Evaluation of the Hepatoprotective Properties of Traditional Formulations Based on Cochlospermum tinctorium Used in Benin

Hepatic diseases represent a public health problem. Among the approaches to their management is the use of traditional treatments based on the use of medicinal plants. In Benin, several recipes based on Cochlospermum tinctorium are used in the treatment of hepatitis without a real scientific basis. This study aimed to evaluate the hepatoprotective effects and acute oral toxicity of 10 of these recipes. The variables studied were the variety of C. tinctorium (wild form vs. cultivated form), the species associated with C. tinctorium (Combretum micranthum vs. Chromolaena odorata), and the proportion of C. tinctorium in the recipe (1; 4/5; 1/2). The hepatoprotective effect of these extracts at doses of 100, 200, and 400 mg/kg/bw was evaluated in Wistar rats subjected to hepatotoxicity induction through the administration of 5 g/kg of paracetamol. Acute oral toxicity was assessed following the OECD 423 protocol. The results revealed an absence of acute oral toxicity for the 10 recipes. The hepatoprotective tests conducted indicated that the hepatoprotective effect of C. tinctorium is dose dependent. The wild variety of C. tinctorium had a better hepatoprotective effect than the cultivated one. The association with C. micranthum enhances the hepatoprotective effect of C. tinctorium, unlike that with C. odorata. This study emphasizes that the combination of C. tinctorium with C. micranthum in the treatment of hepatitis is scientifically justified and it exhibits a dose-dependent hepatoprotective effect.


Introduction
Chronic liver diseases pose a substantial global health challenge, with liver cirrhosis being the ninth leading cause of mortality in Western nations [1].Tese conditions encompass chronic viral hepatitis B and C, alcohol-related liver diseases, nonalcoholic fatty liver disease, and hepatocellular carcinoma, with numerous unresolved challenges.Terapies rooted in Western medical practices frequently demonstrate restricted efectiveness come with potential adverse efects and are often fnancially out of reach, particularly in developing regions [2].
In the context of viral hepatitis, current therapeutic approaches focus on the sustained suppression of hepatitis B virus (HBV) replication as the primary treatment goal.Te preferred treatment involves the prolonged administration of nucleotide reverse transcriptase inhibitors such as entecavir (ETV), tenofovir disoproxil fumarate (TDF), or tenofovir alafenamide (TAF) due to their low risk of resistance [3].However, achieving the desired outcome of hepatitis B surface antigen (HBsAg) loss, which signifes functional cure, is only realized in approximately 1% of chronic hepatitis B patients treated with existing therapies [4].Consequently, future treatment approaches should prioritize HBsAg elimination in the management of viral hepatitis [3].
In the pursuit of efective and safe therapies for liver diseases, medicinal plants have surfaced as a promising avenue.Numerous plants have exhibited hepatoprotective qualities.Notably, an extract derived from Rumex abyssinicus demonstrated a reduction in CCl4-induced elevation of liver enzyme markers at a dosage of 500 mg/kg [5].Te study examined the hepatoprotective potential of the methanolic extract obtained from the fruit pulp of Adansonia digitata L. (100 and 200 mg/kg) against CCl4-induced liver damage in rats.Results indicated a notable reduction (p < 0.05) in serum levels of AST, ALT, ALP, and bilirubin, accompanied by fewer abnormalities in the liver tissue when compared to the group treated solely with CCl4 [6].Moreover, certain plants have exhibited antiviral properties against viral hepatitis.For instance, hyperoside extracted from the ethanolic extract of Abelmoschus manihot was observed to reduce secretion of HBsAg and hepatitis B antigen (HBeAg) in Hep G2.2.15 cells.In addition, it inhibited HBV DNA concentration dependently on days 5, 10, and 13.Histopathological examination further validated signifcant enhancement in hepatocellular architecture [7].In addition, polyphenols extracted from Camellia sinensis (green tea) demonstrated inhibition of HBeAg secretion in a manner dependent on both dosage and time, exhibiting an IC50 value (for HBeAg) of 7.34 μg/mL.Moreover, it notably decreased HBV DNA expression in a dose-dependent manner, with an IC50 of 2.54 μg/mL [8].
In Benin, Cochlospermum tinctorium A. Rich. is commonly incorporated into diverse medicinal formulations targeting various health conditions, notably liver diseases [9].It is utilized in both its wild and cultivated forms, frequently in conjunction with Chromolaena odorata and Combretum micranthum, particularly for addressing liver disease [10].Te scientifc literature has documented the hepatoprotective properties of Cochlospermum tinctorium [11,12].Currently, there is a lack of scientifc evidence regarding the hepatoprotective efcacy and safety/toxicity of combining Cochlospermum tinctorium with Chromolaena odorata and Combretum micranthum.Furthermore, limited research exists on the contrasting biological activities between the wild and cultivated forms of Cochlospermum tinctorium.Gathering scientifc data on these two varieties is crucial and could inform the selection of appropriate sources for particular medicinal uses.Tis study aimed to assess the hepatoprotective efects and acute oral toxicity of recipes combining wild and cultivated varieties of Cochlospermum tinctorium, Chromolaena odorata, and Combretum micranthum used in the treatment of liver diseases in Benin.

Animal Material.
Female albino Wistar rats, aged 12 weeks and weighing between 180 and 200 g, were selected as the experimental animal models.Tese rats were sourced from the animal facility at the Institute of Applied Biomedical Sciences (ISBA).Following a 14-day acclimatization period, the rats were randomly allocated to standard cages, providing them with ad libitum access to water and food.Troughout the study, the rats were housed in an environment maintained at a constant temperature of 22 °C, with a regular light/dark cycle of 12 hours each.

Preparation of Extracts.
Te freshly harvested leaves underwent a thorough washing with distilled water, followed by a drying process in the shade at a controlled temperature of 16 °C for a duration of 14 days within the facilities of the Research Unit in Applied Microbiology and Pharmacology of natural substances [13].Similarly, the rhizomes were initially crushed and then subjected to the same drying conditions.Te resulting powders were achieved through grinding using an electric mill (RETSCH SM 2000/1430/ Upm/Smf ) and subsequently stored in labeled glass vials at room temperature.Following this, diferent formulations were prepared for each of the three recipes, utilizing proportions of 50%, 80%, and 100% of C. tinctorium rhizome powder.Tese formulations were tailored for both wild and cultivated variants of C. tinctorium [10].Te composition of each of the 10 recipes included in the study is detailed in Table 1.
Each formulation underwent the production of a hydroethanolic extract through maceration, utilizing the method outlined by Fanou et al. [14].Tis process involved dispersing a mass of 50 grams of powder within a water-2 Advances in Pharmacological and Pharmaceutical Sciences ethanol mixture (v/v).Te suspension underwent continuous agitation on an automatic shaker for a period of three days at the laboratory's ambient temperature.Following this, the macerate was collected through fltration using hydrophilic cotton and Whatman No. 1 paper, before being evaporated in an oven set at 40 °C.

Hepatoprotective Test.
Te hepatoprotective assay followed a methodology inspired by the research of Ayenew and Wasihun [15] and involved a total of one hundred and sixty-fve rats, divided into 33 groups consisting of 5 rats each (Table 2).Among these groups, 3 served as control batches.Te frst control group (LT) received no treatment.Te remaining 32 groups were orally administered diferent substances for 7 consecutive days: distilled water for the positive control (LTP), silymarin for the reference control (LR), and recipes R1 to R10 at doses of 100, 200, and 400 mg/ kg for the 30 test groups.On the 8th day, all 32 groups received a single dose of paracetamol at a dosage of 5 g/kg.24 hours after paracetamol administration, blood samples were taken from fasted rats anesthetized with thiopental.Tiopental was administered to rats at a dose of 30 mg/kg/ body weight by an intravenous route.Following this administration, blood was drawn from the retro-orbital sinus of Wistar rats.Biochemical parameters such as transaminases, direct and total bilirubin, and alkaline phosphatase were evaluated.
A histological study was carried out on the livers removed from the animals after euthanasia with thiopental.Tiopental was administered to Wistar rats intravenously at a dose of 100 mg/kg body weight.After administration, the death of the animals, placed in individual cages, was confrmed by the absence of respiration, heartbeat, or refexes.In each group, two rats were sacrifced using this method.Following death, the rats were dissected and their livers removed.After removal, the rat body was placed in an animal-type biological waste bag and the appropriate animal carcass disposal procedure was followed.Finally, all equipment was cleaned and disinfected, including cages and work surfaces, and needles and syringes were properly eliminated.
2.6.Acute Oral Toxicity.Te research adhered to OECD guideline 423 [16] for the acute oral toxicity class assessment.A total of thirty-three female albino Wistar rats were utilized and divided into 11 groups based on their weight (refer to Table 3).Te test groups received a single oral dose of 2000 mg/kg simultaneously on the frst day (D0) via oral gavage.Te control group received distilled water under identical conditions.Each rat in every group was individually marked and closely monitored throughout the 14day experiment duration.Blood samples were taken from Wistar rats at the start and end of the experiment to assess biochemical and hematological parameters.For each sampling, rats were anesthetized with thiopental.Tiopental was administered to rats at a dose of 30 mg/kg/body weight by an intravenous route.Following this administration, blood was drawn from the retro-orbital sinus of Wistar rats.

Statistical Data Analysis.
Te statistical analysis was performed using SPSS software version 26.0.Mean values and standard deviations were calculated for each parameter.To assess hepatoprotective efects, univariate analysis of variance was employed to compare data among batches tested at varying doses, as well as between test and control batches (normal, reference, and paracetamol batches).In the toxicity test, the t-test was utilized to compare data from the test batches to the normal batch.A signifcance level of α � 0.05 was set for all statistical tests performed.

Efect of Cochlospermum tinctorium Associated with
Chromolaena odorata and Combretum micranthum on the Biochemical Parameters of Liver Function.Figure 1 illustrates the data concerning ALT levels in Wistar rats across various experimental groups.It is apparent from the fgure that rats in the paracetamol control group exhibited signifcantly higher ALT levels (p < 0.05) compared to the normal control group, indicating liver function impairment due to paracetamol administration.Conversely, silymarin signifcantly reduced ALT levels compared to rats receiving extracts from both cultivated and wild forms of C. tinctorium.Among the two varieties of C. tinctorium Advances in Pharmacological and Pharmaceutical Sciences tested, the extract from the wild form signifcantly decreased ALT levels compared to the cultivated form (p < 0.05) (Figure 1   Advances in Pharmacological and Pharmaceutical Sciences Te combination of C. micranthum with the wild form of C. tinctorium enhances this reduction efect on alkaline phosphatase levels, particularly at doses of 200 and 400 mg/ kg/body weight.However, when combined with C. odorata, there is an increase in the alkaline phosphatase levels of Wistar rats. Regarding total bilirubin, rats treated with paracetamol alone display an increase in the level of this biochemical parameter (Figure 4).Conversely, administration of silymarin and extracts from both the wild and cultivated forms of 100% C. tinctorium induce a reduction in total bilirubin levels.Concerning 5).

Efect of Cochlospermum tinctorium Associated with
Chromolaena odorata and Combretum micranthum on the Histology of Wistar Rat Livers.the silymarin reference group, eosinophilic polymorphonuclear cells are noted around the centrolobular veins, along with rare infammatory infltrates comprising lymphocytes and eosinophilic polymorphonuclear cells in the portal spaces.Te liver retains a subnormal histological architecture.
However, in groups combining C. tinctorium with C. odorata and all other remaining groups, liver histology displays slight congestion of sinusoids, portal spaces, and centrolobular veins.In addition, rare eosinophilic polymorphonuclear cells are observed within the lobules, along with vacuolar degeneration of hepatocytes.Advances in Pharmacological and Pharmaceutical Sciences  Table 5 displays data concerning the biochemical parameters of Wistar rats subjected to various treatments.Upon examination of this table, it becomes apparent that, compared to the control group, the groups treated with C. tinctorium (alone), regardless of whether it was in the wild or cultivated form, as well as the combinations with C. odorata and C. micranthum, exhibited no signifcant diferences for each of these parameters (ALT, AST, urea, and creatinine).On the other hand, Table 6 provides information regarding hematological parameters.Analysis of this table indicates that there were no signifcant diferences between the values of the test groups and the control group for each parameter (p > 0.05).Table 7 presents the values of body weight in Wistar rats from various groups.Tis table highlights a noticeable increase in body weight among rats across all groups.Nevertheless, there was no signifcant variation observed in the weight gain of Wistar rats among the diferent groups.
Mortality of Wistar Rats in Diferent Groups.Troughout the 14-day experimental period, no mortality occurred among rats in the various experimental groups.Tis implies that the LD50 of the tested extracts exceeds 2000 mg/kg/bw.

Discussion
Liver diseases are a signifcant global health concern, attributed to various factors including medication use, alcohol consumption, infections (viral, bacterial, or parasitic), and exposure to hepatotoxic agents.In Africa, traditional treatments often involve medicinal plants, emphasizing the importance of evaluating their efcacy.Medicinal plants may contain bioactive compounds with hepatoprotective properties, making them promising candidates for new treatments.Tis study aimed to assess the hepatoprotective efects and acute oral toxicity of medicinal formulations combining Cochlospermum tinctorium with Chromolaena odorata on one hand and with Combretum micranthum on the other used in the treatment of liver diseases in Benin.
Herbal formulations hold deep cultural signifcance in the African society, often serving as traditional remedies.With continued reliance on these treatments, it is essential to assess their efcacy and safety to promote proper healthcare practices.In Benin, medicinal blends containing Cochlospermum tinctorium, Chromolaena odorata, and Combretum micranthum are utilized for liver disease treatment without scientifc validation.Findings from this study revealed that among the tested C. tinctorium varieties, the wild form notably reduced ALT levels compared to the cultivated type (p < 0.05) (Figure 1(a)).In addition, a dose-dependent decrease in ALT levels was observed.Similar reports highlighting the hepatoprotective properties of Cochlospermum tinctorium have been documented in the literature [17,18].Tis efectiveness can be attributed to the polyphenolic compounds identifed in the plant, such as favonoids and total polyphenols [19].
Te variance in hepatoprotective efcacy observed between the wild and cultivated varieties of Cochlospermum tinctorium may stem from multiple factors, such as diferences in chemical composition, active ingredient content, and growth conditions.Plants often display variations in their chemical makeup depending on environmental factors such as soil quality, climate conditions, sunlight exposure, and nutrient availability.Consequently, the chemical profles of wild and cultivated forms of Cochlospermum tinctorium may diverge, potentially infuencing their respective hepatoprotective properties [19].Furthermore, the wild variant of the plant may encounter a wider array of soil microorganisms in comparison to its cultivated counterpart.Tis biodiversity can impact the symbiotic relationship between the plant and benefcial microorganisms, which might contribute to the production of health-promoting active metabolites.In addition, wild plants often contend  In the scientifc literature, comparable fndings have been documented for a blend of plants, including Cochlospermum tinctorium, Terminalia macroptera, Leptadenia hastata, and Commiphora africana, in Burkina Faso [23].Te authors noted that administering this blend of plants to hepatotoxic Wistar rats resulted in a marked decrease in plasma transaminases and alkaline phosphatase levels compared to the negative control group.Histologically treated rats displayed liver tissues that appeared normal or near-normal, varying with the dosage, in contrast to the control group.Likewise, similar fndings have been documented for a herbal medicine comprising a standardized combination of three extracts from Myristica fragrans, Astragalus membranaceus, and Poria cocos [24].Te authors noted that this herbal medicine, administered at doses ranging from 150 to 400 mg/kg, exhibited statistically signifcant dose-dependent suppression of serum alanine aminotransferase (ALT) levels in the acetaminophen model, ranging from 30.8% (p ≤ 0.05) to 88.1% (p � 0.0001), and in the carbon tetrachloride model, ranging from 66.9% (p � 0.002) to 83.7% (p � 0.0002), respectively.In addition, this herbal medicine led to reductions of up to 75.7%, 60.9%, and 33.3% in serum levels of aspartate aminotransferase (AST), bile acids, and total bilirubin, respectively.Tese fndings suggest a potential additive efect of C. tinctorium and C. micranthum.However, in regard to the formulation blending of C. tinctorium and C. odorata, rats treated with extracts from various formulations did not exhibit normal hepatic marker activity, unlike the combination of C. tinctorium and C. micranthum, particularly regarding ALT activity.Tis fnding contradicts the anticipated synergistic or additive efect.It is well documented in the scientifc literature that C. odorata possesses hepatoprotective properties [25,26].Tis observation may be elucidated by factors such as receptor competition, chemical incompatibility, insufcient dosage, pharmacokinetic interactions, or the intricacies of action mechanisms.Active compounds sourced from diferent plants might engage in competitive inhibition if they target the same liver receptors, or steric hindrance inhibition of active sites if the receptors difer.In instances where two compounds act on the same receptor, one may inhibit or attenuate the efect of the other [27].Certain chemical compounds found in diferent plants can interact unfavorably with each other, resulting in an overall decrease in efectiveness.Concerning inadequate dosage, concentrations of active compounds in each plant within the mixture may difer.While one plant may have a high concentration of a specifc compound, another plant may contain a lower amount.Tis variance can result in insufcient dosages of certain compounds, ultimately compromising the overall hepatoprotective efect [28].Moreover, it is essential to acknowledge that the mechanisms through which plants exert their hepatoprotective efects can be intricated and multifaceted.When two plants target distinct aspects of the hepatoprotective process, their combined efects may not necessarily be synergistic and could even oppose each other.It is crucial to highlight that research on plant interactions and their hepatoprotective efects constitutes a complex and evolving feld.
Concerning acute oral toxicity, the fndings revealed no instances of mortality or changes in biochemical and hematological parameters.Comparable observations are documented for all the plants tested [29,30].Tis study represents the initial demonstration that combinations of C. tinctorium with C. micranthum, as well as with C. odorata, are devoid of toxicity, as determined by the assessment of hematological and biochemical parameters.

Conclusion
Tis study highlighted that the combination of C. tinctorium with C. micranthum presents a better hepatoprotective efect and is nontoxic.Tis may justify the use of this medicinal recipe in the traditional Beninese medicine.However, although nontoxic, the combination of C. tinctorium with C. odorata does not maintain the activity of hepatic markers in Wistar rats making hepatotoxic via paracetamol.Further studies are necessary for more scientifc evidence.
(a)).Regarding the diferent combinations of plants composing the multiplant medicinal recipes (Figure1(b)), the data indicated that the extract derived from an equal combination (50%) of the wild form of C. tinctorium and C. micranthum signifcantly reduced ALT levels compared to the combination of 80% C. tinctorium and 20% C. micranthum.However, concerning the recipe combining C. tinctorium and C. odorata, rats treated with extracts from diferent formulations exhibited an increase in ALT levels.Tis increase was more pronounced for the equal combinations (Figure1(c)).In contrast, Figure2displays data concerning the AST levels of Wistar rats across various groups.Examination of this fgure reveals a notable increase in AST levels (p < 0.05) in rats treated with paracetamol.Conversely, the group treated with silymarin, serving as a reference compound, demonstrated
2.1.Ethical Consideration.Te research protocol received approval from the Ethics Committee of the Research Unit in Applied Microbiology and Pharmacology of natural substances at the University of Abomey-Calavi in Benin (approval no.0022/2021/CE/URMAPha/UAC). All procedures were carried out following the guidelines of the National Institute of Health (NIH) for the care and use of laboratory animals.2.2.Plant Material.Te botanical specimens comprised leaves of Chromolaena odorata, leaves of Combretum micranthum, and rhizomes of Cochlospermum tinctorium (wild and cultivated varieties).Tese plant components were authenticated at the National Herbarium of Benin, located at the University of Abomey-Calavi, with the following identifers: YH 356/HNB for Combretum micranthum G. Don, YH621/HNB for Chromolaena odorata (L.) R. M. King, and YH 622/HNB for Cochlospermum tinctorium ex A. Rich.

Table 1 :
Composition of medicinal recipes.

Table 2 :
Diferent batches of Wistar rats of the hepatoprotective tests.

Table 3 :
Composition of Wistar rat groups of the toxicity test.
a signifcant decrease in AST levels (p < 0.05).Similarly, groups treated with extracts of C. tinctorium exhibited reduced AST levels.Particularly, the extract derived from the wild form of C. tinctorium displayed a signifcant decrease in AST levels compared to the cultivated form of C. tinctorium.Regarding extracts from plant combinations, the extract obtained from the combination of C. micranthum and C. tinctorium induced a signifcant reduction in AST at a dose of 400 mg/kg/body weight.From Figure3, which illustrates the alkaline phosphatase levels of Wistar rats, it becomes apparent that there is a signifcant increase in the level of this biochemical parameter observed in rats treated solely with paracetamol.Conversely, silymarin administration leads to a notable reduction in alkaline phosphatase levels.In terms of the two forms of C. tinctorium, the extract derived from the wild form demonstrates a decrease in the level of this biochemical parameter compared to the cultivated form.

Table 4 :
Hepatic histology of Wistar rats from diferent groups.

Table 5 :
Values of biochemical parameters in Wistar rats subjected to extracts of Cochlospermum tinctorium alone and combined with Chromolaena odorata and Combretum micranthum.

Table 6 :
Values of hematological parameters in Wistar rats subjected to extracts of Cochlospermum tinctorium alone and combined with Chromolaena odorata and Combretum micranthum.