Rumex vesicarius (R. vesicarius or RV) is an annual plant having rounded leaves, with flowers containing seeds. RV protects liver, resists cancer, and removes free radicals in cells. The aim of this study was to illustrate the effect of the R. vesicarius water seeds extract (RVWSE) treatment on mice before and during pregnancy, and its action on the liver and kidney histology and enzymes, aspartate aminotransferase and alanine aminotransferase, the blood urea nitrogen, the creatinine, and the uric acid. The seeds of R. vesicarius were collected, extracted, and its component analyzed via Gas chromatography mass spectrometry. Mice treated with 10 mg kg−1 RVWSE via feeding tube for 1 week before mating, or during pregnancy. The number of offspring or litter size was recorded and the blood sample was collected at the end of the experiment to test the kidney and liver enzymes, and their histology. Results showed that RVWSE contains different phytochemicals consisting of some hexane and chloroform compounds. The fertility rate of 1st treated pre-pregnancy group is 30% and the 2nd group is 35%. The offspring rate of 1st treated group showed higher new born rate (8 new born/female) than 2nd treated group (5.4 new born/female). The blood enzyme levels of the kidney and liver showed some variation between the two groups and their histology illustrates some non-significant variation between the treated and control groups of mice. The implications of the results of this study illustrate the safe use of the RVWSE, its effectiveness in improving mice fertility, and positive impact on biomarker of serum enzymes of liver and kidney with their histopathology.
Rumex vesicarius (R. vesicarius or RV) is a medicinal plant used especially in some Asian countries. The RV plant is found in several Asian countries, North Africa, and South Europe. R. vesicarius can grow in dry and wet areas, in different soils, especially wet and good drainage soils. R. vesicarius is an annual plant with bi-branched and rounded juice leaves. RV flowers have conjoined seeds with white or pink transparent shutters. The flowering period is between January and March. The length of the RV plant is about 11–44 cm, the width is 8 mm, and the length of its roots is about 5–10 cm. Author of ref.  made a Gas Chromatography-Mass spectrometry (GC-MS) analysis of R. vesicarius L. The chemical analysis of R. vesicarius indicates that it contains several biochemical materials, such as anthraquinones, flavonoids, proteins, ascorbic acid, and a low quantity of tocopherol, lipids, and minerals [2,3,4]. The R. vesicarius is a good source of anthraquinones and ß carotenes epically in the roots such as chrysophanol, emodin, vitamins, proteins, lipids, and organic acids , while the other parts contain rumicine, lapathine , and some glycosides: orientin, isoorientin, and vitexin, isovitexin . Some researchers indicate that the R. vesicarius is rich in minerals such as Na, K, Mg, Ca, Fe, Cu, and Mn [2,8]. The R. vesicarius is widely used in Saudi Arabia as food and salad, for medicinal treatments, and as an antidote against snake and scorpion bites . The whole R. vesicarius plant and its seeds are used as a diuretic and an aperitif, and it can be used in anemia treatments [10,11]. The R. vesicarius leaf part is edible, its fresh juice is used for toothache, as a cooling agent, as an appetizer, for nausea, as an astringent, as an anti-venom, and for insect bite . The seeds help in dysentery treatments, such as stomachic treatments . There are also studies on liver cirrhosis , protection from cancer and microbial infection, and free radical scavenger [14,15,16,17,18], its use for treating diabetic rats . The R. vesicarius is used against scabies and leukoderma, as tonic and a diuretic. In addition, it is used in the treatment of constipation, hiccup, and piles . R. vesicarius is used in the treatment of urinary infection, dysmenorrhea, chronic catarrh, renal disorders, dyspepsia, bloody dysentery, coronary, and vomiting , and as alkalinity hepatoprotective, depurative, sedative blood purifier . In addition, some researchers stated that R. vesicarius is used as an antiviral, and for the treatment of leukoderma, lymphatic glandular system disease, also for the treatment of rectal prolapses, antidiabetic, aphrodisiac, anti-cholesterol, impetigo, and carbuncles, and as an antioxidant . Leaf extract of R. vesicarius L. is used for treatment of cancer and inflammation , as spasmogenic and spasmolytic and antipyretic , and for wound healing activity in rabbit . In addition, there are several investigators who studied the soil analysis in Riyadh area, Saudi Arabia, where the R. vesicarius was collected [24,25,26,27]. The soil analysis and correlation test revealed significant variations in the content of salinity, moisture, CO3 −, Cl, SO4, Ca, Mg, and Na among the plant communities .
The liver is one of the largest organs in the body, and its function in many metabolic functions . It performs several detoxifications and complex biological activities that are essential for metabolism in the human and animal bodies. Usually, the histology damage of the hepatic tissue is associated with distortion of metabolic functions . In addition, the kidney’s function is to keep and balance the body’s fluids, electrolytes, and organic solutes in a healthy balance. Some dietary changes can also help control hypertension, edema, and hyperlipidemia, and slow the progression of renal disease . However according to the reviewer notes that there are several of studies proved the role of plant extracts and in the cytotoxicity of human cells in vitro such as the recent study of the synthetic chalcones (3a–3f) to evaluate its cytotoxicity assay as antineoplastic activity by using in vitro glioblastoma models and molecular modeling . Another study illustrates the antiproliferative, genotoxic activities and quantification of extracts and cucurbitacin B obtained from Luffa operculata (L.) Cogn. . In addition, Castro and his co-investigator (2021) studied the genotoxicity and mutagenicity of isoeleutherin and eleutherin isolated from Eleutherine plicata herb using bioassays and in silico approaches .
Since there is very limited information about the seeds that help in dysentery treatments and no information or studies about the impact of uses of the R. vesicarius seeds extract either on animals or human, this study is designed to illustrate the influence of R. vesicarius water seeds extract (RVWSE) on female mice (before and during pregnancy) and its potential effect on the kidney and liver enzymes and their histology structure.
2 Materials and methods
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) . 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 :
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 .
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.
|Chemical contents||Molecular weight|
|Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, (1.alpha.,2.beta.,5.alpha.)-||138.141|
|Hexadecanoic acid, ethyl ester||284.272|
|Octadecanoic acid, ethyl ester||312.303|
|Eicosanoic acid, ethyl ester||340.334|
|Nonadecanoic acid, ethyl ester||326.318|
|Chemical contents||Molecular weight|
|9-Borabicyclo [3.3.1]nonane, 9-(2-propen-1-yloxy)-||178.153|
|Methyl octyl ether||144.151|
|1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester||278.152|
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).
|Treated and control female group (no.) pre and during pregnancy||Female mean body weight and SEM±||Female fertility, pregnancy no and %||Offspring no. and rate/female||Mean body weight of new born in g|
|A. Females treated before pregnancy (20)||27 ± 2.35 g||6/20 = 30%||48/6 = 8/female||1.5|
|A. Control females before pregnancy (20)||30 ± 1.03 g||7/20 = 35%||44/7 = 7.3/female||1.58|
|B. Females treated during pregnancy (20)||27.7 ± 1.78 g||7/20 = 35%||*38/7 = 5.4||1.57|
|B. Control females during pregnancy (20)||29.5 ± 1.91 g||8/20 = 30%||68/8 = 8.5||1.55|
*Statically lower than the other in the same column P < 0.05).
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.
|Mice group and their no.||AST mean value and ± SEM IU mL−1||ALT mean value and ± SEM IU mL−1||BUN mean value and ± SEM mg dL−1||Creatinine mean value and ± SEM mmol L−1||Uric acid mean value and ± SEM mg dL−1|
|Control females before pregnancy (10)||174.70 ± 21.96||65.10 ± 5.12||100.20 ± 4.54||0.42 ± 0.048||5.03 ± 0.77|
|Treated females before pregnancy (9)||172.44 ± 30.26||66.11 ± 7.09||122.00 ± 12.72||0.36 ± 0.066||5.21 ± 0.71|
|Control females during pregnancy (10)||269.20* ± 64.44||90.20 * ± 21.81||128.10 ± 23.33||0.45 ± 0.15||4.53 ± 0.60|
|Treated females during pregnancy (10)||184.10 ± 37.65||70.80 ± 10.16||111.30 ± 12.26||0.33 ± 0.033||4.86 ± 0.53|
|Control males (6)||254.83* ± 41.54||101.83* ± 19.22||146.00 ± 4.62||0.40 ± 0.063||7.81* ± 0.66|
|Treated males (7)||160.57 ± 24.89||69.10 ± 5.98||147.86 ± 12.60||0.71* ± 0.055||8.51* ± 0.66|
(AST): aspartate aminotransferase, (ALT): alanine aminotransferase, (BUN): blood urea nitrogen.
*significantly higher (P < 0.05) compared to the other levels within the same column.
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.
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).
The chemical analysis of the RVWSE showed some variation between the two-solvent extracts of EAE and CE. In addition, the seed extract shows some variations in the components of essential oil obtained from samples of R. vesicarius that were studied in the same region by the author . Also, the RVWSE component showed some variation in studies from Al-Jouf area (north of Saud Arabia), which indicated that the seeds extract exhibited higher phenolic and total flavonoid than leaves or leaves plus seeds extract , and the study by author of refs.  from Al-Zulfi, Majmaah area, central region Saudi Arabia.
The female fecundity rates were measured by dividing the total number of offspring sired by the total number of pregnant females. This is different from the index of Silver’s production efficiency of mice from trial mating, which calculates the number of pups born/female/week, during the experiment . Also, it is unlike ref.  in which the fertility index was counted as the slope of the regression of a cumulative number of pups produced by a female over elapsed time in a monogamous mating trial by using a robust resampling technique. The current results showed some differences in the fecundity rate of female mice between the 1st and the 2nd group; however, the rate of the offspring in the 1st female group is higher than the 2nd group. The offspring rate of mice (5–8 in both groups) than the  study that controls delivered an average of (4.75 litters per fertile pair). The RVWSE improved the fertility of mice  compared with the alcoholic extract of Rumex steudelli roots, which led to abortion in rats (95%) , because the extracts of Rumex steudelli roots were used as abortion agent at a dose of 200–400 mg kg−1 for long-term treatments.
The results of the enzymatic (AST and ALT) levels showed some differences between the 1st pre-pregnant group compared to 2nd during pregnancy groups, especially in the control groups, this could be due to high blood flow to the liver and kidney during pregnancy, either to feed the embryos and to get rid of the embryo waste by these two. Although the results of the enzymatic levels showed some variation in the AST and ALT, between the 2nd control group and the control male group compared to the 1st pre-pregnant group, the treatment with RVWSE in both male and female groups reduced it to a normal level. The biochemical profiles of AST, ALT, and creatinine of the SWR serum blood treated with RVWSE 10 mg kg−1 were about the same as that of the Balb/c mice treated with or without WEHI-3 cells by intra-vein transplantation following treatment of MJ-29 intra-peritoneal injection , but their uric acid level showed less than our study results. Although ref.  indicate that a variety of enzymes such as AST and ALT are released into the blood after hepatocytes damage, both are considered potential indicators of hepatocytes disorder. However, the treatment with RVWSE showed slight morphological differences in the histology of the liver and kidney structure compared with the control mice. So the current findings agree with several experimental studies on R. vesicarius extract which proved its safe use [13,43,44]. It is worth to do a correlation of the different plant communities and plant diversity to soil moisture, salinity, and other soil properties, so that it can be concluded that soil moisture and salinity factors were the fundamental driving forces for plant community structure as stated by author of ref. . So it is a good idea in the future studies to make an analysis of the soil where the plant was collected and compare its implication and correlation of the soil content and the plant extract content. In addition, the study on the in vitro effect of the R. vesicarius seeds extract on the in vitro fertilization and embryo development, and its action as anti-oxidant or apoptosis action on cells will be carried out in future.
So the implications of the results of this study illustrate the safe use of the RVWSE, with its effect for improving mice fertility and positive impact on biomarker of serum enzymes of liver and kidney with their histopathology. The result of this study suggest more investigation on the analysis of the soil and its correlation with the plant extraction, epically the seeds; also, to study the in vitro effect of the R. vesicarius seeds extract, and its action as anti-oxidant or apoptosis action on cells, in addition to the study on the blood flow to the reproductive system during the R. vesicarius treatment of seeds extract.
The authors thank the Researchers Supporting Project for funding this work, project number (RSP-2021/232) at King Saud University, Riyadh, Saudi Arabia.
Funding information: This research was funded by the Researchers Supporting Project number (RSP-2021/232), King Saud University, Riyadh, Saudi Arabia.
Author contributions: R.A.A. and A.A.A., and MAA; validation: A.R.A., R.A.A., and A.M.R.; formal analysis: A.R.A and N.M.A.; investigation: R.A.A. and A.R.A.; data curation: A.R.A. and M.Q.A.; writing – original draft preparation: A.A.A. and A.R.A.; writing – review and editing: R.A.A., A.R.A., and NMA.; visualization: A.R.A., A.A.A., and M.Q.A.; supervision: A.R.A. and M.K.O. All authors have read and agreed to the published version of the manuscript.
Conflict of interest: The authors of the manuscript declare that they have no competing or financial interests
Ethical approval: All animal treatments were done according to the ethics committee, the Institutional Animal Care at King Saud University regulation; and the CITI program, Lab Animal Research ID no. Record ID 35011831. All animals used were weighed before and after the experiment.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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