CN108883121B - 3,6-无水-l-半乳糖用于预防龋齿的用途 - Google Patents
3,6-无水-l-半乳糖用于预防龋齿的用途 Download PDFInfo
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Abstract
本发明涉及3,6‑无水‑L‑半乳糖用于预防龋齿的用途。更具体地说,3,6‑无水‑L‑半乳糖抑制口腔微生物的生长,并显示出抗龋活性,以抑制由口腔微生物消耗碳源所引起的产酸。因此,3,6‑无水‑L‑半乳糖可用于药物、食品、口腔卫生制剂等,用于预防、改善或治疗口腔微生物引起的口腔疾病,如龋齿、牙龈炎、牙周炎、口腔粘膜溃疡、口臭或口干症。
Description
技术领域
本发明涉及3,6-无水-L-乳糖用于通过抑制口腔微生物的生长和抑制酸产生来预防或治疗口腔疾病的抗龋齿用途。
背景技术
龋齿是一种造成人们拜访牙医的主要原因之一的口腔疾病,并且随着人类使用糖作为甜味剂,其发病率增加。变形链球菌(Streptococcus mutans)是龋齿的主要致病细菌(Loesche WJ et al.(1975)Infect Immun.11(6):1252-60)。变形链球菌在口腔中分解糖分,并且分泌葡糖基转移酶(GTF)以在牙齿表面形成不溶性葡聚糖,这导致变形链球菌或各种其他细菌附着到牙齿表面以在其上增殖。牙齿表面上的细菌引起龋齿,这是由于牙齿表面的牙釉质被有机酸如作为碳水化合物代谢物的乳酸(Dashper SG et al.(1996)Microbiol.142,33-29) 脱钙。作为防止龋齿的代表性材料,已知通过减少牙菌斑中变异链球菌数目来预防龋齿的氟化合物和木糖醇(Trahan L et al.(1985)Caries Res.19:53-63)。木糖醇是存在于自然界的5碳醇,由于与糖类似的甜味和清新口感而作为口香糖制造并广泛使用,但其不利之处在于只有在高浓度的木糖醇的情况下才可以抑制变形链球菌的生长。
目前,具有对抗变形链球菌的抗微生物活性的天然材料的研究越来越多地进行,并且越来越关注天然糖作为抗龋齿甜味剂,但是研究发现的具有与木糖醇相仿抗致龋作用的天然糖不足。当变异链球菌摄取木糖醇时,经由磷酸烯醇式丙酮酸-木糖醇磷酸激酶系统(PEP-木糖醇PTS)的活动形成木糖醇-5-磷酸酯。该产品抑制糖酵解酶的活性,不再被代谢,因此随后以木糖醇的形式在细胞外排出,从而通过不产生能量且仅消耗能量的无效循环来抑制细菌生长和酸生产。此时,只有浓度为25g/L或更高的木糖醇才能抑制变形链球菌的生长,并且研究还表明,当在含木糖醇的培养基中连续培养变形链球菌时,产生耐药菌株,其生长未被木糖醇抑制(Knuuttila et al.(1975)Caries Research,9(3), 177-189)。
因此,需要开发即使在低浓度下也能有效抑制口腔细菌生长而不形成耐药菌株的防龋剂。
发明内容
技术问题
本发明的一个目的是提供用于预防或治疗口腔疾病的药物组合物,其包含作为有效成分的具有抑制口腔生物生长和酸产生活性的3,6-无水-L-半乳糖,或用于预防或治疗口腔疾病的药物组合物。
本发明的另一个目的是提供一种通过将3,6-无水-L-半乳糖施用于有需要的对象来治疗口腔疾病的方法。
本发明的另一个目的是提供一种口腔卫生组合物,用于预防或缓解口腔疾病,其包含3,6-无水-L-半乳糖作为活性成分。
本发明的另一个目的是提供一种用于预防或缓解口腔疾病的食物组合物,其包含3,6-无水-L-半乳糖作为活性成分。
技术方案
为了实现上述技术目的,本发明提供了用于预防或治疗口腔疾病的药物组合物,其包含3,6-无水-L-半乳糖。
本发明还提供用于预防或治疗口腔疾病的药物组合物,其包含3,6-无水- L-半乳糖。
本发明还提供了3,6-无水-L-半乳糖在制备用于预防或治疗口腔疾病的药物组合物中的用途。
本发明还提供了一种治疗口腔疾病的方法,包括向有需要的对象施用药学有效量的用于预防或治疗口腔疾病的药物组合物。
本发明还提供了用于预防或缓解口腔疾病的口腔卫生组合物,其包含3,6- 无水-L-半乳糖。
本发明还提供用于预防或缓解口腔疾病的食物组合物,其包含3,6-无水- L-半乳糖。
3,6-无水-L-半乳糖可以由如下式1表示
<式1>
3,6-无水-L-半乳糖可通过使预处理琼脂糖与琼脂酶、琼脂分解β-半乳糖苷酶和α-新琼二糖水解酶的酶混合物反应来得到,或通过依次使预处理的琼脂糖与琼脂糖酶、琼脂分解β-半乳糖苷酶和α-新琼二糖水解酶反应,然后分离和纯化其反应产物来获得。
所述预处理可以通过令琼脂糖与弱酸在40℃至150℃的温度下反应5分钟至6小时来进行。
所述酶反应可以在20℃至40℃的温度下进行30分钟至7天。
3,6-无水-L-半乳糖可以通过抑制一种或多种口腔微生物的生长和产酸来预防、减轻或治疗口腔疾病,所述口腔微生物选自变形链球菌、口腔链球菌 (Streptococcusoralis)、血链球菌(Streptococcus sanguinis)、具核梭杆菌 (Fusobacteriumnulcleatum)、牙龈卟啉单胞菌(Porphyromonas gingivalis)、粘性放线菌(Actinomycesviscosus)、伴放线放线杆菌(Actinobacillus actinomycetemcomitans),以及白色念珠菌(Candida albicans)。
口腔微生物可以使用选自葡萄糖、蔗糖、果糖和麦芽糖中的一种或多种作为碳源。
口腔疾病可以是选自龋齿、牙龈炎、牙周炎、口腔粘膜溃疡、口臭和口腔干燥症中的任何一种。
有益效果
本发明的3,6-无水-L-半乳糖抑制口腔微生物的生长和口腔微生物消耗碳源产酸,因此具有防龋活性。
因此,可以将3,6-无水-L-半乳糖用于预防,缓解或治疗龋齿,牙龈炎,牙周炎,口腔粘膜溃疡,口臭等口腔疾病的药物,食品,口腔卫生剂等中和由口腔微生物引起的口腔干燥症。
附图说明
图1展示了单一碳源对液体基本培养基或分别补充有10g/L葡萄糖、10g/L 3,6-无水-L-半乳糖、10g/L木糖醇作为碳源的固体基本培养基中的变形链球菌生长的影响 (底物条件:葡萄糖10g/L,3,6-无水-L-半乳糖10g/L,木糖醇 10g/L;稀释倍数:10倍,100倍,1000倍,10000倍和100000倍)。
图2展示了单一碳源对分别补充有10g/L葡萄糖、10g/L 3,6-无水-L-半乳糖和10g/L木糖醇作为碳源的液体基本培养基中变形链球菌生长的影响。
图3a至3f示出了在含有10g/L葡萄糖作为碳源的基本培养基中不同浓度的3,6-无水-L-半乳糖(图3a)和木糖醇(图3b)对变形链球菌的抑制作用,以及对口腔链球菌(图3c:3,6-无水-L-半乳糖;图3d:木糖醇)和血链球菌 (图3e:3,6-无水-L-半乳糖;图3f:木糖醇)的浓度依赖性抑制作用。
图4a至图4c示出了在含有10g/L葡萄糖作为碳源并分别补充有0g/L(图 4a)、5g/L(图4b)和10g/L 3,6-无水-L-半乳糖(图4c)的基本培养基中的分析结果:变形链球菌生长、剩余碳源的浓度以及产生的酸(乳酸、甲酸和乙酸)的量。
图5a和5b示出了在含有10g/L葡萄糖作为碳源并分别补充有20g/L(图 5a)和40g/L(图5b)的木糖醇的基本培养基中的分析结果:变形链球菌生长、剩余碳源的浓度和产生的酸(乳酸、甲酸和乙酸)的量。
图6a至6f示出了对在含有10g/L蔗糖(图6a和6d)、10g/L果糖(图6b 和6e)或10g/L的麦芽糖(图6c和6f)作为碳源并补充有3,6-无水-L-半乳糖 (0-10g/L;图6a至6c)或木糖醇(0-40g/L;6d至6f)的基础培养基中的变形链球菌生长的验证。
具体实施方式
本发明的发明人通过使用三种酶的酶促水解来预处理琼脂糖,产生了半乳糖和3,6-无水-L-半乳糖(均为单糖),然后仅分离和纯化3,6-无水-L-半乳糖,用作变形链球菌的碳源。本发明人通过观察3,6-无水-L-半乳糖是否抑制变异链球菌的生长和产酸,来分析3,6-无水-L-半乳糖的防龋效果,并使用葡萄糖以及木糖醇和可发酵糖(如蔗糖,果糖,麦芽糖等)作为碳源作为对照,进行比较实验。
结果,与葡萄糖条件相比,在3,6-无水-L-半乳糖或木糖醇条件下菌落形成受到抑制;并且在高度稀释的碳源的情况下,在3,6-无水-L-半乳糖条件下无法形成菌落。另外,3,6-无水-L-半乳糖不影响变形链球菌的生长促进。因此,证实3,6-无水-L-半乳糖是变形链球菌的不可发酵糖(如木糖醇),并且具有比木糖醇更强的生长抑制作用。另外,在3,6-无水-L-半乳糖和葡萄糖、蔗糖、果糖、麦芽糖等的碳源混合物的条件下,3,6-无水-L-半乳糖在低浓度的时候抑制变形链球菌的生长,同时在高浓度的3,6-无水-L-半乳糖时变形链球菌不生长。另外,确认了在3,6-无水-L-半乳糖的低浓度(5g/L)下,变异链球菌的滞后期延长且其生长速度降低,生成酸的浓度降低,因此,3,6-无水 -L-半乳糖对变异链球菌的细胞生长和产酸有弱抑制效果,而在3,6-无水-L-半乳糖的高浓度(10g/L)下,完全没有观察到变形链球菌的生长和产酸,并且 3,6-无水-L-半乳糖的浓度没有变化,因此变异链球菌不能使用3,6-无水-L-半乳糖作为碳源。同时,随着木糖醇(已知其为防止龋齿的糖)浓度增加,变形链球菌的生长受到更多抑制。然而,即使在浓度比3,6-无水-L-半乳糖的浓度高得多即40g/L的情况下,也观察到变形链球菌的相当高的生长,并且酸产生没有被完全抑制。这表明即使浓度低于木糖醇,3,6-无水-L-半乳糖也具有更强的抑制变形链球菌生长和产酸的效果。
因此,本发明提供了一种用于预防或治疗口腔疾病的药物组合物,其包含 3,6-无水-L-半乳糖。
本发明还提供用于预防或治疗口腔疾病的药物组合物,其包含3,6-无水- L-半乳糖。
本发明还提供了3,6-无水-L-半乳糖在制备用于预防或治疗口腔疾病的药物组合物中的用途。
3,6-无水-L-半乳糖是构成琼脂的单糖,其是构成红藻生物质的代表性碳水化合物组分,并且可以通过化学合成获得;或通过使预处理的琼脂糖与琼脂糖酶、琼脂分解β-半乳糖苷酶和α-新琼二糖水解酶的酶混合物反应并分离和纯化反应产物获得;或者通过使预处理的琼脂糖与琼脂糖酶、琼脂分解β-半乳糖苷酶和α-新琼二糖水解酶依次反应而获得。
琼脂糖的预处理可以通过琼脂糖与弱酸在40℃至150℃的温度下反应5分钟至6小时来进行。当预处理条件在上述范围内时,由弱酸和残留乙酸引起的琼脂糖过量分解产物可以被最小化。
作为弱酸,可以单独使用乙酸、甲酸、琥珀酸、柠檬酸、苹果酸、马来酸、草酸等,也可以使用其中两种以上,但不特别限定于此。
考虑到生产成本和中和弱酸后产生的盐的分离,弱酸的使用浓度可以是0. 5%(w/v)至60%(w/v)。更特别地,弱酸的浓度可以在20%(w/v)至4 0%(w/v)的范围内。
在上述预处理之后产生的琼脂寡糖与产生二糖的外切型琼脂糖酶反应,以产生琼脂三糖和新琼二糖。
琼脂三糖用琼脂分解β-半乳糖苷酶(分解琼脂三糖非还原末端的β-1,4连接键的酶)处理该酶是,产生3,6-无水-L-半乳糖和新琼二糖,最后,使用α- 新琼二糖水解酶将新琼二糖分解为D-半乳糖和3,6-无水-L-半乳糖,其为单糖。
所述酶促反应可以在20℃至40℃的温度下进行30分钟至7天。
如下进一步详细描述该酶促反应。
首先,作为使琼脂寡糖分解为新琼二糖(二糖)的琼脂糖酶,可以使用使琼脂糖的D-半乳糖与3,6-无水-L-半乳糖之间的β-1,4-糖苷键断裂的酶(以下称为“Aga50D”)。
琼脂酶不仅包括SEQ ID NO:1所示的氨基酸序列,而且还包括作为突变蛋白质的具有琼脂寡聚糖水解活性的蛋白质,例如该酶的一个或多个取代、缺失、易位、添加等,其均在本发明的酶范围内,并且优选包括与SEQ ID N O:1所示的氨基酸序列具有至少80%、至少85%、至少90%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列同源性的氨基酸序列。
琼脂糖酶可以来源于Saccharophagus degradans 2-40T,但不特别限于此。
可以从Saccharophagus degradans 2-40T培养物的上清液中分离和纯化琼脂糖酶;并且可以使用基因重组技术从Saccharophagus degradans以外的菌株生产和分离;或通过人造化学合成方法等。
当使用重组技术时,可以使用用于促进典型重组蛋白的表达的因子,例如抗生素抗性基因,和可用于亲和柱色谱的报道蛋白或肽,并且这样的技术属于本发明的范围内,其可以由本发明所属领域的技术人员容易地实施。例如,琼脂糖酶可以是通过转化可食用菌株例如酵母获得的转化体的培养上清液。在韩国专利申请公开No.2010-0040438(2010年4月20日公开)中可以找到更详细的构建技术。
使用琼脂糖酶进行琼脂寡糖的酶水解反应,可以在20℃至40℃的温度下进行30分钟至7天。更具体地,该反应可以在25℃至35℃的温度下进行1天至 4天。
接着,将琼脂三糖(其为琼脂寡糖)水解为新琼二糖和D-半乳糖的琼脂分解β半乳糖苷酶(也称为“VejABG”)寡聚糖D-半乳糖,有效分解常规方法不水解并仍然存在的琼脂三糖,并且因此可以显著增强由琼脂糖生产3,6-无水- L-半乳糖的效率。
该琼脂分解β-半乳糖苷酶是属于糖苷水解酶(GH)家族2的酶,且当其活性与属于GH家族2的其他β-半乳糖苷酶的活性进行比较时,常规报道的β-半乳糖苷酶不表现出分解琼脂三糖来产生D-半乳糖和新琼二糖的活性,但是来自弧菌(Vibrio)sp.EJY3的琼脂分解β-半乳糖苷酶可以表现出对琼脂三糖的裂解活性。此外,琼脂分解β半乳糖苷酶作用于各种琼脂寡聚糖(n)的非还原性末端寡聚糖,例如琼脂戊糖和琼脂庚糖,从而水解琼脂寡聚糖为D-半乳糖和新琼二糖(n-1)。
可以通过与多肽生成相关的DNA片段来转录和翻译琼脂分解β-半乳糖苷酶,所述多肽包括所述酶的编码区的上游和下游区域以及各编码片段即编码基因之间的插入序列。例如,可以从SEQ ID NO:2所示的序列转录并翻译琼脂分解β-半乳糖苷酶,但不特别限于此。另外,具有作为突变蛋白质的琼脂三糖水解活性的蛋白质,例如所述酶的一个或多个取代、缺失、易位、添加等,也可以在本发明的酶的范围内,并且所述酶优选包括与SEQ ID NO:2所示的氨基酸序列具有至少80%、至少85%、至少90%、至少93%、至少94%、至少 95%、至少96%、至少97%、至少98%、至少98%或至少99%的序列同源性的氨基酸序列。
可以从弧菌sp.EJY3培养物的上清液中分离和纯化琼脂分解β-半乳糖苷酶,并且可以使用基因重组技术从除了弧菌sp.EJY3以外的菌株生产和分离;或通过人工化学合成方法等。当使用重组技术时,可以使用通过大肠杆菌转化获得的转化大肠杆菌的培养上清液或上清液,但不特别限于此。在韩国专利申请公开号2015-0043040(2015年4月22日公开)中可以找到更详细的生产技术。
琼脂三糖和琼脂分解β-半乳糖苷酶的反应可以在20℃至40℃的温度下进行 30分钟至7天。更具体地说,该反应可以在25℃至35℃的温度下进行1天至4 天。
在约30℃至约40℃和约5至约9.6的pH下,琼脂分解β-半乳糖苷酶相对于琼脂三糖可表现出最佳的琼脂分解活性。
最后,能够将新琼二糖分解成3,6-无水-L-半乳糖和D-半乳糖的α-新琼二糖水解酶(也称为“SdNABH”),不仅包括SEQ ID NO:3所示的氨基酸序列,还包括作为突变蛋白质的具有琼脂三糖水解活性的蛋白质,如所述酶的一个或多个取代、缺失、易位、添加等,其在本发明酶的范围内,并且优选与SE Q ID NO:3所示的氨基酸序列具有至少80%、至少85%、至少90%、至少9 3%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的序列同源性的氨基酸序列。
α-新琼二糖水解酶可以来源于Saccharophagus degradans 2-40T,但不特别限于此。
可以从Saccharophagus degradans 2-40T的培养物上清液或上清液中分离和纯化α-新琼二糖水解酶,并且可以使用基因重组技术从Saccharophagus degr adans以外的菌株生产和分离α-新琼二糖水解酶;或通过人工化学合成方法等。当使用重组技术时,可以使用通过大肠杆菌转化获得的转化大肠杆菌的培养上清液或上清液,但不特别限于此。在韩国专利申请公开号2013-0085017(2 013年7月26日公开)中可以找到更详细的生产技术。
新琼二糖和α-新琼二糖水解酶之间的反应可以在20℃至40℃的温度下进行 30分钟至7天。更具体地说,该反应可以在25℃至35℃的温度下进行1天至4 天。
可以依次进行硅胶色谱法(其为吸附色谱法)和Bio-gel P2色谱法(其为凝胶渗透色谱法),对新琼二糖的分解产物进行分离纯化,从而分离和纯化大约95%高纯度的3,6-无水-L-半乳糖。
在本说明书中,术语“蛋白质”和“多肽”可以互换使用。
在本发明中,相对于另一个序列具有特定百分比(例如,80%,85%,9 0%,95%或99%)的序列同源性的多肽,意味着在两个序列相互比对的序列比较时,两个序列具有以上述百分比表示的相同的氨基酸残基。比对和百分比同源性或同一性,可以使用本领域已知的任何合适的软件程序来确定,例如,如Current Protocols in Molecular Biology(F.M.Ausubel,et al.,(eds)198 7Supplement 30section 7.7.18)中所述。优选的程序包括GCG Pileup程序,例如FASTA(Pearson,et al.,1988Proc.Natl.Acad.Sci.USA 85:2444-2 448)和BLAST(BLAST Manual,Altschul et al.,Natl.Cent.Biotechnol.I nf.,NatlLib.Med.(NCIB NLM NIH),Bethesda,Md.,and Altschul et al.,1997NAR25:3389-3402)。另一个优选的比对程序是ALIGN Plus(Scie ntific and Educational Software,PA),其优选使用基本参数。可以在此使用的另一个优选的序列软件程序是在序列软件包(Sequence Software Package) 版本6.0(Genetics Computer Group,University ofWisconsin,Madison WI) 中可用的TFASTA数据搜索程序。
在本发明中,术语“重组”与细胞、核酸、蛋白质或载体一起使用时,表示通过引入异源核酸或蛋白质或改变先天性核酸或蛋白来修饰所述细胞、核酸、蛋白质或载体酸或蛋白质,或者细胞来源于如此修饰的细胞。也就是说,重组细胞,例如表达在原始非重组形式的细胞中未发现的基因,或表达在表达时异常表达或完全不表达的原始基因。
在本说明书中,术语“核酸”涵盖所有类型的单链或双链DNA、RNA及其化学变体。术语“核酸”和“多核苷酸”在本文中可互换使用。由于遗传密码是简并的,因此可以使用一个或多个密码子来编码特定的氨基酸,本发明涵盖编码某些氨基酸序列的多核苷酸。
术语“引入”在用于描述将核酸序列插入细胞中时,是指“转染”、“转化”或“转导”,并且涵盖将核酸序列整合到真核或原核细胞中。在这种情况下,核酸序列被整合到细胞的基因组(例如,染色体、质粒、质体或线粒体DNA)中,并转化成自主复制子或暂时表达。
硅胶色谱法(其为吸附色谱法)和Bio-gel P2色谱法(其为凝胶渗透色谱法),可用于从上述酶反应后获得的反应产物中仅分离和纯化3,6-无水-L-半乳糖,并且可以通过气相色谱/质谱(GC/MS)对3,6-无水-L-半乳糖进行定量。
本发明的药物组合物可以进一步包含药学上可接受的载体。
药学上可接受的载体包括在医学领域中常用的载体和赋形剂,其实例包括但不限于离子交换树脂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(例如人血清白蛋白)、缓冲物质(例如磷酸盐、甘氨酸、山梨酸、山梨酸钾,和饱和植物脂肪酸的部分甘油酯混合物)、水、盐或电解质(例如硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠和锌盐)、胶体二氧化硅、三硅酸镁、聚乙烯吡咯烷酮、基于纤维素的基质、聚乙二醇、羧甲基纤维素钠、多芳基化合物、蜡、聚乙二醇和羊毛脂。
另外,除了上述成分之外,本发明的药物组合物还可以包含润滑剂、润湿剂、乳化剂、悬浮剂、防腐剂等。
在一个实施方案中,根据本发明所述的药物组合物可以以适合于口腔或胃肠外给药的各种制剂的形式配制。
口腔给药制剂的非限制性实例包括锭剂、丸剂、片剂、水性混悬剂、油性混悬剂、制备的粉剂、颗粒剂、乳剂、硬胶囊剂、软胶囊剂、糖浆剂和酏剂。
为了配制用于口服给药的本发明的药物组合物,可以使用粘合剂如乳糖、蔗糖、山梨糖醇、甘露醇、淀粉、支链淀粉、纤维素、明胶等;诸如磷酸二钙等的赋形剂;崩解剂如玉米淀粉、甘薯淀粉等;润滑剂如硬脂酸镁、硬脂酸钙、富马酸硬脂酯钠、聚乙二醇蜡等;或使用类似物,也可以使用甜味剂、香料、糖浆等。
此外,在胶囊的情况下,除了上述物质之外,还可以使用液体载体如脂肪油。
用于肠胃外给药的制剂的非限制性实例包括注射剂、栓剂、呼吸吸入粉剂、喷雾用气雾剂、口腔喷雾剂、口腔清洗剂、牙膏、软膏、施用粉剂、油剂和乳膏剂。
为了配制用于肠胃外给药的本发明的药物组合物,可以使用无菌水溶液、非水溶剂、悬浮液、乳剂、冷冻干燥制剂、外用药剂等;且作为非水溶剂和悬浮液,可以使用丙二醇、聚乙二醇、植物油如橄榄油、可注射酯如油酸乙酯等。
此外,更具体地说,当本发明的药物组合物配制成注射剂时,本发明的组合物可以在水中与稳定剂或缓冲剂混合以制备成溶液或悬浮液,然后配制成单位剂量形式如安瓿或小瓶。此外,当本发明的药物组合物配制成气溶胶时,可以将推进剂等与添加剂混合以分散水分散性浓缩物或湿粉末。
另外,将本发明的药物组合物配制成软膏剂、乳膏剂等时,可以使用如动物油、植物油、蜡、石蜡、淀粉、黄蓍胶、纤维素衍生物、聚乙二醇、硅酮、膨润土、二氧化硅、滑石、氧化锌等载体来配制药物组合物。
本发明的药学部位的药学有效量和有效剂量可以根据配制方法、给药方式、给药方案和/或给药途径等而变化,并且可以根据各种因素而变化,所述因素包括通过施用药物组合物实现的反应的类型和程度,施用组合物的个体的类型、年龄、体重、一般健康状况、疾病的症状或严重程度、性别、饮食、排泄,在相应个体中同时使用或不同时间使用的药物,其他组合物的成分等等,以及医学领域中众所周知的类似因素。本领域普通技术人员可以容易地确定和规定有效剂量以进行期望的治疗。本发明的药物组合物可以每天给药一次或数次。因此,剂量并不意在以任何方式限制本发明的范围。
本发明药物组合物的给药途径和给药方式可以彼此独立,给药方法没有特别限制,且给药途径和给药方式可以是任意给药途径和给药途径,只要它们使药物组合物能够到达相应的部位。药物组合物可以口服或胃肠外给药。
非肠道给药可以是例如静脉内给药、腹腔内给药、肌肉内给药、经皮给药、皮下给药等,并且组合物可以施用于或喷洒于疾病部位,或吸入,但不限于此。
本发明的药物组合物可优选口服或经注射给药。
本文使用的术语“预防”是指通过将本发明的药物组合物给予个体来抑制或延缓口腔疾病发作的所有行为。
本文使用的术语“治疗”意指通过将本发明的药物组合物给予个体以减轻或有益地改变口腔疾病症状的所有行为。
在本发明中,口腔疾病是涵盖口腔中发生的所有疾病而不管其症状如何的概念,并且可以包括例如主要来源于口腔微生物的口腔疾病,所述口腔微生物例如变形链球菌、口腔链球菌、血链球菌、具核梭杆菌、牙龈卟啉单胞菌、粘性放线菌、伴放线放线杆菌、白色念珠菌等,或由口腔免疫退化引起的口腔疾病。口腔疾病的非限制性实例包括龋齿、牙龈炎、牙周炎、口腔粘膜溃疡、口臭和口腔干燥症。
本发明还提供了一种治疗口腔疾病的方法,包括向有需要的对象施用药学有效量的用于预防或治疗口腔疾病的药物组合物。
如本文所用,术语“对象”包括含哺乳动物在内的所有动物,哺乳动物包括小鼠、家畜、人类等。
在本发明的口腔疾病的治疗方法中,药物组合物的剂量、给药途径、给药方式等的描述与本发明的药物组合物相关描述相同。
术语“有效量”是指至少部分实现期望的免疫应答或延迟或完全停止待治疗的特定疾病的发作或进展所需的量。该量取决于待治疗对象的健康和身体状况、受治疗者的分类群、对象的免疫系统合成抗体的能力、期望的保护程度、疫苗制剂、医疗条件评估和其他相关因素。该量将会在通常的尝试中可以测量的相对广泛的范围内。
本发明还涉及用于预防或缓解口腔疾病的口腔卫生组合物,其包含3,6-无水-L-半乳糖。
在本发明中,3,6-无水-L-半乳糖的描述,口腔疾病和预防与以上关于本发明的药物组合物所述的相同。
在本发明中,口腔卫生组合物包括可用于口腔卫生的所有类型和制剂。例如,口腔卫生组合物可包含在选自以下的口腔产品中:牙膏、口腔清洗剂、口腔清洁剂、口腔喷雾剂、口腔软膏、口腔保护漆,口腔漱口液和口香糖。
本发明的口腔卫生组合物,可以配制成适合于口服给药的各种制剂或待使用的肠胃外给药的形式,并且与其相关的描述与以上关于本发明的药物组合物所述的相同。
如本文所用的术语“减轻”意指至少减少与正在治疗的病症相关的参数(例如症状)的程度的所有动作。
本发明还涉及用于预防或缓解口腔疾病的食物组合物,其包含3,6-无水- L-半乳糖。
在本发明中,对3,6-无水-L-半乳糖、口腔疾病、预防和缓解的描述与以上关于本发明的药物组合物所述的相同。
本发明的食品组合物没有特别限制,并且包括健康功能食品组合物。
术语“保健功能食品”是指通过将3,6-无水-L-半乳糖添加到诸如饮料、茶、调味品、口香糖、糖果等的食物物质中而制备的食物,或以胶囊、粉末、悬浮液等形式制备的食物,并且是指当摄入时赋予特定健康效果的食物。
当将本发明的健康功能食品组合物用作食品添加剂时,所述组合物可以直接添加或可以与其他食品或食品成分组合使用,并且可以根据一般方法适当地使用。
食物的种类没有特别限定,包含所有通常意义上的食物。该材料适用的食品的非限制性实例包括肉、香肠、面包、巧克力、糖果、点心、糕饼、披萨、拉面、其他面条、口香糖、乳制品(包括冰淇淋)、各种汤、饮料、茶、酒水、含酒精饮料和维生素复合物。
当本发明的健康功能食品组合物是饮料组合物时,饮料组合物可以如普通饮料般包括各种增味剂、天然碳水化合物等作为附加成分。天然碳水化合物的非限制性实例包括单糖如葡萄糖和果糖;二糖如麦芽糖和蔗糖;天然甜味剂如糊精和环糊精;和合成甜味剂如糖精和阿斯巴甜。所添加的附加成分的比例可以由本领域普通技术人员适当选择和确定。
另外,本发明的健康功能食品组合物可以包含各种营养素、维生素、电解质、调味剂、着色剂、果胶酸及其盐、藻酸及其盐、有机酸、保护性胶体增稠剂、pH调节剂、稳定剂、防腐剂、甘油、醇类、碳酸饮料中使用的碳酸化剂等。另外,本发明的健康功能食品组合物可以包括用于制备天然果汁、果汁饮料、蔬菜饮料等的果肉。这些成分可以单独使用或者可以使用这些成分中的两种或更多种的组合。这些添加剂的比例也可以由本领域普通技术人员适当选择。
实施例
在下文中,将参照以下实施例详细描述本发明。提供这些实施例仅用于说明的目的,并不意图限制本发明的范围。
<实施例1>琼脂糖的预处理
琼脂糖用3N乙酸溶解至浓度为7%(w/v),使用微波消化器在130℃下反应10分钟,然后将反应产物用1.5L的94%乙醇洗涤两次以去除残留的乙酸和过量的分解产物。将反应产物冷冻干燥24小时以获得粉末型琼脂寡聚糖。
<实施例2>通过琼脂寡聚糖的酶水解生成3,6-无水-L-半乳糖
使实施例1中制作的酸水解物(琼脂寡聚糖)与外切型β-琼脂糖酶(即Ag a50D)(参照2010年4月20日公开的韩国专利申请公开第2010-0040438号) 反应,以令其分解为D-半乳糖和3,6-无水-L-半乳糖(均为单糖),且作为反应产物生成琼脂三糖和新琼二糖。
在Aga50D反应完成后,将琼脂三糖用酶VejABG(可分解非还原性末端的β-1,4键的酶)(参见2015年4月22日公开的韩国专利申请公开号2015-004 3040)处理,以产生3,6-无水-L-半乳糖和新琼二糖。
为了从新琼二糖产生D-半乳糖和3,6-无水-L-半乳糖(单糖),使VejAB G的反应产物与SdNABH酶(参见2013年7月26日公布的韩国专利申请公开号2013-0085017)反应。
在大肠杆菌BL21(DE3)中表达三种重组酶并用HisTrap柱纯化。通过向 200mL的50mM Tris-HCl缓冲液(pH7.4)中添加5%(w/v)琼脂寡聚糖,并使得到的溶液在30℃下依次与Aga50D、VejABG和SdNABH反应而进行酶促反应3天。在酶促反应中使用酶(即Aga50D),VejABG和SdNABH,其量分别为20mg、1mg和2mg,总量为200mL。
<实施例3>使用硅胶色谱和Bio-Gel P2色谱进行分离和纯化
进行色谱层析以从实施例2中制备的反应产物中仅分离和纯化3,6-无水-L -半乳糖。将反应产物吸附到硅藻土上以形成粉末形式的样品,然后进行硅胶色谱层析(吸附色谱法)。使用将比例为78:20:2(v/v/v)的氯仿、甲醇和水混合而获得的溶剂作为流动相,并且作为流动相的溶剂的总体积为4L。一部分的体积为20mL,并且通过TLC分析由总共200个部分组成的样品,由此在其中收集含有3,6-无水-L-半乳糖的部分,并且使用旋转式真空浓缩器从中除去有机溶剂。
<实施例4>使用GC/MS分析定性和定量3,6-无水-L-半乳糖
通过GC/MS分析对实施例3中制得的高纯度3,6-无水-L-半乳糖进行定量。GC/MS分析的衍生程序如下。将纯化的样品在真空浓缩器中干燥,加入 10μl含4%(w/v)O-甲基羟胺盐酸盐的吡啶,然后在30℃反应120分钟。然后,向所得产物中加入45μl N-甲基-N-(三甲基硅烷基)三氟乙酰胺,并在3 7℃下反应30分钟。GC/MS分析的设备条件如下。用于分析的色谱柱是DB5- MS毛细管色谱柱。首先,在GC色谱柱温度条件下,将样品保持在100℃的温度3.5分钟,加热至160℃并保持20分钟。之后,将样品加热至200℃,保持1 5分钟,最后加热至280℃并保持5分钟。以9.6的分流比分析1微升样品。
<实施例5>细胞培养条件
本实验中使用的菌株是变形链球菌ATCC 25175、口腔链球菌和血链球菌。代表性地,变形链球菌ATCC 25175在脑心浸液(BHI)培养基中预培养 12小时,并用2mM磷酸钾缓冲液洗涤两次,然后在参考先前报道的文献制备的基本培养基中(Fujiwara et al(1978)Arch Oral Biol.23,601-602)在37℃和 180rpm下进行主培养。基本培养基的组成包括:5mL的50mM Tris-HCl缓冲液(pH7.4)、10mg L-谷氨酸、1mg半胱氨酸盐酸盐、4.5mg L-亮氨酸、5mg 氯化铵、17.5mg磷酸氢钾、7.5mg磷酸二氢钾、21mg碳酸盐、6mg七水合硫酸镁、0.1mg氯化锰四水合物、3mg丙酮酸钠、5μg核黄素、2.5μg硫胺素盐酸盐、0.5μg生物素、5μg烟酸、0.5μg对氨基苯甲酸、2.5μg泛酸钙和5μg吡哆醇盐酸盐。
<实施例6>在单一碳源条件下对变形链球菌生长的抑制作用
为了验证在单一碳源条件下抑制变形链球菌生长的效果,将变形链球菌在固体基本培养基和含有3,6-无水-L-半乳糖、葡萄糖和木糖醇分别10g/L的液体基本培养基中培养。预培养的变形链球菌在2mM磷酸钾缓冲液中稀释10 倍、102倍,103倍,104倍或105倍,接着在固体培养基中点样并培养30小时。
如图1所示,与葡萄糖条件相比,在3,6-无水-L-半乳糖和葡萄糖的条件下菌落的形成受到抑制。特别是,在3,6-无水-L-半乳糖的情况下,在104倍和105倍的稀释条件下完全没有观察到菌落。
另外,通过在含有10g/L各碳源(3,6-无水-L-半乳糖、葡萄糖和木糖醇) 的液体基本培养基中培养变形链球菌,观察到抑制细胞生长的效果。使用实施例5的方法将细菌主要培养35小时,通过每5小时测量600nm波长的培养物的吸光度来测量细胞生长的程度。
如图2所示,在葡萄糖条件下,在进入固定相后20小时测量变形链球菌的干细胞重量(DCW)为4.05g/L;在木糖醇的情况下,其干细胞重量(DCW) 显著降低并且测量其浓度为1.46g/L;对于3,6-无水-L-半乳糖,完全没有观察到变异链球菌的生长。因此,证实3,6-无水-L-半乳糖类似于木糖醇,是变形链球菌的不可发酵糖,并且具有比木糖醇更强的生长抑制作用。
<实施例7>在混合碳源条件下抑制变形链球菌生长的效果
使用实施例5的方法,在96孔板中于200μl含有根据浓度为10g/L葡萄糖和3,6-无水-L-半乳糖或者木糖醇的液体基本培养基中培养少量变形链球菌、口腔链球菌和血链球菌,并随着时间测量600nm波长处的吸光度来观察其生长抑制效果。3,6-无水-L-半乳糖的处理浓度为0g/L至20g/L之间,木糖醇的处理浓度为0g/L至60g/L之间。
如图3a和3b所示,在1.3g/L和2.5g/L的3,6-无水-L-半乳糖下,变形链球菌的生长没有显著变化,在5g/L和10g/L3,6-无水-L-半乳糖时观察到显著的细胞生长抑制效果,20g/L的3,6-无水-L-半乳糖下没有发生细胞生长。在木糖醇的情况下,随着木糖醇处理浓度的增加,变异链球菌的生长受到抑制,但即使在显著高浓度即40g/L下,也观察到相当多的细胞生长。由此证实,即使在浓度低于木糖醇浓度的情况下,3,6-无水-L-半乳糖也具有更强的细胞生长抑制效果。
另外,即使对于口腔链球菌和血链球菌,3,6-无水-L-半乳糖随着其浓度增加也显示更大的细胞生长抑制效果(参见图3c和3d)。高浓度下的木糖醇 (即60g/L的木糖醇)对血链球菌显示出强烈的抑制作用(参见图3e和3f)。
<实施例8>在混合碳源条件下抑制变形链球菌生长和产酸的效果
为了验证3,6-无水-L-半乳糖或木糖醇对变异链球菌生长和产酸的影响,使用实施例5的方法,在5mL含有10g/L葡萄糖和0g/L、5g/L或10g/L的 3,6-无水-L-半乳糖或20g/L或40g/L的木糖醇的液体基本培养基中培养变形链球菌35小时,每5小时使用吸光度测量细胞生长,并通过HPLC测量细胞外底物浓度和产酸量。将仅添加葡萄糖的条件设定为阴性对照,将添加葡萄糖和木糖醇的条件设定为阳性对照。用于分析的柱是Aminex HPX-87H,并且作为流动相的0.01N硫酸以0.5mL/min的速率和65℃流动。
如图4a所示,在仅含葡萄糖作为碳源的条件下,测得干细胞重量为3.34g/ L,非生长速率为0.086h-1。葡萄糖在30小时内全部消耗,测得乳酸浓度为5.2 4g/L。另外,少量产生的甲酸和乙酸的浓度分别为0.82g/L和0.71g/L。
在10g/L的葡萄糖中含有5g/L的3,6-无水-L-半乳糖的条件下,变形链球菌的最终干细胞重量浓度没有降低,但滞后期延长。从该结果,即非生长速度降低至0.075h-1和生成的酸浓度降低,证实混合碳源具有抑制细胞生长和酸生成的弱作用(参见图4b)。
在10g/L的葡萄糖中含有10g/L的3,6-无水-L-半乳糖的条件下,完全没有观察到变异链球菌的生长和产酸。另外,在这两种条件下,3,6-无水-L-半乳糖的浓度没有变化,从中证实变异链球菌不能使用3,6-无水-L-半乳糖作为碳源(参见图4c)。
另外,在10g/L葡萄糖中含有20g/L木糖醇的条件(阳性对照)下,最终的干细胞重量为1.42g/L,这表明抑制了变异链球菌的生长,但是与阴性对照的非生长率(即0.086h-1)相比,其非生长率(即0.087h-1)没有变化。消耗总共 5.28g/L的葡萄糖,产生的乳酸和乙酸的浓度分别为3.67g/L和0.55g/L,其低于阴性对照(参见图5a)。在包含在高浓度的条件下(即含40g/L木糖醇的10g/ L葡萄糖),观察到比20g/L木糖醇更长的滞后期,并且消耗的葡萄糖的浓度相似地测量为5.42g/L。由于葡萄糖的消耗,产生的乳酸和乙酸的浓度分别测量为3.67g/L和0.55g/L(参见图5b)。
另外,在变异链球菌发酵过程中,木糖醇浓度一直保持不变,从中证实木糖醇对变形链球菌表现出非发酵性质。
从这些结果可以确认,木糖醇虽然已知是防止龋齿的糖,但即使在远高出 3,6-无水-L-半乳糖的浓度下,(即40g/L),也不会完全抑制变形链球菌的生长和酸产生(如在10g/L的3,6-无水-L-半乳糖的条件下的结果所示)。这表明3,6-无水-L-半乳糖即使在低于木糖醇的浓度下也具有更强的抑制变形链球菌生长和产酸的效果。
<实施例9>在葡萄糖以外的各种糖的条件下AHG和木糖醇对变形链球菌的抑制作用
在葡萄糖以外的可发酵糖例如蔗糖、果糖和麦芽糖的条件下,研究了AHG 和木糖醇对变形链球菌的抑制作用。在含有0g/L至10g/L AHG或0g/L至40g/ L木糖醇的96孔板中,在分别含有10g/L蔗糖,果糖和麦芽糖的规定基本培养基中,进行了实验。
在蔗糖、果糖和麦芽糖的条件下,变形链球菌的生长受到AHG的抑制。在蔗糖条件下,在1.3g/L的AHG的情况下未观察到细胞生长的显著降低,但是随着AHG的浓度增加(2.5g/L AHG),细胞生长受到显著抑制。在高浓度下,即5g/L和10g/L的AHG,变形链球菌的细胞生长被完全抑制(参见图6 a)。即使在果糖和麦芽糖条件下,也观察到类似的AHG对变形链球菌细胞生长抑制作用的结果(参见图6b和6c)。
在AHG和蔗糖2.5g/L的条件下的非生长速率(0.040h-1)低于40g/L木糖醇和蔗糖的条件下的非生长速率(0.061h-1)(参见图6a和6d)。此外,在1.3g/L AHG和果糖的条件下的非生长速率(0.052h-1)远低于在40g/L木糖醇和果糖的条件下的非生长速率(0.040h-1)(参见图6b和6e)。同样,在2.5g/L AHG和麦芽糖条件下的非生长速率(0.031h-1)远低于在40g/L木糖醇和麦芽糖的条件下的非生长速率(0.045h-1)(参见图6c和6f)。
在木糖醇和蔗糖的条件下,随着木糖醇浓度增加,变异链球菌的细胞生长降低(参见图6d)。然而,木糖醇即使在最大浓度即本实验中使用的40g/L时也不能完全抑制变形链球菌的细胞生长(参见图6d)。相反,在蔗糖条件下,变形链球菌的细胞生长在低得多的浓度即5g/L AHG下被完全抑制(参见图6 a)。
从以上结果可以看出,AHG不仅抑制葡萄糖代谢,还抑制其他可发酵糖即蔗糖、果糖和麦芽糖的代谢。不寻常的是,在四种可发酵糖中,AHG的抑制效果在果糖条件下最显著。这似乎是由于AHG和果糖之间的结构相似性比其他糖(即具有呋喃糖环的酮糖)具有更大的结构相似性。
【工业实用性】
本发明可以用作预防、缓解或治疗口腔疾病的领域中的药物、食品、口腔卫生剂等。
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Claims (6)
1.3,6-无水-L-半乳糖在制备用于治疗口腔疾病的口服药物上的用途,其特征在于,所述口腔疾病是选自由一种或多种口腔微生物引起的龋齿、牙龈炎、牙周炎、口腔粘膜溃疡、口臭和口腔干燥症中的任何一种,所述口腔微生物选自:变异链球菌、口腔链球菌和血链球菌。
2.根据权利要求1所述的用途,其中所述口腔微生物使用选自葡萄糖、蔗糖、果糖和麦芽糖的一种或多种碳源。
3.3,6-无水-L-半乳糖在制备用于缓解口腔疾病的口腔卫生产品上的用途,其特征在于,所述口腔疾病是选自由一种或多种口腔微生物引起的龋齿、牙龈炎、牙周炎、口腔粘膜溃疡、口臭和口腔干燥症中的任何一种,所述口腔微生物选自变异链球菌、口腔链球菌和血链球菌。
4.根据权利要求3所述的用途,其中所述口腔卫生产品为以下形式:牙膏、口腔喷雾剂、口腔软膏、口腔保护漆或口腔漱口液。
5.根据权利要求3所述的用途,其中所述口腔卫生产品为口腔清洗剂。
6.根据权利要求3所述的用途,其中所述口腔卫生产品为口腔清洁剂。
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KR101316084B1 (ko) | 2011-01-24 | 2013-11-13 | 고려대학교 산학협력단 | 3,6-안하이드로-l-갈락토오스에 작용하는 신규한 3,6-안하이드로-l-갈락토오스 데히드로게나제와 이 효소를 이용한 3,6-안하이드로 갈락토닉산의 생산 |
CA2854548C (en) | 2011-11-07 | 2018-11-20 | Wm. Wrigley Jr. Company | Chewing gum base containing substituted polysaccharides and chewing gum products made there from |
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KR101521711B1 (ko) | 2013-10-14 | 2015-05-19 | 고려대학교 산학협력단 | 신규한 아가로올리고당 분해효소 및 이를 이용한 아가로오스로부터 3,6-안하이드로-l-갈락토오스와 갈락토오스의 생산방법 |
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WO2014142507A1 (ko) * | 2013-03-15 | 2014-09-18 | 고려대학교 산학협력단 | 3,6-안하이드로-l-갈락토오스를 포함하는 대장암 예방 또는 치료용 조성물 |
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KR101743421B1 (ko) | 2017-06-05 |
US10548828B2 (en) | 2020-02-04 |
US20180280272A1 (en) | 2018-10-04 |
WO2017065546A1 (ko) | 2017-04-20 |
CN108883121A (zh) | 2018-11-23 |
KR20170044945A (ko) | 2017-04-26 |
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