The Possible Side Effects of Ziziphus spina-christi Extract on the Liver, Kidneys of Female Rats

Throughout the ages, the use of natural plants and herbal medicines has become widespread for ailments treatment and human health improvement. These extensive practices of herbal products are supported by the prevailing belief among people that they are safer than conventional drugs since they are natural products. It has become necessary to provide advice and education to users of medicinal herbs to increase evidence-based awareness about the possible side effects of these herbs (Alghadir et al., 2022). The unpredictable harmful effects of some herbal products used as complementary and alternative medicines on the liver, kidneys  and other organs have been reported (Başaran et al., 2022; Jain and Olivero, 2019; Philips et al., 2020; Seeff et al., 2015).

The Ziziphus genus (Sidr in Arabic) encompasses approximately 135 species belonging to the Rhamnaceae family (Ara et al., 2008). They are found across warm-temperate, subtropical and tropical regions and are used as alternative folk remedies. The members of the Ziziphus genus have effective pharmaceuticals as an anti-inflammatory (Alsayari and Wahab, 2021), antipyretic (Jalung et al., 2023), analgesic (Henneh et al., 2021), antidiabetic (Abdel-Zaher et al., 2005), antimicrobial (Yahia et al., 2020), antifungal (El-Shahir et al., 2022), prevention of hypertension (Mohebbati et al., 2018) and treatment of malaria (Hafiz et al., 2019). More specifically, diverse methods of extract and various fractions of Ziziphus spina-christi (ZSC) have been employed for various remedial attributes such as immune enhancers and antioxidants (Abduljawad, 2020; Al-Ali and Jewad, 2019), decreasing obesity and metabolic inflammation (Yagoub et al., 2021), against bacterial infections (El-Kamali and Mahjoub, 2009) and as antidiarrheal agents (Adzu et al., 2003). The phytochemicals (like triterpenes, phenolics, flavonoids and saponin glycoside) of ZSC were discussed in various reports (Sakna et al., 2022).

Although, numerous investigations have proven the therapeutic importance and pharmacological activities of ZSC, however, fully investigating its side effects still requires further research. This project emphasizes on the side effects of the ZSC aqueous extract on biochemical indicators and histopathological examination of the liver, kidneys of female rats.

Twenty grams of ZSC leaves were macerated in distilled water (200 ml) at 30°C with shaking at 150 rpm for 24h. The suspensions were filtered with Whatman filter paper and the residue was re-extracted and then re-filtered as above. The two filtrates were pooled together and evaporated in a rotary evaporator at 40°C. The concentrated filtrate of each plant (water crude extract) was weighed and re-dissolved in normal saline at a final concentration of 100 mg, 50 mg, 10 mg /kg and stored at -20°C until use.

The twenty of healthy female rats (180-220 g) with ages between 12 and 14 weeks were sourced from the animal facility at the Zoology Department of the Science College, King Saud University (KSU). These rats were acclimated to a well-ventilated environment at a room temperature of 25±2°C, with a regular 12-hour light and dark cycle. They were provided with a standard diet and access to water. All experimental procedures adhered to the guidelines set by the ethics committee and the Institutional Animal Care at KSU (Approval no: KSU-SE-23-06). The four separate groups of female rats, each consisting of five animals, were exposure as the following: the first group, the control, received water, while the second, third and fourth groups were administered with the extract at dosages of 10, 50 and 100 mg/kg bw/day for 14 days.

Female rats were fasted for 12 hours to examine blood glucose levels using a Glucometer device. The blood was drawn directly from the heart of female rats into plain tubes, then centrifuged at 3500 rpm for 15 minutes to obtain the serum and frozen in the refrigerator until analysis. The clinical biochemical biomarkers of liver (ALT and ALP enzymes) and kidneys (creatinine, uric acid and blood urea nitrogen) were evaluated in blood serum.

The data collected from the experiment underwent analysis using the GraphPad Prism (version 10.1.1) software. To assess the normal distribution of our data, the Shapiro-Wilk test was employed. Various parameters such as liver enzymes, kidney parameters, organ weights, were subjected to one-way ANOVA, followed by Tukey’s test. The results are reported as mean values accompanied by their standard deviations.

No signs of poisoning or death were observed among female rats during the fourteen days of oral administration of ZSC aqueous extract. In addition, there was no significant difference between the daily body weights of female rats during the experimental period (Fig 1). The average weights of kidneys of animals treated with the ZSC aqueous extract did not show significant differences compared to the control group (Fig 2).





Fig 3 shows no significant differences in the serum glucose concentrations (mg/dl) among female rats after two weeks of exposure to the ZSC extract. BUN, creatinine and uric acid levels displayed no statistical difference (P>0.05) between female rats groups (Fig 3). The alkaline phosphatase was elevated in female rats who received 50 mg (p<0.05) and 100 mg (p<0.01) of ZSC aqueous extract in comparison with the control group. In addition, alanine aminotransferase was slightly increased at a dosage of 100 mg group with no significant differences (P>0.05) compared to rats in the control group (Fig 3).



No pathological changes were revealed in the hepatocytes, portal sinuses and central veins of hepatic lobules (Fig 4). In renal histological sections, glomerular capsules, renal tubules and renal capillary structures were normal in all groups (Fig 5).





The lack of reports discussing the toxicity of ZSC extract justifies the need for more research to confirm its safety and to grow evidence-based awareness of its potential side effects. This research explores the side effects of the ZSC aqueous extract on biochemical indicators and histopathological changes of the liver, kidneys and ovary of female rats.

The liver is the main site metabolism of xenobiotics and toxic substances and therefore the liver is considered one of the organs most affected by these substances. The hepatocytes contain high levels of the ALT and ALP enzymes compared to other organs; therefore, they are considered biomarkers for detecting hepatobiliary damage (Henneh et al., 2022; Islam et al., 2021; Ozer et al., 2008).

Scientists are putting a lot of effort into finding natural substitutes that humans may use without risk. As a result, research on a variety of natural alternatives persisted, and our investigation thereafter concentrated on assessing the use of Ziziphus spina-christi extract. Although our research showed some minor adverse effects on enzymes, this is not thought to be conclusive evidence for its intended application. Studies have demonstrated its efficacy in treating a variety of illnesses and infections. Similarly, weights were not considerably impacted by our findings. Additional many researches are necessary since there could be other variables, such the length of exposure, the extract’s concentration, the dose, and several other issues.

The authors of the manuscript declare that they have no competing or financial interests.