Antimicrobial Activity of Serbian Propolis Evaluated by Means of MIC, HPTLC, Bioautography and Chemometrics

New information has come to light about the biological activity of propolis and the quality of natural products which requires a rapid and reliable assessment method such as High Performance Thin-Layer Chromatography (HPTLC) fingerprinting. This study investigates chromatographic and chemometric approaches for determining the antimicrobial activity of propolis of Serbian origin against various bacterial species. A linear multivariate calibration technique, using Partial Least Squares, was used to extract the relevant information from the chromatographic fingerprints, i.e. to indicate peaks which represent phenolic compounds that are potentially responsible for the antimicrobial capacity of the samples. In addition, direct bioautography was performed to localize the antibacterial activity on chromatograms. The biological activity of the propolis samples against various bacterial species was determined by a minimum inhibitory concentration assay, confirming their affiliation with the European poplar type of propolis and revealing the existence of two types (blue and orange) according to botanical origin. The strongest antibacterial activity was exhibited by sample 26 against Staphylococcus aureus, with a MIC value of 0.5 mg/mL, and Listeria monocytogenes, with a MIC as low as 0.1 mg/mL, which was also the lowest effective concentration observed in our study. Generally, the orange type of propolis shows higher antimicrobial activity compared to the blue type. PLS modelling was performed on the HPTLC data set and the resulting models might qualitatively indicate compounds that play an important role in the activity exhibited by the propolis samples. The most relevant peaks influencing the antimicrobial activity of propolis against all bacterial strains were phenolic compounds at RF values of 0.37, 0.40, 0.45, 0.51, 0.60 and 0.70. The knowledge gained through this study could be important for attributing the antimicrobial activity of propolis to specific chemical compounds, as well as the verification of HPTLC fingerprinting as a reliable method for the identification of compounds that are potentially responsible for antimicrobial activity. This is the first report on the activity of Serbian propolis as determined by several combined methods, including the modelling of antimicrobial activity by HPTLC fingerprinting.关于蜂胶的生物活性与天然产物品质的相关新发现已被揭示，而该领域亟需一种快速可靠的评估手段，例如高效薄层色谱法（High Performance Thin-Layer Chromatography，HPTLC）指纹图谱技术。本研究针对塞尔维亚来源蜂胶对多种细菌的抗菌活性，探究了色谱与化学计量学相结合的分析方法。本研究采用偏最小二乘（Partial Least Squares，PLS）这一线性多元校正技术，从色谱指纹图谱中提取有效信息，即识别出代表酚类化合物的色谱峰——这些酚类化合物可能是样本抗菌性能的关键贡献物质。此外，本研究通过直接生物自显影技术，在色谱图上定位抗菌活性位点。本研究通过最低抑菌浓度（minimum inhibitory concentration，MIC）实验测定了各蜂胶样本对多种细菌的生物活性，结果证实这些样本属于欧洲杨属来源蜂胶类型，并根据植物来源将其划分为蓝色、橙色两类。其中，样本26的抗菌活性最强：对金黄色葡萄球菌（Staphylococcus aureus）的MIC值为0.5 mg/mL，对单核细胞增生李斯特菌（Listeria monocytogenes）的MIC值低至0.1 mg/mL，该浓度也是本研究中测得的最低有效抑菌浓度。总体而言，橙色类蜂胶的抗菌活性优于蓝色类蜂胶。本研究基于上述HPTLC数据集构建了PLS模型，所得模型可定性识别出对蜂胶样本抗菌活性具有关键作用的化合物。影响蜂胶对所有受试菌株抗菌活性的最关键色谱峰，为比移值（retardation factor，RF）分别为0.37、0.40、0.45、0.51、0.60及0.70的酚类化合物。本研究所得结论可用于将蜂胶的抗菌活性归因于特定化学成分，同时验证了HPTLC指纹图谱技术作为可靠手段，用于识别具有潜在抗菌活性的化合物。本研究是首次通过多种联用方法（包括基于HPTLC指纹图谱的抗菌活性建模）对塞尔维亚蜂胶的活性进行测定的相关报道。