Abstract:

Earthworms are a commonly used animal-derived traditional Chinese medicine in China， with proteins and polypeptides as their main active ingredients. However， the existing methods for separating and detecting earthworm polypeptides have problems such as insufficient preparation capacity and poor separation reproducibility. To address these challenges， this study used the iron porphyrin metal-organic framework material PCN-222（Fe） to specifically adsorb the earthworm polypeptide component L-PeakⅡ， with an adsorption capacity of 139 mg/g. First of all， PCN-222（Fe） material was successfully prepared via the one-pot method. The earthworm protease hydrolysate was obtained through alkali extraction and acid precipitation， and trypsin enzymatic hydrolysis. Then， PCN-222（Fe） was further used as a solid-phase adsorbent and combined with liquid chromatography to rapidly separate and prepare the polypeptide component L-PeakⅡ from complex earthworm enzymatic hydrolysate of the Chinese medicine. Qualitative analysis was performed by HPLC， and quantitative analysis was conducted with a BCA assay kit. HPLC analysis of the earthworm protease hydrolysate revealed multiple chromatographic peaks. After enrichment with PCN‑222（Fe） and subsequent elution， the eluted sample exhibited fewer peaks， with a well‑defined peak appearing at a retention time of approximately 15 min. In order to obtain the optimal experimental conditions for the enrichment of earthworm polypeptides， influencing factors including enzyme hydrolysate mass concentration， pH value， elution solvent type， and elution time were investigated and optimized. The results showed that optimal enrichment was achieved by incubating at an enzymatic hydrolysate mass concentration of 3.0 mg/mL and pH of 9， followed by elution with 50% acetonitrile containing 0.2% trifluoroacetic acid for 2 h. Finally， the earthworm-derived polypeptide component was obtained using a high-pressure preparative chromatographic system， achieving a purity exceeding 95%. The integrated approach employed in this study， which combines PCN-222（Fe） material with preparative chromatography， enables the efficient preparation of the target polypeptide L-PeakⅡ from medicinal earthworm samples. This strategy provides a valuable reference for the separation and purification of polypeptide components from complex animal-derived traditional Chinese medicines. Moreover， it offers a more robust， scalable， and reproducible pathway for harnessing the therapeutic potential of natural product peptides， thereby bridging the gap between traditional medicine and modern pharmaceutical analysis.

Key words: earthworm, polypeptides, separation and purification, metal-organic frameworks （MOFs）, high performance liquid chromatography （HPLC）