Antiurolithic activity and biotransformation of galloylquinic acids by Aspergillus alliaceus ATCC10060, Aspergillus brasiliensis ATCC 16404, and Cunninghamella elegans ATCC 10028b

Highlights

• Galloylquinic acids were transformed by A. alliaceus ATCC10060 into M1.

• Galloylquinic acids and M1 significantly inhibited COM binding to renal cell surface.

• Galloylquinic acids and M1 inhibited HSP90 and ANXA1 surface expressions.

• Galloylquinic acids and M1 exhibited free radical scavenging activity.

Abstract

Copaifera lucens n-butanolic fraction (BF) was used as a source of galloylquinic acids, and aerobically incubated with Aspergillus alliaceus ATCC10060, Aspergillus brasiliensis ATCC 16404, and Cunninghamella elegans ATCC 10028b cultures for 60 and 120 h. Out of the three studied filamentous fungi, A. alliaceus ATCC10060 was able to degrade galloylquinic acids into one major metabolite, 3-O-methylgallic acid (M1). The product was identified by ¹H NMR, UPLC-MS/MS, and its potential effect on calcium oxalate monohydrate (COM) crystal binding to Madin-Darby canine kidney cells type I surface was studied. Renal cells pretreatment with BF and M1 for 3 h significantly decreased COM crystaladherence at 50 μg/mL and 5 μM, respectively. Moreover, both M1 and BF significantly reduced the surface expression of COM-binding proteins annexin A1 (ANXA1) and heat shock protein 90 (HSP90), respectively as evidenced by Western blot analysis of membrane, cytosolic, and whole cell lysate fractions. The compounds also showed antioxidant activities in a DPPH assay.

Introduction

Different species of Copaifera species (Fabaceae), such as C. lucens and C. langsdorffii are widely distributed in Brazil (Motta et al., 2017) and they possess promising biological activities mainly their use in the modulation of renal stones, also known as urolithiasis (Oliveira et al., 2013). The major secondary metabolites (Nogueira et al., 2015) in Copaifera leaves extracts were identified as galloylquinic acids (Fig. 1), and flavonoids (Brancalion et al., 2012; Nogueira et al., 2015), that contribute to their antiurolithic effect. We previously reported that the 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME) inhibited calcium oxalate crystal growth in a Drosophila melanogaster model, and downregulated renal cell surface expression of annexin A1 (ANXA1), α-enolase and heat shock protein 90 (HSP90), which are potential calcium oxalate monohydrate (COM) binding receptors, and it also decreased cells crystal adhesion, a key factor in renal stones pathogenesis (Abd El-Salam et al., 2018). However, there is very little information on the metabolic pathway and pharmacokinetic of galloylquinic acids in Copaifera extracts. Therefore, studies on the metabolism of such phytochemicals by filamentous fungi are important for predicting their metabolic behavior and better understanding their biological effects. In this study, the n-butanolic fraction of C. lucens (BF) and gallic acid (GA) were cultured aerobically with Aspergillus alliaceus ATCC10060, Aspergillus brasiliensis ATCC 16404, and Cunninghamella elegans ATCC 10028b cultures for 60 h and 120 h. One major compound 3-O-methyl gallic acid (M1) was detected and identified in the fungal culture of Aspergillus alliaceus ATCC10060 by UPLC-MS/MS and ¹H NMR. The two other fungal strains showed no apparent biotransformation. The resulting metabolite M1 and the BF were tested for their potential to inhibit COM crystal adherence to the surface of Madin-Darby Canine Kidney type I (MDCKI) cells as well as COM-binding proteins, using crystal binding assay and Western blot analysis, respectively. The compounds were also investigated for their ability to scavenge free radicals in a DPPH assay.