Supplementary Material for: Systematic Screening of Plant Extracts from the Brazilian Pantanal with Antimicrobial Activity against Bacteria with Cariogenic Relevance

This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants - (1) <i>Jatropha weddelliana</i>, (2) <i>Attalea phalerata</i>, (3) <i>Buchenavia tomentosa</i>, (4) <i>Croton doctoris</i>, (5) <i>Mouriri elliptica</i>, (6) <i>Mascagnia benthamiana</i>, (7) <i>Senna aculeata</i>, (8) <i>Unonopsis guatterioides</i>, (9) <i>Allagoptera leucocalyx</i> and (10) <i>Bactris glaucescens</i> - using different extraction methods - (A) 70° ethanol 72 h/25°C, (B) water 5 min/100°C, (C) water 1 h/55°C, (D) water 72 h/25°C, (E) hexane 72 h/25°C and (F) 90° ethanol 72 h/25°C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against <i>Actinomyces naeslundii</i> ATCC 19039, <i>Lactobacillus acidophilus</i> ATCC 4356, <i>Streptococcus gordonii</i> ATCC 10558, <i>Streptococcus mutans</i> ATCC 35688, <i>Streptococcus sanguinis</i> ATCC 10556, <i>Streptococcus sobrinus</i> ATCC 33478 and <i>Streptococcus mitis</i> ATCC 9811. The active extracts were tested to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except <i>S. gordonii</i> and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from <i>C. doctoris</i> were the most active against bacteria involved in dental caries disease.

本研究提出了一种针对植物提取物抗致龋相关细菌抗菌潜力的生物勘探方法。本研究从10种植物中制备了60份提取物——(1) 麻疯树（<i>Jatropha weddelliana</i>）、(2) 鳞苞椰（<i>Attalea phalerata</i>）、(3) 绒毛比乌木（<i>Buchenavia tomentosa</i>）、(4) 克罗顿木（<i>Croton doctoris</i>）、(5) 椭圆叶莫利里木（<i>Mouriri elliptica</i>）、(6) 本特汉姆马斯卡尼亚藤（<i>Mascagnia benthamiana</i>）、(7) 刺决明（<i>Senna aculeata</i>）、(8) 桂木状翁诺普西斯藤（<i>Unonopsis guatterioides</i>）、(9) 白鞘银叶椰子（<i>Allagoptera leucocalyx</i>）、(10) 淡色贝叶棕（<i>Bactris glaucescens</i>）——并采用6种不同的提取工艺：(A) 70%乙醇、72 h/25℃，(B) 蒸馏水、5 min/100℃，(C) 蒸馏水、1 h/55℃，(D) 蒸馏水、72 h/25℃，(E) 正己烷、72 h/25℃，(F) 90%乙醇、72 h/25℃。采用琼脂孔扩散试验，以50 mg/ml的浓度，针对7种致龋相关细菌进行抗菌活性筛选，受试菌株包括：<i>Actinomyces naeslundii</i> ATCC 19039、<i>Lactobacillus acidophilus</i> ATCC 4356、<i>Streptococcus gordonii</i> ATCC 10558、<i>Streptococcus mutans</i> ATCC 35688、<i>Streptococcus sanguinis</i> ATCC 10556、<i>Streptococcus sobrinus</i> ATCC 33478以及<i>Streptococcus mitis</i> ATCC 9811。对具有抗菌活性的提取物，进一步测定其最低抑菌浓度（minimum inhibitory concentration, MIC）、最低杀菌浓度（minimum bactericidal concentration, MBC）、细胞毒性，并开展化学表征。最终有47份提取物（占比78%）对至少一种受试微生物具有抗菌活性。其中，提取物4A对除<i>Streptococcus gordonii</i>以外的所有受试菌株均表现出最低的MIC与MBC值，且其MIC浓度下的提取物无细胞毒性；而浓缩提取物则呈现轻度细胞毒性。电喷雾串联质谱分析结果显示，该提取物的成分分子量与萜类及酚类化合物的分子量相吻合。整体而言，采用提取工艺A、B、C所得到的提取物抗菌效果最优。本研究证实了多种植物具备抗菌活性，其中，来自<i>Croton doctoris</i>的提取物对致龋相关细菌的抗菌活性最为突出。