Many novel therapeutic compounds have been developed as a result of the long-standing practice of using plants for medical purposes [1]. Particularly in poor nations where orthodox medications are frequently utilized, traditional medicine continues to be the backbone of the healthcare system. Around the world, medicinal plants are used to treat a variety of illnesses that conventional treatments are unable to effectively treat [2]. Years of fighting diseases with crippling effects have led to an awareness of the medicinal potential of plants. This has prompted the quest for pharmaceuticals from unconventional sources, especially natural products from plant components including bark, seeds, fruits, roots, leaves, and wood [3]. Modern science has recognized their potency and included a variety of plant-based medications into pharmacotherapy [4]. However, these plants are continually being evaluated for their bioconstituents and pharmacological activities. More so, researchers are making progressive efforts to determine the advantages of herbal medicines in modern science aimed at adopting efficient medical practice and overcoming the harmful ones [5].

Widespread throughout West Africa, Cassia sieberiana (Caesalpiniaceae) is ubiquitous in all savannah woods or shrubs of the Sudanian zones, extending up to the border of the Guinean forest in Casamance. This herb is commonly used in traditional medicine as an analgesic for microbial infections, human body discomfort, dysmenorrhea, and veterinary medicine [6]. Most customers find the herb to be readily available, reasonably priced, and agreeable [7].

In this study, we examined the stem bark extract from C. sieberiana DC (Caesalpiniaceae) for its antibacterial properties as well as its toxicological implications.

Gathering and classifying plant material

 In Chaza, Niger State, Nigeria, a farm was used to gather the stem bark of C. sieberiana. The plant was verified at the National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, herbarium division. Additionally, a voucher specimen bearing the voucher number NIPRD/H/6736 was placed in the herbarium.

Processing of plant

After carefully cleaning the obtained C. sieberiana stem bark with distilled and running tap water, it was allowed to air dry for two weeks. Using an electric blender (Kenwood Blender: BL440), the dried bark was ground into a fine powder. It was then sealed in plastic containers and stored in a cool, dry location until needed for extraction.

Getting the extract ready

Using a Soxhlet device, 500 grams of stem bark powder was extracted in 2 litres of 100 % ethanol and vigorously shaken 48 hours. The yield of the ethanol extract solution was 20. 2% w/w on a dried weight basis after it was dried on a water bath at 40º C. To use in the experiment, the extract was reconstituted in distilled water.

Analysis of phytochemistry

The presence of bioactive constituents from the ethanol stem bark extract of C. sieberiana was carried out using standard chemical methods [8, 9].

Microbial strains

Six clinical pathogenic microorganisms were employed to evaluate the antibacterial activity of the ethanol stem bark extract: Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus and Proteus mirabilis. The Elex Ekwueme University Teaching Hospital's microbiology department in Abakaliki, Ebonyi State, Nigeria, was the source of all isolates.

Antimicrobial assay

The antibacterial activity of the ethanol extract was assessed using Akuodor et al.'s disc agar diffusion method [10]. The required microbial isolates were divided into purity plates prior to the assay, and biochemical examinations were carried out to confirm the identity of the organisms. Furthermore, distinct colonies of the fresh cultures derived from the bacterial isolates were mixed with five milliliters of nourishing broth and incubated at 37 degrees Celsius for a whole day. In accordance with the manufacturer's instructions, 15 mL of Muller-Hinton agar and 0.5 mL of isolate broth cultures with 105 cfu/mL of organism were properly reconstituted and poured into a sterile petri dish. The mixture was thoroughly mixed and allowed to harden. The stem bark extract obtained from the ethanol was reconstituted in distilled water with concentrations of 25, 50, 100, 150, 200, and 250 μg/mL. Standard antibacterial agent cefoxitin was applied in separate wells of the growth plates. A conventional sterile cork borer with a diameter of 8 mm was used to bore holes in the plates. The studies were carried out in duplicate, and the plates were correctly mixed for two hours before being incubated for 48 hours at 37°C. The zones of inhibition were then assessed in respect to the organisms under investigation. Pure water was used as the control, and cefoxitin, at a concentration of 20 µg/mL, was the conventional medication used. Next, the width of the area of inhibition was compared between the antibacterial activity and standard treatment.

Establishing the lowest inhibitory concentration

The minimum inhibitory concentration (MIC) of C. sieberiana ethanol stem bark extract was obtained using Essiet et al., [11]'s approach. A rack holding six sterile tubes was filled with 0.5 mL of nutritional broth for each test tube. Subsequently, the extract was diluted in steps of 200, 150, 100, 50, and 25 μg/mL. Following aspiration, 0.5 mL of the organism was added to test tubes holding the extract and broth, and they were cultured for 24 hours at 37°C. The stem bark extract concentration that prevented the test organism from growing was known as the minimum inhibitory concentration, or MIC.

Experimental animals

For the tests, adult male Wistar rats weighing 180–190 g were obtained from the University of Nigeria, Nsukka, Department of Veterinary Medicine. Every animal was kept in plastic cages with the tops covered with wire mesh and the bedding being dust. For seven days, they were acclimated to laboratory settings, which included a temperature of 28±20 C, a 12-hour light/dark cycle, and a relative humidity of 50±5%. These conditions persisted throughout the experiments. Ad libitum access to clean water and chow pellets (Agro Feeds, Abakaliki) were provided for the rats. The International Guidelines for Care and care of Experimental Animals [12] were followed in the care of the animals.

Toxicological evaluation

Acute toxicity test: Lorke's method [13] was used to examine the safety of the agent in the stem bark extract of C. sieberiana. Nine mice total were randomly assigned to three groups, with three mice in each group, during the two phases of the study. The mice were given the stem bark extract orally at doses of 10 mg/kg, 100 mg/kg, and 1000 mg/kg. For the first four hours and then for the next twenty-four hours, the animals were watched for any signs of toxicity or fatality. The next four groups of one rat per cage were given 20 mL/kg of distilled water and 1600 mg/kg, 2900 mg/kg, and 5000 mg/kg of the extract during the second phase. For late toxicity, the indicators of toxicity and mortality were monitored for 24 and 72 hours, respectively.

Sub-acute toxicity study

With a few minor adjustments, the procedure as outlined in Organization Economic Community Development guidelines number 407 [14] was followed. For the study, twenty-four male Wistar rats were weighed and randomly assigned to groups of four, with six rats in each group. For 28 days, the control rats received 10 mL mg of distilled water, whereas the treatment groups received 100 mg/kg, 200 mg/kg, and 400 mg/kg body weight of the ethanol stem bark extract of C. sieberiana via orogastric gastric cannula. Rats were put to sleep with halothane at the conclusion of the experiment, and blood samples were taken via retroorbital plexus and placed into plain and EDTA-containing sample bottles for hematological and biochemical analysis. The organs—liver, kidney, heart, and lungs—were taken out, weighed, and microscopically inspected.

Analyses haematologic

This was carried out at the Alex Ekwueme University Teaching Hospital in Abakaliki, Ebonyi State, Nigeria, using an automated hematology analyzer (Sysmex-XT-1800, Kobe, Japan).

Investigationsonbiochemistry
In order to produce serum, the blood in the plain bottles was allowed to stand for three hours at room temperature to guarantee full clotting. The clotted samples were then centrifuged at 3500 rpm for ten minutes, and the clear serum that was produced was then aspirated and kept for the biochemical investigations. Using standard ready-to-use reagent kits (Randox Ltd., UK) and the manufacturer's instructions, biochemical indices were calculated. The hepatic indices that were evaluated included uric acid, creatinine, and electrolytes; the kidney indices included creatinine, uric acid, and total proteins/albumin levels, as well as bilirubin levels (total, direct, and indirect).

Weights of organs

The heart, lungs, liver, and kidneys of the rats were removed, their blood washed out, they were weighed, macroscopically inspected, and their relative weights were computed as follows: Weight of organ / rat body weight × 100 is the relative organweight, or ROW (%).

Analytical Statistics

The data was processed using SPSS version 26, and descriptive statistics values were reported as means ± SEM. Tukey's post hoc multiple comparison tests were used after one-way analysis of variance (ANOVA) to establish statistical significance. When P<0.05 was reached, differences were deemed significant.

Components of phytochemistry

Polyphenols, tannins, flavonoids, saponins, steroids, glycosides, and alkaloids were among the phytochemicals found in the ethanol stem bark extract of C. sieberiana; anthraquinones were not present.

Test for acute toxicity

The ethanol stem bark extract of C. sieberiana did not produce any lethality or visible signs of toxicity in rats up to the oral dose level of 5000 mg/kg body weight 24 hours after treatment. Further monitoring for seven days did not still yield mortality or visible toxic signs. Therefore, the LD50 value exceeded 5000 mg/kg of body weight.

Antibacterial activity

The potency of C. sieberiana ethanol stem bark extract against pathogenic bacterial organisms was investigated. As a reference drug, Cefoxitin at 20 μg/ml demonstrated high activity against all organisms assayed (Table 1), and the extract showed strong activity against all tested bacterial strains. C. sieberiana extract exerted lowest activity against E. coli and Proteus mirabilis and the highest activity against P. aeruginosa, K. Pneumonea, S. aureus, and S. pyrogenes. The results of the reported minimum inhibitory concentration (MIC) are shown in Table 2.

Impacts on the indicators of haematology

Table 3 depicts the effects of ethanol stem bark extract of C. sieberiana on haematological indices. There were minimal variations in haematological parameters in rats exposed to all doses of the ethanol stem bark extract compared to control.

Effect of the of extract on kidney indices

The stem bark extract of C. sieberiana did not show much significant difference in activity on the kidney parameters after administration of the extract except significant (p< 0.05 and p<0.01) difference on Chloride and uric acid in treated rats compared to the control (Table 4).

Effects on biochemical parameters

The effect of C. sieberiana ethanol stem bark extract assessment on biochemical parameters in rats are shown in Table 5. The extract caused non-significant effects on the plasma levels of liver enzymes (ALT, AST and ALP), total proteins and albumin compared to the control group.

Impact of the extract on rats' essential organs

After being administered daily for 21 days, the ethanol stem bark extract of C. sieberiana did not significantly affect the weight of the rats' various essential organs (Table 6). In terms of macroscopy, every organ was similar to the control.

Table 1: Cefoxitin and C. sieberiana leaf extracts in ethanol have antibacterial properties.

Pseudomonas Organisms                                                  Inhibition zones (mm)  

                                                                     Extract         Cefoxitin

­­­­­­­­­­­­­­­­­­­Escherichia coli                                            16                    20                                                

aeruginosa                                                    20                    26                             

Staphylococcus aureus                                 18                    24                             

Klebsiella pneumonia                                   20                    30                             

Streptococcus pyogenes                               18                     26                            

 Proteus mirabilis                                         15                     22                           

Table 2: The ethanol stem bark extract of C. sieberiana at its minimum inhibitory concentration (MIC)

Organisms                                                   Minimum inhibitory concentration (μg/ml)

Escherichia coli                                                                   50

Pseudomonas aeruginosa                                                    22

Staphylococcus aureus                                                        45

Klebsiella pneumonia                                                          28

Streptococcus pyogenes                                                       30

 Proteus mirabilis                                                                35

Table 3: Impact of C. sieberiana stem bark extract on ethanol on haematological markers in rats

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                                                                        Dose (mg/kg)

Indices                  Control             100                          200                        400

 RBC (x1012/L)         8.42±2.17             8.62±2.55                     9.11±1.71                      8.80±2.52

HGB (g/dL)               16.50±2.20           16.62±2.81                  17.19±1.91^a                     17.51±2.16^a

PCV (%)                    51.51±0.31           51.80±0.11                  52.20±0.22^a                     52.80±0.03^a

MCV (fl)                   32.16±0.33            32.91±0.25                 32.06±0.19                     35.84±0.54^b

MCHC (g/dL)            31.29±0.51             31.60±0.88                31.95±0.59^a                    31.70±0.22^a

WBC (x109 /L)          10.43±0.75             11.61±0.51^a              11.42±0.94^a                   11.72±0.66^a

PLT (103 /L)              945.11±42.81         944.00±44.1             943.93±50.81                950.03±50.11^a

Neutrophils (%)        25.03±1.61             25.11±1.33                26.06±2.01a                    25.56±1.11^a

Lymphocytes (%)     70.54±11.10           70.33±11.06              70.59±10.9                    70.31±12.21

Monocytes (%)        1.06±0.32                0.91±0.42                  1.05±0.29                     0.94±0.42

Eosinophil (%)        1.23±0.16                 1.31±0.11              1.22±0.14               1.30±0.12

Data are means ± SEM (n = 6). ^a significantly different from control at P<0.05;   ^bsignificantly different from control at P<0.01.

RBC, red blood cells; HGB, hemoglobin; PCV, packed cell volume; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; WBC , white blood cells; PLT, platelets

Table 4: Rat kidney parameters are affected by C. sieberiana leaf extract in ethanol.

______________________________________________________________________________                                                               

                                                                           Doses (mg/kg) 

_____________________________________________________________________________________________   

   Indices                                 Control                    100                       200                        400

Na (mmol/L)                         141.60±10.30      140.90±11.20       142.40±10.60^a        142.3±15.2a

Ca (mmol/L)                          2.60±0.30            2.60±0.40              2.7±0.30                  2.70±0.70

K (mmol/L)                            5.50±1.20          5.40±1.70                5.8±0.60                  5.9±1.00

Cl (mmol/L)                          90.40±6.30        91.60±7.10               95.0±5.80^a                97.30±6.80^b

Creatinine (µmol/L)              41.60±2.10        41.30±1.80                 41.6±6.30              42.6±4.00^a

Urea (mmol/L)                      8.30±1.00          8.20±1.50                    8.00±2.50             8.40±1.00

Uric acid (mmol/L)              159.30±11.7    160.80±10.600            161.40±9.5^a              162.9±16.80^b

_____________________________________________________________________________________________

Data are means ± SEM (n = 6).  Data are means ± SEM (n = 6). ^a significantly different from control at P<0.05;   ^bsignificantly different from control at P<0.01.

Table 5: Rat liver parameters were affected by the ethanol stem bark extract of C. sieberiana.

____________________________________________________________________________

                                                                                           Dose (mg/kg)

Indices                         Control                                 100                          200                           400

AST (IU/L)                 170.10±14.20           171.40±13.20            175.00±6.20^b               175.6±4.60^b

ALT (IU/L)                  46.90±9.50              47.50±10.70              47.40±6.40                   48.60±7.40^a

ALP (IU/L)                  192.71±10.10          192.40±10.10            194.40±11.30a              196.30±12.50^b

TP (g/dL)                     6.01±3.20                 6.45±4.40                  6.61±3.50                    6.70±2.40

Alb. (g/dL)                   2.15±11.10              2.70±9.20                  2.67±8.50                    2.34±6.40

Data are means ± SEM (n = 6). Data are means ± SEM (n = 6). ^a significantly different from control at P<0.05;   ^bsignificantly different from control at P<0.01.

AST, Aspartate aminotransferase, ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; Alb, albumin

Table 6: Impact of C. sieberiana stem bark extract on the relative weight of the rats' essential organs

______________________________________________________________________________

                                                                                                 Dose (mg/kg

_____________________________________________________________________________________

Organs                       Control                                100                             200                                  400

Heart                          0.38±0.01                           0.39±0.01                   0.40±0.01                     0.37±0.03

Lungs                         0.57±0.04                            0.58±0.05                   0.56±0.07                   0.57±1.00

Kidneys                     0.76±0.02                            0.75±0.05                   0.77±0.04                    0.78±0.03

Liver                          3.51±0.13                            3.50±0.10                   3.49±0.25                     3.53±0.11

Data are means ± SEM (n = 6).

Herbal medicines have a lot of promise to offer new therapeutic agents and are becoming more and more popular in primary healthcare across the globe. The lack of scientific data on the safety characteristics of the majority of these products, however, is a source of concern [15]. The chemical components of herbal recipes are responsible for their pharmacological actions as well as their positive and negative consequences [16]. There are several ways to extract the components of every medicinal plant. The ethanol stem bark extract of C. sieberiana underwent phytochemical examination, which showed the presence of anthraquinones, steroids, glycosides, alkaloids, saponins, tannins, presence polyphenols, and flavonoids in varying amounts. Primary screening of medicinal plants for secondary metabolites aids in the identification of bioactive chemicals that, on the one hand, may lead to drug discovery and, on the other, may have unfavorable effects [17]. These biochemicals could be the cause of the impacts this study saw. Global reports of medicinal plants' antimicrobial qualities are growing. The stem bark extract had strong activity against the pathogenic bacteria under investigation in this work, indicating the presence of bioactive chemicals that may operate as an antibacterial agent or as a lead compound in the manufacture of an antibacterial agent. Although the antibacterial property of the stem bark extract is related to its phytoconstituents especially alkaloids and also others, some alkaloids specifically those belonging to betacarboline group possess antimicrobial, anti-HIV and antiparasitic activities [18]. The potency of C. sieberiana against tested bacteria gives a piece of evidence for the medicinal value and provides the basis for its traditional usage as a remedy for skin infections.

With the use of an acute toxicity study, a researcher can ascertain the kind and intensity of toxic effects that could result from a single, typically high dose exposure to a chemical, as could happen in a clinical overdose. Even at a dosage of 5000 mg/kg body weight, there was no indication of toxicity in rats subjected individually to varying doses of the stem bark extract in this investigation. Thus, after acute ingestion, the ethanol stem bark extract can be regarded as non-toxic. The plant's stem bark is often employed in folk medicine, which may be explained by this observation of a strong safety profile. Conversely, sub-acute toxicity studies aid in locating any toxic effects directed towards specific organs. The tests also assist in determining the proper dose regimens for long-term investigations and the level of non-observable adverse effects [19]. Animals exposed to xenobiotics may exhibit changes in their body and organ weights, which are recognized markers of the agents' effects [20].

The weights of essential organs are sensitive indicators of a xenobiotic's effects in toxicological evaluations since they define toxicity as the manifestation of notable alterations in those organs [21]. The study found that the extract did not affect normal growth, as evidenced by the lack of gross abnormalities and negligible alterations in the relative important organs. Both conventional and herbal medicines can have an impact on hematopoiesis, a process that is essential to life.

The assessment of haematological indices in animal models yields insights into the beneficial effects of plant extracts on blood, as well as their potential hazards [22].

Additionally, the technique is helpful in determining human risk because translated data from animal haematological alterations has a strong predictive value for human toxicity. Haematological indices did not significantly change, according to the study's hematological assessment results. The erythrocyte indices, which are a component of the previously stated, were unaffected. It can be concluded that the red blood cells' shape, osmotic fragility, and erythropoiesis were unaffected by the stem bark extract [23]. There were no notable changes to the overall white blood cell count or its variations. It can be assumed that the stem bark extract has no effect on immunological responses because these cells are the primary effectors of both innate and adaptive immunity [24]. This study's findings on platelet counts also suggest that the stem bark extract has no effect on hemostasis, which platelets regulate. Since all blood cells come from the hematopoietic stem cells in the bone marrow, the absence of changes in haematological indices may indicate that the extract does not negatively impact the bone marrow [25]. However, the fact that the extract did not change the primary stimulants in the pathway—erythropoietin for erythropoiesis and thrombopoietin for thrombocytosis—could account for the observed results. Every observation indicates that the extract is nonhematotoxic at the levels employed in this investigation. Similar results were also seen by other researchers who studied a variety of plant extracts [26, 27, 28, 29]. But since haematological parameter changes happen gradually, it's possible that the length of the experiment was insufficient to detect any changes; this was the case with Lychnophora trichocarpha [30]. The extract contains certain phytochemicals that have been shown to have stimulating effects on haematopoiesis, although it's possible that the quantities utilized were insufficient to produce these effects. The purpose of the serum chemistry analysis was to determine how the extract affected the treated rats' hepatic and renal functions in comparison to the controls.

Analyzing the functions of the liver and kidney, two significant synthesis and elimination organs, is crucial for the hazardous evaluation of xenobiotics [31]. None of the evaluated liver and kidney parameters were significantly changed by the C. sieberiana ethanol stem bark extract. Hepatotoxicity or liver disorders can be detected by elevated serum values of AST, ALT, and ALP [32]. Because hepatocytes generate serum proteins, particularly albumin, their levels are used to measure the liver's capacity for synthesis [33]. It appears that the extract had no effect on the hepatocytes because there were no appreciable changes in liver indices between the extract-treated and control groups in this investigation. Serum potassium is the most persuasive electrolyte measure and serum creatinine is the most sensitive marker when it comes to assessing renal function [34, 35]. None of these markers changed in this investigation, indicating that the extract is not nephrotoxic.

According to the study's findings, C. sieberiana's ethanol stem bark extract possesses a wide range of antibacterial properties that can be used to treat infections in humans caused by the bacterium under investigation. Results from acute and sub-acute investigations also suggest that oral administration of the ethanol stem bark extract is reasonably safe. Because of the stem bark extract from C. sieberiana that has been shown to have antibacterial properties and to be nontoxic, it is a good candidate for bioassay-guided chemical isolation, which may result in the creation of novel lead structures for disease-fighting drug development initiatives.

The authors have no conflict of interest.

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