Isolation, Structural Elucidation and Biological Evaluation of Labdane Diterpenes from Seeds of Alpinia nigra (Gaertn.) B.L. Burtt

Abstract

The current investigation unveils the potential uses of Alpinia nigra (Gaertn.) B. L. Burtt as a source of bioactive compounds towards development of therapeutic agent against various diseases. The plant is used as vegetable and food flavouring agent in various parts of Northeast India apart from other uses in folk ethnomedicines. In the present study, the mature seeds of A. nigra were collected, processed and subjected to soxhlet extraction in a polarity gradient with three organic solvents viz. n-Hexane, Ethylacetate and Methanol respectively. All the three extracts were characterized by GC-MS and chemical fingerprints (NMR and FTIR) were recorded. Qualitative phytochemical screening was performed to detect the presence of secondary metabolites (saponins, alkaloids, phenolics and terpenoids) in each extract. Moreover, study on DPPH free radical scavenging activity suggested ethyl acetate and methanolic extracts as most potential ones to explore further. Principal components from these extracts were further isolated and characterized with spectroscopic techniques viz. 1H NMR, 13C NMR, FT-IR and HRMS that established the identity of compounds as (E)-labda-8(17),12-diene-15, 16-dial (I) and (E)-8 ,17-epoxylabd-12-ene-15,16-dial (II). From the hexane extract another major compound was purified and established as 1,2- dihexadecanoyl-3-(9Z-hexadecenoyl)- sn-glycerol (III). All the purified compounds were tested for Lipinski drug likeliness properties, where, compound I and II were found as suitable candidate for biological uses. Further, biocompatibility of these compounds (I and II) were determined by using RBC hemolysis assay. These two labdane diterpenes were critically evaluated for their antibacterial activities and tested in seven pathogenic bacterial indicators. Minimum inhibitory concentration (MIC) was determined for each compound for respective bacteria. Among the Gram positive bacteria, Staphylococcus aureus (ATCC6538) and among the Gram negative bacteria, Yersinia enterocolitica (MTCC 859) were found most susceptible to these compounds. Flow cytometry (FC) and cell leakage analysis showed the compromised state of bacterial cell membrane. Pore formation and damage of bacterial cells were confirmed by field emission scanning electron microscope (FESEM) imaging. Antidiabetic activity of I and II were investigated which showed dose dependant inhibition of -amylase and -glucosidase in a non-competitive manner. Molecular docking was performed for human pancreatic -amylase (HPA) and maltase-glucoamylase (MGAM) with both the compounds and standard drug acarbose. In case of HPA and MGAM, both the compounds interacted in the active site similar to acarbose which might lead to non-competitive mode of inhibitions. To understand the effects of I and II on matrix metalloproteinase (MMPs) activity, HT1080 human fibrosarcoma cells was used as a source of MMPs. In vitro treatment with I and II showed no significant modulation of MMPs activity. However, when HT1080 culture supernatants were directly incubated with I and II they were found to inhibit the MMP-2 and MMP-9 activities. These compounds also induced cell cycle arrest in the S phase and nuclear condensation leading to apoptosis in HT1080 cells. The current research has enabled identification of two principal bioac..