Ethnobotanical, Phytochemical, Toxicological, and Pharmacological Properties of Ziziphus lotus (L.) Lam.: A Comprehensive Review

Ziziphus lotus (L.) Lam. (Rhamnaceae) is a plant species found across the Mediterranean area. This comprehensive overview aims to summarize the botanical description and ethnobotanical uses of Z. lotus and its phytochemical compounds derived with recent updates on its pharmacological and toxicological properties. The data were collected from electronic databases including the Web of Science, PubMed, ScienceDirect, Scopus, SpringerLink, and Google Scholars. It can be seen from the literature that Z. lotus is traditionally used to treat and prevent several diseases including diabetes, digestive problems, urinary tract problems, infectious diseases, cardiovascular disorders, neurological diseases, and dermal problems. The extracts of Z. lotus demonstrated several pharmacological properties in vitro and in vivo such as antidiabetic, anticancer, anti-oxidant, antimicrobials, anti-inflammatory, immunomodulatory, analgesic, anti-proliferative, anti-spasmodic, hepatoprotective, and nephroprotective effects. The phytochemical characterization of Z. lotus extracts revealed the presence of over 181 bioactive compounds including terpenoids, polyphenols, flavonoids, alkaloids, and fatty acids. Toxicity studies on Z. lotus showed that extracts from this plant are safe and free from toxicity. Thus, further research is needed to establish a possible relationship between traditional uses, plant chemistry, and pharmacological properties. Furthermore, Z. lotus is quite promising as a medicinal agent, so further clinical trials should be conducted to prove its efficacy.


Introduction
It is well-known that plants have been used in popular medicine since ancient times to treat and protect against a variety of human diseases [1,2]. According to the World Health Organization, the percentage of people using traditional medicine including plants is estimated to be 80% [3]. The security, efficacy, economic feasibility, and accessibility are the most important criteria justifying these traditional practices [4,5]. Recently, the traditional uses of medicinal herbs in drug therapies have grown in importance. The therapeutic efficacy of these herbs is mainly due to the presence of secondary metabolites that have

Eco-Geographical Features
Typically, Z. lotus is found in dry and semi-arid regions, particularly in the Mediterranean region [16]. It grows in a few southern European nations including Greece, Italy, and Spain. It is also found in western Asia and is very common in northern Africa from Morocco to the Egyptian Sahara [31]. It reappears on the island of Socotra, in Yemen, and throughout the Middle East including Cyprus, Turkey, Palestine, and Syria [32]. It is also distributed in Iran, China, and South Korea [13]. Z. lotus can adapt to a wide range of climatic circumstances. Due to its late blooming, it is more resistant to winter frosts, up to −15 °C, than spring frosts (May-July) [11]. It is a sun plant reserved for warm and dry climates. It can withstand drought well and needs a lot of heat (maximum 45 °C) to bear

Eco-Geographical Features
Typically, Z. lotus is found in dry and semi-arid regions, particularly in the Mediterranean region [16]. It grows in a few southern European nations including Greece, Italy, and Spain. It is also found in western Asia and is very common in northern Africa from Morocco to the Egyptian Sahara [31]. It reappears on the island of Socotra, in Yemen, and throughout the Middle East including Cyprus, Turkey, Palestine, and Syria [32]. It is also distributed in Iran, China, and South Korea [13]. Z. lotus can adapt to a wide range of climatic circumstances. Due to its late blooming, it is more resistant to winter frosts, up to −15 • C, than spring frosts (May-July) [11]. It is a sun plant reserved for warm and dry climates. It can withstand drought well and needs a lot of heat (maximum 45 • C) to bear fruit [31]. This species is adapted to areas with low rainfall [33]. A variety of soil types are tolerated by Z. lotus. It favors deep, sandy soils that are neutral to slightly alkaline and can withstand salt [31]. This plant can be encountered in desert areas with very low rainfall and grows in rocky areas, cliffs, and foothills. It reproduces vegetatively with a low Digestive problems, skin problems, nervous system disorders, diabetes, urinary tract problems, endocrine and metabolic disorders, and muscles diseases [8] Northeastern Morocco including eight province districts -Flowers, leaves, roots Decoction, infusion, powder -Diabetes, urinary infections, antispasmodic, kidney diseases, hair care, circulatory disorders, and respiratory problems [9] Region of Fez-Meknes Nbeg Fruits, leaves Decoction -Kidney stones [37] High Atlas Central Morocco Ssedra, Azgour Fruits, leaves Decoction powder -Antiulcer, antidiarrheal, anorexia [55] High Atlas Central of Morocco Ssedra, Azgour Fruits, leaves Decoction, infusion, powder -Antidiarrheal, promotes the healing of wounds, antiulcer, aperitif, antidiabetic, [38] High Atlas Central of Morocco Ssedra, Azougar Fruits, leaves, roots Decoction, infusion, powder -Diabetes [55] Middle Oum Rbia Sdar, Nbeg Fruits, leaves, seeds --Digestive, dermatological, genitourinary, cardiovascular, metabolic [56] Guercif Province Sadra Roots Maceration -Diabetes, intestinal pain [57] Northeastern Morocco Asadra, Nbeg Leaves, fruits, roots Decoction, infusion, powder Oral Urine retention, diuretic, renal colic, pyelonephritis, polycystic kidney disease, and kidney stones [36] Markets of Salé Prefecture, Northwestern Morocco Sedr -Decoction -COVID-19 [58] Rif, Northern Morocco Nbeg, Tazart Seeds --Digestive system disorders [59] Region of Tadla Azilal Nbeg Fruits, leaves Powder -Gastrointestinal disorder [60] Rabat-Sale-Kenitr Nbeg Fruits, leaves Decoction -Chronic kidney diseases [61] Taza Sadra Fruits, leaves Infusion, powder Externally, oral Kidney problems, digestive system, diabetes, antimicrobial, hair care [62] Moulay Yacoub Region Asadra Fruits Infusion, powder Oral Stomach ache, hair care [63] Tarfaya Province Seder Leaves Powder Oral Kidney stones [64] Province of Tarfaya Ssder Fruits, leaves Powder, poultice Oral, externally Kidney stones, stomach pain, hair loss [65] Province of Sidi Kacem Ssedra Seeds Raw -Digestive infection [66]

Pharmacological Activities
Modern pharmacological research has revealed in recent years that Z. lotus extracts contain a range of pharmacological features including anti-diabetic, anti-cancer, hepatoprotective, nephroprotective, anti-inflammatory, analgesic, anti-oxidant, and antibacterial actions. Numerous studies have revealed that the chemical constituents and raw extracts of Z. lotus have significant levels of biological properties. Figure 7 provides an overview of the mechanisms of the pharmacological activities demonstrated by Z. lotus extracts.

Pharmacological Activities
Modern pharmacological research has revealed in recent years that Z. lotus extracts contain a range of pharmacological features including anti-diabetic, anti-cancer, hepatoprotective, nephroprotective, anti-inflammatory, analgesic, anti-oxidant, and antibacterial actions. Numerous studies have revealed that the chemical constituents and raw extracts of Z. lotus have significant levels of biological properties. Figure 7 provides an overview of the mechanisms of the pharmacological activities demonstrated by Z. lotus extracts.

Antidiabetic Activity
Numerous diabetic patients have employed conventional herbal treatments in a variety of formulations as a supplemental therapy to manage problems with diabetes since Antiquity [112,113]. As indicated in Table 1, the Z. lotus plant is frequently used in traditional medicine to treat diabetes. The anti-diabetic activities of these plant extracts have been confirmed by several preclinical studies on animals. In fact, an in vivo investigation discovered that giving diabetic hamsters an aqueous extract of Z. lotus fruit at a concentration of 300 mg/kg controlled the blood glucose levels [114]. The authors of this study indicated that the anti-diabetic activity of the aqueous extract of Z. lotus fruit

Antidiabetic Activity
Numerous diabetic patients have employed conventional herbal treatments in a variety of formulations as a supplemental therapy to manage problems with diabetes since Antiquity [112,113]. As indicated in Table 1, the Z. lotus plant is frequently used in tra-ditional medicine to treat diabetes. The anti-diabetic activities of these plant extracts have been confirmed by several preclinical studies on animals. In fact, an in vivo investigation discovered that giving diabetic hamsters an aqueous extract of Z. lotus fruit at a concentration of 300 mg/kg controlled the blood glucose levels [114]. The authors of this study indicated that the anti-diabetic activity of the aqueous extract of Z. lotus fruit (300 mg/kg) was comparable to that of the drug glucophage 50 mg (metformin). In addition, the effect of the Z. lotus leaf and fruit extract on the inhibition of α-amylase and α-glycosidase was evaluated in vitro [20]. The finding of this survey indicates that the Z. lotus leaf and fruit extract has potent in vitro anti-diabetic effects via α-amylase inhibition (leaves: IC 50 = 20.40 ± 1.30 µg/mL; fruits: IC 50 = 31.91 ± 1.53 µg/mL), and α-glycosidase (leaves: IC 50 = 8.66 ± 0.62 µg/mL; fruits: IC 50 = 27.95 ± 2.45 µg/mL). The effect of this extract was stronger to that of the common acarbose prescription. The data gathered from this text indicate the anti-diabetic potential of Z. lotus extracts, and support their traditional uses as anti-diabetic drugs.

Anti-Obesity and Dyslipidemic Activity
In recent years, hyperlipidemia has been considered as a major health illness. This factor is well-known to be the primary risk factor for the development of atherosclerosis as well as other related cardiovascular and brain vascular diseases [115]. Several previous investigations have been conducted to confirm the anti-hyperlipidemia activities of Z. lotus in the preclinical stage. Indeed, it has been demonstrated that the aqueous extract of Z. lotus fruits exerts anti-hyperlipidemic effects in albino mice fed a prolonged, fat-rich diet [35]. Results from this study indicate that the administration of the aqueous extract of Z. lotus fruit at 200 and 400 mg/kg for 30 days improved abnormal changes in the lipid profile (total cholesterol, HDL-cholesterol/total cholesterol, triglycerides, HDL-cholesterol, and atherogenic index) and blood glucose in albino mice subjected to a chronic high-fat diet. In addition, one study assessed the effects of Z. lotus fruits on diet-induced obesity in mice [116]. This study showed that taking 10% (w/w) of Z. lotus fruit powder supplemented with a high-fat diet for six weeks improved the plasma lipid concentrations, and thus the expression of key genes involved in energy metabolism and inflammation. In addition, an in vivo study demonstrated the anti-cholesterolemic effect of aqueous Z. lotus fruit extract in hamsters exposed to a high-fat diet [114]. Daily administration of an aqueous extract of Z. lotus fruit at a dose of 300 mg/kg in obese hamsters for 30 days substantially reduced the plasma level of bad cholesterol compared to obese animals not treated with the Z. lotus extract.

Antiulcerogenic and Anti-Spasmodic Activities
As indicated in Table 1, Z. lotus is utilized in herbal medicine in several countries to treat digestive tract pathologies such as constipation, diarrhea, and spasms. Several previous preclinical studies have shown the beneficial effects of Z. lotus extracts against some digestive problems. Bakhtaoui et al. (2014) showed that the administration of Z. lotus fruit methanol extract at 500 mg/kg had anti-ulcerogenic effects provoked by HCl/ethanol, pyloric ligature, and aspirin in Wistar rats [117]. Another study found that the oral administration of aqueous extracts of Z. lotus root bark, leaves, and fruits resulted in a substantial and dose-dependent inhibition of acute ulcers caused by the HCl/ethanol solution [118]. According to the same authors, this effect of Z. lotus extracts is comparable to that of cimetidine and omeprazole (standard drugs). Furthermore, the antispasmodic effects of aqueous and methanol extracts of Z. lotus leaves and root bark were tested on male rats [119]. The results of this investigation showed that Z. lotus root bark and leaf extracts were able to relax the tone of spontaneous duodenum contractions in rats and antagonize the spasmogenic effects caused by agonists such as acetylcholine, KCl, and BaCl 2 . The authors explain this effect by the fact that the extracts of Z. lotus clumped on the cholinergic receptors and blocked the influx of Ca 2+ .

Anti-Inflammatory and Immunomodulatory Activities
Inflammation is typically characterized as a reaction to an injury or infection [120]. It is now widely recognized that chronic inflammation is always linked to diseases of wealth and prolonged longevity including cancer, obesity, cardiovascular, and neurological disorders [120][121][122][123]. Inflammation is characterized by four primary symptoms: redness, heat, swelling, and pain. The body naturally responds to damaging stimuli by inducing inflammation, which is accomplished by the migration of plasma and leukocytes into damaged tissues. This particular immune response, which is categorized as acute inflammation, is crucial for the body to fend off dangerous microorganisms [124]. It is critical to identify chemicals that can aid in the resolution of inflammation in this situation in a way that is homeostatic, modulatory, effective, and well-tolerated by the body. In this context, a study revealed that Z. lotus extracts had an anti-inflammatory effect against carrageenan-caused edema in rats [29]. According to the findings of the study, administering an aqueous extract of the Z. lotus root bark intraperitoneally at doses of 50, 100, and 200 mg/kg significantly reduced the paw swelling caused by carrageenan three hours later by 37.81%, 69.18%, and 72.90%, respectively. Additionally, a considerable activity occurred at a dose of 200 mg/kg, three hours after the injection of carrageenan, after taking the methanolic extract, with a reduction of 67.57% in the volume of the paw. The authors of this study showed that this anti-inflammatory effect was close to that of the standard drugs (piroxicam). Furthermore, previous research indicates that extracts from various parts of Z. lotus have immunosuppressive properties. The methanolic extract of Z. lotus has the important property of modulating the changes in intracellular calcium concentrations caused by thapsigargine in human T Jurkat lymphocytes. It also has the ability to reduce the phosphorylation of the 1/2 kinase controlled by the extracellular signal (ERK1/2) as well as the proliferation of T-lymphocytes by slowing their progression from the S phase to the G2/M phase of the cell cycle and the expression of interleukin-2 (IL-2) [16]. Similarly, the aqueous extract of Z. lotus pulp, seeds, leaves, roots, and stems had a significant immunosuppressive effect, inhibiting the T lymphocyte proliferation [125].

Analgesic Activity
As a sensory modality, pain frequently serves as the sole indicator for the diagnosis of a number of disorders. It frequently serves a defensive purpose. Humans have employed a variety of therapies throughout history to relieve pain, with medicinal herbs standing out due to their widespread use [126]. In this regard, Borgi et al. (2007) reported that Z. lotus extracts have an analgesic activity in the preclinical stage [29]. In this investigation, it was shown that intraperitoneal injection of the aqueous, methanol, chloroform, and ethyl acetate extracts at doses of 50, 100, and 200 mg/kg caused a decrease in acetic acidprovoked writhing in mice. The same authors claimed that the analgesic effects of the ethyl acetate extract were extremely effective in comparison to the other tested extracts.

Anti-Cancer and Anti-Proliferative Activities
Cancer establishes when cells divide rapidly and invade the surrounding tissue before spreading to other parts of the body [127]. The key aim of anticancer therapy is to cure the illness while also attempting to extend and boost the quality of a patient's condition [128]. Since chemotherapy has harmful effects on cancer treatment, alternative therapies based on bioactive cytotoxic substances are an important help in cancer prevention. Preclinical studies conducted using methanolic, aqueous, petroleum ether, dichloromethane, and acetonic extracts of Z. lotus have revealed the potential mechanisms of anticancer activity.  [104]. The results of this study showed that this extract inhibited the proliferation of CaCo-2 and K-562 with an IC 50 below 50 µg/mL. Moreover, the Z. lotus root bark lipophilic extract displayed promising antiproliferative effects against MDA-MB-231, a triple negative breast cancer cell line, with an IC 50 = 4.23 ± 0.18 µg/mL [102].

Hepato-Renoprotective Effects
Bencheikh et al. (2021), investigated the nephroprotective efficacy of an aqueous extract of Z. lotus fruits [21]. The authors utilized gentamicin (GM), an antibiotic aminoglycoside used to treat severe acute illnesses, to produce nephrotoxicity in rats, and examined the preventative efficacy of the aqueous extract of Z. lotus fruits as a result. GM caused a significant biochemical imbalance (an increase in blood urea, creatinine, and uric acid as well as a decrease in urine) and significant deterioration in renal function was evident in this imbalance. However, daily administration of the aqueous extract from Z. lotus fruits three hours prior to GM injection successfully restored these GM-induced defects. The plant extract's efficacy was dose-dependent, with the highest effect reported at 400 mg/kg. These findings suggest the use of Z. lotus fruits as a nephroprotective agent due to their activities in improving the altered parameters during a nephrotoxicity condition [21]. In addition, Bencheikh et al. (2019) reported that Z. lotus fruits had hepatoprotective properties [22]. The authors utilized carbon tetrachloride (CCl 4 ) as a toxic agent in this investigation to induce oxidative stress and hepatotoxicity in rats. This treatment led to a significant decrease (p < 0.001) in body weight as well as a large rise (p < 0.001) in the relative liver weight. Furthermore, the activity of plasma liver indicators (AST, ALT, and ALP) rose considerably (p < 0.001). It also had the ability to dramatically increase (p < 0.001) the concentration of direct and total plasma bilirubin as well as the levels of triglycerides (p < 0.05) among other liver biomarkers. Moreover, the authors assessed indicators of renal excretory function by assessing the plasma levels of creatinine, urea, and uric acid. Interestingly, the addition of the aqueous extract of Z. lotus fruits demonstrated substantial protection against CCl 4 -induced hepatotoxicity and nephrotoxicity. Indeed, the aqueous extract Z. lotus fruits restored almost all serum indicator enzymes and antioxidant status, bringing all values back to normal, indicating that the extract's protective action in rats was mediated by reducing oxidative damage and liver injury [22]. The litholytic activity of the aqueous extracts of Z. lotus fruits and leaves was assessed in vitro and in silico [101]. Furthermore, the aqueous extract of Z. lotus fruits and leaves was found to suppress the production of CaOx crystals, induced by the addition of 0.1 mol/L oxalic acid in human urine, to form calcium oxalate (CaOx) crystals. The existence of bioactive chemicals detected by HPLC such as adenosine, isorhamnetin 3-O-rutinoside, p-hydroxybenzoic acid, and neoechinulin A was demonstrated by this litholytic action. In silico tests revealed that the discovered compounds work by targeting enzymes involved in calcium control, urate management, and acid-base homeostasis maintenance as well as having anti-inflammatory characteristics [101]. A study by Kouchalaa et al. (2017) evaluated the litholytic effects of the aqueous extract of Z. lotus on the dissolution of calcium oxalate and uric acid calculations in vitro [129]. The findings showed that at the end of the experiment, the ability of the aqueous extract to dissolve the calcium oxalate calculation was 7.65%, while the dissolution of the uric acid calculation was 10.75%. The high quantities of polyphenols and flavonoids in the extract were linked to the findings, according to the authors. Chakit et al. (2022) demonstrated that the aqueous extract of Z. lotus fruit had an anti-urolithic action in rats induced by ethylene glycol [130]. In the same ethylene glycol-induced urolithiatic model of rats, the administration of the aqueous extract of Z. lotus fruits dramatically decreased and prevented the formation of kidney stones and significantly alleviated renal impairment. The observed findings may be attributed to the tendency for urine alkalinization in Z. lotus fruit aqueous extract-treated mice is likely to have a role in oxalate crystal solubilization, which might potentially be one of the processes via which plant components act [130]. Calcium oxalate (CaOx) crystals were formed after the addition of 0.1 mol/L oxalic acid. The effect of aqueous extracts was compared to two reference antagonists (citrate and magnesium).

Antimicrobial Activity
Worldwide, bacterial diseases account for a significant portion of mortality and morbidity. Inappropriate and excessive use of antibiotics has resulted in the development of resistance, which is making treatment more difficult as antibiotic resistance rises [131]. Therefore, it has become more crucial than ever to create novel antibiotics that can withstand the array of bacterial resistance mechanisms. To this end, the intention has been focused in recent years on the search for natural-based new therapeutic agents, particularly medicinal plants. The latter could be a viable area for researching the capacity of natural antimicrobials to suppress and/or destroy bacteria [132]. In this direction, Z. lotus extracts have demonstrated antimicrobial effects against a wide range of bacteria, namely, Bacillus pumilus, Enterococcus faecalis, Listeria monocytogenes, Micrococcus luteus, Rhizobium sp., Staphylococcus aureus, Staphylococcus epidermidis, Agrobacterium sp., E. coli, Helicobacter pylori, Pseudomonas aeruginosa, and Salmonella Typhimurium and also show effects against two fungal strains (e.g., Candida albicans and Candida tropicalis). Table 3 summarizes the antimicrobial properties of the Z. lotus extracts. Evidently, the acetonic extract of Z. lotus leaves had significant antibacterial activity against S. aureus, S. aureus methicillin-resistant, S. epidermidis, S. epidermidis methicillin-resistant, and L. monocytogenes, with the MIC ranging from 250 to 1000 µg/mL and MBC ranging from 500 to 2000 µg/mL [105]. Another study revealed that lipophilic extracts of various parts of Z. lotus have antibacterial potential against E. coli, S. aureus, and S. epidermidis strains, with MICs ranging from 1024 to 2048 µg/mL [102]. At a concentration of 10 mg/mL, the methanolic extract of Z. lotus leaves inhibits the growth of S. aureus, L. monocytogenes, S. typhimurium, and E. coli, with inhibition zones ranging from 10 to 13 mm [19]. Furthermore, the results of a study on ethanolic, methanolic, and aqueous extracts of Z. lotus seeds showed that these extracts have significant antibacterial activity against E. coli, P. aeruginosa, S. aureus, and E. faecalis with MICs ranging from 50 to 200 mg/mL [18]. Thus, the antimicrobial activity of the methanolic extract of Z. lotus stems was evaluated using the agar-well diffusion method against pathogenic microbial species S. aureus, E. coli, and P. aeruginosa [133]. The results of this study indicate a potential antibacterial activity of this extract with MIC values ranging from 6 to 7 mg/mL. In addition, the study of the antibacterial activity of the methanolic extract of Z. lotus fruits through the disc diffusion and micro-dilution method showed that this extract had an interesting activity against E. coli, Agrobacterium sp., Rhizobium sp., B. pumilus, and B. subtilis with a MIC range from 3.2 to 400 µg/mL [134]. Growth inhibition activities of Z. lotus leaf methanol extract against bacterial species, notably B. subtilis, S. aureus, E. coli, P. aeruginosa, S. Typhimurium, were assessed using the conventional paper disk test [135]. The findings of this study indicate that the Z. lotus leaf methanol extract inhibited bacterial growth with a MIC in the ranges of 12.5 to 1000 µg/mL.

Antioxidant Activity
An imbalance between the quantity of reactive oxygen species and free radicals, which have unpleasant side effects, and the body's natural anti-oxidative defense mechanisms, results in oxidative stress [137,138]. In recent decades, researchers have concentrated on discovering naturally occurring anti-oxidizing chemicals that can counteract the potentially dangerous effects of free radicals [139]. In this respect, studies on the ability of Z. lotus extracts to bind free radicals have revealed that the various organs (twigs, leaves, fruits, seeds, and roots) have a notable anti-oxidative activity. The anti-oxidative capacity tests were performed using the DPPH, FRAP, TAC, β-carotene bleaching assay, and ABTS methods. The following table (Table 4) summarizes the results obtained in various countries throughout the world. Several studies have been conducted to assess the anti-oxidative potential of extracts from different parts of Z. lotus in Morocco, Algeria, Tunisia, and Italy. In Morocco, the anti-oxidant ability of the aqueous extract of Z. lotus fruits was found to be interesting with IC 50 = 116 ± 0.02 µg/mL, and an important inhibition of β-carotene oxidation, 21.11% at 100 µg/mL, tested by the mean of DPPH and the βcarotene assays, respectively [35]. Aya et al. (2020) highlighted that the various extracts (namely, hexane, methanol, and dichloromethane extracts) of the fruits and the leaves of Z. lotus possessed an interesting DPPH radical scavenging ability, ranging from an IC 50 equal to 0.70 mg/mL, and an IC 50 >40 mg/mL, with the methanolic extract of Z. lotus leaves being the most potent antioxidant (IC 50 = 0.70 mg/mL) among the other tested extracts [101]. Another report found that the methanolic, and ethanolic extracts from Z. lotus seeds expressed an anti-oxidant activity, corresponding to the IC 50 = 1.33 ± 0.01 mg/mL, and IC 50 = 1.32 ± 0.09 mg/mL, respectively [18]. A comparative study of the anti-oxidant abilities of the aqueous extract of Z. lotus fruits and leaves was conducted by [20] by the mean of three anti-oxidant methods (DPPH, ABTS, and FRAP). In this sense, the radical scavenging potential of the aqueous extract of the fruits was higher than that of the leaves in the three anti-oxidant assays, as shown in Table 4. The observed results were attributed to the high level of phenolics and flavonoid compounds in the fruits. Other Moroccan, Tunisian, Algerian, and Italian research teams have confirmed that the radical-scavenging ability is more important in Z. lotus fruits, regardless of the nature of the solvent. In this context, the crude methanol extract of Z. lotus fruits from Oued Esseder (southeastern Tunisia) showed an important scavenging effect against DPPH radicals with an IC 50 = 15.15 ± 0.90 µg/mL as well as an important total antioxidant capacity of 25.02 ± 0.55 mg GAE/g EDW. The same results were obtained for the methanolic extracts of the fruits from Bengardane (southeastern Tunisia) [19]. Ait Abderrahim et al. (2017) examined the anti-oxidant capacity of the methanolic extract of Z. lotus stems in vitro using DPPH [133] and reported a remarkable anti-oxidant result of 480.20 ± 40.64 mg AAE/g EDW. An Italian team investigated the antioxidant effect of the methanolic extract of stem bark from Z. lotus (from Addaura, Palermo, Italy) using three different methods [140]. They recorded a strong capacity to chelate ferrous ions from the ferrozine complex (39.01 ± 4.30 mg ethylenediaminetetraacetic acid equivalents, EDTA/g extract). The tested extract exhibited a potent DPPH free radical-scavenging effect displayed by the equivalent of ascorbic acid (304.02 ± 4.80 mg AAE/g EDW). For the FRAP test, a strong lowering power in the FRAP test was observed (296.68 ± 1.81 mg TE/g EDW) [140].

Others Activities
Human skin enzymatic browning entails the production of melanin, which proceeds through numerous steps involving multiple enzymes [144]. Within these enzymes, tyrosinase is implicated in the first two steps, and its inhibition could be used to build skin-protective medicines. Inhibiting the tyrosinase activity is an essential skin protection approach. The dermatoprotective activity of the extracts of Z. lotus fruit and leaves was investigated in the work of Marmouzi et al. (2019) [20]. The aqueous extract of the Z. lotus fruits demonstrated a higher tyrosinase inhibitory activity than the leaf extract, with IC 50 values of 70.23 ± 5.94 µg/mL and 129.11 ± 9.40 µg/mL, respectively. Both extracts were more effective than the reference substance utilized (quercetin) with 246.90 ± 1.90 µg/mL. The difference in inhibitory activities between the Z. lotus fruit and leaf extracts is mostly related to chemical functional component differences. The extract of Z. lotus fruits is high in phenolic components such as catechin, gallic acid, and rutin. These substances are recognized tyrosinase inhibitors [144]. Khazri et al. (2017) evaluated the neuroprotective effect of the Z. lotus fruit extract against the neurotoxicity-induced by cypermethrin (CYP), a synthesized pyrethroid employed as an insecticide in large-scale agricultural applications [145]. CYP administration in mice resulted in a substantial increase (p < 0.05) in the heart, liver, and kidney oxidative markers (H 2 O 2 and catalase) as well as an increase (p < 0.001) in the MDA levels in the heart, liver, and kidney. The current study also found that once 150 µg/L of CYP administered to mice significantly inhibited the AChE activity, when referred to healthy mice, this decrease (p < 0.05) in AChE activity was observed in all organs. The administration of the Z. lotus fruit extract successfully preserved standard biochemical parameters in mice against the toxicity generated by CYP. These findings support the pharmacological efficacy of the Z. lotus fruit extract in CYP-induced oxidative stress, suggesting the use of the extract as a neuroprotective agent

Toxicology
The plant's security was proposed by its widespread use as a food and in ethnomedicine for a range of ailments, with no mentioned harmful effects. Bencheikh et al. (2019) investigated the acute oral toxicity of the aqueous extract of Z. lotus fruit in mice. After 14 days of observation, they found that a single oral dosage of this extract at 2000 mg/kg body weight did not cause any animal fatalities or changes in animal behavior [22]. Bekkar et al. (2021) found that a single dose of 5000 mg/kg body weight did not exhibit a toxic effect, death, or change in behavior after 14 days [23]. According to the findings of this plant's acute toxicity, an acute use of this plant could be safe. To validate the safety of the long-term application of Z. lotus species, more studies on sub-chronic and chronic toxicity assessment should be carried out.

Concluding Remarks and Future Prospects
We emphasized the studies on the ethnobotanical, phytochemical, toxicological, and pharmacological properties of Z. lotus in this review. Several countries, especially those in North Africa, employ this plant extensively in herbal medicine to cure a broad range of illnesses including diabetes, digestive system issues, urinary tract issues infectious diseases, cardiovascular disorders, neurological diseases, skin issues, and others. According to the most recent pertinent data, several bioactive substances have been identified and isolated from Z. lotus extracts. These chemicals are secondary metabolites that belong to the flavonoids, phenolic acids, terpenoids, alkaloids, and other classes. In various scientific studies, the pharmacological assessment of Z. lotus revealed interesting medicinal activities. Indeed, Z. lotus fruit was observed to be the most efficient component of the plant in terms of treating diabetes, obesity, dyslipidemia, ulcers, and spasms. Additionally, it was found to have an anti-urolithic effect, preventing the formation of kidney stones. Furthermore, Z. lotus fruits showed significant protective effects against both CCl 4 -induced hepatotoxicity and GM-induced nephrotoxicity in rats. It was found that the fruit extract of Z. lotus demonstrated a higher inhibitory activity against tyrosinase compared to the leaf extract. For the neuroprotective effect of the plant against neurotoxicity induced by CYP, the Z. lotus fruit extract helped preserve standard biochemical parameters in mice against the toxicity generated by CYP, suggesting the potential use of the extract as a neuroprotective agent. The fruits and leaves also exhibited the ability to inhibit the production of CaOx crystals, induced by the addition of oxalic acid in humans. Research has demonstrated that the root bark of Z. lotus possesses properties that can help to reduce inflammation, and has immunosuppressive abilities as it can regulate intracellular calcium levels and reduce T-lymphocyte proliferation. Regarding the antimicrobial activity, all parts of the plant including the leaves, stems, seeds, and fruits displayed noteworthy antibacterial activity against a variety of bacterial strains including S. aureus, E. coli, and P. aeruginosa. However, the efficacy of each part may vary depending on the specific bacteria and extraction method used. Several studies have shown that different parts of the Z. lotus plant such as twigs, leaves, fruits, seeds, and roots possess significant antioxidant properties. The antioxidant capacity of these extracts was tested using various methods such as DPPH, FRAP, TAC, β-carotene bleaching assay, and ABTS, which demonstrated varying levels of antioxidant activity. These in vivo and in vitro pharmacological confirmations confirm the traditional uses of Z. lotus extracts. However, the studies on the pharmacological properties discussed in this paper did not show the underlying pathways by which Z. lotus extracts act. Using data from the literature, some studies suggest a positive relationship between pharmacological properties and some of the isolated Z. lotus compounds in some bioactivities. To fully understand the mechanism of action of the bioactive compounds found in Z. lotus, additional study on this topic is necessary. The toxicological testing of Z. lotus extracts on animal models revealed no significant acute toxicity. However, further research is required to assess the toxicity at various dosages and over various time periods. Clinical investigations are urgently required to support the usage of this herb since controlled trials were not performed.

Conflicts of Interest:
The authors declare no conflict of interest.