Optimization of Ultrasonic-assisted Extraction Conditions of Melleolides from Wild Armillaria mellea and Liquid Culture Mycelium, and Analysis of Their Compounds in Extracts

Objective: To analyze the differences of compounds in Armillaria mellea and mycelium, this study was conducted to investigate the optimal extraction process conditions of Melleolides and analyze the compounds in the extracts using northeastern wild Armillaria mellea fruit body and liquid mycelium as the research objects. Methods: The indexes of this study were extracted yield and Melleolides content. Ultrasonic cell crushing-assisted petroleum ether was used for extraction, and the extraction process parameters were optimized by single-factor experiments and orthogonal tests. The compounds in the liquid mycelium and Armillaria mellea fruit body were analyzed and identified by ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results: The optimal extraction process conditions were determined as material-liquid ratio 1:20 g/mL, ultrasonic power 300 W, ultrasonic time 20 min, solvent reflux time 50 min, under which the yield of liquid culture mycelium extract was 26.8% and the content of Melleolides was 0.74 mg/g. 305 and 592 compounds were analyzed and identified in the liquid mycelium and wild Armillaria mellea, respectively. 16 Melleolides and 5 fragments of suspected Melleolides were identified, including 15 Melleolides and 3 fragments in the mycelium, with total contents of 2.551 and 0.588 µg/mL, respectively. 7 Melleolides and 4 fragments in fruit body, with total contents of 2.413 and 2.124 µg/mL, respectively. 6 Melleolides and 2 fragments were found in both; The liquid mycelium had 9 unique Melleolides and 1 fragment: 10-dehydroxymelleoloede, Nectarine, Arnamiol, Bexagliflozin, A52a, 4-dehydroxyarmillarin, 9-(2-Chloro-4-hydroxy-5-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione, (4R,5S,7R,9S,13R)-2',5-epoxy-4-dehydroxyarmillarin, 4'-demethoxyarmillaribin, and fragment C. Wild Armillaria mellea uniquely possessed one Melleolides and two fragments as: Melledonal as well as fragments D and E. Conclusion: The results showed that Melleolides were more abundant in the liquid mycelium than in the fruit body, providing reference for the development of complementary functional foods and drugs.