Chromolaena odorata (L.) R. M. King and H. Robinson Leaves Aqueous Extract Improves the Femoral Head in Ethanol-Induced Osteonecrosis in Rats

Chronic alcohol consumption damages bone formation and causes bone pathology, including osteonecrosis of the femoral head. The aim of this work was to evaluate the effects of the leaf aqueous extract of Chromolaena odorata (C. odorata) on the femoral head in ethanol-induced osteonecrosis in rats. Animals received alcohol (40°) at 3 g/kg for 12 weeks. A group of animals were sacrificed to attest to the instalment of osteonecrosis by using histopathological analysis. The remaining animals received alcohol concomitantly with the plant extract (150, 300, or 600 mg/kg) or diclofenac (1 mg/kg) for 28 additional days. At the end of the experimental period, biochemical parameters including total cholesterol, triglycerides, calcium, alkaline phosphatase (ALP), reduced glutathione (GSH), malondialdehyde (MDA), nitrite, superoxide dismutase (SOD), and catalase activities were measured. Histopathological and histomorphometry analyses of femurs were also assessed. The administration of alcohol, irrespective of the experimental period, induced a significant increase in total cholesterol (p < 0.05) and triglyceride (p < 0.01) and a decrease in ALP (p < 0.05) and calcium (p < 0.05–p < 0.001) levels. Intoxicated animals showed an alteration in oxidative stress parameters accompanied by a significant drop in bone cortical thickness and density with necrosis and marked bone resorption. The concomitant administration of the plant with ethanol reversed the alcohol-induced bone defect, characterized by the improvement of the lipid profile (p < 0.001), bone calcium concentration (p < 0.05), bone ALP activity (p < 0.001), oxidative stress parameters, improved cortical bone thickness (p < 0.01), and bone density (p < 0.05). These results are supported by the absence of bone resorption with an obvious effect at a dose of 300 mg/kg. The pharmacological effect of the extract on ethanol-induced osteonecrosis of the femoral head is probably due to its osteogenic, hypolipidemic, and antioxidant properties, justifying its use in Cameroonian folk medicine for articulation and bone pain management.


Introduction
Alcohol consumption has become one of the most serious substance abuse disorders worldwide [1]. Chronic alcohol consumption has a variety of harmful efects on several organs, including the brain, heart, liver, muscles, and bones [2]. Although recent studies have indicated that moderate levels of alcohol consumption may have benefcial efects on bone parameters and mineral density [3]. However, excessive alcohol consumption may be associated with impaired bone turnover and consequently bone fragility and a high risk of bone fracture [4]. Epidemiological investigations have shown that alcohol increases the risk of developing aseptic osteonecrosis of the femoral head [1]. Excessive alcohol intake has been associated with alterations in circulating lipids [5]. In fact, ethanol induces the diferentiation of stromal cells in the bone marrow into adipocytes; likewise, it causes a signifcant increase in serum triglyceride and cholesterol levels. Te deposition of triglyceride in osteocytes leads to pyknosis and an accumulated percentage of empty osteocytes, ultimately leading to osteocyte death [6]. Te main purpose of the treatments used to manage this pathology is to reduce pain, prevent the progression towards the loss of sphericity of the femoral head, and give indications for conservative or prosthetic replacement surgeries.

Plant Material and Extraction Preparation.
Te leaves of C. odorata were harvested at Yaoundé III in the Center Region of Cameroon in May 2019. Te plant was authenticated at the Cameroon National Herbarium in comparison with the specimen voucher N°8680/SRF/cam. Te fresh leaves were cleaned, cut into pieces, and dried under a shade at room temperature. Te decoction was carried out by boiling 600 g of fresh leaves in 3 L of tap water for 10 min following the traditional healer's instructions. Te fltrate obtained was evaporated at 45°C in a drying cupboard to yield a 7.10% extract, which was kept at room temperature until use. Te experimental doses used were obtained by measuring two cups of the fltrate extract according to the traditional healer's daily dose, dried, and quantifed it corresponding to an average dose of 300 mg/kg. Tis dose was then fanked by the lower (150 mg/kg) and the higher (600 mg/kg) doses.

Animals.
Six weeks old Wistar strain male rats weighing between 90 and 100 g were used in the present study. Tey were obtained from the animal house of the Faculty of Science at the University of Yaoundé I (Cameroon). Animals were given normal laboratory rat food and they received water ad libitum. Te procedures followed the principles of laboratory animal use and care of the "European Community Guidelines (EEC Directive 2010/63/EEC) and were approved by the "Animal Ethical Committee" of the Faculty of Science, University of Yaoundé I.

Osteonecrosis Induction and Study
Design. Animals received daily administration of ethanol (0.3 g/mL) at a dose of 3 g/kg for 12 weeks. A group of 5 animals (alcohol control group 1) were sacrifced to attest to the instalment of osteonecrosis. Te remaining animals received ethanol and diferent treatments simultaneously as follows: alcohol control group 2 received ethanol and distilled water (10 mL/ kg), one group received ethanol with diclofenac (1 mg/kg) as a reference drug, and three groups received ethanol with the plant extract at doses of 150, 300, and 600 mg/kg. All substances were administered by the oral route for twentyeight days. At the end of the experimental period, all animals were sacrifced under anaesthesia using ketamine (30 mg/kg) and diazepam (10 mg/kg) via the intraperitoneal route. Arteriovenous blood was collected, and the serum was prepared for biochemical analysis (total cholesterol, triglyceride, and calcium) assessed using a commercial Kit (BIOLABO, France). Bones (femur) were carefully removed and weighted, and the volume and density were measured using the method of Oumarou et al. [17]. Bones' biochemical analysis was performed to determine the level of calcium and alkaline phosphatase (ALP) using a commercial kit from BIOLABO (France).

Assessment of the Aqueous Extract from C. odorata on
Some Bone Parameters of Oxidative Stress. Te femur was carefully removed, stripped of muscle tissue, and then weighted. Te bones (0.2 g) were homogenised using 3 mL of phosphate bufer saline (pH, 7.4). Te mixture was then centrifuged at 3000 rpm at 4°C for 30 min. Te supernatant obtained was used to determine superoxide dismutase (SOD) activity using the method of Sinha [18], catalase activity according to the Misra and Fridovich protocol [19], reduced glutathione (GSH) concentration by the Ellman method [20], malondialdehyde (MDA), and nitrite concentrations according to Wilbur et al. [21] and Green et al. [22] procedures, respectively.

Histopathological Analysis.
Histological analyses of the femurs were assessed by the hematoxylin-eosin staining method. Te bones were conserved in 10% formalin and demineralized in a 10% HCl solution for 5 days. Isolated femur samples were embedded in parafn, and sections with a thickness of 10 μm were made using a microtome (Reichert-Jung 2030). Photographs of the sections were taken using a digital camera for the microscope (DCM, 35 : 350 K Pixels, USB 2.0) aided with appropriate flters. Te cortical bone thickness was measured using Image J software version 1.49.

Statistical Analysis.
Results are expressed as mean-± standard error mean. Statistical signifcance was determined by the one-way analysis of variance followed by the Tukey post-test using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, California, USA). Diference was considered signifcant if p < 0.05.

Efects of the C. odorata Leaves Aqueous Extract on Total
Cholesterol and Triglyceride Concentrations in Alcohol-Intoxicated Rats. Te efect of C. odorata on cholesterol and triglyceride concentrations is shown in Figure 1. Te administration of alcohol provoked a signifcant increase in total cholesterol (p < 0.05) and triglyceride levels (p < 0.01) irrespective of the administration period (before and after treatment). Te simultaneous administration of alcohol and the plant extract for 28 days signifcantly reduced the total cholesterol level (p < 0.01) by 34.85%, 51.05%, and 55.58% at respective doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg compared to intoxicated animals. At the same doses, the plant extract also signifcantly dropped the triglyceride concentration, respectively, by 48.33% (p < 0.05), 35.76% (p < 0.01), and 36.36% (p < 0.01). Te diclofenac used as an anti-infammatory drug provoked a signifcant decrease (p < 0.001) in total cholesterol and triglyceride (p < 0.05) concentrations.

Efects of C. odorata Leaves Aqueous Extract on Serum and
Bone Calcium in Alcohol-Intoxicated Rats. Alcohol treatedrats showed a signifcant decrease (p < 0.05) in serum and bone calcium concentrations by 19.89% and 38.24%, respectively, after three months of administration compared to normal rats (Figure 2(a)). Additional administration of alcohol for twenty-eight more days signifcantly emphasized the drop in serum calcium concentration by 50.00% (p < 0.05) and bone calcium by 73.91% (p < 0.001) as compared to their respective controls (Figure 2(b)). Concomitant administration of alcohol and plant extract resulted in an increase in serum calcium concentration by 65.90% (p < 0.05) and 109.84% (p < 0.01), bone calcium by 79.76% (p < 0.05) and 154.16% (p < 0.001) at respective doses of 300 and 600 mg/kg compared to their corresponding alcohol control groups. Te plant extract at a dose of 150 mg/kg and diclofenac administered at 1 mg/kg failed to increase serum and bone calcium.

Efects of C. odorata Leaves Aqueous Extract on Bone ALP
Activity. Te efect of C. odorata on ethanol-induced osteonecrosis is shown in Figure 3. Te administration of alcohol for 12 weeks did not signifcantly decrease ALP activity; while a signifcant decrease in bone ALP activity was observed with the cumulative administration of alcohol for an additional 28 days as compared to the normal control. Te plant extract caused a signifcant increase in bone ALP activity irrespective of the doses, with the highest efect at a dose of 300 mg/kg compared to the alcohol control group. Te extract at all doses as well as diclofenac, caused a signifcant increase (p < 0.01 − p < 0.001) in bone ALP activity in comparison to the normal control.

Efects of C. odorata Leaves Aqueous Extract on Non-Enzymatic Bone Oxidative Stress Parameters in Alcohol-Intoxicated Rats.
Te alcohol supplementation induced a signifcant decrease in glutathione (p < 0.05) and nitrite (p < 0.01) concentrations, whereas a signifcant increase was observed in MDA (p < 0.05) concentrations regardless of the administration time (Figures 4(a) and 4(b)). Alcohol consumption for an additional 28 days also signifcantly increased the MDA concentration by 71.77% (p < 0.001). In comparison to the alcohol group, daily administration of the plant extract inhibited the decrease in GSH concentration, though nonsignifcant (150 mg/kg and 300 mg/kg). Simultaneous administration of ethanol and the extract at doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg resulted in a signifcant decrease (p < 0.001) in MDA concentration respectively by 60.16%, 65.25%, and 63.38%. Te nitrite content was signifcantly increased (p < 0.05), with a marked efect at a dose of 300 mg/kg compared to the ethanol group. Diclofenac administered under the same conditions as the plant extract caused a signifcant decrease (p < 0.01) in MDA and an increase (p < 0.05) in nitrite concentration as compared to the alcohol control.

Efects of C. odorata Leaves Aqueous Extract on Enzymatic Bone Oxidative Stress Parameters in Alcohol-Intoxicated Rats.
Te administration of alcohol at a dose of 3 g/kg for three months resulted in a signifcant increase (p < 0.001) in the SOD and catalase activities before the treatment ( Figure 5(a)). Whereas, the supplementation of alcohol for an additional 28 days induced a signifcant decrease (p < 0.05) of SOD and catalase activities compared to the normal control ( Figure 5(b)). Te administration of the plant extract for 28 days signifcantly reversed the deleterious efect of alcohol on SOD (p < 0.05) and catalase (p < 0.001) activities at doses of 150 mg/kg and 600 mg/kg. At a dose of 150 mg/kg, the extract also improved (p < 0.05) SOD and catalase activities as compared to the alcohol group; and interestingly, the values were close to those of normal rats.

Efects of C. odorata Leaves Aqueous Extract on the Cortical Bone Tickness and Bone Density of Alcohol-Intoxicated Rats.
Te efect of the aqueous extract of C. odorata leaves on bone cortical thickness and bone density is shown in Figure 6. Te oral administration of alcohol for three months induced a signifcant decrease in bone cortical thickness (p < 0.001) and bone density (p < 0.01) as compared to normal control rats. Concomitant administration of the plant extract with alcohol for more than 28 days signifcantly increased cortical thickness (p < 0.01) and bone density (p < 0.05) at a dose of 300 mg/ kg in comparison with the alcohol control (Figures 6(a) and 6(b)). Te plant extract failed to improve cortical thickness at doses of 150 and 600 mg/kg but signifcantly increased bone density at a dose of 600 mg/kg (p < 0.01) as compared to the ethanol control group. Figure 7 shows the efect of the extract on the femoral head of alcoholintoxicated rats. Te femoral head section of normal control shows a distinct perichondrium and Tidemark with a normal aspect of chondrocytes (normal control). Te administration of alcohol provoked bone resorption which was accentuated Evidence-Based Complementary and Alternative Medicine when alcohol administration was prolonged for 28 additional days (alcohol control 1 group). In addition, there was marked necrosis in the alcohol treated group (alcohol control 2 group). Te plant extract prevented the instalment of bone damage with a marked efect at a dose of 300 mg/kg (C. odorata 150-300 mg/kg). Te highest dose (600 mg/kg) failed to protect the bone against alcohol-induced bone resorption.  Figure 1: Efects of C. odorata leaf aqueous extract on total cholesterol and triglycerides in alcohol-intoxicated rat. Each bar represents the mean ± SEM (n � 5). * p < 0.05, * * p < 0.01: signifcantly diferent compared to the normal control (1 or 2) group; a p < 0.05, c p < 0.001: signifcantly diferent compared to alcohol control (1 or 2) group. C. odorata 150, C. odorata 300, and C. odorata 600: rats treated concomitantly with the plant extract and alcohol at doses of 150, 300, and 600 mg/kg; Diclofenac1: rats treated concomitantly with alcohol and diclofenac at a dose of 1 mg/kg.

Discussion
Te present study was carried out to investigate the efects of C. odorata aqueous extract on ethanol-induced osteonecrosis of the femoral head of a rat. In the present study, the administration of ethanol (40°) at a dose of 3 g/kg irrespective of the period induced a signifcant decrease in bone mineral density. Tese results suggest that ethanol has harmful efects on bone metabolism. Indeed, alcohol slows down the bone remodeling process [23] by several mechanisms: the increase in sclerostin serum (an inhibitor of bone formation), which in turn increases osteoblast apoptosis through activation of caspase pathways [24], the stimulation of osteoclastogenesis by increasing the RANKL/OPG  Figure 2: Efects of C. odorata leaves aqueous extract on serum and bone calcium. Each bar represents the mean ± SEM (n � 5). * p < 0.05, * * p < 0.01, * * * p < 0.001: signifcantly diferent compared to the normal control group; a p < 0.05, c p < 0.001: signifcantly diferent compared to the alcohol control (1 or 2) group. $ p < 0.05, $$ p < 0.01: signifcantly diferent compared to diclofenac, C. odorata 150, C. odorata 300, and C. odorata 600: rats treated concomitantly with plant extract and alcohol at the respective doses of 150, 300, and 600 mg/ kg; Dicloenac1: rats treated concomitantly with alcohol and diclofenac at a dose of 1 mg/kg.

Evidence-Based Complementary and Alternative Medicine 5
(receptor activator of NF-κB ligand-osteoprotegerin) ratio [25,26]. In addition, ethanol induces the diferentiation of mesenchymal cells into adipocytes [27,28], which increase adipose cells within the bone marrow contributing to the decrease in bone mass. Te administration of the extract at diferent doses increased bone density, suggesting that C. odorata aqueous extract could inhibit ethanol-induced necrosis and/or stimulate the bone formation process. ALP is a marker of bone formation secreted by osteoblasts and playing a role in bone mineralization [29]. Te ethanol administration for 12 weeks led to a signifcant decrease in serum and bone ALP, attesting once more to the negative impact of ethanol on the bone formation process [30]. Indeed, ethanol inhibits the diferentiation of mesenchymal stem cells into osteoblasts [6] by suppressing the Wnt/ β-Catenin signaling pathway [31], thus promoting the differentiation of these pluripotent cells into adipocytes [32].
Te results obtained in this study confrm the efects of alcohol on the levels of ALP and bone mineralization. Te administration of the extract of C. odorata led to an increase in the concentration of bone ALP at diferent doses, indicating that the extract would have lifted the inhibitory efect of ethanol on the transformation of mesenchymal stem cells into osteoblasts. Calcium and phosphorus are necessary building blocks for bone formation and are stored in the bone as hydroxyapatite, which ensures bone rigidity. As observed in the present study, administration of ethanol signifcantly decreased serum and bone calcium levels. In fact, excessive alcohol consumption has been demonstrated as a cause of malnutrition [33] expressing by harmful efects on the absorption processes, metabolism, and excretion of micronutrients [23,34], thereby increasing the risk of the occurrence of osteonecrosis. Tus, in this context, the vitamin D defciency caused by ethanol induces a decrease in the intestinal reabsorption of calcium, hence its negative impact on bone metabolism. Administration of the extract at the gradual doses of 300 mg/kg and 600 mg/kg resulted in a signifcant increase in serum and bone calcium, testifying its ability to promote bone mineralization. Tis result would be explained not only by the capacity of the extract to reduce the deleterious efects of ethanol on the metabolism of micronutrients but also by the presence of minerals in the plant extract such as calcium, phosphorus, magnesium and, sodium which play a role in the process of bone mineralization [35][36][37]. Oxidative stress is known as one of the mechanisms involved in osteonecrosis [38,39]. In the femur, MDA concentration increased while catalase SOD activity and GSH level decreased, expressing the establishment of oxidative stress in ethanol-induced osteonecrosis. It is well demonstrated that ethanol increases the activity of the  Figure 3: Efects of C. odorata leaves aqueous extract on bone ALP. Each bar represents the mean ± SEM (n � 5). * p < 0.05, * * p < 0.01, and * * * p < 0.001: signifcantly diferent compared to the normal control group; c p < 0.001: signifcantly diferent compared to the alcohol control (1 or 2) group. C. odorata 150, C. odorata 300, and C. odorata 600: rats treated concomitantly with the plant extract and alcohol at doses of 150, 300, and 600 mg/kg; Diclofenac1: rats treated concomitantly with alcohol and diclofenac at a dose of 1 mg/kg. enzyme NADPH oxidase (NOX), thus leading to an increase in the production of free radicals within osteoblasts and the expression of RANKL, which activates osteoclastogenesis [40]. Ethanol-induced lipoperoxidation contributes to cytomembrane lesions; degeneration of the arterioles, and arteriosclerosis, which lead to ischemia in the target organ. In fact, lipid peroxidation induced by alcohol and its metabolites also leads to worsening of the ischemic state of osteocytes and therefore leads to osteonecrosis [41]. Te decrease in nitrite levels could result from the disruption of the release of nitric oxide (NO). Indeed, ethanol could have acted by stimulating modulators of apoptosis, such as inducible nitric oxide synthase (iNOS), leading to locally toxic levels of NO in osteoblasts and osteocytes, followed by cell death and consequently the development of osteonecrosis of the femoral head [42]. Te treatment of animals with the extract at doses of 150 and 300 mg/kg has resulted in the correction of these parameters indicating the antioxidant activity of the plant extract. Te diferent osteogenic and antioxidant properties demonstrated by the aqueous extract of C. odorata could be due to secondary metabolites within the plant. In fact, phytochemical screening of this extract revealed the presence of tannin, saponin, favonoids, phenols, and coumarins [43,44]. Most of these biometabolites, such as favonoid [45,46] and saponins [47], promote bone regeneration, while coumarins increase bone morphogenic protein-2 expression and thus enhance bone formation [48]. Moreover, these secondary metabolites particularly  Figure 5: Efects of C. odorata leaves aqueous extract on SOD and catalase activities. Each bar represents mean ± SEM (n � 5). * p < 0.05, * * p < 0.01, and * * * p < 0.001: signifcantly diferent compared to the normal control group; a p < 0.05, c p < 0.001: signifcantly diferent compared to the alcohol control (1 or 2) group. $ p < 0.05: signifcantly diferent compared to diclofenac, C. odorata 150, C. odorata 300, and C. odorata 600: rats treated concomitantly with the plant extract and alcohol at doses of 150, 300, and 600 mg/kg; Diclo1: rats treated concomitantly with alcohol and diclofenac at a dose of 1 mg/kg. epiphysis and the presence of empty lacunae testifying bone resorption in animals receiving ethanol [41]. Te absence of lacunae in animals receiving the extract suggests the inhibitory activity of the extract against the deleterious impact of ethanol on the femoral head, with marked activity in animals receiving the extract at a dose of 300 mg/kg.  Figure 6: Efects of C. odorata leaves aqueous extract on bone cortical thickness and density. Each bar represents mean ± SEM (n � 5). * p < 0.05, * * p < 0.01, and * * * p < 0.001: signifcantly diferent compared to the normal control group; a p < 0.05, c p < 0.001: signifcantly diferent compared to the alcohol control (1 or 2) group. C. odorata 150, C. odorata 300, and C. odorata 600: rats treated concomitantly with the plant extract and alcohol at doses of 150, 300, and 600 mg/kg; Diclo1: rats treated concomitantly with alcohol and diclofenac at a dose of 1 mg/kg. Furthermore, the reduction in cortical bone thickness in animals receiving ethanol demonstrates its negative impact on the process of bone metabolism by stimulating bone degeneration, justifying the reduction in bone density in these animals. Te administration of the extract induced an improvement of these parameters suggesting the capacity of the extract to counteract the efects of ethanol on the bone damage by lifting the inhibitory efect of ethanol on the diferentiation of stem cells into the osteoblastic line.

Conclusion
Te concomitant administration of the plant extract with ethanol reversed alcohol-induced bone defects, characterized by the improvement of the lipid profle, bone calcium concentration, and bone ALP activity. Te extract also reduced oxidative stress parameters increased cortical bone thickness and bone density. Te extract protected the bone against resorption induced by alcohol, with a pronounced efect at a dose of 300 mg/kg. Tese results confrm the use of this plant extract for the management of articulation and bone pain in Cameroonian folk medicine. Supplementary studies are needed to determine the mechanism of action of the C. odorata aqueous extract-induced-osteoprotective efect.  NO:

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
FTN and TD guided the study design. FTN and JH analysed the data and drafted the manuscript. RKG, JPD, and RF performed the histopathological and histomorphometry studies. SMS and JJ harvested the plant sample and performed the plant extraction performed in vivo experiment. CND, FTN, DPDD, and AK conducted the pharmacological and biochemical; All authors contributed substantially to the manuscript, read, and approved its fnal version.