UPLC-MS/MS identification of terpenoid metabolites in different organs of Eriobotrya japonica

Abstract:【Objective】Loquat (Eriobotrya japonica Lindl.) is a rosaceous subtropical and evergreen fruit tree native to China. It has been used as a pharmaceutical plant for a long time. Its leaves, flowers as well as other organs have been used for the treatment of different diseases. Dry loquat leaves are widely used in traditional Chinese medicine for treatment of cough, asthma, diabetes, mellitus, chronic bronchitis and skin diseases. Terpenoids have been found in the roots, stems, leaves, flowers, peel, fleshe, and seeds of loquat, and some of the terpenoid compounds have been proved to have bioactivities such as anti-inflammatory, antitussive, diuretic, anti-tumor and diabetes. The aim of this work was to identify the compounds of terpenoids in different organs of loquat, and provide a basis for resource identification and functional utilization of this species.【Methods】The components of terpenoids in the anhydrous ethanol extract of loquat (Eriobotrya japonica Lindl.‘Anhuidahongpao’) leaves, flowers, peel, fleshe and seeds were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analytical conditions were as follows, UPLC: column, Agilent SB-C18 (1.8 µm, 2.1 mm´100 mm); The mobile phase was consisted of solvent A, pure water with 0.1% formic acid, and solvent B, acetonitrile with 0.1% formic acid. The sample measurements were per-formed with a gradient program employing the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.1 min and kept for 2.9 min. The flow velocity was set as 0.35 mL per minute; The column oven was set to 40 ℃; The injection volume was 4 μL. The effluent was alternatively connected to an ESI- triple quadrupole- linear ion trap (QTRAP)-MS. The LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), AB4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550 ℃; ion spray voltage (IS) 5500 V (positive ion mode) / -4500 V (negative ion mode); ion source gas Ⅰ (GSⅠ), gas Ⅱ (GSⅡ), curtain gas (CUR) were set at 50, 60, and 25.0 psi, respectively; the collision-activated dissociation (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol·L^-1 polypropylene glycol solutions in QQQ and LIT modes, respectively. The QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to medium. DP and CE for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period. The differences compounds of terpenoids in different organs were compared, and the R software was used to perform principal component analysis and cluster analysis of the measurement results (principal component analysis, PCA), and screening for substances with relative contents of more than 1%.【Results】The results showed that a total of 51 compounds of terpenoids in the leaves, flowers, peel, fleshe and seeds were detected, including 1 kinds of monoterpene, 1 kinds of Sesquiterpe-no, 3 kinds of diterpene, 42 kinds of triterpenes and 4 kinds of triterpene saponin. There were 35 kinds of compounds such as perillyl alcohol, Dehydroabietic acid, betulin, camaldulenic etc. were isolated from loquat for the first time. There were 50, 49, 49, 45 and 40 kinds of compounds of terpenoids were detected in the leaves, flowers, peel, fleshe and seeds, respectively. There were 38 kinds of compounds were shared by all organs, accounting for 74.5% of 51 terpenoids. The 11-keto-ursolic acid was the spe-cific substance in the leaves, and 2α, 3α, 19α-trihydroxyurs-12-en-23-formyl-28-oic acid not founded in the leaves. There were 14, 15, 11, 13 and 10 kinds of compounds with relative contents of more than 1% in the leaves, flowers, peel, fleshe and seeds, respectively. The most main compounds were triterpenes, accounting for 88.46% , 92.78% , 92.37% , 94.62% , and 96.28% of the total relative contents in the leaves, flowers, peel, fleshe and seeds, respectively. The 2α, 19α-dihydroxy-3-oxours-12-en-28-oic acid, corosolic acid, maslinic acid, 2-hydroxyoleanolic acid, 2α, 3α, 23-trihydroxyolean-12-en-28-oic acid, eu-scaphic acid, asiatic acid and rosamultic acid were the most important metabolites in each organ of loquat. The relative contents of 2Α, 19α-dihydroxy-3-oxursolic-12-ene-28-acid were the highest in all or-gans, accounting for 18.32%, 32.57%, 39.79%, 37.98% and 38.75% of the total terpenoids in the leaves, flowers, peel, fleshe and seeds, respectively. The cluster analysis showed the accumulation characteris-tics of the terpenoids in different organs.【Conclusion】There were abundant kinds of terpenoids in loquat leaves, flowers, peel, fleshe and seeds, and the highest contents of the compounds was found in the leaves, the next in the flowers and peel, the third in the freshe, and the lowest content of terpenoids was found in the seeds. Many kinds of terpenoid metabolites with potential functions have been found.