Testicular Caspase-3 and β-Catenin Regulators Predicted via Comparative Metabolomics and Docking Studies

Many routes have been explored to search for effective, safe, and affordable alternatives to hazardous female contraceptives. Herbal extracts and their secondary metabolites are some of the interesting research areas to address this growing issue. This study aims to investigate the effects of ten different plant extracts on testicular spermatogenesis. The correlation between the chemical profile of these extracts and their in vivo effect on male reproductive system was evaluated using various techniques. Approximately 10% of LD50 of hydro-methanolic extracts were orally administrated to rats for 60 days. Semen parameters, sexual organ weights, and serum levels of male sex hormones in addition to testes histopathology, were evaluated. Moreover, metabolomic analysis using (LC-HRESIMS), multivariate analysis (PCA), immunohistochemistry (caspase-3 and β-catenin), and a docking study were performed. Results indicated that three plant extracts significantly decreased epididymal sperm density and motility. Moreover, their effects on testicular cells were also assured by histopathological evaluations. Metabolomic profiling of the bioactive plant extracts showed the presence of diverse phytochemicals, mostly oleanane saponins, phenolic diterpenes, and lupane triterpenes. A docking study on caspase-3 enzyme showed that oleanane saponins possessed the highest binding affinity. An immunohistochemistry assay on β-catenin and caspase-3 indicated that Albizzia lebbeck was the most active extract for decreasing immunoexpression of β-catenin, while Rosmarinus officinalis showed the highest activity for increasing immunoexpression of caspase-3. The spermatogenesis decreasing the activity of A. lebbeck, Anagallis arvensis, and R. officinalis can be mediated via up-regulation of caspase-3 and down-regulation of β-catenin existing in testis cells.

The only reported article concerning Lettuce seeds on male reproductive system utilized 50-200 mg/kg of seeds extract (i.p for 10 days), and no adverse effects, toxicities or mortalities were provided. [2]

Apium graveolens leaves
The only reported article concerning celery oral LD50 was demonstrated by Al-Howiriny, et al on aerial parts which was = 7500 mg/kg in rats, indicating its high safety margin [3,4] Acute oral toxicity test revealed no deleterious or toxic symptoms or mortality over a period of 14 days by a dose of 250-500 mg/kg in rats upon investigating studying gastric antiulcer, antisecretory and cytoprotective properties of celery, indicating high safety properties of celery aerial parts. [4] The only reported article concerning A. graveolens leaves on male reproductive system utilized 50-150 mg/kg/2 days of leaves extract was injected (IP) for 20 days to rats, and no adverse effects, toxicities or mortalities study were provided. [4]

Anethum graveolens seeds
The only reported article concerning LD50 of dill seeds was demonstrated by Al-Hosseinzadeh, et al which was = 3004 mg/kg, i.p., (1500, 6016) and 6098 mg/kg, i.p., (5069, 8056) of aqueous and ethanolic extract, respectively in mice (~2102 mg/kg, and 4269 mg/kg in rats*), indicating its high safety margin when taken orally. [5] It was reported that aqueous and hydroalcoholic extracts of seeds possess male contraceptive effects at doses of 45, 450 and 500, 5000 mg/kg/orally, respectively in rats. And no toxicity study was provided. [6] Calendula officinalis flowers A LD50 of 375 mg/kg and a LD100 of 580 mg/kg has been determined in mice by intravenous and intraperitoneal administration of aqueous extracts. In hydro-alcoholic extracts a LD50 of 45mg/ mouse (sub-cutaneous) and LD50 of 5260 mg/ kg in rats (intravenous) have been reported. [7] In the oral acute study of aqueous extract (2000 mg/kg) in rats, there were no mortality and signs of toxicity. In the subchronic study (250, 1000 mg/kg), several of the blood elements were significantly affected in males and females after 90 days; hemoglobin, erythrocytes, leukocytes and blood clotting time. For blood chemistry parameters, ALT, AST and alkaline phosphatase were affected. Histopathological examination of tissues showed slight abnormalities in hepatic parenchyma that were consistent with biochemical variations observed. These studies indicate that the acute and subchronic toxicities of C. Officinalis aqueous extract are low. [8] In the acute toxicity test, hydro-alcoholic extract failed to cause death in the animals after administration of oral doses up to 5000 mg/kg in rats. Oral treatment with hydro-alcoholic extract at 25, 250, 500 and 1000 mg/kg/day for 30 days did not induce hematological alterations. In the biochemical parameters, there was an increase in blood urea nitrogen (BUN) and in alanine transaminase (ALT) levels.
Morphological examination of the brain, kidney and heart did not show any alteration. [9] Oral LD50 of the ethanolic extract was found to be 2450 mg/Kg in mice (~1715 mg/kg in rats*) [10] It was reported that butanol fraction of flowers possess spermicidal activity, and no toxicity studies concerning male reproductive system were reported. [11]

Menthae piperitae leaves
Aqueous extract above 5000 mg/kg orally in rats revealed no mortality (LD50), in which the histological changes observed in the selected organs and the biochemical deviation of blood compared to the normal range level were minimal after 14 days (sub-chronic). So, it is suggested to be highly safe. [12] The oral LD50 of ethanolic extract of leaves was found to be 3700 mg/kg in rats [13] Aqueous tea was investigated to affect male reproductive system through free administration to rats randomly without fixed daily dose. No toxicity study was provided. [14]

Rosmarinus officinalis leaves
The median lethal dose (LD50) value of methanolic extract of Rosmarinus officinalis leaves was 4125 mg/kg in mice intraperitoneally (~2888 mg/kg in rats*). [15] The oral LD 50 of rosemary leaves extract has been described as > 8500 mg/kg in rats.
[16] The article which discussed the effect of hydroethanolic extract of Rosmarinus officinalis leaves has used 500 mg/kg daily fixed dose, and no toxicity study was provided. [17] Calotropis procera aerial parts Oral LD50 of ethanolic extract of C. procera leaves is 95.52 mg/kg in rats. ` [18] C. procera ethanolic extract of leaves could induce marked toxicity in heart and testis with 1/10 or 1/20 LD50 (95.52 mg/kg in rats) for a period of 4-8 weeks. [18] 200 mg/kg body weight of the aqueous leaves extract was daily administered orally by gavages during 42 [19] days in rabbits (~375in rats*). All the rabbits gained weight during the administration period, with an appreciable gain for smaller animals. Significant decrease of ALT and RBC were noticed in the youngest rabbits. A significant increase of serum creatinine level and lymphocytes were also noticed within the group of the juvenile rabbits. Necropsy revealed lesions in kidney and liver, these lesions were further confirmed by histopathology observations that revealed more pronounced pathology with the youngest animals. Although animals in different test groups show some toxic effects; small animals of eight weeks exhibit more effects with more severe lesions. The aqueous leaf extract (50, 75 mg/kg/orally in rats) for 15 day produced significant increase in packed call volume (PCV) but did not influence coagulation time. The extract also produced hypoproteinaemia reflected as hypoalbuminaemia. Similarly the leaf extract also caused elevation in the activities of aspartate aminotransferase and alanine aminotransferase. Although the extract did not produce lesions in the heart, spleen and liver examined, the increase in liver enzyme activities could be due to early liver damage. [20] C. procera ethanolic extract of leaves could induce marked toxicity in kidney by using 1/10 0r 1/20 LD50 (~95.52 mg/kg in rats*) for a period of 4-8 weeks, orally. [21] Oral LD50 was estimated (940 mg/kg) in rabbits (~1725 mg/kg in rats*) of fresh leaves aqueous extract. 80, 40 and 20 mg/kg/day of the extract were administered orally during sub-acute toxicity study for 14 days in rabbits (~150, 75, 40 mg/kg in rats*), Statistical analysis of aspartate amino transferase (AST), ala-nine amino transferase (ALT), alkaline phosphatase (ALP), albumin and protein showed no significant changes. Changes in packed cell volume (PCV), white blood cells (WBC), haemoglobin (Hb), platelets, and differential leucocyte count (lymphocytes, monocytes, eosinophils, heterophils/neutrophils and basophils) were equally statistically insignificant. However, gross and histopathological examination of some organs and tissues (heart, liver, kidney, brain, small intestine and lungs) revealed lesions. [22] Oral LD50 was = 533 mg/kg of leaves ethanolic extract in mice (~373 mg/kg in rats*). Hepato-and renaltoxicities (1/10 or 1/20 LD50) were occurred (~40 or 20 mg/kg in rats*). Also, the oral treatment with the ethanolic crude extract of leaves of Calotropis procera at a high dose (1/5 of the LD50) for a prolonged time could inhibit or arrest the spermatogenesis process leading to infertility in male albino mice (~80 mg/kg min rats*). [23] It was observed that the hydroalcholic extract of the leaves of C. procera (5000 mg/kg, p.o.) did not induce changes in the behavior of male mice during the first 30 min and for a period of up to 4 h after [24] administration. No death was recorded during the fourteen days of observation. No significant changes in intake of food and water or in body weight were observed throughout the period. The LD50 could not therefore be estimated and is possibly higher than 5000 mg/kg The oral LD50 of the hydroalcoholic extract of stem bark with no signs of acute toxicity at 2000 mg/kg in rats [25] The LD50 of leaves aqueous extract could not therefore be estimated and is possibly higher than 5000 mg/kg [26] The Oral LD50 dose of the flower methanolic extract on mice is found to be 1660 mg/kg [27] Flower aqueous and ethanolic extracts were evaluated for their effect on male reproductive system via using an intraperitoneal dose of 5, 10 mg/30 gm in mice per two days for 20 days. No general toxicity study was provided. [28]

Hibiscus sabdariffa calyces
The plant extracts are characterized by a very low degree of toxicity. The LD50 of H. sabdariffa calyx extract in rats was found to be above 5000 mg / kg. A single report has suggested that excessive doses for relatively long periods could have a deleterious effect on the testes of rats [29] After 14 days of a single oral administration of aqueous extract (5,000 mg/kg). No signs and differences of the weights or behavior compared to the control rats were observed. An oral administration of aqueous extract at the doses of 50, 100 and 200 mg/kg body weight for 270 days does not cause chronic toxicity in rat. [30] The intraperitoneal LD50 of the aqueous extract of Hibiscus sabdariffa calyx was found to be greater than 5000 mg/kg in mice [31] The LD50 of rosell calyx aqueous extract was found to be above 5000 mg kg−1 IN rats [32] Hibiscus sabdariffa calyces aqueous extract was investigated to affect male reproductive system by using oral doses of 1100-4600 mg/kg in rats/ day for 60 days. No other toxicological parameters were measured (other body organs). [33] Hibiscus sabdariffa calyces aqueous extract was also investigated to affect the sperm morphology by using 200 mg/kg/orally in mice per day for 30 days. No other toxicological parameters were measured (other body organs). [34]

Anagallis arvensis
The intraperitoneal LD50 was 10718 mg/kg of alcoholic extract of A. arvensis [35] (1/5 and 1/10 LD50) IP [10718 mg/kg.b.wt] for 15 days i.e. 2650 mg/kg causes clinical signs included [35] anorexia, restlessness, diarrhea, thirst, difficult breathing, tremors and ended by coma and death. Hematologically, there were a significant reduction in PCV%, Hb concentration and RBCs count of the intoxicated rats. Concerning kidney function tests, there were a significant increase in urea and creatinine level of the intoxicated rats. Pathologically, the lesions were primarily confined to the urinary system. No reported toxicity studies of Anagallis arvensis extracts on male reproductive system. Our article is considered the first article that investigated the effect of this herb on spermatogenesis.

Albizzia lebbeck pods
The acute oral LD50 of aqueous extract of stem in bark albino rats of either sex was found to be 2000 mg/kg. LD0 was found to be 400 g/kg, and LD100 approximately 3200 mg/kg. [36] The intraperitoneal LD50 of the aqueous methanol extract of seeds of Albizzia lebbeck was found to be 82 mg/kg in rats. [37] Oral LD50 of methanolic extract of Albizia lebbeck leaves was considered as 2000 mg/kg in rats.
[38] Oral LD50 of 70% ethanolic extract of Albizzia lebbeck bark. Since no mortality was observed at 2000 mg/kg in rats. [39] Oral LD50 more than 2000 mg/kg without any toxic symptoms in stem bark methanolic extract in mice [40] The LD50 (mice, oral) is therefore estimated to be beyond 5000 mg/kg body weight of aqueous or alcoholic extract of flowers [41] Oral LD50 more than 5000 mg/kg without any toxic symptoms in stem bark methanolic extract in mice [42] Its toxicity on male reproductive system was reported when using a fixed daily dose of 200 mg/kg/day/orally of ethanolic extract of pods in rats for 60 days. The RBC and WBC counts, haemoglobin, haematocrit and blood sugar were within the normal range at this dose. [43] The other article that investigated the toxicity of pods on male reproductive system has used 250 mg/kg/orally of triterpene fraction of methanolic extract of pods in rate for 60 days. There were no significant changes in RBC and WBC count, haemoglobin, haematocrit, blood glucose, cholesterol, protein, triglycerides, phospolipids and HDL-cholesterol. [44]