Yuxin XIE ¹ Na Lin ¹ Pingping Song ¹ Xiangyan Ni ² Yakun Wang ¹ Peng Huang ¹ Zhili Han ¹ Dianlei Wang ^1* Nianxia Sun ^1*

• ¹ Anhui University of Chinese Medicine, Hefei, China
• ² Beijing Agricultural Product Quality and Safety Center, Beijing, China

The rhizome of Atractylodes macrocephala Koidz. (Compositae), commonly known as Atractylodes Macrocephala Rhizome (AMR), has been shown in pharmacological studies to modulate immunity. Nevertheless, its resources have been largely depleted and the pharmacological activity of artificial AMR is relatively modest. We hypothesized that the fermented crude extracts of the rhizosphere fungi of AMR would likewise have immunomodulatory effects since the metabolites generated by the rhizosphere fungi were similar to those of the host plant due to long-term synergistic evolution. In this study, the rhizosphere fungi of AMR were isolated, identified, and evaluated the in vitro immunological activity. The biologically active secondary metabolites produced by the rhizosphere fungi were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Three rhizosphere fungi, namely Penicillium (MK-1), Penicillium glaucoroseum (MN-1), and Purpureocillium lilalium (MG-1), were isolated from the rhizosphere soil of AMR and cultured in order to produce secondary metabolites. Metabolites were extracted in sequence with four solvents of increasing polarity (petroleum ether, ethyl acetate, n-butanol and water). The assays for cell proliferation capacity, cell phagocytosis activity, and NO secretion capacity stated that all of the metabolite extracts exhibited in vitro immunomodulatory activity. The crude extracts of MG-1 exhibited the highest levels of in vitro immunomodulatory activity in comparison to the other extracts. Furthermore, it was demonstrated that the fermented extracts of MG-1 could facilitate immunological enhancement in vitro by altering cell morphology of the resting state and increasing the secretion of TNF-α, IL-1β and IL-6. Meanwhile, there was no endotoxin contamination. The metabolite profiling of MG-1 by UHPLC-Q-TOF-MS revealed the presence of several compounds with established immunoreactive activity, including L-arginine, prostaglandin I2, deoxyguanosine, bestatin, and osthole. The present study demonstrated that the metabolite extracts of the rhizosphere fungi isolated from the rhizosphere soil of AMR exhibited in vitro immunoreactive activities and that the rhizosphere fungi could produce several bioactive metabolites. The crude extracts of the rhizosphere fungi might serve to extend the medicinal utility of AMR and could provide a basis for further development of natural plant immunomodulators.