Zygo-Albuside A: New Saponin from Zygophyllum album L. with Significant Antioxidant, Anti-Inflammatory and Antiapoptotic Effects against Methotrexate-Induced Testicular Damage

Abstract

Chemical investigation of the crude extract of the aerial part of Zygophyllum album L. (Z. album) led to the isolation of a new saponin, Zygo-albuside A (7), together with seven known compounds, one of them (caffeic acid, compound 4) is reported in the genus for the first time. NMR (1D and 2D) and mass spectrometric analysis, including high-resolution mass spectrometry (HRMS), were utilized to set up the chemical structures of these compounds. The present biological study aimed to investigate the protective antioxidant, anti-inflammatory, and antiapoptotic activities of the crude extract from the aerial part of Z. album and two of its isolated compounds, rutin and the new saponin zygo-albuside A, against methotrexate (MTX)-induced testicular injury, considering the role of miRNA-29a. In all groups except for the normal control group, which received a mixture of distilled water and DMSO (2:1) as vehicle orally every day for ten days, testicular damage was induced on the fifth day by intraperitoneal administration of MTX at a single dose of 20 mg/kg. Histopathological examination showed that pre-treatment with the crude extract of Z. album, zygo-albuside A, or rutin reversed the testicular damage induced by MTX. In addition, biochemical analysis in the protected groups showed a decrease in malondialdehyde (MDA), interleukin-6 (IL-6) and IL-1β, Bcl-2-associated-protein (Bax), and an increase in B-cell lymphoma 2 (Bcl-2) protein, catalase (CAT), superoxide dismutase (SOD) in the testis, along with an increase in serum testosterone levels compared with the unprotected (positive control) group. The mRNA expression levels of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), p53, and miRNA-29a were downregulated in the testicular tissues of the protected groups compared with the unprotected group. In conclusion, the study provides sufficient evidence that Z. album extract, and its isolated compounds, zygo-albuside A and rutin, could alleviate testicular damage caused by the chemotherapeutic agent MTX.

Keywords: Zygophyllum album L.; antiapoptotic; antioxidant activity; saponins; testicular damage.

Figure 1

Chemical structures of the isolated compounds.

Figure 2

Selected COSY and HMBC correlations of compound 7.

Figure 3

Effect of administration of Z. album crude extract, zygo-albuside A and rutin on serum levels of (A) testosterone, and testicular levels of (B) MDA, (C) CAT and (D) SOD in MTX-induced testicular injury mice. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. Values are expressed as mean ± SD (n = 8). A statistically significant difference was assumed at p < 0.01 and denoted by * vs. NC; # vs. MTX. MDA; malondialdehyde, CAT; catalase, SOD; superoxide dismutase.

Figure 4

Testicular gene expression of (A) NF-κB, (B) TNF-α and tissue levels of (C) IL1-β and (D) IL-6 in the normal control (NC) group and experimental groups (MTX, crude extract + MTX, zygo-albuside A + MTX, rutin + MTX). Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. Values are expressed as mean ± SD (n = 8). A statistically significant difference was assumed at p < 0.01 and denoted by * vs. NC; # vs. MTX. NF-κB; nuclear factor kappa B, TNF-α; tumor necrosis factor-α, IL-1β; interleukin-1β, IL-6; interleukin-6.

Figure 5

Effect of administration of crude extract, zygo-albuside A, or rutin on testicular expression of (A) p53, (B) miRNA-29a, mRNA and testicular levels of (C) Bax and (D) Bcl-2 in mice with MTX-induced testicular injury. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. Values are expressed as mean ± SD (n = 8). A statistically significant difference was assumed at p < 0.01 and denoted by * vs. NC; # vs. MTX.

Figure 6

Representative photomicrographs of testis from control and experimentally treated mice. Testicular tissue sections stained with hematoxylin and eosin (40×) and scored by Johnsen. The normal control group (NC) showed complete spermatogenesis (black arrow) in all tubules, up to the sperm count (score 10). In the MTX group, some tubules are hyalinized, while other tubules show a marked reduction in spermatogenesis with few spermatocytes (black arrows) (score 2). The Crude extract + MTX group showed tubules with complete spermatogenesis (black arrows) and slightly impaired spermatogenic cells (score 9). The Zygo-albuside A + MTX group showed the most pronounced effect; complete spermatogenesis is evident in all tubules up to spermatids (black arrow) (score 10). The Rutin + MTX group also showed complete spermatogenesis in all tubules, up to the spermatids (black arrow) (score 10). Scores are expressed as mean ± SD (n = 8). Statistical analysis was performed by one-way analysis ANOVA followed by Tukey’s post hoc test. A statistically significant difference was assumed at p < 0.01 and marked with * vs. NC; # vs. MTX.

Figure 7

Protective mechanism of zygo-albuside A against methotrexate-induced testicular damage. Red arrows indicate the effects of MTX injection. Green arrows indicate the effects of zygo-albuside A administration. MTX; methotrexate, NF-κB; nuclear factor kappa B, TNF- α; tumor necrosis factor-α, IL-1β; interleukin 1β, IL-6; interleukin-6, SOD; superoxide dismutase, ROS; reactive oxygen species, MDA; malondialdehyde, Cyt C; cytochrome C, Casp; caspase.