CN1411813A - 具有抗癌活性的可可碱 - Google Patents
具有抗癌活性的可可碱 Download PDFInfo
- Publication number
- CN1411813A CN1411813A CN02127608A CN02127608A CN1411813A CN 1411813 A CN1411813 A CN 1411813A CN 02127608 A CN02127608 A CN 02127608A CN 02127608 A CN02127608 A CN 02127608A CN 1411813 A CN1411813 A CN 1411813A
- Authority
- CN
- China
- Prior art keywords
- theobromine
- cancer
- cell
- cacao bean
- gjic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G4/00—Chewing gum
- A23G4/06—Chewing gum characterised by the composition containing organic or inorganic compounds
- A23G4/068—Chewing gum characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
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Abstract
本发明公开了具有抗癌活性的可可碱，它抑制属于包括肝癌在内的各种类型癌症发展过程中出现的病理现象的GJIC(缺口连接细胞通讯)抑制和癌细胞DNA的合成，由此抑制肝、胃和结肠癌细胞的增殖。
Description
发明领域
本发明涉及具有抗癌作用的可可碱，且更具体地说，本发明涉及可抑制属于在包括肝癌在内的各种类型癌症发展过程中出现的病理现象的缺口连接细胞通讯(GJIC)抑制以及癌细胞DNA合成的可可碱，由此抑制癌细胞的增殖。
发明背景
尽管已经在近30年中进行了广泛尝试来治疗癌症，但是癌症的发生率和死亡率并没有下降。(Sporn，M.B.，Lancet，347：1377-1381，1996)。看起来这种情况是过去30年来针对癌症的手段大部分集中在治疗观点而非预防观点上所导致的。正如已知的，摄取含有可有效抑制或延缓癌症多个发展阶段的成分的某一食品或药物有助于降低癌症的危害和随后由它导致的死亡率且已经进行了许多研究(Kang等，Chemical prevention of cancer，Korea Medicine，2000；Surh，Y.J.，Mutat.Res.，428：305-327，1999；Sporn，M.B.，Lancet，347：1377-1381，1996；Caragay，A.B.，Food Technol.，65-68，1992)。
癌发生是由刺激、助长和发展3个阶段组成的多阶段过程。在研究可有效预防或抑制癌症的活性剂的过程中，近期的研究更多地涉及到鉴定可以抑制助长和发展阶段而非刺激阶段的物质，刺激阶段是一个相对短而不可逆转的阶段(Kang等，Chemical prevention ofcancer，Korea Medicine，2000；Surh，Y.J.，Mutat.Res.，428：305-327，1999；Sporn，M.B.，Lancet，347：1377-1381，1996)。特别地，用食物提取物和级分预防和抑制癌症不同于药物而成为更有效的手段，条件是它们以癌发生助长阶段为靶，这一阶段一般发展了20年以上且还可逆转(Kang等，Chemical prevention of cancer，Korea Medicine，2000；Yamasaki，H.等，Carcinogenesis 11：1051-1058，1990；Kelloff.G.J.等.，Eur.J.Cancer，35：1755-1762，1999；Surh，Y.J.，Mutat.Res.，428：305-327，1999)。
缺口连接细胞通讯(GJIC)对通过调节多细胞生物体内细胞增殖和分化来调节内环境平衡来说是必不可少的。将GJIC抑制看作在癌发生时、特别是在肿瘤助长阶段观察到的关键生化指标；因此，预计可以抑制这类过程的物质可抑制癌发生的助长阶段，由此预防并抑制癌症的发展。此外，将由DNA合成导致癌细胞增殖看作癌发生发展阶段时观察到的关键生物指标；因此，预计可以抑制这类过程的物质可抑制癌发生的发展阶段，由此预防并抑制癌症的发展。(Kang等，Chemoprevention of cancer，Korea Medicine，2000；Yamasaki，H.等，Carcinogenesis 11：1051-1058，1990；Kelloff.G.J.等.，Eur.J.Cancer，35：1755-1762，1999；Surh，Y.J.，Mutat.Res.，428：305-327，1999；Holder.J.W.等Cancer Res.，53，3475-3485，1993)。
诸如咖啡、茶、可口可乐和巧克力这样不同的食品中含有一种黄嘌呤衍生物咖啡因(Lee，W.J.，Pharmacology Lecture，Medicineculture，195-198，1993)。特别在可可豆和可可豆壳中富含可可碱，它也是一种不同于咖啡因的黄嘌呤衍生物。一般来说，可可豆含有约1.5-3％的可可碱且可可豆壳含有约2％的可可碱。诸如由可可豆制成的巧克力这样的加工食品含有约0.2-0.5％的可可碱且该含量高于咖啡因含量7-10倍(Barry，L.Z.，J.Food Sci.，45：314-316，1980)。黄嘌呤衍生物的一般药物功能是刺激中枢神经系统、骨骼肌和心肌以及松弛平滑肌和冠状动脉、增强胃液分泌并利尿。然而，已知可可碱的药理活性远弱于咖啡因的药理活性(Lee，W.J.，PharmacologyLecture，Medicine culture，195-198，1993)。相反，咖啡因存在于不同食品中且具有广泛的药理活性，可可碱仅大量存在于可可豆和可可豆壳中且由此没有对可可碱进行深入研究。已经报导咖啡因具有抗癌活性；例如对胃癌治疗剂的协同作用(Takahashi，M.等，Anticancer Res.18：4399-4402，1998)、对肺癌的抑制作用(Jeffrey，C.等，Cancer Res.38：1757-1761，1978；Chung，F.L.等，CancerRes.58：4096-4101，1998)和对TPA(12-O-十四酰基佛波醇-13-乙酸酯)诱导的癌发生的抑制作用(Perchellet，J.P.等，Cancer Res.41：3927-3925，1981)。在绿茶和红茶的预防作用中，还报导咖啡因与多酚组合起重要作用(Yang.C.S.，Biofactor 13：73-79，1999；Huang，M.T.等，Cancer Res.57：2623-2629，1977；Lu，Y.P.等，Cancer Res.61：5002-5009，2001)。
然而，尚没有研究揭示出可可豆和可可豆壳中含有的可可碱在预防和抑制癌症中的作用。特别是尚无有关可可碱对在亚洲以及韩国中发生率相对较高的癌症的作用的报导，所述的癌症诸如肝癌、胃癌和结肠癌。
发明概述
本发明的发明者在基于安全原因而研究的天然食物中的抗癌剂的有潜力候选物的通式发现大量存在于可可豆和可可豆壳中的可可碱具有抗癌活性，诸如抑制属于癌发生助长和发展阶段过程中发生的GJIC抑制和癌细胞的DNA合成。
因此，本发明的目的是提供一种包括可可碱作为活性组分的抗癌剂。
附图说明
附图1是表示可可碱对大鼠肝脏上皮细胞中由H2O2产生的GJIC抑制的抑制作用的一组图，其中H2O2是人体内的癌症助长剂和最强活性氧种类(ROS)之一(a：未治疗的对照组；b：使用400ΠM H2O2治疗的组；c：使用200ΠM/ml可可碱和400ΠM H2O2治疗的组)。
附图2是表示可可碱对大鼠肝脏上皮细胞中由H2O2产生的控制GJIC的主要蛋白质连接蛋白43的超磷酸化抑制作用的图(a：对照组；b：使用400ΠM H2O2治疗的组；c：使用200ΠM/ml可可碱和400ΠM H2O2治疗的组)。
附图3是表示可可碱通过抑制一种肝癌细胞HepG2的DNA合成对增殖的抑制作用的示意图。
附图4是表示可可碱通过抑制一种胃癌细胞SNU1的DNA合成对增殖的抑制作用的示意图。
附图5是表示可可碱通过抑制一种结肠癌细胞SNUC2A的DNA合成对增殖的抑制作用的示意图。
本发明详细描述
本发明涉及一种包括下列通式1的可可碱作为活性组分的抗癌剂。
本发明具体描述如下。
本发明的可可碱用于预防并抑制癌发生，这一过程不仅通过抑制癌细胞DNA的合成、而且通过抑制GJIC的抑制来进行，所抑制的这些现象是癌发生助长和发展阶段过程中发生的特征现象。因此，本发明还涉及包括可可碱作为活性组分的药物或食品添加剂。
从可可豆和可可豆壳中分离可可碱的方法如下。
首先，从干燥的可可豆中除去可可油并将1重量份剩余的可可豆部分(可可质量)或可可豆壳加入到4-10重量份的50％丙酮、50％乙醇或50％甲醇的水溶液(Duksan Co.，Ltd.，Korea)中。然后对该混合物进行搅拌提取，同时在40-70℃下回流4-6小时。在将所得提取物离心后回收上清液并再次重复提取剩余的量。将上述提取物合并、干燥、过滤并获得可可豆和可可豆壳的提取物。最终通过HPLC、按照Kreiser的方法(Barry，L.Z.等，J.Food Sci.，45：314-316，1980)从该提取物中分离可可碱。对由此获得的可可碱或化学合成的可可碱检验它们对GJIC抑制和癌细胞增殖的抑制作用且我们发现它们抑制癌发生助长和发展阶段时发生的GJIC抑制和癌细胞增殖。
本发明还涉及有包括可可碱作为活性组分的药物或食品添加剂且按照一种公知的生产方法制备它们。
在生产包括可可碱的药物的过程中，可以用可可碱本身来制备可可碱或可以通过将可可碱与药物上可接受的载体、成形剂、稀释剂等混合而将它们制成粉剂、颗粒、胶囊和注射剂的形式。此外，可可碱的给药水平可以根据患者身体吸收率、体重、年龄、性别和健康情况、饮食、给药时间和方法、排泄率、疾病的严重性等的不同而改变；且一般优选给予0.1-5mg/kg体重。因此，制备包括本发明可可碱的药物应考虑到其有效范围。可以在专家的监督下按照特定的医疗方案给予由此制备的给药用单位制剂或根据患者的需求给药或在规定时间间隔给药几次。
如上所述，包括可可碱作为活性组分的药物或食品添加剂在抑制和诱发癌发生方面具有极佳的作用。
在下文中使用下列实施例来具体描述本发明，不过，不应将这些实施例看作用来限定本发明的范围。
参比实施例1：从可可豆中分离可可碱
从含有可可豆壳的可可豆中除去可可油，向1重量份剩余的部分(可可质量)中加入6重量份的50％丙酮溶液(Duksan Co.，Ltd.，Korea)并在60℃下进行搅拌提取5小时，同时回流。在4℃下以8,000rpm的速率(Vision Co.，Ltd.，Korea)离心该提取物并收集所得的上清液。将残余物重复提取1次并合并全部上清液。按照Kreiser法、通过HPLC从该提取物中分离可可碱。
参比实施例2：从可可豆壳中分离可可碱
使用与上述参比实施例1中相同分方法分离可可碱，但用可可豆壳替换可可豆。
参比实施例3：合成的可可碱的应用
可可碱商购自Sigma Co.，Ltd.(USA)且由此使用。
检验本发明分离和合成的可可碱对GJIC抑制和由H2O2使Cx43超磷酸化(P3)以及对肝癌、胃癌和结肠癌的癌细胞增殖的抑制作用。
实施例1：可可碱对GJIC抑制的抑制作用
人体内一种癌症助长剂和最强ROS之一的H2O2通过主要调节GJIC的连接蛋白43蛋白质(Cx43)的超磷酸化诱导缺口连接通道的抑制。检验可可碱对GJIC抑制和对由H2O2使连接蛋白43超磷酸化的作用。1-1：
可可碱对H2O2 抑制缺口连接通道的抑制作用
正如下面进一步所解释的，按照Scrape Loading/Dye Transfer(SL/DL)的已知测定法(Upham，B.L.，Kang，K.S.，Cho，H.Y.，& Trosko，J.E.，Carcinogenesis，18：37-42，1997)检测可可碱对由H2O2诱导的缺口连接通道抑制的抑制作用。
将WB-F344细胞用作肝细胞以便检测其对肝细胞中缺口连接通道抑制的抑制作用。在37℃和5％ CO2下的培养箱(Forma Scientific Co.，Marjetta，OH，USA)中将上述肝细胞在含有10％ FBS、添加了100IU/ml青霉素和100Πg/ml链霉素的MEM培养基中培养。用于上述培养物的培养基组成商购自GIBCO BRL(Grand Island，NY，U.S.)。将培养基中由此培养的肝细胞转入各为2ml的培养皿(1×105个细胞/mL)并培养44小时。然后用含有不同浓度可可碱的新鲜培养基取代上述培养基，同时仅用新鲜培养基替换对照组培养基。在该步骤后4小时用不同浓度的可可碱与400Πg H2O2一起处理细胞，同时仅用新鲜培养基替换对照组培养基。上述处理后1小时通过聚焦显微镜(BioRad，Hercules，CA，USA)、使用荧光黄染色观察可可碱对由H2O2抑制缺口连接通道的抑制水平。正如附图1中所示，与仅用H2O2处理相比，用200Πg可可碱处理完全抑制了由H2O2对缺口连接通道的抑制。
1-2：
可可碱对H2O2 产生的连接蛋白43超磷酸化的抑制作用
人体内一种癌症助长剂和最强ROS之一的H2O2通过主要调节GJIC的连接蛋白43蛋白质(Cx43)的超磷酸化诱导缺口连接通道的抑制。通过使用蛋白质印迹分析(Upham，B.L.，Kang，K.S.，Cho，H.Y.，& Trosko，J.E.，Carcinogenesis，18：37-42，1997)检验可可碱对连接蛋白43超磷酸化的抑制作用。
通过使用含有1mM苯甲基磺酰氟(PMSF)的20％ SDS从与上述1-1中相同方式培养的细胞中提取蛋白质。使用DC测定试剂盒(BioRadCorp.，U.S.)测定蛋白质含量。使各约15Πg的提取的蛋白质上12.5％SDS-PAGE凝胶并通过凝胶电泳分离。在与单克隆抗体(Zymed，U.S.)反应后通过使用ECL试剂盒(Amersham，Life Science，U.S.)检测连接蛋白43。
正如附图2中所示，可可碱抑制了由H2O2对连接蛋白43的超磷酸化。正如附图1和2中所示，经证实可可碱通过抑制GJIC抑制而具有抗癌作用。
实施例2：可可碱对肝癌细胞增殖(DNA合成)的抑制作用
通过使用3H-胸苷吸收测定法(Marshall，E.S.等European J.Cancer，30A：1370-1376，1994)检验可可碱对肝癌细胞增殖的抑制作用。
将HepG2用作肝癌细胞。在37℃和5％CO2下的培养箱(FormaScientific Co.，Marjetta，OH，USA)中将HepG2肝癌细胞在含有10％FBS、100IU/ml青霉素和100Πg/ml链霉素的RPMI-1640培养基中培养。该培养基商购自GIBCO BRL(Grand Island，NY，U.S.)。将由此培养的HepG2肝癌细胞转入96孔平板，其中各自含有2×104个细胞并加入获自参比实施例1-3的不同浓度的可可碱且培养72小时。在回收上述细胞前6小时向各孔中加入1ΠCi的3H-胸苷(Sigma，St.Louis，MO，USA)。在培养完成后，通过使用回收仪器(CambridgeScientific Inc.，cambridge，MA，USA)将细胞回收入玻璃纤维滤器(Brandel Inc.，Gaithersburg，MA，USA)。用液体闪烁计数器(Wallac，Turku，Finland)检测与3mL闪烁混合物溶液(Wallac，Turku，Finland)混合后回收细胞中导入的3H-胸苷。按照上述方法将整个过程重复3次。
正如附图3中所示，对使用可可碱处理的肝癌细胞的DNA合成的抑制率以剂量依赖性方式增加，这表明对肝癌细胞增殖的抑制作用依赖于可可碱的浓度。
实施例3：可可碱对胃癌细胞增殖(DNA合成)的作用
将胃癌细胞SNU1用与实施例2相同的不同浓度的可可碱处理且结果如附图4中所示。正如附图4中所示，对使用可可碱处理的胃癌细胞的DNA合成的抑制率以剂量依赖性方式增加，这表明对胃癌细胞增殖的抑制作用依赖于可可碱的浓度。
实施例4：可可碱对结肠癌细胞增殖(DNA合成)的抑制作用
将结肠癌细胞SNUC2A用与实施例2相同的不同浓度的可可碱处理且结果如附图5中所示。正如附图5中所示，对使用可可碱处理的结肠癌细胞的DNA合成的抑制率以剂量依赖性方式增加，这表明对结肠癌细胞增殖的抑制作用依赖于可可碱的浓度。
因此，从上述实施例中可以证实可可碱增加GJIC、抑制肝癌细胞、胃癌细胞和结肠癌细胞的DNA合成，这表明可可碱对癌发生具有预防和抑制作用。
实施例5：片剂的制备
活性组分 10g
乳糖 70g
结晶纤维素 15g
硬脂酸镁 5g
总计 100g
通过粉碎成小碎块且将上述组分混合且然后通过直接压片法制备。制备含有总量为100mg的各片且每片中活性组分的量为10mg。
实施例6：粉剂的制备
活性组分 10g
玉米淀粉 50g
羧基纤维素 40g
总计 100g
通过粉碎成小碎块且将上述组分混合并制备成粉剂。将由此制备的100克粉末装填入软胶囊并制成胶囊制剂。
实施例7：毒性试验
如下进行本发明可可碱毒性测试：将溶于二甲亚砜(DMSO)并用水稀释的可可碱以0.5g/kg的浓度对鼠给药(10只小鼠/组)并对小鼠观察7天。结果表明全部小鼠均存活。
实施例8
通过使用包括20wt％胶质软糖基质、76.9wt％的糖、1wt％食用香料、2wt％水和0.1wt％的可可碱的组合物、按照常规方法生产口香糖。
实施例9
通过使用包括60wt％糖、39.8wt％淀粉浆、0.1wt％食用香料和0.1wt％的可可碱的组合物、按照常规方法生产糖果。
实施例10
通过使用包括50wt％糖醇、49.8wt％麦芽糖、0.1wt％食用香料和0.1wt％的可可碱的组合物、按照常规方法生产口香糖。
实施例11
通过使用包括下列成分的的组合物、按照常规方法生产口饼干：25.59wt％的一级筋力弱的面粉、22.22wt％的一级中等面粉、4.80wt％的糖、0.73wt％的盐、0.78wt％的葡萄糖、11.78wt％的棕榈起酥、1.54wt％的碳酸氢铵、0.17wt％的碳酸氢钠、0.16wt％的焦亚硫酸钠、1.45wt％的米粉、0.0001wt％的维生素B1、0.0001wt％的维生素B2、0.04wt％的奶粉、20.6998wt％的水、1.16wt％的全脂奶粉、0.29wt％的乳粉替代品、0.03wt％的磷酸二氢钙、0.29wt％的雾化盐、7.27wt％的雾化乳和1wt％的可可碱。
实施例12
通过使用包括下列成分的组合物、按照常规方法生产饮料：0.26wt％的蜂蜜、0.0002wt％的硫辛酸酰胺、0.0004wt％的烟酸酰胺、0.0001wt％的核黄素-5’-磷酸钠、0.0001wt％的HCl吡哆素、0.001wt％的肌醇、0.002wt％的原酸、98.7362wt％的水和1wt％的可可碱。
实施例13
通过使用包括下列成分的组合物、按照常规方法生产饮料：3.5wt％的水果提取物、4.8wt％的果泥、7.78wt％的糖、0.11wt％的柠檬酸、82.71wt％的纯水和1wt％的可可碱。
实施例14
通过使用包括下列成分的组合物、按照常规方法生产香肠：65.18wt％的猪肉、25wt％的鸡肉、3.5wt％的淀粉、1.7wt％的大豆蛋白、1.62wt％的盐、0.5wt％的葡萄糖；1.5wt％的甘油和1wt％的可可碱。
实施例15
通过使用包括下列成分的组合物、按照常规方法生产片型的保健食品：55wt％的spirurina、10wt％的胍尔豆胶的酶降解产品、0.01wt％的盐酸维生素B1、0.01wt％的盐酸维生素B6、0.23wt％的DL-甲硫氨酸、0.7wt％的硬脂酸镁；22.2wt％的乳糖、1.85wt％的玉米淀粉和10wt％的可可碱。
实施例16
通过使用包括下列成分的组合物、按照常规方法生产胶囊型的保健食品：11.26wt％的壳寡糖、0.2wt％的大蒜粉、0.2wt％的银杏提取物粉、0.9wt％的E-胡萝卜素(30％混悬液)、1.2wt％的Δ-生育酚、1.2wt％的卵磷脂；4.5wt％的精制棕榈油、1.6wt％的黄蜂蜡、18.994wt％的大豆油、37.83wt％的明胶、16.51wt％的甘油、0.09wt％的乙基香草醛、0.076wt％的二氧化钛、0.44wt％的食用色素和10wt％的可可碱。
如上所述，本发明的可可碱表现出它可抑制属于癌发生助长和发展阶段时出现的主要病理现象的GJIC抑制和癌细胞增殖，由此抑制癌症发展。可以将按照本发明生产的可可碱制剂看作食品等同物且由此可以安全地给予而不再使用另外的纯化步骤。此外，通过单纯分离生产可可碱制剂的方法能够比那些常规抗癌剂的生产方法降低单位生产成本；特别从经济的观点来看，已经用于丢弃的大量可可豆壳的再循环利用可能极有价值。
Claims (3)
1.一种包括下列通式1的可可碱作为活性组分的抗癌剂：
2.权利要求1所述的抗癌剂，其中所述的可可碱分离自可可豆或可可豆壳或通过化学合成来制备。
3.一种具有抗癌作用的包括下列通式1的可可碱的食品添加剂：
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| KR1020010064287A KR20030032484A (ko) | 2001-10-18 | 2001-10-18 | 발암 억제효능을 갖는 데오브로민 |
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| DE602005011939D1 (de) * | 2005-05-31 | 2009-02-05 | Kraft Foods R & D Inc | Verfahren zur Herstellung von Polyphenol angereicherten Zusammensetzungen aus Kakaoschale |
| WO2007016953A1 (en) * | 2005-07-29 | 2007-02-15 | Matuschka-Greiffenclau Markus | Composition for reducing alcohol induced liver cancer risk |
| CA2611389C (en) | 2005-07-29 | 2015-02-17 | Tima Foundation | Composition for moderating alcohol metabolism and for reducing the risk of alcohol induced diseases |
| RU2591084C2 (ru) * | 2010-02-12 | 2016-07-10 | Теокорп Холдинг Ко., Ллс | Способы и композиции для улучшения механической прочности зубов |
| DE102012022778A1 (de) * | 2012-11-22 | 2014-06-05 | Siegfried Wegener | Arzneimittel gegen krebs, herzinfarkt und tuberkulose sowie gegen sämtliche krankheiten |
| CN105189736A (zh) * | 2013-03-15 | 2015-12-23 | 斯欧考普控股公司 | 适用于提高胎儿体重增加并增强骨特性的可可碱组合物 |
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| CA2066951A1 (en) * | 1991-04-26 | 1992-10-27 | Kou Moriyama | Edible composition |
| HUP9700654A2 (hu) * | 1997-03-26 | 1999-09-28 | Dezső Korbonits | Teobromin tartalmú köhögéscsillapító készítmények |
| KR100319251B1 (ko) | 1999-06-30 | 2002-01-05 | 한수길 | 충치 억제 효능을 지닌 카카오 콩껍질 분획물의 제조방법 |
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| EP1707207A3 (en) | 2006-12-06 |
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| US6693104B2 (en) | 2004-02-17 |
| CN1823626A (zh) | 2006-08-30 |
| CN100571540C (zh) | 2009-12-23 |
| EP1304046B1 (en) | 2006-05-24 |
| JP2003137786A (ja) | 2003-05-14 |
| EP1304046A1 (en) | 2003-04-23 |
| US20030099686A1 (en) | 2003-05-29 |
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