Polyphenolic Profile of Herniaria hemistemon Aerial Parts Extract and Assessment of Its Anti-Cryptosporidiosis in a Murine Model: In Silico Supported In Vivo Study

Abstract

Herniaria hemistemon J.Gay is widely used in folk medicine to treat hernia. The present study aimed to annotate the phytoconstituents of H. hemistemon aerial-part extract and investigate its in vivo anticryptosporidial activity. The chemical characterization was achieved via the LC-ESI-MS/MS technique resulting in the annotation of 37 phytocompounds comprising flavonoids and phenolic acids. Regarding the anticryptosporidial activity, fifty dexamethasone-immunosuppressed mice were separated into five groups: GI, un-infected (normal control); GII, infected but not treated (model); GIII, infected and received NTZ, the reference drug; GIV, infected and received H. hemistemon extract (100 mg/kg); and GV, infected and received H. hemistemon extract (200 mg/kg). When GIII, GIV, and GV were compared to GII, parasitological analyses displayed highly significant differences in the mean numbers of Cryptosporidium parvum oocysts in the stool between the different groups. GV demonstrated the highest efficacy of 79%. Histopathological analyses displayed improvement in the small intestine and liver pathology in the treated groups (GIII, IV, and V) related to the model (GII), with GV showing the highest efficacy. Moreover, the docking-based study tentatively highlighted the potential of benzoic acid derivatives as lactate dehydrogenase inhibitors. The docked compounds showed the same binding interactions as oxamic acid, where they established H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195. To sum up, H. hemistemon is a promising natural therapeutic agent for cryptosporidiosis.

Keywords: Herniaria hemistemon J.Gay; LC–ESI–MS/MS; anticryptosporidiosis; lactate dehydrogenase inhibitors; nitazoxanide; polyphenols.

Figure 1

LC chromatogram of H. hemistemon aerial-part extract.

Figure 2

MS/MS spectra of esculetin glucoside ((a), compound 7), esculetin sulfate ((b), compound 11), and esculetin ((c), compound 13) from Table 1.

Figure 3

Histological photomicrographs of small intestinal sections (stained with H&E, unless mentioned otherwise, 200×). (a) Sections from normal mice (GI) displayed the normal structure of the villi with a preserved brush border and normal pattern of mucin secretion. (b) Sections from infected mice (GII) revealed a significant broadening of villi (red line), dense infiltration by mononuclear inflammatory cells (yellow arrows), and villous tip-region degeneration (green arrows). (c) Sections from infected mice (GII) revealed numerous adherent (green arrow) and separate cryptosporidium oocysts (red arrow, H&E stain, stained purple, 4–6 µm in diameter, 1000×). (d) Sections from infected mice (GIII) treated with the reference drug (NTZ) demonstrated mild villi broadening with focal tip-region degeneration and mild mononuclear inflammatory cell infiltration. (e) Sections from infected mice (GIV) treated with H. hemistemon extract (100 mg/kg) demonstrated moderate villous broadening (red line), infiltration by mononuclear inflammatory cells (yellow arrow), and focal degeneration of the villous tip regions (green arrow). (f) Sections from infected mice (GV) treated with H. hemistemon extract (200 mg/kg) showed a retained villous architecture, such as the recovery of an almost normal villous pattern with the occasional appearance of inflammatory cells.

Figure 4

Histological photomicrographs of liver tissue (stained with H&E, unless mentioned otherwise, 200×). (a) Sections from normal mice (GI) showing a normal architecture of hepatic lobules. (b) Sections from infected mice (GII) displaying moderate degeneration of hepatocytes (black arrow) and focal infiltration with mononuclear cells (red arrow). (c) Sections from infected mice (GIII) treated with the reference drug, NTZ, displayed mild degeneration of hepatocytes. (d) Sections from infected mice (GIV) treated with H. hemistemon extract (100 mg/kg) showed moderate hepatocellular degeneration (black arrow) and focal infiltration by mononuclear cells (red arrow). (e) Sections from infected mice (GIV) treated with H. hemistemon extract (200 mg/kg) showed a retained hepatic lobular architecture.

Figure 5

(A–D): Binding modes of quinic acid, gallic acid, hydroxybenzoic acid, and methyl gallate inside the active site of C. parvum-derived lactate dehydrogenase (PDB ID: 4ND1). The four structures (in brick-red color) are aligned with the structure of the co-crystalized inhibitor oxamic acid (in orange color). The structure of the co-factor nicotinamide adenine dinucleotide (NAD) is shown in pink color.