Table1_The high-throughput solid-phase extraction of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) from Lactobacillus plantarum demonstrates efficacy against multidrug-resistant bacteria and influenza A (H3N2) virus.pdf

Introduction: 2,5-diketopiperazines are the simplest forms of cyclic dipeptides (CDPs) and have diverse frameworks with chiral side chains that are useful for drug development. Previous research has investigated the antimicrobial properties of proline-linked CDPs and their combinations in the culture filtrate (CF) of Lactobacillus plantarum LBP-K10 using anion exchange chromatography (AEC). However, the quantity of CDPs showcasing notable anti-influenza virus activity derived from AECs was generally lower than those originating from Lactobacillus CF.
Methods: To address this issue, the study aims to propose a more efficient method for isolating CDPs and to introduce the antiviral combinations of CDPs obtained using a new method. The study employed a novel technique entailing high-throughput C18-based solid-phase extraction with a methanol gradient (MeSPE). The MeSPE method involved increasing the methanol concentration from 5% to 50% in 5% increments.
Results: The methanol SPE fractions (MeSPEfs) eluted with methanol concentrations between 35% and 45% evinced substantial efficacy in inhibiting the influenza A/H3N2 virus via plaque-forming assay. MeSPEf-45, the 45% MeSPEf, exhibited exceptional efficacy in preventing viral infections in Madin-Darby kidney cells, surpassing both individual CDPs and the entire set of MeSPEfs. To identify the specific antiviral components of MeSPEf-45, all MeSPEfs were further fractionated through preparative high-performance liquid chromatography (prep-HPLC). MeSPEf-45 fractions S8 and S11 presented the highest activity against multidrug-resistant bacteria and influenza A/H3N2 virus among all MeSPEfs, with 11 common fractions. Antiviral fractions S8 and S11 were identified as proline-based CDPs, specifically cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro), using gas chromatography-mass spectrometry. The combination of MeSPEf-45 fractions S8 and S11 displayed superior antibacterial and anti-influenza virus effects compared to the individual fractions S8 and S11.
Discussion: High-throughput MeSPE-derived MeSPEfs and subsequent HPLC-fractionated fractions presents an innovative approach to selectively purify large amounts of potent antimicrobial CDPs from bacterial CF. The findings also show the effectiveness of physiologically bioactive combinations that utilize fractions not containing CDP. This study provides the initial evidence demonstrating the antimicrobial properties of CDPs acquired through high-throughput SPE techniques.

引言：2,5-二酮哌嗪（2,5-diketopiperazines）是结构最简单的环二肽（cyclic dipeptides, CDPs），其骨架带有可用于药物开发的手性侧链，拥有多样化的分子结构。既往研究已通过阴离子交换色谱（anion exchange chromatography, AEC）探究了植物乳杆菌LBP-K10培养滤液（culture filtrate, CF）中脯氨酸连接的环二肽及其组合的抗菌特性。但经阴离子交换色谱分离得到的、具备显著抗流感病毒活性的环二肽含量，通常低于直接来源于植物乳杆菌培养滤液的环二肽总量。

方法：针对这一问题，本研究旨在提出一种更高效的环二肽分离方法，并介绍通过新方法获得的环二肽抗病毒组合。本研究采用了基于C18的甲醇梯度高通量固相萃取（high-throughput C18-based solid-phase extraction with a methanol gradient, MeSPE）新技术，该方法将甲醇浓度以5%为增量从5%逐步提升至50%。

结果：甲醇浓度介于35%~45%时洗脱得到的甲醇固相萃取组分（methanol SPE fractions, MeSPEfs），经空斑形成试验验证，对甲型流感病毒A/H3N2展现出显著的抑制活性。其中45%甲醇洗脱的MeSPEf-45组分，在马-达二氏犬肾细胞中表现出优异的病毒感染预防效果，优于单一环二肽及所有甲醇固相萃取组分。为明确MeSPEf-45中的特异性抗病毒组分，本研究通过制备型高效液相色谱（preparative high-performance liquid chromatography, prep-HPLC）对所有甲醇固相萃取组分进行了进一步分级。结果显示，MeSPEf-45中的S8与S11组分，在所有甲醇固相萃取组分中抗多重耐药菌与抗甲型流感病毒A/H3N2活性最高，二者共含有11个共有组分。经气相色谱-质谱联用（gas chromatography-mass spectrometry, GC-MS）鉴定，具有抗病毒活性的S8与S11组分为脯氨酸基环二肽，具体为顺式-环(L-亮氨酰-L-脯氨酸)和顺式-环(L-苯丙氨酰-L-脯氨酸)。将S8与S11组分联合使用时，其抗菌与抗流感病毒效果优于单一的S8或S11组分。

讨论：基于高通量甲醇固相萃取得到的甲醇固相萃取组分，结合后续高效液相色谱分级的方法，为从细菌培养滤液中选择性纯化大量高活性抗菌环二肽提供了一种创新路径。本研究结果还证实，不含环二肽的组分所形成的生理活性组合同样具备有效性。本研究首次提供了通过高通量固相萃取技术获取的环二肽具有抗菌特性的初步证据。