Hepatoprotective, nephroprotective, anti-amylase, and anti- glucosidase effects of Ziziphus spina-christi (L.) against carbon tetrachloride-induced toxicity in rats

Purpose: To explore the hepatoprotective, nephroprotective, anti-amylase, and anti-glucosidase effects of the medicinal plant Ziziphus spina-christi (L.). Methods: Ziziphus spina-christi (L.) methanol extract (ZS-1) and its ethyl-acetate (ZS-2), n-butanol (ZS3), and aqueous (ZS-4) fractions were evaluated for their hepatoprotective, anti-amylase, and antiglucosidase activities. Adult male Wister rats were divided into 11 groups (IXI) with 6 mice per group. Group I was normal control, while the treatment groups were as follows: group II, CCl4; group III, Silymarin + CCl4; group IV, Ziziphus spina-christi total methanol extract (ZS-1), 100 mg/kg) + CCl4; group V, ZS-1 (200 mg/kg) + CCl4; group VI, ethyl acetate fraction (ZS-2), 100 mg/kg + CCl4; group VII: ZS-2 (200 mg/kg) + CCl4; group VIII, butanol fraction (ZS-3), 100 mg/kg) + CCl4; group IX, ZS-3 (200 mg/kg) + CCl4; group X, aqueous fraction (ZS-4), 100 mg/kg) + CCl4; group XI: ZS-4 (200 mg/kg) + CCl4. Silymarin was used as the standard. Biomarkers of liver and kidney toxicity and histopathological changes were evaluated. Results: Liver and kidney malondialdehyde (MDA), non-protein sulfhydryls (NP-SH) and total protein levels were elevated in CCl4-treated rats; however, ZS-1 and ZS-4 of Z. spina-christi significantly reduced these levels. ZS-2 and ZS-3 did not significantly improve the studied parameters. These results were confirmed by results from histopathological examination. ZS-1 and ZS-2 showed mild inhibitory activities against α-amylase and α-glucosidase (54 and 43 % at 100 μg/ml, respectively). Conclusion: The results indicate that ZS-1 and ZS-4 samples displayed dose-dependent hepatoprotective and nephroprotective effects, whereas ZS-2 and ZS-3 samples did not exhibit these effects. Similarly, α-amylase and α-glucosidase enzymes are considerably inhibited by ZS-1 and ZS-2.


INTRODUCTION
Many environmental impurities, drugs, chemicals, and antibiotics exert numerous adverse effects on the kidney, liver, intestine, and heart [1]. The liver is a remarkably important organ with the critical function of regulating physiological activities. It is involved in almost all biochemical processes in the body such as development, nutrient delivery, disease progression, reproduction, and energy provision. In addition, the liver supports the metabolism of proteins and carbohydrates, fat detoxification, bile secretion, and vitamin storage [2]. The liver is often damaged by one's surrounding environment, toxins, alcohols, and over-thecounter treatments, ultimately leading to hepatitis, cirrhosis, and liver disorders [3]. Therefore, treating liver diseases is very important. Over the centuries, medicinal plants have been used to manage several human diseases and play very significant roles in the health care system globally [4]. Diabetes is an advanced metabolic disease of glucose metabolism, and in the long-term, leads to microvascular variations [5].
In Saudi Arabia, the local name for Zizyphus spina-christi (L) is Sidr. Sidr is a shrub belonging to the Rhamnaceae family and is indigenous to warm and subtropical areas including North Africa, the South and Middle East, East of Asia, Mediterranean region, South Europe, Australia, and tropical America [6]. The edible fruit of Z. spina-christi is important in the medical field as it is applied to ulcers and cuts. Essentially, the fruit is used to treat pulmonary diseases and fevers, and for healing fresh wounds [7]. In folk medicine, Sidr is used to heal several ailments such as liver complaints, urinary issues, digestive syndromes, weakness, obesity, diabetes, skin infection, appetite loss, fever, bronchitis, pharyngitis, anemia, insomnia and diarrhea [8]. The Sidr leaves are locally applied to sores while the roots are used to treat skin diseases [9]. The seeds are at times ingested with buttermilk to halt vomiting, treat nausea and abdominal problems related to pregnancy, and for their sedative effects [10]. The leaves are used to treat liver diseases, fever and asthma [11]. Additionally, Ziziphus spina-christi leaves have been reported to exhibit significant antioxidant and hypoglycemic activities [12] and are rich in ceanothic, betulinic acids, saponins, various flavonoids, triterpenes, tannins and flavonoids [13]. The aim of the current study was to evaluate the hepatoprotective, nephroprotective, and anti-α-amylase and anti-α-glucosidase activities of Ziziphus spina-christi.

Extract preparation
Air-dried, grinded (1.2 kg leaves) Z. spina-christi material was immersed in 80% methanol for three days, then filtered and evaporated with a rotary evaporator. A greenish residue was obtained as the methanol extract. A portion of this extract (ZS-1) was used for bioscreening purposes while the remaining portion was partitioned using a separating funnel where polarity of the solvent was increased using nhexane, chloroform, ethyl acetate (ZS-2), and nbutanol (ZS-3); an aqueous fraction (ZS-4) was also obtained.

Animals and experimental design
A total of 66 albino male rats weighing 160 ±11 g was retrieved from the Experimental Center of Animal, Faculty of Pharmacy., King Saud University, Riyadh, Kingdom of Saudi Arabia. The rats were supplied with water and food, and housed at ‫52ـ22‬ °C under a 12-h dark-light cycle. All animals were handled as per the approvals of the 'Guide for the Care and Use of Laboratory Animals' permitted by the Institutional Animal Ethics Committee of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia (clearance no. CBR-4538) [12]. Prior to the start of the experiments, rats were adapted to the conditions of the laboratory for 7 days. The animals were randomly divided into 11 groups (6 rats each) with the details of each listed in Table  1. Carbon tetrachloride (CCl 4 ) in liquid paraffin was administered intraperitoneally (IP) to the rodents.

Blood and tissue sampling
At the end of the experimental period, animals from the different groups were starved for 12 h then weighed. Blood samples were collected from the sublingual vein, left to coagulate at room temperature, and centrifuged for 15 min at 3000 rpm. The non-hemolyzed, clear serum was quickly separated and stored at -80 °C for use in biochemical investigations of lipid profile, and liver and kidney function parameters. Animals were sacrificed using diethyl ether anesthesia, and the liver and kidney tissues were rapidly removed and divided into two parts; one was immediately preserved in 10% buffered formalin at 4 °C for histological examination and the remaining part stored at -80 °C for biochemical analysis.

Evaluation of kidney functions
Reflotron Plus Analyzer and Roche kits (Roche Diagnostics GmbH, Mannheim, Germany) were used to determined creatinine and uric acid levels (in mg/dL).

Assessment of liver functions
SGOT enzyme, SGPT activities, alkaline phosphatase (ALP) activity and total bilirubin concentration were measured by the methods described by Ullah et al [14].

Determination of malondialdehyde (MDA)
The method reported by Utley et al was used to determine the level of malondialdehyde (MDA). In brief, the kidney and liver samples were isolated and then immersed in 0.15 M KCl using an electric homogenizer. Color development was observed at 532 nm using a UV/Vis spectrophotometer. Malondialdehyde content was calculated using an MDA standard curve.

Total protein determination (TP)
The total protein was assessed using kit delivered by Crescent Diagnostics, Jeddah, KSA. Protein concentration measured by measuring the developed colour at 546 nm with the help of UV-Vis-Spetrophotometer Model UV-mini-1240, Shimadzu (Japan).

Non-protein sulfhydryls (NP-SH) assay
Renal non-protein sulfhydryls were calculated by the method of Sedlak and Lindsay [15]. Kidney samples were grinded in ethylene diamine tetraacetic acid (0.02 mmol/L) at 4 °C. A 5 mL aliquot of the homogenized kidney was added to 4 mL of water and 1 mL of trichloroacetic acid (TCA) (50%), which were blended spasmodically and then centrifuged at 3000 rpm for 10 min. Two mL of the clear extract was added to 4 mL of 0.4 mmol/L Tris buffer (pH 8.9) and 0.1 mL of 2-nitrobenzoic acid (5, 5'-dithio-bis) (DTNB) added, with shaking, to the sample. Color development was measured at 412 nm.

Assessment of alpha-amylase and alphaglucosidase activity
Inhibition of amylase activity in plant samples was evaluated as described by Sabitha et al [16], while inhibition of glucosidase activity was determined based on the method of Hossan et al [17].

Histopathological investigation
Specimens from liver tissues were taken and fixed in buffered neutral formalin solution (10%) for 24 h. Samples were dehydrated via a graded alcohol series, which was removed using xylol before embedding the samples in paraffin. Tissues were cut into 6 μm-thick sections with a microtome, which were stained with hematoxylin eosin (HE) and photographed using a light microscope.

Statistical analysis
The collected data are expressed as mean ± standard error (SE) and were statistically analyzed using the Student's t-test or one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test. Significant differences between the treatment groups were found at p < 0.05, p < 0.01 or p < 0.001. Table 2, SGOT, SGPT, GGT, ALP, and bilirubin levels were significantly increased in CCl 4 -induced hepatotoxic rats compared to those in the normal controls. Treatment with ZS-1 (100 mg/kg), ZS-1 (200 mg/kg), ZS-4 (100 mg/kg), and ZS-4 (200 mg/kg) reduced these elevated levels compared to the levels in the CCl 4 -only group, and high dose (200 mg/kg of body weight) ZS-1 and ZS-4 treatment showed almost similar effects to silymarin treatment (10 mg/kg body weight). Administering ZS-2 (100 mg/kg), ZS-2 (200 mg/kg), and ZS-3 (100 mg/kg) did not lead to significant results when compared to those observed in the CCl 4 only group ( Table 2).

Effect of Z. spina-christi extract/fraction on NP-SH, MDA, and total protein in liver tissue
MDA levels in the liver tissue of CCl 4 -induced rats were significantly higher (p > 0.001) than those in the controls ( Table 3).

Effects of Z. spina-christi extract/fractions on NP-SH, MDA, and total protein in kidney tissue
As shown in Table 5, the levels of MDA, total protein, and NP-SH in kidney samples from animals administered CCl 4 were significantly increased compared to those in normal control rats.
Treatment with ZS-1 (100, 200 mg/kg) and ZS-4 (100, 200 mg/kg) caused dose-dependent and significant changes in the levels of these markers compared to the levels found in the CCl4 only group. Although administering ZS-2 (100, 200 mg/kg) and ZS-3 (100 mg/kg) did not result in considerable changes, ZS-2 (200 mg/kg) treatment significantly reduced the concentration of MDA, but this was only relative to the CCl4 group.

Morphological features of liver
In the control group, histopathological assessment of the liver sections revealed a normal histological architecture. The central vein was at the center of the lobules bounded by the hepatocytes containing strong eosinophilic granulated cytoplasm, and distinctive nuclei. In addition, hepatic sinusoids were observed within the strands of hepatocytes (Figure 3 A). The livers of rats treated with CCl 4 showed clear histopathological changes characterized by hepatocyte necrosis and hydropic degeneration (Figure 3 B). Moreover, hepatocyte necrosis, inflammatory cell infiltration and congestion of the portal areas with noticeable hemorrhage were found in rats after CCl 4 administration (Figure 3 B).
In the silymarin + CCl 4 group, liver sections appeared more or less normal ( Figure 3C) and microscopic examination of liver from the ZS-1 (100 or 200 mg/kg) + CCl 4 group revealed improvements in the liver structure, except minor hepatocyte necrosis (Figure 3 D and E, respectively).

Morphological features of Kidney
Histopathological examination of kidney sections from the control group showed renal tubules, renal corpuscles, distal convoluted tubules and proximal convoluted tubules. Furthermore, the glomerulus, urinary space and Bowman's capsule appeared as shown in Figure 5 A. Sections of the kidney from the CCl 4 group showed glomerular and tubular degeneration, interstitial hemorrhage, infiltration, and tubular widening of the lumen (Figure 5 B). Sections from the Silymarin + CCl 4 group showed glomerular and tubular structure similar to that of the control (

DISCUSSION
As severe liver damage can be initiated by administering CCl 4 , a hepatotoxin, to rats, this has contributed to its use in the study of liver disorders. The hepatotoxicity effect of CCl 4 is caused by the trichloromethyl free radical. This highly active free radical causes cell death or damage, which completely disturbs the body's lipid profile [14]. Liver enzymes (SGOT, SGPT and ALP) are considered to be the most widespread biochemical markers to assess liver injury as they are present in the cytoplasm of cells and are released into circulation during cellular injury [18]. The activities of such enzymes in serum can therefore reveal the severity of liver alterations [19]. The abundance of these biomarkers compared to that in normal conditions indicates dysfunction of the liver. These impairment can be studied histopathologically with the help of necrotic hepatocytes that appear in liver tissues [20].
By administering CCl 4 in the current study, we reveal a significant enhancement in ALP, SGOT and SGPT levels, indicating that CCl 4 intoxication disrupts the integrity of the hepatic cell membranes [21]. Pretreatment with methanol and aqueous Zizyphus spina-christi L. extract significantly decreased the levels of SGOT, SGPT and ALP toward normal levels. These observations align with the notion that serum concentrations of transaminases revert due to the effect of CCl 4 in repairing hepatic tissue damages and plasma membrane stabilization [22].
The histopathological study revealed extensive architecture distortion, congestion, necrosis and inflammation, which were successfully stimulated by CCl 4 administration. In the adopted mechanism of CCl 4 hepatotoxicity via reductive dehalogenation catalyzed by P 450 , the extremely reactive trichloromethyl (CCl 3 ) free radical readily interacts with molecular oxygen to form the trichlomethyl peroxyl radical (CCl 3 OO) [19]. This radical can then bind to lipids, causing lipid peroxidation and consequently liver destruction to greatly contribute to the pathogenesis of diseases [23].
The histological appearance in the total methanol (ZS-1) and aqueous (ZS-4) Zizyphus spinachristi L. groups was partly similar to that of the control group, and tissue injuries and necrosis occurred at a lesser extent in these groups than in the CCl 4 group. The overall histopathological findings correlate with the biochemical parameters and suggest that total methanol (ZS-1) and aqueous (ZS-4) Zizyphus spina-christi L. may be effective against CCl 4 -induced changes in the liver. The reduction in total protein level could be viewed as a helpful indicator of the degree of hepatocellular injury [24].
In this investigation, CCl 4 intoxication decreased total protein level in the tissue. This was due to the formation and restriction of the preliminary injury to the endoplasmic reticulum, damaging cytochrome P-450 enzymes and reducing their function in the synthesis of protein and triglycerides which causes fatty liver [25]. Preceding studies have shown that administering CCl 4 to different animals results in a rapid reduction in protein synthesis in the liver [14]. Pretreatment with the extract and fractions of ZS-1 and ZS-4 Zizyphus spina-christi L., and CCl 4 restored total protein level, stabilizing the endoplasmic reticulum for protein synthesis [24].
Due to its antioxidant ability, bilirubin is considered a cytoprotectant [21]. At this time, administering CCl 4 raises the possibility of renal malfunction by increasing serum creatinine and total bilirubin [14]. Reduction in bilirubin and serum creatinine concentrations in the groups treated with the different fractions of plant extract enhanced the contributory mechanism of lowered oxidative stress. The kidney assists in the maintenance of homeostasis in the body by reabsorbing vital materials and eliminating waste.
Creatinine is commonly used as a measure of kidney function and its increased level in the blood is considered an indicator of kidney damage. In the current investigation, treatment with CCl 4 significantly increased creatinine level. The observed increase is an investigative indicator of cellular leakage and cell membrane damage in renal tissues [26]. The current study showed that CCl 4 induces renal injury due to the high uric acid and serum creatinine levels, elevated MDA concentration and the reduced contents of protein and NP-SH in the kidney of treated animals, all of which closely agree with previous studies. Elevated levels of uric acid and creatinine are indices of nephrotoxicity [26]. Uric acid and serum creatinine are the final compounds of purine and can modify the glomerular filtration rate. Alterations of the glomerular filtration rate increase serum creatinine and uric acid levels which are associated with renal damage [27].
Co-administering ZS-1 and ZS-4 in the current study successfully prevented harm related to CCl 4 administration in the renal system, as depicted by the renal functioning biomarkers and histopathological test. Methanol and aqueous Zizyphus spina-christi L significantly decreased serum creatinine and uric acid . In addition, the extract evidently enhanced NP-SH and protein depletion in kidney tissue, and significantly reduced MDA concentration, which were increased by CCl 4 treatment.

CONCLUSION
Co-administering ZS-1 and ZS-4 of Zizyphus spina-christi L. results in hepatoprotective effects against liver injury induced by CCl 4 . Therefore, we recommend this plant for further bioactive phytochemical screening in vivo evaluation.