Mature or nearly mature fruits of Piper longum have been applied as a spice with significant commercial and industrial values, as well as a traditional Chinese medicine with multiple effects such as dispelling cold and relieving pain. Based on the long history of medicinal usage, P. longum fruits are attractive to explore their therapeutic material basis. The chemical components of traditional Chinese medicines are normally complex, thus how to efficiently discover novel active components is a difficult problem in natural product research. Prof. Haji Akber Aisa’s lab at Laboratory of Resource Chemistry, Xinjiang Technical Institute of Physics & Chemistry (XTIPC) of Chinese Academy of Sciences isolated 12 dimeric amide alkaloid enantiomers with anti-inflammatory and antidiabetic effects from P. longum fruits with the assistance of a molecular network-based dereplication strategy. This work was published in Journal of Agricultural and Food Chemistry.

The molecular network of a 95% ethanol extract of P. longum fruits was created by the GNPS platform’s Feature-Based Molecular Networking (FBMN) module. A distinct cluster in the network, mainly containing nodes with molecular weights of 486.191−653.255 Da, was not matched to known chemical structures in public databases. A node within this cluster with a molecular weight of 627.3420 Da matching a previously identified amide alkaloid dimer from P. longum, piperchabamide H (m/z 627.3428 [M + H]⁺), was used as a “seed molecule”, leading to the isolation of 12 dimeric amide alkaloids. These 12 dimeric amide alkaloids were successfully separated into 12 pairs of enantiomers by chiral HPLC. The complete structures of these compounds were elucidated by means of comprehensive spectroscopic data, electronic circular dichroism (ECD) calculations, and X-ray diffraction analysis. These dimeric amide alkaloids were identified as eight pairs of cyclobutane-type dimers and four pairs of cyclohexene-type dimers, including five pairs of new cyclobutane-type dimers and one pair of new cyclohexene-type dimer.

In vitro bioactivity screening revealed that three compounds exhibited notable anti-inflammatory effects in an LPS-induced RAW 264.7 macrophage model, one compound demonstrated significant inhibitory activity against α-glucosidase, and three compounds exhibited promising inhibitory activity against protein tyrosine phosphatase-1B (PTP1B). To explore the interaction mechanisms between these dimeric amide alkaloids and the enzymes α-glucosidase and PTP1B, molecular docking studies were conducted using two most active compounds from the series as representatives. The results showed that both two compounds were capable of forming stable complexes with their respective target proteins. Further molecular dynamics simulations provided additional evidence of the stability of the complexes between the two active compounds and corresponding proteins.

This study highlights the promising application of these amide alkaloid dimers in the development of functional foods and pharmaceutical products, thereby expanding the health-promoting potential of P. longum fruits.

This work was supported by the Tianshan Talent Training Program of Science & Technology Department of Xinjiang Uygur Autonomous Region, the Natural Science Foundations of China, and the National Key R&D Program of China.

Figure: Discovery of dimeric amide alkaloid enantiomers by a molecular network-based dereplication strategy from Piper longum fruits and their bioactivities (Provided by Prof. Haji Akber Aisa’s group)