Methods: The crude extracts of green and fallen pine needles were subjected to sequential fractionation to get partially purified fractions. Human peripheral blood lymphocytes (PBL) were used to analyze immunoenhancing potential of these fractions. Microcytotoxicity assay was employed to investigate improved cancer cells killing capabilities of PBL against various cancer cell lines. GC-MS was carried out to identify of the major compounds in bioactive fractions.

               

Results: The lymphocyte proliferation assay depicted the immunoenhancing potential of extracts and fractions of fallen and green needles of P. roxburghii. The ethyl acetate fractions of both fallen and green needles displayed highest mitogenic activity on human PBL. Both fractions heightened the expression of cell surface markers (CD3, CD8, and CD56) and significantly increased the production of cytokines (IL-2, IFN-γ and TNF-α). The enhanced intracellular granulysin (immunomarker for activated CTLs and NK cells) expression also confirmed immune stimulatory potential of these fractions on human lymphocytes. The ethyl acetate fractions of pine needles enhanced the cytotoxicity of PBL towards various cancer cells (HCT-116, HeLa, PC-3 and A549) as compared to untreated PBL. GC-MS analysis showed presence of 3-α-mannobiose, octakis (trimethylsilyl) ether, methyloxime and cyclodecasiloxane, eicosamethyl as major compounds ethyl acetate fractions of green and fallen needles, respectively.

 

Conclusion: The bioactive fractions of both fallen and green needles of P. roxburghii showed immunostimulatory potential on human PBL leading to enhanced cancer cells killing capabilities of these lymphocytes.

 

Key words: Anticancer, Immunomodulation, Granulysin, GC-MS, Pine needles