Bio-guided isolation of potential anti-inflammatory constituents of some endophytes isolated from the leaves of ground cherry (Physalis pruinosa L.) via ex-vivo and in-silico studies

Abstract

Background: Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus Physalis as a source of anti-inflammatory constituents, the present study aimed at isolation for the first time some endophytic fungi from the medicinal plant Physalis pruinosa.

Methods: The endophytic fungi were isolated from the fresh leaves of P. pruinosa then purified and identified by both morphological and molecular methods. Comparative evaluation of the cytotoxic and ex vivo anti-inflammatory activity in addition to gene expression of the three pro-inflammatory indicators (TNF-α, IL-1β and INF-γ) was performed in WBCs treated with lipopolysaccharide (LPS) for the identified endophytes, isolated compounds and the standard anti-inflammatory drug (piroxicam). For prediction of the binding mode of the top-scoring constituents-targets complexes, the Schrödinger Maestro 11.8 package (LLC, New York, NY) was employed in the docking study.

Results: A total of 50 endophytic fungal isolates were separated from P. pruinosa leaves. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters, which were then identified as Stemphylium simmonsii MN401378, Stemphylium sp. MT084051, Alternaria infectoria MT573465, Alternaria alternata MZ066724, Alternaria alternata MN615420 and Fusarium equiseti MK968015. It could be observed that A. alternata MN615420 extract was the most potent anti-inflammatory candidate with a significant downregulation of TNF-α. Moreover, six secondary metabolites, alternariol monomethyl ether (1), 3'-hydroxyalternariol monomethyl ether (2), alternariol (3), α-acetylorcinol (4), tenuazonic acid (5) and allo-tenuazonic acid (6) were isolated from the most potent candidate (A. alternata MN615420). Among the tested isolated compounds, 3'-hydroxyalternariol monomethyl ether showed the highest anti-inflammatory potential with the most considerable reductions in the level of INF-γ and IL-1β. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein (IL-1β, TNF-α and INF-γ)-ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis.

Conclusions: The results obtained suggested alternariol derivatives may serve as naturally occurring potent anti-inflammatory candidates. This study opens new avenues for the design and development of innovative anti-inflammatory drugs that specifically target INF-γ, IL-1β and INF-γ.

Keywords: Alternaria; Anti-inflammatory; Endophytes; Ex vivo; Molecular docking; Physalis pruinosa.

Fig. 1

The endophytic fungi colonies on PDA medium after 7 days incubation. (A) Alternaria alternata MN615420, (B) Stemphylium sp. MT084051, (C) Fusarium equiseti MK968015, (D) Alternaria alternata MZ066724, (E) Stemphylium simmonsii MN401378, (F) Alternaria infectoria MT573465

Fig. 2

Phylogenetic tree based on neighbour-joining method using MEGA X of the identified endophytic fungus (Stemphylium simmonsii MN401378) isolated from P. pruinosa leaves and related sequences

Fig. 3

Phylogenetic tree based on neighbour-joining method using MEGA X of the identified endophytic fungus (Alternaria infectoria MT573465) isolated from P. pruinosa leaves and related sequences

Fig. 4

Phylogenetic tree based on neighbour-joining method using MEGA X of the identified endophytic fungus (Alternaria alternata MZ066724) isolated from P. pruinosa leaves and related sequences

Fig. 5

Phylogenetic tree based on neighbour-joining method using MEGA X of the identified endophytic fungus (Stemphylium sp. MT084051) isolated from P. pruinosa leaves and related sequences

Fig. 6

Phylogenetic tree based on neighbour-joining method using MEGA X of the identified endophytic fungus (Fusarium equiseti MK968015) isolated from P. pruinosa leaves and related sequences

Fig. 7

Phylogenetic tree based on neighbour-joining method using MEGA X of the identified endophytic fungus (Alternaria alternata MN615420) isolated from P. pruinosa leaves and related sequences

Fig. 8

Structures of compounds (1–6) isolated from A. alternata MN615420

Fig. 9

(A) 2D and 3D interaction diagrams of alternariol monomethyl ether in the active site of TNF-α (2AZ5). (B) 2D and 3D interaction diagrams of 3’-hydroxyalternariol monomethyl ether in the active site of IL-1β (4DEP). (C) 2D and 3D interaction diagrams of 3’-hydroxyalternariol monomethyl ether in the active site of IFN-γ (6E3K)