Division of Oral Maxillofacial Surgery, Pathology & Anesthesiology, College of Dentistry, 2191B Postle Hall, 305 W. 12th Ave, The Ohio State University, Columbus, OH 43210, USA.
Cancer Prev Res (Phila). 2011 Aug;4(8):1209-21. doi: 10.1158/1940-6207.CAPR-11-0040. Epub 2011 May 10.
Our oral cancer chemoprevention trial data implied that patient-specific differences in local retention and metabolism of freeze-dried components of black raspberries (BRB) affected therapeutic responsiveness. Subsequent studies have confirmed that anthocyanins are key contributors to BRB's chemopreventive effects. Consequently, functional assays, immunoblotting, and immunohistochemical analyses to evaluate levels and distribution of BRB anthocyanin-relevant metabolic enzymes in human oral tissues were conducted. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) analyses of time course saliva samples collected following BRB rinses were conducted to assess local pharmacokinetics and compare the capacities of three different BRB rinse formulations to provide sustained intraoral levels of anthocyanins. Protein profiles showed the presence of key metabolic enzymes in all 15 oral mucosal tissues evaluated, whereas immunohistochemistry confirmed these enzymes were distributed within surface oral epithelia and terminal salivary ducts. β-Glucosidase assays confirmed that whole and microflora-reduced saliva can deglycosylate BRB anthocyanins, enabling generation of the bioactive aglycone, cyanidin. LC/MS-MS analyses showed retention of parent anthocyanins and their functional, stable metabolite, protocatechuic acid, in saliva for up to 4 hours after rinsing. Furthermore, postrinse saliva samples contained glucuronidated anthocyanin conjugates, consistent with intracellular uptake and phase II conversion of BRB anthocyanins into forms amenable to local recycling. Our data show that comparable to the small intestine, the requisite hydrolytic, phase II and efflux transporting enzymes necessary for local enteric recycling are present and functional in human oral mucosa. Notably, interpatient differences in anthocyanin bioactivation and capacities for enteric recycling would impact treatment as retention of bioactivated chemopreventives at the target site would sustain therapeutic effectiveness.
我们的口腔癌化学预防试验数据表明，患者对冷冻干燥黑莓（BRB）成分的局部保留和代谢的个体差异影响治疗反应。随后的研究证实，花色苷是 BRB 化学预防作用的关键贡献者。因此，进行了功能测定、免疫印迹和免疫组织化学分析，以评估人类口腔组织中 BRB 花色苷相关代谢酶的水平和分布。对 BRB 冲洗后收集的时间过程唾液样本进行液相色谱/串联质谱（LC/MS-MS）分析，以评估局部药代动力学，并比较三种不同 BRB 冲洗配方提供持续口腔内花色苷水平的能力。蛋白质谱显示，在评估的所有 15 个口腔黏膜组织中均存在关键代谢酶，而免疫组织化学证实这些酶分布在口腔表面上皮和末端唾液导管内。β-葡萄糖苷酶测定法证实，全唾液和减少微生物的唾液可以使 BRB 花色苷去糖基化，从而生成生物活性的糖苷元，矢车菊素。LC/MS-MS 分析表明，冲洗后 4 小时内，原花青素及其功能稳定的代谢物原儿茶酸仍保留在唾液中。此外，冲洗后唾液样本中含有葡萄糖醛酸化的花色苷缀合物，与 BRB 花色苷的细胞内摄取和相 II 转化为可局部再循环的形式一致。我们的数据表明，与小肠相似，人类口腔黏膜中存在并具有功能性，需要局部肠内再循环的必需水解、相 II 和外排转运酶。值得注意的是，花色苷生物活化和肠内再循环能力的个体间差异会影响治疗效果，因为在靶部位保留生物活化的化学预防剂将维持治疗效果。
