Perstwong Naruemon, Binalee Asma, Kobtrakul Khwanlada, Phongsopitanun Wongsakorn, Sanookpan Kittipong, Areecheewakul Sudartip, Buranasudja Visarut, Vimolmangkang Sornkanok
Graduate Program in Pharmaceutical Science and Technology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
Phyto Analytica Testing Laboratory, Leapdelab Company Limited, Bangkok, 10330, Thailand.
BMC Complement Med Ther. 2025 Apr 25;25(1):159. doi: 10.1186/s12906-025-04873-3.
Ya-Kao (YK) is a traditional Thai medicine used to relieve fever and consists of 14 crude drugs. It has been recommended by Thai folk healers and the Department of Thai Traditional and Alternative Medicine for COVID-19 treatment, with clinical studies conducted to assess its efficacy. However, scientific evidence on its biological properties remains limited. This study aims to explore the quality aspect of YK and evaluate its antibacterial and anti-inflammatory effects.
The quality aspect of the YK formula was assessed using high-performance thin-layer chromatography (HPTLC). Agar disc diffusion and TLC bioautography were used for antibacterial activity, while anti-inflammatory effects were evaluated by measuring nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages.
The HPTLC method, utilizing a developing system of toluene, ethyl acetate, and formic acid (70:30:1, v/v/v), was successfully developed for the authentication of YK crude drugs. This method provides the most distinct fingerprint pattern of the components within the YK formulation, enabling clear visualization of its constituent. Additionally, it enables preliminary identification of phenolics, terpenes, and steroids, providing valuable insight into the formulation's chemical composition. The antibacterial activity of YK was evaluated using the disk diffusion assay, and it was found that the lime juice extract of the YK formula (LYK) exhibited the largest zone of inhibition against both Gram-positive and Gram-negative bacterial strains, particularly those pathogenic to the respiratory tract. Additionally, the antibacterial activity was evaluated using TLC bioautography, and the results indicated that the methanolic extract of YK (MYK) exhibited an inhibition zone against Streptococcus pyogenes and Klebsiella pneumoniae. It was found that several compounds displayed an inhibition zone. Furthermore, the YK extract with methanol, water, and lime juice exhibited significant anti-inflammatory properties by suppressing NO accumulation in LPS-stimulated macrophage cells (p < 0.05).
This study is the first to standardize YK raw materials using HPTLC and evaluate the biological properties of each crude drug and the combined formula. The developed HPTLC method ensures accurate identification of YK raw materials, preventing the use of incorrect ingredients. Additionally, the pharmacological findings confirm YK's anti-inflammatory and antibacterial activities, particularly against respiratory pathogens linked to COVID-19.
亚考（YK）是一种用于退热的泰国传统药物，由14种生药组成。泰国民间治疗师和泰国传统与替代医学部已推荐将其用于治疗新冠肺炎，并开展了临床研究以评估其疗效。然而，关于其生物学特性的科学证据仍然有限。本研究旨在探索YK的质量方面，并评估其抗菌和抗炎作用。
采用高效薄层色谱法（HPTLC）评估YK配方的质量方面。采用琼脂平板扩散法和薄层色谱生物自显影法检测抗菌活性，通过测量脂多糖刺激的RAW264.7巨噬细胞中一氧化氮（NO）的产生来评估抗炎作用。
成功开发了一种利用甲苯、乙酸乙酯和甲酸（70:30:1，v/v/v）展开系统的HPTLC方法，用于YK生药的鉴定。该方法提供了YK制剂中成分最清晰的指纹图谱，能够清晰地观察其组成成分。此外，它能够初步鉴定酚类、萜类和甾体类化合物，为制剂的化学成分提供有价值的见解。采用纸片扩散法评估YK的抗菌活性，发现YK配方的酸橙汁提取物（LYK）对革兰氏阳性和革兰氏阴性细菌菌株，特别是对呼吸道致病菌株的抑菌圈最大。此外，采用薄层色谱生物自显影法评估抗菌活性，结果表明YK的甲醇提取物（MYK）对化脓性链球菌和肺炎克雷伯菌有抑菌圈。发现几种化合物有抑菌圈。此外，用甲醇、水和酸橙汁提取的YK提取物通过抑制脂多糖刺激的巨噬细胞中NO的积累表现出显著的抗炎特性（p<0.05）。
本研究首次采用HPTLC对YK原料进行标准化，并评估了每种生药及其复方制剂的生物学特性。所开发的HPTLC方法确保了YK原料的准确鉴定，防止使用错误的成分。此外，药理学研究结果证实了YK的抗炎和抗菌活性，特别是对与新冠肺炎相关的呼吸道病原体的活性。
