Toxicity studies of Acacia nilotica ( L . ) : A review of the published scientific literature

Introduction The use of plants for the treatment of diseases dates back to the history of human life. In all parts of the world and more precisely in Africa and other least developed countries from Asia, plants are used in traditional medicine to treat different communicable and non-communicable diseases (1–3). It is estimated that more that 80% of people living in developing countries frequently use traditional practices for their primary health care needs (1,4). This situation has given rise to inquietude among health professionals and consumers on the issue of safety. Indeed, many plants investigated elsewhere were found to contain toxic substances (5) like certain secondary metabolites. Among them are tannins, saponins, terpenoid, cyanogenic, toxic amino acids, glycosides, alkaloids, coumarins, flavonoids (6–8). Other factors such as the quantity consumed, the time of exposure, different parts of the plant, and genetic differences within the species may also cause certain side effects (9,10). Diarrhea, weight loss, agitation, convulsions, tremors, dyspnea, and mortality are the most http://www.herbmedpharmacol.com doi: 10.15171/jhp.2019.xx


Introduction
The use of plants for the treatment of diseases dates back to the history of human life.In all parts of the world and more precisely in Africa and other least developed countries from Asia, plants are used in traditional medicine to treat different communicable and non-communicable diseases (1)(2)(3).It is estimated that more that 80% of people living in developing countries frequently use traditional practices for their primary health care needs (1,4).This situation has given rise to inquietude among health professionals and consumers on the issue of safety.Indeed, many plants investigated elsewhere were found to contain toxic substances (5) like certain secondary metabolites.Among them are tannins, saponins, terpenoid, cyanogenic, toxic amino acids, glycosides, alkaloids, coumarins, flavonoids (6)(7)(8).Other factors such as the quantity consumed, the time of exposure, different parts of the plant, and genetic differences within the species may also cause certain side effects (9,10).Diarrhea, weight loss, agitation, convulsions, tremors, dyspnea, and mortality are the most indicative clinical signs of toxicity.Life-threatening effects of certain toxins on organ systems such as liver, kidney, heart, spleen, brain, testicles, etc were also reported in many toxicological studies.Therefore, in the present study, toxicological studies related to Acacia nilotica (L.), a tree belonging to the family Mimosaceae were reviewed.
The species is native of Egypt and is widely distributed in various tropical and sub-tropical countries around the world.In Niger, the plant is frequently found in clay-sandy sites flooded by ponds where it flowers in the rainy season.It is common in the Delta and Senegal valley (11).The tree is 5 to 20 m (in humid zone) in high with a spherical crown.The stems and branches are usually black, cracked bark, gray-pink, exuding a reddish gum of low quality but which is edible, and is used in confectionery making.The tree has fine gray spines in axillary pairs (usually 3 to 12), 5 to 7.5 cm long in young trees.Mature trees are often thorn less.The leaves are bi-pinnate, with 3-6 pairs of pinnacles and 10-30 pairs of leaflets each, rachis with a gland at the bottom of the last pair of pinnae.Flowers with globular head of 1.2 to 1.5 cm in diameter with a bright golden yellow color, present on pedicels of 2-3 cm long are located at the ends of the branches.The pods are strongly constricted, white-gray, thick and soft (12).
Different parts of A. nilotica are widely used in traditional medicine for the treatment of various ailments.In different countries of the West Africa, A. nilotica pods, bark, gum, root, flowers and leaves are very solicited for the treatment of several diseases, such as gastrointestinal disorders (diarrhea, dysentery, hemorrhoid, abdominal aches, toothaches, sore throat, etc), diabetes, asthma, hypertension, etc (11,13).
Acacia nilotica has been used since time immemorial for its multiple pharmacological properties.The antibacterial potential of different plant parts has been reported by several investigators (20)(21)(22)(23)(24). Anti-platelet activity of the plant extract was reported (25).A study conducted by Kalaivani et al showed that the plant is potentially rich in antioxidant substances.The standardization and or formulation of these various components as supplements will enhance the therapy of several diseases such as cancer, diabetes and other inflammatory diseases (26).Tahir et al reported ethyl acetate extracts to have a higher activity on Plasmodium falciparum (27).Jigam et al reported total methanol extract of the roots of A. nilotica to be significantly active against Plasmodium berghei (28).
Although the traditional use of different parts of A. nilotica is widespread by maceration, decoction or infusion, there is lack of systematic review that comprehensively reports all about what is known about its level of safety.Thus, the objective of this study was to review and evaluate the existing scientific information on the toxicity of A. nilotica (L.) by bibliographic literature review.

Materials and Methods
The literature review and search strategies were designed to provide an overview of the scientific information about the toxicity profile of A. nilotica.The relevant information were collected from different scientific studies.A blind search using Web search engines such as Google, Bing and Baidu and other databases of scientific journals such as PubMed (https://www.ncbi.nlm.nih.gov/pubmed),Scopus (https:/www.scopus.com),CAS (https://www.cas.org),CABI (https://www.cabi.org),HINARI (http:// www.who.int/hinari/en/) and AJOL (https://www.ajol.info) were used to retrieve valuable publications from 1999 to 2017.Keywords such as ' Acacia' , 'Acacia nilotica' , 'toxic Acacia nilotica' , 'Acacia nilotica toxicity' , 'medicinal plant toxicity' and 'toxicity' were used to collect relevant articles.University library (China Pharmaceutical University Library) and local or international institutions (e.g.IRD, CCFN, INRAN, LASDEL, and ENSP) present in Niger republic were also visited to include books, thesis and other scientific write-up with known academic rating.Criteria were set to screen the search results for relevance in the study.Only scientific journals published in English or French that reported toxicities effects of A. nilotica were considered.All information on toxicities of A. nilotica were extracted from the exploited literature.

Results
A total of 29 valuable studies out of 75 initially identified through database searching were included in this review.Criteria for study inclusion such as data accessibility, consistency, reliability, uniformity, well precised objectives, etc have permitted to exclude 47 (38.6%) studies.Toxicological study of medicinal plants is conducted in vitro or in vivo.Toxicity of a plant is appreciated on the basis of certain parameters or characteristics such as the parts of plant used, the type of extract, the concentration of the extract, the mode of administration, the organism under consideration, and the LD 50 value expected to cause death in 50% in the treated animals in a given period (29,30).In addition, histological or genetic modifications are among the most relevant indicators for chronic or subchronic toxicity studies.Information on toxicity or safety of A. nilotica reported by different studies are presented in Table 1.Of the 29 studies, 14 (48.3%)have reported the toxicity potential of A. nilotica with 7 (50.0%)reporting chronic injuries to organ systems and 7 (50.0%)reporting cytotoxic effects.Of the three parts (stem bark, root, fruits) of A. nilotica that have been reported in 7 different studies to cause organ systems injury, stem bark was found cited by up to 3 (42.8%)studies followed by root and fruits each cited by 2 studies.For the plant parts extraction, 14 (50.0%)studies have reported the use of water (aqueous) as the only extractor, 12 (42.8%)studies reported the use of either organic solvents (ethanol, methanol, hexane, acetone, n-butanl, ethyl acetate, and petroleum ether), and 2 (7.1%) studies accounting for parts identified not been extracted by any solvent.Methods used to evaluate the safety of A. nilotica included in vivo acute (AT) and sub-acute toxicity (SAT) tests and in vitro cytotoxicity tests.Took together, 15 (51.7%) studies have reported the use of in vivo acute toxicity as the only model for evaluation, while 7 (24.1%) reported the use of both in vivo acute and SAT assays, 6 (20.7%) reported the use of in vitro cytotoxicity assays and 1 (3.4%) study for both in vitro and in vivo acute toxicity assay.The mammalian and non-mammalian in vivo animals such as rats, mice, got, cat, and brine shrimp and the in vitro animal cell lines such as lymphocytes, erythrocytes, HepG2, Caco-2, and HeLa were used as experimental models.The route of extract and row plant administration in experimental animals included oral, intraperitoneal, intravenous, and intramuscular.Oral route alone was found as the most privileged option for acute cytotoxicity studies followed by intraperitoneal option for SAT studies.Complex active ingredients present in most important plants were reported to cause varying degrees of side effects (31).Details on the studies that reported both indicative clinical signs, organ systems, and cell toxicity potential of A. nilotica are exhaustively reviewed.
Indicative clinical signs of toxicity Administration of any toxic substances may lead to the development of early (1-2 hours for immediate release) or late (4-8 hours in sustained release) observable clinical signs alterations in the exposed living animals.Indicative observations of toxicity include tremors, convulsions, salivation, diarrhea, lethargy, sleep, coma, and mortality (58).Most of these signs of discomfort and disease appears when external circumstances or internal conditions due to toxic substances cannot be adjusted by normal body mechanisms.A single change in any one system may result in numerous effects in other systems and thus clinical signs of disease are often quite similar for different diseases (Figure 1).Plants are reported to content diverse secondary metabolites that can be toxic to body systems when took without cautious (1,5).Several studies have reported the contribution of certain medicinal plants in the development of various observable side effects after administration.Mohammed et al reported acute behavioral changes such as slight decrease of alertness and locomotion (at half an hour post treatment), slight animals' spontaneous activity (at 1 and 2 hours post treatment), and a slight rise in passivity (evident at 2 hours) during the first 24 hours post treatment of rats with fruits ethanol extract of A. nilotica at lower doses.At last, on day 21 all the earlier noted behavioral changes disappeared.In contrast, 20-100% mortality was reported in rats treated acutely with 50-500 mg/kg of extract on intraperitoneal administration (35).The methanol seeds extract of A. nilotica was investigated for acute toxicity in mice at doses of 50, 100, 200, 500, 1000 mg/ kg body weight by Munira et al.The results showed little behavioral changes such as locomotor ataxia, diarrhea and weight loss (53).In 2015, Tanko et al reported a decrease in locomotion, a decrease in sensitivity to touch, and prostration as clinical signs of toxicity of the leaves extract of A. nilotica in rats after 12-18 hours post treatment (48).Also, in march 2015, in another study, the same author reported the same major observable clinical signs in rats that are treated with ethyl acetate and n-Butanol extracts of leaves of A. nilotica for 14 days with early deaths recorded after 12 hours and late deaths 48 hours after fractions administration (46).Umaru et al reported clinical signs of toxicity in rats treated with the aqueous pod extract of A. nilotica.Depression, anorexia and dyspnea indicative of respiratory and nervous-impairment were the altered clinical signs reported due to the treatment (42).Medani et al, on the other hand reported clinical signs of toxicity such as salivation, staggered gait, intermittent loss of voice, low appetite and death (between day 4 and day 8 at dose of 5 g/kg/d) in Nubian goats fed with whole pods of A. nilotica at doses of 1 to 5 g/kg/day for 35 days (47).Weight loss can be intentional, or unintentional and be a manifestation of illness due to the exposure to certain toxic substances (59,60).Different parts of A. nilotica were reported to significantly contribute to loss of appetite and thus decrease in body weight gain in the exposed animals.This was suggested to happen due to high tannin levels content in most parts of the plants (14,18,55).Al-Mustafa and Dafallah investigated the potential toxicity of aqueous pod extract of A. nilotica in rats maintained on 2% and 8% acacia diet for 2 and 4 weeks.The study showed a significantly decrease in weight gain in treated animals suggesting the presence of growth impairing substances such as tannin in acacia pods (55).In a another study, Mohan et al, also reported a decrease in body weight in rats fed with 2% and 8% leaves aqueous extract of A. nilotica in the diet for 2 and 4 weeks (40).While on the other hand, the methanol root extract of A. nilotica was reported by Jigam et al to significantly contribute to decrease in the body weight of mice treated over a 5 week period (51).

Kidney and liver injury
Animal exposure to toxic substances can impair a particular organ or organs function in many ways.Like modern pharmaceutical medicines, medicinal plants were also reported to have the potential to cause organ injury.
Possible reasons of such toxic effects are diverse, including the use of inherently toxic medicinal plants, improper intake (over dosage or longer than required duration of use) of plants, drug-plant medicine interactions, and contamination by toxic heavy metals (31) (Figure 1).
Fortunately, the body has mechanisms, mainly via certain key organs, to process and eliminate many of these substances.The functions of these organs are so vast and very indispensable that they alone, are testaments to the ingenuity of the body.Failure to remove toxins from the body system organs can lead to nephrotoxicity (61), hepatotoxicity (62) and more.Many research studies around the world have demonstrated and reported the implication of certain medicinal plants in the development of kidney, liver, heart, lungs, spleen, intestines, brain and testicles injury in vivo (62)(63)(64)(65)(66)(67)(68)(69)(70).Very few studies have reported the toxic effects of different parts of A. nilotica in different organs of animal models such as rat, goat, and mice.Medani et al reported the administration of the aqueous pod extracts of A. nilotica at doses of 1 and 5 g/ kg body weight to Nubian goats dispatched in two groups for 35 days.Clinical signs and changes in hematological and histology were reported.The histopathological testing revealed the presence of hepatic centrolobular necrosis lesions and fatty changes associated with the significant changes in γ-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were indicating hepatic dysfunction; renal malfunction was indicated by hemorrhages in addition to the change in urea concentration (47).Alli et al studied the toxicological effects of a single dose (acute) and of repeated doses (sub-acute) of aqueous root extract of A. nilotica in rodents.In the acute toxicity test, Swiss albino mice were orally administered aqueous extract of A. nilotica at doses 50, 300 and 2000 mg/kg body weight for 14 days.In the SAT study, rats received 125, 250 and 500 mg/kg b.w of the extract for 28 days by oral gavage.Changes in clinical signs, hematological and biochemical parameters and histology were recorded.Increased level of alanine aminotransferase, aspartate aminotransferase and ALP activities were recorded at 500 mg/kg b.w.Single dose administration of the aqueous extract in mice was found safe at doses higher than 250 mg/kg b.w., while repeated administration of doses higher than 250 mg/kg b.w of the extract for 28 days in rats caused hepatotoxicity.Renal function parameters analyzed at any of the doses of the extract administered were found unaltered.This implies that the extract does not have adverse effects on renal function at the doses tested (52).Juma et al in another study reported the effects of A. nilotica on enzyme and non-enzyme makers of liver and kidney injury.In this study, the aqueous stem bark extract of A. nilotica at 50, 100, 200, and 300 mg/kg body weight were orally and intraperitoneally administered to male Swiss white albino mice daily for 28 days.Changes in body and organ weight, hematological and biochemical parameters and histology were recorded.Intraperitoneal administration of the extract at 1 g/kg body weight significantly reduced body weight gain, percent organ to body weight of testes, while oral administration at the same dose decreased levels of platelets.Oral administration of the extract at 1 g/kg b.w. has led to the development of certain abnormalities in the levels of γ-glutamyltransferase, creatine kinase, alanine aminotransferase, aspartate aminotransferase, α-amylase, ALP and total bilirubin.These conditions may explain the hepatotoxic, cardiovascular toxicity, and nephrotoxic effects of A. nilotica extract on biomakers of liver, heart, and kidney function.The histopathological study carried out on formalin fixed kidney tissue extracted from a sacrificed treated mice indicated that long term exposure to doses of aqueous extract of A. nilotica had no obvious histopathological lesions on the liver, kidney, and other organs (32).In a thesis research study, Mwangi evaluated six plants including A. nilotica for their toxicity using aqueous stem bark extracts in mice treated with a doses of 1000 mg/kg b.w.orally for 28 days.Significant increase in the level of uric acid, an important marker of kidney function, was recorded.The intraperitoneal administration of the extract indicated an increase in kidney weight as compared to normal control mice.Histopathological observations of kidney, liver, heart, and other organs extracted from sacrificed treated mice for 28 days showed normal cellular architecture, indicating that the extracts were not toxic to the organs at the dosage level used (50).Jigam et al investigated the toxicity potential of the crude methanol root extract of A. nilotica in mice gavaged with 300 mg/kg body weight for 35 days.The results indicated significant elevation in oxaloacetate transaminase and Chloride.Histopathological examinations indicated a feathery degeneration of hepatocytes and destruction of nephrons (51).Mohammed et al investigated the toxicity potential of the ethanol fruit extract of A. nilotica in rats treated at doses of 75, 100, 112.5, 125, 187.5, 250 and 500 mg/kg for 21 days.Nephrotoxicity and hepatotoxicity assessments were based on elevated urea, creatinine, ALT (GPT) and AST (GOT) in plasma.On day 21 no behavioral changes were recorded.However, a treatment for three weeks induced significant elevation in urea and ALT.The increase in the level of both biomarkers (urea and ALT) suggested that kidney and liver functions are respectively impaired in the treated rats, particularly at high dose (35).

Cytotoxicity effect
Several in vitro assays involving different types of mammalian cells have been used for high-throughput screening of toxicity effects of potential medicinal compounds as well as to identify potential safety (39,71).Certain chemical constituents of medicinal plants are found to have a hemolytic or anti-hemolytic effect on animal erythrocytes (44,72,73).Few number of studies conducted elsewhere have reported erythrocyte toxicity caused by different plant extracts of A. nilotica.Sulaiman and Gopalakrishnan studied the hemolytic activity of the aqueous extracts of different Acacia species including A. nilotica at doses of 50 μg/mL, 100 μg/mL, 150 μg/mL and 200 μg/mL.A. nilotica (at dose 200 μg/mL) possesses highest hemolytic activity (8.9 ±0.16).Hemolytic percentage was found to be increasing with increase in dose (44).In another study, Kalaivani et al reported the in vitro cytotoxicity effect of the aqueous leaves extracts of A. nilotica at different doses (5-500 μg/mL) against rat and human erythrocytes.The test aqueous leaves extracts showed hemolytic effect with an IC 50 <200 μg/mL (26).While Rasool et al reported the hemolytic effect of either organic (Chloroform, ethyl acetate, ethanol, and methanol) root extracts of A. nilotica.The percentage lysis of the erythrocytes by plant extracts was found to be in the range of 1.27-3.59%(43).Tahir et al used human peripheral blood mononuclear cells (PBMC) to evaluate the toxicity effect of aqueous and organic extracts of A. nilotica in vitro (27).The methanol extracts of seed and husk showed low toxicity effects to the tested human lymphocyte at 100 ug/mL.Dikti Vildina et al used Caco-2 cell lines as model to evaluate the cytotoxicity effect of fruit extracts of A. nilotica (increasing concentrations up to 100 or 150 µg/mL) prior to in vivo toxicological studies (39).The results demonstrated that the viability of the tested human intestinal Caco-2 cells is inhibited by the crude extract of A. nilotica (mean CC 50 93.2± 1.1 µg/mL (µM)).van den Bout-van den Beukel et al evaluated the effects of stem extracts of A. nilotica at doses of 8 to 500 μg/mL on mitochondrial activity, cellular proliferation, damage to the cellular membrane, glutathione depletion and the electron transport chain activity with the aid of Alamar Blue, Hoechst 33342, calcein-AM uptake, glutathione depletion and O2-consumption assays, respectively.Hep-G2 and HeLa human cell lines were used as models.On the basis of the results obtained from these five assays, the plant was found to be highly toxic (10).Adoum used the brine shrimp in vitro lethality bioassay to predict the cytotoxic effect of the organic stem bark extract of A. nilotica.The results demonstrated moderate toxicity of the plant extract on brine shrimps (57).

Conclusion
Are there sufficient knowledge available to justify the safety of A. nilotica?This inquietude is the key goal of this review.Several ethnobotanical and ethnopharmacological studies conducted around the developing world have emphasized on the importance of A. nilotica in most indigenous communities to treat different diseases.From most of these surveys, it follows that local communities especially women have used A. nilotica for generations without any reports of pathologic effects (56).However, the increasing demands in the use of A. nilotica as alternative agent to treat various diseases have incited the curiosity of numerous scientists to further investigate its potentials.Found to be very rich in secondary metabolites and sufficiently active, scientists have hypothesized on its probable potentials to cause toxicity and though pursed several studies for justification.In general, chemical compounds present in the plant at certain doses could cause damage to the body systems; thereby the higher the dose administered the more the injuries caused are important especially if the consumption is regular.It has been noted that studies reporting the toxicity potential of A. nilotica are very few, most with very limited information and corresponding to short-term period of less than 15 days.Of the studies that are reviewed in this paper, only 3 have reported serious deleterious toxic effects of certain parts of A. nilotica on major organ systems such as liver and kidney.Alterations in certain hematological and biochemical parameters in relation to organs injuries but with no histopathological lesions were also reported by other 4 studies.All of these anomalies were observed after long-term exposure and at relatively higher doses.Stem bark as part of A. nilotica appeared to be the most cited to cause observable clinical signs of toxicity and organs lesions.
Overall, because of the remarkable increasing demand that the therapeutic usage of different parts of A. nilotica has attend, as well as its enormous potential in the near future, more advanced long-term-based toxicological studies are clearly needed.

Figure 1 .
Figure 1.Possible reasons for organ injury as a result of medicinal plant intake.

Table 1 .
Results of literature search: Acacia nilotica parts and their toxicities IntraperitonealSignificant loss of body weight; no significant change in the hematological parameters; no significant changes for most enzymes except a decline in ALP; no major histopathological changes in the organs examined that is liver, heart, kidney, lungs, brain, testis, intestines and spleen AT: acute toxicity; SAT: sub-acute toxicity; GGT: γ-Glutamyl Transferase; ALP: Alkaline Phosphatase; α-AMYL: α-Amylase.