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

A BSTRACT Background: It has become important to provide advice and education to herbal remedies users to increase evidence-based awareness of their potential side effects. This research explores the side effects of the Ziziphus spina-christi (ZSC) aqueous extract on biochemical indicators and histopathological changes of the liver, kidneys of female rats. Methods: Fifteen female rats (five in each group) were orally administrated with the extract of ZSC at dosages of 10, 50 and 100 mg/ kg bw/day for 14 days. Five other females were considered a control group, given only water. Clinical observations of toxicity were recorded during fifteen days. The biochemical biomarkers of liver (ALT and ALP enzymes) and kidneys (creatinine, uric acid and blood urea nitrogen) were evaluated in blood serum. Result: In addition, the histopathological alterations in organs were evaluated. The results did not show any clear toxicity on the animal’s weight or body organs. The alkaline phosphatase was elevated in female rats who received 50 mg and 100 mg of ZSC aqueous extract in comparison with the control group. The results did not show any significant changes in the serum glucose, BUN, creatinine, uric acid, alanine aminotransferase levels in female rats who received ZSC extract for 14 days. In conclusion, the results showed indications of side effects of the ZSC extract on liver enzymes after treating female rats with ZSC extract for 14 days.


INTRODUCTION
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).
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.

MATERIALS AND METHODS
Twenty grams of ZSC leaves were macerated in distilled water (200 ml) at 30C with shaking at 150 rpm for 24 h.The suspensions were filtered with W hatman filter paper and the residue was re-extracted and then re-filtered as above.
The Possible Side Effects of Ziziphus spina-christi Extract on the Liver, Kidneys of Female Rats 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-W ilk 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.

RESULTS AND DISCUSSION
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   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).

CONCLUSION
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.

Fig 1 :
Fig 1: Results of female rat mean body weight of treated with Ziziphus spina-christi compared to control group.

Fig 2 :
Fig 2: Results of female rat mean kidney weight of treated with Ziziphus spina-christi compared to control group.

Fig 3 :
Fig 3: Means of blood glucose, uric acid, creatinine, BUN, ALP and ALT enzymes level of female rats group treated with Ziziphus spina-christi extract for two weeks.

Fig 5 :
Fig 5: Histology of the Kidney of the female rat groups treated with Ziziphus spina-christi extract for two weeks compared to the normal.