Triterpene Profiling of Neem Exudate (Toddy) Using UHPLC-QTOF-MS and its Anti-inflammatory Activity

Neem tree (Azadirachta indica) is one of the richest sources of secondary metabolites. More than 250 natural products have been characterized from various parts of the neem tree. These include diterpenoids, triterpenoids, steroids, flavonoids, coumarins, hydrocarbons, and fatty acids. Many of these products possess therapeutic properties. Neem exudate or toddy is a milky white liquid with a strong smell secreted from the angle between the 2 main branches of old trees. Profiling of neem toddy for the presence of active metabolites was done by an in-house database using UHPLC-QTOF-MS. Fifty-seven metabolites were identified from the full scan of electrospray ionization positive and negative mode from the neem toddy extract by using UHPLC-QTOF-MS. Further confirmation of 31 of these metabolites was done by obtaining MS/MS spectrum from UHPLC-QTOF-MS. Principal component analysis study of metabolites from neem toddy with leaves, seed, seed coat, and bark revealed that they are closely related to those contained in neem seeds and seed coats. Azadirachtin, nimbidiol, 22,23-dihydroazadirachtin, nimbonone, nimbonolone, nimosone, and 6-deacetylnimbinene were found to be some of the most abundant metabolites in neem toddy. The neem toddy extract showed significant anti-inflammatory activity when tested in N9 murine microglial cells with 25 ng of recombinant mouse tumor necrosis factor alpha protein (active) using qRT-PCR. The active metabolites in neem toddy could be further explored for their therapeutic potentials.