The potential effect of the Rumex vesicarius water seeds extract treatment on mice before and during pregnancy on the serum enzymes and the histology of kidney and liver

2.1 The water extraction from Rumex vesicarius seeds

The Plant samples were collected from different parts of the north and east area of Riyadh city, Saudi Arabia, between January and March 2020. The taxonomy of R. vesicarius was done by Mohammad K. Okla, a plant taxonomy specialist and was confirmed by the head of the herbarium unit of the Botany department, at the University of King Saud. The ingredients of the R. vesicarius seeds extract were analyzed at the college of pharmacy (Pharmacology Department, University of King Saud) via two solvents, ethyl alcohol extract (EAE) and chloroform extract, through GC-MS, via the Perkin Elmer instrument.

The R. seeds were collected from different parts around Riyadh city area during Feb and April 2020. The collected plant was kept in a dried room at 25–28°C until it dried for 2 weeks. Then, the seeds were separated and ground to powder of 200 g. The ground seeds (100 g) was added to 1 L of distilled water in the separation tube for 48 h with repeated stirring, and the solution was separated via a steam filter. To calculate the concentration of water seed extract of R. vesicarius, a 50 mL beaker was weighed and then filled with 5 mL of water seed extract RV filtered fluid and then evaporated in an incubator (60°C for 5 h, the remaining water seed extract in the beaker was weighed and ×100) [19]. Afterward, to reach the (50 mg kg⁻¹) desired concentration of water seed extracts RV, each mice was fed via an oral feeding tube with the desired dose, according to the following formula [34]:

2.2 Animal treatment

Adult female SWR mice (20–30 g) and male mice (35–48 g) were obtained from the Animal House at the Department of Zoology, Faculty of Science, the University of King Saud. The mice were kept under a 12 h light/dark cycle in a ventilated room at a temperature of 25 ± 2°C. The mice were fed with standard laboratory feeding and water ad libitum.

2.3 Experimental design

All animal treatments were done according to the ethics committee, the Institutional Animal Care at King Saud University regulation; and the Collaborative Institutional Training Initiative (CITI) program, Lab Animal Research ID no. Record ID 35011831. All animals used were weighed before and after the experiment.

2.4 Experimental animals divided into two groups

Treated animal Group I: treatment with water seed extract RV before mating: Female SWR mice (20 females) treated with 50 mg kg⁻¹ water seed extract R. vesicarius via an oral feeding tube for 1 week before mating, while a control group of 20 female mice were treated with water (0 mg kg⁻¹). Then, the SWR female mice were caged with SWR male mice (3–4 females + 1 male) for 1 week. Then, the females were checked daily in the morning for vaginal plug observation to confirm mating, and to determine the effect of mice fecundity due to the treatment with water seed extract R. vesicarius before mating.

Treated animal Group II: treatment after mating: (40 females SWR mice) were caged with SWR male mice (3–4 females per male per cage). Then, vaginal plug of each female was checked daily in the morning to confirm copulation. Then, 7 days post-copulation, the pregnant SWR mice (20 females) were treated orally via a feeding tube with water seed extracts R. vesicarius (50 mg kg⁻¹) for 1 week conceded as the treated group. While the other 20 pregnant SWR female mice treated with water, only (0 mg kg⁻¹), were conceded as a control group to detect the effect of the water seed extracts on pregnancy rate. Data from mice groups I and II were collected including the female body weight, offspring produced from each female group, and newborn mice weight, and recorded.

2.5 Analysis of kidney and liver functions from mice blood samples

At the end of the experiment, the blood samples were collected from female and male SWR mice in heparinized glass tubes. Then, serum was separated via centrifugation at 3,000 rpm for 15 min. The enzymatic activities of Aspartate aminotransferase (AST) and the Alanine aminotransferase (ALT), and the amount of the blood urea nitrogen (BUN), creatinine, and uric acid were measured using the diagnostic kits from the Bio-System interment BTS-350.

2.6 Kidney and liver samples

For the histology of the liver and kidney at the end of the experiment of all treated and control female groups before mating and during pregnancy treatments groups, 10 females from each group were kept in a CO₂ chamber container for 5–10 min until death. Then, their liver and kidney were collected for histological preparation at the histology lab unit of the Zoology Department. The organ samples were fixed in 10% neutral formalin, for 2 days, and processed for routine paraffin block preparation by dehydration in different alcoholic levels (30, 50, 70, and 100%). They were cleared with xylene and later with paraffin wax used for impeding, and then sectioned to a size of 5 mm and mounted in glass slides, and stained with hematoxylin and eosin stain. Then, the sections were examined under a microscope by an experienced histopathologist, to evaluate the hepatotoxicity and nephron structure of the liver and kidney.

2.7 Statistical analysis

The mean body weight of females and offspring of mice, with the offspring not produced recorded data were computed via the Mine Tab InStat program. Analysis of variance was performed using the methods of Kolmogorov and Smirnov. The histology damage rates were tested using the Chi-square analysis [34].

3.1 Components of R. vesicarius seeds extract

The analysis of the R. vesicarius seed extract components in two solvents, EAE, and CE, through GCMS showed some variation between the (EAE) and the (CE) extraction solvents, such as the CE contained caffeine, while the EAE did not, Tables 1 and 2. Phytochemical content of the R. vesicarius seed extract consisting of some hexane compounds, with a peak of chemical compound 352,163 MW of Hexadecane, 1-iodo., and 340.334 MW of Eicsanic acid, ethyl ester of the ethanol extract compounds, while the CE peak compounds of the 4,8,12,16, tetramethyheptadecan-4-olide 324.303 MW and Diisooctyl adipate 370.308 MW, Tables 1 and 2 and Figures 1 and 2.

Figure 1

The analysis of the chemical contents of the Rumex vesicarius seed extract by the chloroformed solvent.

Figure 2

The analysis of the chemical contents of the Rumex vesicarius seed extract by the chloroformed solvent.

3.2 Effect of RVWSE on mice body weight and fertility

Table 3 illustrates the result obtained from both groups I and II treated with RVWSE before or during pregnancy compared to the control group and their offspring data. The body weight of the treated groups’ female mice are close to the control and vary from (27–30 g). In addition, the females’ fecundity rate in both the treated group and control are close to each other and vary from 30 to 35%. Also, in the offspring, the mean body weight of new born vary from 1.5 to 1.58 g, Table 3. While the number of sired litters per female in the 2nd treated group shows statically lower rate (5.5 new born/female) which is lower than the 2nd control group sired (8.5 new born/female) and the 1st treated and control (8 and 7.3 new born/female), respectively (Table 3).

3.3 Effect of RVWSE on serum parameters of treated mice

The enzymatic levels of AST, ALT, BUN, creatinine, and uric acid, between the control and the pre-pregnancy treated female group showed no significant differences as shown in Table 4. While the 2nd group (during pregnancy) showed higher levels (p < 0.05) in AST and ALT, compared to the 1st group and the control of second group (Table 4). The creatinine showed a lower level in the treated 1st and 2nd group compared to both the control groups, Table 4. The mean value of BUN and uric acid in the two treated and control groups showed no significant differences in their levels among all females. The BUN in all groups (control and treated) vary from 100 to 128 mg dL⁻¹, and the uric acid also varies from 4.53 to 5.21 mg dL⁻¹ in all females, Table 4.

In the enzymatic analysis of the male groups, the AST (254.83 + 41.45 IU mL⁻¹), and the ALT (101.83 + 19.22 IU mL⁻¹) levels were significantly higher in control groups compared to the treated male group (AST: 160.57 + 24.88 IU mL⁻¹ and ALT: 69.15 + 5.98 IU mL⁻¹). While the creatinine in the treated group show higher level (0.70 + 0.05 mmol L⁻¹) (p < 0.05) compared to the control group (0.40 + 0.06 mmol L⁻¹). The uric acid in males showed a close level between the treated males compared to the control males. When comparing the female groups and male groups, the AST level of the control during pregnancy 2nd group and the control males showed a higher value (female 269.2 + 64.44  IU mL⁻¹ and males 254.83 + 41.54 IU mL⁻¹) compared to all other groups (AST varied from 160.45 + 24.88 to 184.10 + 37.68 IU mL⁻¹), Table 4. While the ALT showed the highest level in the control male group (101.83 + 19.22 IU mL⁻¹) compared to the lowest level in the control 1st female group (65.10 + 5.12 IU mL⁻¹). The BUN levels showed the highest in the treated male group (147.86 + 12.60 mg dL⁻¹) compared to the lowest control 1st female group (100.20 + 4.54 mg dL⁻¹). Also, the treated male groups showed the highest level of creatinine (0.71 + 0.055 mmol L⁻¹) compared to the lowest creatinine level in the 2nd treated female group (0.33 + 0.048 mmol L⁻¹). While the uric acid showed very high in both males groups (p < 0.05) compared to the female groups (8.51 + 0.66 mg dL⁻¹, 4.53 + 0.66 mg dL, respectively), Table 4.

3.4 Histology of the murine liver

The liver histology sections shown in Figure 3a–c illustrate the structure of two different after treated with RVWSE groups compared to the control groups. The liver section of the treated mice with the RVWSE showed no disarrangement and no degeneration of normal hepatic cells with intense centrilobular and no necrosis or swelling, and no fatty degeneration of hepatocytes, it is the same as the histology of the control group with no visible changes Figure 3a and b.

Figure 3

(a–c) Photomicrograph of liver sections of male and female mice treated with RVWSE before mating (group I), and during pregnancy (group II) (×100 or ×200 magnification). [CV = central vein, BD = bile duct, Hc = hepatocytes, CT = connective tissue. Si = sinusoid, Ha = Hepatic artery]. (a) Group I female liver control (×100) and Group I female liver treated (×200). (b) Group II female liver control (×100) and Group II female liver treated liver (×100). (c) Male liver control (×100) and male liver treated (×100).

3.5 Histology of mice kidney

The kidney histology sections of the SWR mice (Figure 1a–c) illustrate the structure of two different groups treated with RVWSE, compared to the control groups or the 1st treated group I before pregnancy, or 2nd group during pregnancy. Areas of both kidney sections, the cortex (C) (with darker stain) and the medulla (M) (with paler stained area) of the treated and control groups showed a close similar gross structure. The examination of the gross structure of the renal tissue reflects no readily apparent difference in the arrangement of the nephron structure or histopathology in both groups of treated and control females and male mice (Figure 4).

Figure 4

(a–c) Sections picture of the kidney of male and female mice treated with RVWSE: before mating (group 1) and during pregnancy (group II) (100× or 200× magnification). [Pt = proximal collecting tubules, Dt = distal collecting tubules, G = glomerulus, Bc = Bowman capsule, Kc = kidney cortex, Km = kidney medulla, Aa = afferent arterioles]. (a) Group I female kidney control (×100) and Group I female kidney treated (×200). (b) Group II female kidney control (×100) and Group II female kidney treated (×100). (c) Male kidney control (×100) and male kidney treated (×100).