention and treatment of tumors.

[0019] 选择活性氧H2O2诱导的细胞毒性模型,评价樟属植物提取物对人正常肺上皮HBE细胞的保护作用。 [0019] Model selection cytotoxic reactive oxygen species induced by H2O2, evaluated the protective effect of plant Cinnamomum extract HBE normal human lung epithelial cells. 结果表明植物提取物能够抑制H2O2产生的毒性,提高细胞生存率(图5)。 The results show that plant extracts can suppress generation of H2O2 toxicity, increased cell survival (FIG. 5). 肿瘤的发生多与活性氧产生的毒性密切相关,该结果证明樟属植物提取物可用于肿瘤的预防和治疗。 Closely related toxicity cancers are produced by the active oxygen, the results demonstrate the prevention and treatment of Cinnamomum plant extract may be used for tumors.

附图说明 BRIEF DESCRIPTION

[0020] 图1:ARE依赖的荧光素酶报告试验表明樟属植物提取物对能够激活Nrf2信号通路,图中的樟属植物提取物浓度单位为Ug/mL,萝卜硫素2.5 μΜ为阳性对照,其中A为肉桂,B为坚叶樟,C为毛叶樟,D为芳樟,E为阴香,F为黄樟。 [0020] FIG. 1: ARE-dependent luciferase reporter assay showed Cinnamomum plant extract can activate Nrf2 signal path, FIG. Cinnamomum plant extract concentration units Ug / mL, sulforaphane 2.5 μΜ positive control where A is cinnamon, B is Zhang Jian Ye, C for the hair-leaved Zhang, D is linalool, E Yin Hong, F is sassafras.

[0021]图2:蛋白质印迹分析表明樟属植物提取物能够在蛋白水平激活Nrf2其下游基因,图中的樟属植物提取物浓度单位为μ g/mL,萝卜硫素2.5 μ M为阳性对照,其中A为肉桂,B为坚叶樟,C为毛叶樟,D为芳樟,E为阴香,F为黄樟。 [0021] Figure 2: Western blot analysis showed that cinnamon plant extract can activate Nrf2 downstream gene at the protein level, the figure Cinnamomum plant extract concentration in units of μ g / mL, sulforaphane 2.5 μ M for the positive control where A is cinnamon, B is Zhang Jian Ye, C for the hair-leaved Zhang, D is linalool, E Yin Hong, F is sassafras.

[0022] 图3:樟属植物提取物能够增加细胞内谷胱甘肽水平,图中萝卜硫素2.5 μΜ为阳性对照,樟属植物浓度分别为:肉桂(60 μ g/mL),坚叶樟(60 μ g/mL),毛叶樟(90 μ g/mL),芳樟(60 μ g/mL),阴香(90 μ g/mL),黄樟(400 μ g/mL)。 [0022] FIG. 3: Cinnamomum plant extract capable of increasing the intracellular glutathione levels, FIG sulforaphane 2.5 μΜ as a positive control, the genus Cinnamomum concentrations were: cinnamon (60 μ g / mL), Kennedy leaves Zhang (60 μ g / mL), hairy Cinnamomum (90 μ g / mL), linalool (60 μ g / mL), burmannii (90 μ g / mL), sassafras (400 μ g / mL).

[0023] 图4:樟属植物提取物能够降低砷(As)诱导的细胞毒性(*p〈0.05),其中A为肉桂(60 μ g/mL),B 为坚叶樟(60 μ g/mL),C 为毛叶樟(90 μ g/mL),D 为芳樟(60 μ g/mL),E为阴香(90 μ g/mL),F 为黄樟(400 μ g/mL)。 [0023] FIG. 4: Cinnamomum plant extract can be reduced cytotoxicity arsenic (As) induced (* p <0.05), where A is a cinnamon (60 μ g / mL), B kin Cinnamomum (60 μ g / mL), C for the hair Cinnamomum (90 μ g / mL), D is linalool (60 μ g / mL), E is burmannii (90 μ g / mL), F is sassafras (400 μ g / mL ).

[0024] 图5:樟属植物提取物能够降低双氧水(H2O2)诱导的细胞毒性(*p〈0.05),其中A为肉桂(60 μ g/mL),13为坚叶樟(60 μ g/mL),(3为毛叶樟(90 μ g/mL),D 为芳樟(60 μ g/mL),E 为阴香(90 μ g/mL),F 为黄樟(400 μ g/mL)。 [0024] FIG. 5: Cinnamomum plant extract can reduce the cytotoxicity of hydrogen peroxide (H2O2) - induced (* p <0.05), where A is a cinnamon (60 μ g / mL), 13 kin Cinnamomum (60 μ g / mL), (3 Cinnamomum for the hair (90 μ g / mL), D is linalool (60 μ g / mL), E is burmannii (90 μ g / mL), F is sassafras (400 μ g / mL).

具体实施方式 detailed description

[0025] 下面结合实施例对本发明作进一步的说明,下述实施例仅为说明之目的,并非用于限制本发明。 [0025] The following embodiments in conjunction with embodiments of the present invention will be further explained, the purpose of the following examples are illustrative only and not intended to limit the present invention.

[0026] 实施例1:樟属植物提取物对Nrf2信号通路的激活作用 [0026] Example 1: Activation of the genus Cinnamomum extract Nrf2 signaling pathway

[0027] 方法:(I)樟属植物提取物的制备 [0027] Method: (I) preparing a plant extract of Cinnamomum

[0028] 樟属植物(分别为肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟,共进行6种樟树植物提取物的制备)地上部分200g,粉碎,75%乙醇800mL,提取2次,每次I小时,合并提取液,浓缩干燥即得6种樟树植物提取物,用于活性评价。 [0028] Cinnamomum plants (respectively cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras, were prepared six kinds of plant extracts is camphor) aboveground 200g, pulverized, 800 mL 75% ethanol, extracted twice, each time I hour, the combined extracts were dried and concentrated to obtain 6 kinds of camphor plant extracts for activity evaluation.

[0029] (2)人乳腺癌细胞MDA-MB-231细胞系的培养 [0029] (2) Culture human MDA-MB-231 breast cancer cell lines Cell

[0030] 人乳腺癌细胞MDA-MB-231购自美国模式培养物集存库(ATCC),采用含10%胎牛血清(FBS)、2mMHEPES和6ng/mL胰岛素的MEM培养基,置于37°C,5%0)2培养箱中培养。 [0030] Human breast cancer MDA-MB-231 cells were purchased from American Type culture collection deposit library (ATCC), containing 10% fetal bovine serum employed (FBS), MEM medium 2mMHEPES and 6ng / mL insulin at 37 ° C, 5% 0) 2 incubator.

[0031] (3) ARE依赖的荧光素酶报告试验 [0031] (3) ARE-dependent luciferase reporter assays

[0032] 将MDA-MB-231-Luc细胞接种于96孔板上,24小时后加入不同浓度的待测化合物,处理16小时,采用细胞裂解液[0.1M磷酸钾和ImM 二硫苏糖醇]裂解细胞,用检测液(25mM双甘氨肽,15mM硫酸镁,500 μ MATP, 250 μ M荧光素和250 μ M辅酶Α),测定荧光强度。 [0032] The MDA-MB-231-Luc cells were seeded in 96-well plates, 24 hours after the addition of different concentrations of test compound, 16 hours, cell lysates using [0.1M potassium phosphate and ImM dithiothreitol ] cells were lysed with test solution (25mM glycylglycine, 15mM magnesium sulfate, 500 μ MATP, 250 μ M 250 μ M fluorescein and coenzyme [alpha]), fluorescence intensity was measured.

[0033] 结果:如图1所示,樟属植物提取物能够显著增强ARE依赖的荧光素酶活性,在任一提取物最佳浓度下,强度是空白对照组的4倍,阳性对照药物萝卜硫素(2.5μΜ)是空白对照组的2倍。 [0033] Results: As shown in FIG. 1, Cinnamomum plant extract can significantly enhance luciferase activity of ARE-dependent, in either an extract optimum concentration, four times the intensity of the blank control group, positive control sulphoraphone Su (2.5μΜ) is 2 times of the control group. 上述结果表明,六种樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物能够诱导Nrf2信号通路,对机体具有更好的保护作用。 The above results show that the six genus Cinnamomum (cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract capable of inducing Nrf2 signal path, having a better protection of the body.

[0034] 实施例2:樟属植物提取物能够上调Nrf2及以下游基因蛋白水平的表达 [0034] Example 2: genus Cinnamomum extract can increase the expression of a downstream gene Nrf2 protein and the

[0035] 方法:蛋白质印迹分析(Westernblot)检测细胞中蛋白水平的变化 [0035] Method: Western Blot Analysis Change (Western blot) protein levels in the cell detected

[0036] 将MDA-MB-231细胞接种于直径35mm培养皿中,培养至密度达到70%〜80%后,加入不同浓度的樟属植物提取物(樟属植物提取物为实施例1制备)处理16h,PBS洗涤2次,加入细胞裂解液(50 μ g/ml抑肽酶,0.5mM苯甲基磺酰氟,ImM正钒酸钠,1mM氟化钠,ΙΟπιΜβ-磷酸甘油),收集蛋白并采用Bradford法测定蛋白浓度。 [0036] The MDA-MB-231 cells were seeded in 35mm diameter culture dish and cultured until the density reaches 70% ~ 80%, with different concentrations of plant extract cinnamon (Cinnamomum plant extract prepared in Example 1) treatment for 16 h, washed with PBS 2 times, cell lysate (50 μ g / ml aprotinin, 0.5mM PMSF, ImM sodium orthovanadate, 1mM sodium fluoride, ΙΟπιΜβ- glycerol phosphate), protein was collected and the protein concentration was determined using the Bradford method. 各取样品蛋白(100 μ g)上样,SDS-PAGE分离蛋白组分并采用电转移法将蛋白条带转移至硝酸纤维素薄膜上。 Loaded onto a protein from each sample (100 μ g), SDS-PAGE and the protein fractions isolated by electric transfer method with protein bands transferred to nitrocellulose membrane. 薄膜经TBS配制5%的脱脂奶粉溶液室温封闭Ih后,分别与各待测蛋白抗体4°C孵育过夜。 Skim milk powder solution prepared at room temperature the film was 5% after TBS blocking Ih, respectively, with each test antibody overnight at 4 ° C incubation. 经TBS洗涤后分别加入辣根过氧化物酶偶联的二抗孵育Ih后,用增强型ECL化学发光进行蛋白分析。 After washed with TBS were added to the horseradish peroxidase-conjugated secondary antibody for Ih, analyzed for protein by ECL enhanced chemiluminescence.

[0037] 结果:如图2所示,细胞经樟属植物提取物处理16h后,Nrf2及其下游抗氧化和II相解毒酶蛋白NQOl和γ GCS蛋白水平呈现剂量依赖性增加,证实六种樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物能够在蛋白水平上计划Nrf2信号通路。 [0037] Results: As shown, the cells were treated plant extract Cinnamomum 16h 2, Nrf2 its downstream antioxidant and phase II detoxification enzyme NQOl and γ GCS protein levels in a dose-dependent increase in protein, confirmed six kinds of camphor plants (including cinnamon, Zhang Jian Ye, Zhang Ye Mao, linalool, Yin Hong, sassafras) extract can plan Nrf2 signaling pathway at the protein level.

[0038] 实施例3:樟属植物提取物增加细胞内谷胱甘肽水平 [0038] Example 3: Cinnamomum plant extract increased intracellular glutathione levels in

[0039] 方法:(I)人正常肺上皮细胞HBE细胞的培养 [0039] Method: (I) of normal human lung epithelial cells in culture HBE

[0040] 人正常肺上皮细胞HBE购自美国模式培养物集存库(ATCC),采用MEM培养基,并向其中加入10%胎牛血清(FBS),5%谷氨酰胺,置于37°C,5%0)2培养箱中培养。 [0040] HBE normal human lung epithelial cells were purchased from American Type Culture Collection library (ATCC), using the MEM medium to which 10% fetal bovine serum (FBS), 5% glutamine, disposed 37 ° C, 5% 0) 2 incubator.

[0041] (2)细胞内谷胱甘肽含量的测定 (2) Measurement of intracellular glutathione levels [0041]

[0042] 将HBE细胞接种于直径35mm培养皿中,培养至密度达到70%〜80%后,加入不同浓度的樟属植物提取物(樟属植物提取物为实施例1制备)处理24小时,PBS洗涤2次,加入0.5mL50mM磷酸钠和ImMEDTA缓冲液收细胞,超声lmin,1000g离心15分钟,取上清液,按照谷胱甘肽测定试剂盒说明书操作,412nm测定吸光度并计算谷胱甘肽含量。 [0042] The HBE cells were seeded in 35mm diameter petri dishes, cultured to a density of 70% ~ 80%, with different concentrations of plant extract cinnamon (Cinnamomum plant extract as prepared in Example 1) for 24 hours. washed with PBS 2 times, and added 0.5mL50mM sodium phosphate buffer ImMEDTA closed cells, ultrasonic lmin, 1000g centrifugation for 15 minutes, and the supernatant, glutathione assay according to the kit instructions, and the absorbance was measured at 412nm calculated glutathione content.

[0043] 结果:如图3所示,六种樟属植物(包括肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟)提取物能够显著增加细胞内谷胱甘肽水平,增强细胞内还原能力。 [0043] Results: As shown in FIG 3, six kinds genus Cinnamomum (cinnamon, Cinnamomum Kennedy, hairy leaves camphor, linalyl burmannii, sassafras) extract can significantly increase the intracellular glutathione levels, enhanced intracellular reduction capability.

[0044] 实施例4:樟属植物提取物能够抑制三价砷对人肺支气管上皮HBE细胞的毒性 [0044] Example 4: cinnamon extract can inhibit plant trivalent arsenic toxicity to human bronchial epithelial cells HBE

[0045] 方法:MTT法测定化合物对砷(As)诱导的细胞毒性的保护作用 [0045] Method: MTT Determination of the protective effect of the compound of arsenic (As) induced cytotoxicity

[0046] 将HBE细胞接种于96孔板上,加入不同浓度待测樟属植物提取物处理16小时,然后加入不同浓度的砷和待测浓度樟属植物提取物(樟属植物提取物为实施例1制备)处理24小时,加入MTT3小时后,590nm测定吸光度并计算细胞生存率。 [0046] The HBE cells were seeded in 96-well plates with different concentrations tested Cinnamomum plant extract for 16 hours, followed by addition of different concentrations of test concentrations of arsenic and plant extract cinnamon (Cinnamomum embodiment of a plant extract after preparation Example 1) for 24 h, added MTT3 hours, 590nm absorbance was measured and calculate the cell viability.

[0047] 结果:如图4所示,采用樟属植物提取物预处理细胞16小时,然后加入20 μM、40 μΜ的砷和樟属植物提取物处理细胞24小时,结果表明樟属植物提取物对HBE细胞具有保护作用,能够抑制砷诱导的细胞毒性。 [0047] Results: As shown in FIG. 4, using a plant extract Cinnamomum Cells were pretreated for 16 hours followed by addition of 20 μM, 40 μΜ arsenic and Cinnamomum plant extract treated for 24 hours. The results showed that the plant extract Cinnamomum HBE cells has a protective effect, can be suppressed arsenic-induced cytotoxicity. 樟属植物提取物处理组的细胞生存率明显高于未加药物处理组。 Cinnamomum plant extract treated cell survival rate was significantly higher than group plus drug treated groups. 结果证明,樟属植物提取物(包括肉桂、坚叶樟、芳樟、阴香、黄樟)能够抑制致癌物砷诱导的毒性,能够用于肿瘤的预防和治疗。 The results demonstrate, cinnamon plant extract (cinnamon, Cinnamomum Kennedy, linalyl burmannii, sassafras) capable of inhibiting carcinogen-induced toxicity of arsenic, can be used for the prevention and treatment of tumors.

[0048] 实施例5:樟属植物提取物能够抑制活性氧双氧水(H2O2)对人肺支气管上皮HBE细胞的毒性 [0048] Example 5: Cinnamomum plant extract capable of inhibiting active oxygen of hydrogen peroxide (H2O2) toxicity to human bronchial epithelial cells HBE

[0049] 方法同实施例4。 [0049] The same method as in Example 4.

[0050] 结果:图5所示,采用相应浓度樟属植物提取物预处理细胞16小时,然后加入100 μ Μ,200 μ M的H2O2和相应浓度樟属植物提取物处理细胞24小时,结果表明樟属植物提取物对HBE细胞具有保护作用,能够抑制H2O2诱导的细胞毒性。 [0050] Results: As shown in FIG. 5, using the appropriate concentration of plant extract Cinnamomum Cells were pretreated for 16 hours followed by addition of 100 μ Μ, H2O2, and the corresponding concentration of 200 μ M Cinnamomum plant extract treated for 24 hours. The results show that Cinnamomum plant extract has protective effects on HBE cells, capable of inhibiting cytotoxicity induced by H2O2. 樟属植物提取物处理组的细胞生存率明显高于未加药物处理组。 Cinnamomum plant extract treated cell survival rate was significantly higher than group plus drug treated groups. 结果证明,樟属植物提取物(包括肉桂、坚叶樟、芳樟、阴香、黄樟)能够抑制活性氧H2O2产生的神经细胞毒性,能够用于肿瘤的预防和治疗。 The results demonstrate, cinnamon plant extract (cinnamon, Cinnamomum Kennedy, linalyl burmannii, sassafras) capable of inhibiting neurotoxicity H2O2 generated active oxygen, can be used for the prevention and treatment of tumors.

[0051] 实施例6:肉桂提取物的制备 Cinnamon extract prepared: Example 6 [0051] Embodiment

[0052] 肉桂地上部分5kg,粉碎,用药材重量的3倍的90%乙醇回流提取2次,每次提取时间为4小时,合并提取液,过滤,回收乙醇,减压干燥,即得樟属植物提取物315g,收率为 [0052] cinnamic aboveground 5kg, pulverized, and extracted twice with 90% ethanol at reflux for 3 times the weight of the medicine, each extraction time was 4 hours, the combined extracts, filtration, recovery of ethanol, dried under reduced pressure to obtain cinnamon plant extracts 315g, yield

6.3%ο 6.3% ο

[0053] 实施例7:芳樟提取物的制备 Linalyl extract prepared: 7 cases of [0053] Embodiment

[0054] 芳樟地上部分5kg,粉碎,用药材重量的5倍的75%乙醇回流提取3次,每次提取时间为3小时,合并提取液,过滤,回收乙醇,喷雾干燥,即得樟属植物提取物350g,收率为 [0054] linalyl aboveground 5kg, pulverized and extracted three times with 75% ethanol was refluxed for 5 times by weight of herbs, extraction time is 3 hours, combined extracts, filtration, recover ethanol, spray-dried, to obtain cinnamon plant extracts 350g, yield

7.0%。 7.0%.

[0055] 实施例8:黄樟提取物的制备 [0055] Example 8: Preparation of extract of sassafras

[0056] 黄樟地上部分5kg,粉碎,用药材重量的7倍的35%乙醇回流提取4次,每次提取时间为2.5小时,合并提取液,过滤,回收乙醇,冷冻干燥,即得樟属植物提取物412g,收率为8.24%ο [0056] sassafras aboveground 5kg, pulverized and extracted four times with 35% ethanol was refluxed for 7 times by weight of herbs, extraction time was 2.5 hours and the combined extracts, filtration, recover ethanol, freeze-dried, to obtain cinnamon plant extracts 412g, a yield of 8.24% ο

[0057] 实施例9:片剂的制备 Tablets were prepared: Example 9 [0057] Embodiment

[0058] 黄樟提取物(实施例8制备)20g,加入玉米淀粉30g,糊精15g,充分混勾,制颗粒,干燥,整粒,加适量滑石粉,压片,即得。 [0058] sassafras extract (Preparation Example 8) 20g, corn starch is added 30g, dextrin 15g, mixed well hook, granulation, dried and sieved, add appropriate amount of talcum powder, tableting, i.e., too.

[0059] 实施例10:胶囊剂的制备 Preparation of capsules: Example 10 [0059] Embodiment

[0060] 黄樟提取物(实施例8制备)20g,加入淀粉20g、糊精15g,微晶纤维素8g,充分混匀,制颗粒,干燥,整粒,加入适量硬脂酸镁,装胶囊,即得。 [0060] sassafras extract (Preparation Example 8) 20g, added starch 20g, dextrin 15g, microcrystalline cellulose 8g, thoroughly mixed, granulation, dried and sieved, were added appropriate amount of magnesium stearate, encapsulating , that is, too.

Claims (4)

  1. 1.樟属植物提取物在制备预防肿瘤的药物或保健品中的应用,其特征在于:所述樟属植物为肉桂{Cinnamomum cassia Presl.)、坚叶棒{Cinnamomum chartophyllum Η.ff.Li)、毛叶棒(Cinnamomummolli foli unR.ff.Li)、芳棒{Cinnamomum camphora var.1inalooliferd1.Fujita) > Ifj^ (Cinnamomumburmannii(CGet Th.Nees) B1.)、黄棒(Cinnamomum porrecturn (Roxb.) Kosterm)。 1. Cinnamomum plant extract in the pharmaceutical and health products in the preparation of an application or preventing tumors, wherein: the plant of the genus Cinnamomum cinnamon {Cinnamomum cassia Presl), Kennedy leaf rod {Cinnamomum chartophyllum Η.ff.Li). , hairy leaves rods (Cinnamomummolli foli unR.ff.Li), aryl bar {Cinnamomum camphora var.1inalooliferd1.Fujita)> Ifj ^ (Cinnamomumburmannii (cGet Th.Nees) B1.), yellow bar (Cinnamomum porrecturn (Roxb.) Kosterm ).
  2. 2.根据权利要求1所述的应用,其特征在于:具体应用时,以樟属植物提取物为原料,添加医学上可接受的辅料,制成胶囊剂、片剂、散剂、颗粒剂、口服液、酒剂、丸剂、合剂或酊剂;或者将樟属植物提取物纯化制成注射制剂;或者:将樟属植物提取物与其它对肿瘤具有预防作用的药物混合,再添加或不添加医学上可接受的辅料,制成胶囊剂、片剂、散剂、颗粒剂、口服液、酒剂、丸剂、合剂、酊剂或注射剂;所述樟属植物提取物为该植物地上部分的提取物。 2. Use according to claim 1, wherein: specific application to the genus Cinnamomum extract as raw material, medically acceptable materials to prepare capsules, tablets, powders, granules, oral liquid, liquor, pills, agent or tincture; Cinnamomum or purified plant extract prepared injectable formulations; or: a medicine by mixing the agent Cinnamomum plant extract has a preventive effect on other tumors, again with or without added acceptable excipients to prepare capsules, tablets, powders, granules, oral liquid, wine, pills, cement, tincture or injections; Cinnamomum the plant extract for extract plant parts above ground.
  3. 3.如权利要求1或2所述的应用,其特征在于:所述樟属植物提取物是通过以下方法制备得到的:将樟属植物地上部分粉碎,用O〜95%乙醇回流提取I〜8次,每次溶剂用量为药材重量的I〜20倍,每次提取时间为0.5〜10小时;合并提取液,过滤,回收乙醇,进行浓缩、干燥,即得樟属植物提取物,所述樟属植物选自肉桂、坚叶樟、毛叶樟、芳樟、阴香、黄樟。 3. The use of claim 1 or claim 2, wherein: said Cinnamomum plant extract was prepared by the following method: the aerial parts of the plant Cinnamomum pulverized, extracted with O~95% ethanol was refluxed I~ 8 times the amount of the solvent I~20 times the weight of the herbs, extraction time is 0.5~10 hours; the combined extracts, filtration, recover ethanol, then concentrated and dried to obtain cinnamon plant extract, the Cinnamomum genus selected from cinnamon, Zhang Jian Ye, Zhang Ye Mao, linalool, Yin Hong, sassafras.
  4. 4.如权利要求1或2所述的应用,其特征在于:所述樟属植物提取物是通过以下方法制备得到的:将樟属植物地上部分粉碎,用30〜90%乙醇回流提取2〜4次,每次溶剂用量为药材重量的3〜7倍,每次提取时间为2〜4小时;合并提取液,过滤,回收乙醇,进行浓缩、干燥,即得樟属植物提取物。 4. The use of claim 1 or claim 2, wherein: said Cinnamomum plant extract was prepared by the following method: the aerial parts of the plant Cinnamomum pulverized, extracted with 30~90% ethanol was refluxed for 2 ~ 4, the solvent in an amount of 3~7 times per weight of herbs, extraction time is 2 ~ 4 hours; the combined extracts, filtration, recover ethanol, then concentrated and dried to obtain a plant extract cinnamon.
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