Ethanol Extract of Lannea Acida A. Rich (Anacardiaceae) Stem Bark Accelerates Bone Fracture Healing in Ovariectomized Wistar Rat Volume 45 - Issue 2

Oumarou Riepouo Mouchili¹, Sylvin Benjamin Ateba²*, Edwin Mmutlane³, Derek Tantoh Ndinteh³, Constant Anatole Pieme⁴, Stéphane Zingue^3,5,6 and Dieudonné Njamen^1,3

• ¹Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé 1, Cameroon
• ²Department of Biology of Animal Organisms, Faculty of Science, University of Douala, Cameroon
• ³Department of Chemical Sciences, Faculty of Science, University of Johannesburg, South Africa
• ⁴Department of Biochemistry and Physiological Sciences, Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1, Cameroon
• ⁵Department of Life and Earth Sciences, Higher Teachers’ Training College, University of Maroua, Cameroon
• ⁶Department of Medical and Biomedical Engineering, Higher Technical Teachers’ Training College, University of Yaoundé 1, Cameroon

Received: June 27, 2022;   Published: July 20, 2022

*Corresponding author: Sylvin Benjamin Ateba, Department of Biology of Animal Organisms, Faculty of Science, University of Douala, P.O. Box 24157 Douala, Cameroon

DOI: 10.26717/BJSTR.2022.45.007172

Abstract PDF

Background: Osteoporotic fractures are a leading cause of morbidity and mortality in post-menopausal women. Lannea acida (Anacardiaceae) is a plant traditionally used to treat bone related disorders. In our previous study, an ethanolic stem bark extract of Lannea acida prevented postmenopausal osteoporosis in ovariectomized Wistar rat model. Based on this preceded results, the present study has been designed to evaluate the capacity of that extract to improve bone-fracture healing in estrogendeficient Wistar rat model of osteoporosis.

Material and Methods: Sham-operated (SHAM) and ovariectomized (OVX) female rats were fractured (30 days post-ovariectomy), divided into 6 groups (n = 5) and then treated by gavage for 60 days. SHAM and OVX controls receiving distilled water, OVX rats treated with estradiol valerate (E2V) at 1 mg/kg BW/day, and three OVX groups respectively treated with L. acida at 50, 100 and 200 mg/kg BW/day.

Results: Ethanol extract of L. acida extract prevented the OVX-induced decrease in femur weights compared to OVX group. It increased calcium and inorganic phosphorus levels and reduced ALP activity in both serum and bone of OVX-fractured animals. Compared to the OVX-fractured animals, it promoted a bone callus formation and union. It increased the trabecular thickness and a quasi-normal trabecular bone network was observed at the fracture site. Moreover, it also improved the fractured bone oxidative stress status.

Conclusion: This study demonstrated for the first time that L. acida extract promotes bone fracture healing in osteoporotic Wistar rats.

Keywords: Lannea Acida; Bone Fracture Healing; Postmenopausal Osteoporosis; Drill-Hole; Oxidative Stress

Osteoporosis is a systemic skeletal disease that affects bone quality through a reduction of bone mineral contents and density, and an alteration of the architecture. These events result in increased bone fragility and higher fracture risk [1,2]. In postmenopausal women, endogen estrogen deficiency plays a central role in the pathogenesis of osteoporosis and resulting fractures represent the major cause of morbidity and mortality in that women [3]. On a global scale, the increase in women life expectancy induces a dramatic rise in osteoporotic fracture incidence. Yet in 2013, the annual number of osteoporotic fractures worldwide was estimated to 10 million, with two-thirds in women [4]. This number is expected to increase over the coming years or decades as populations steadily age. Colossal medico-economic consequences are associated with osteoporotic fractures. Williamson, et al. [5] estimated to US$43669 per patient, with immediate inpatient care costing on average $13331, the global health and social care cost in the first year following a hip fracture. For this period, fracture incurs healthcare costs of more than $30,000 in US, of which $3000 will be paid by the patient [6]. In UK, in addition to the costs of disability (long-term social support for survivors), direct health costs are estimated to £4.3 billion for the 50,0000 fragility fractures occurring annually [7]. Apart the heavy economic burden, osteoporotic fractures cause chronic pain, loss of mobility, and loss of independence and death [1].

By contrast to high-income settings, no study has been published on the health costs of fragility fractures in sub-Saharan Africa. The health and socio-economic burden of fractures would likely be much heavier in this low-income setting where there is a lack of health and social systems that facilitate long-term care, a substantial part falling on family and friends. Rather than treat, it is recommended to prevent osteoporotic fractures. In case they occur, comorbidities specific to the patient such osteoporosis strongly interfere with the healing [8,9]. In addition to the mechanical stability, bone fracture healing can be positively modulated by systemic drug therapies [10]. Nowadays, anti-osteoporotic drugs including anti-resorptive and anabolic agents alone or in combination with calcium/vitamin D remain the main treatments [10-12]. However, they display a variable efficacy depending on the type of fracture. For instance, bisphosphonates do not influence the healing after distal radius, hip and vertebral fractures [13]. Anabolic agents accelerate the healing of long bone fractures, while clinical evidence for antiresorptive agents remains unclear [10]. Moreover, safety concerns as well as the poor long-term adherence of these agents substantially contributed to their reduction in prescriptions. Long-term use of bisphosphonates, the most popular antiosteoporotic agents, is associated with increased risk of atypical femur fracture, upper gastrointestinal symptoms and risk of venous thrombosis [14,15]. Nowadays, seeking alternative treatments has become more and more important. Accordingly, medicinal plants have been suggested as an alternative to promote a fast and efficient (without recurrence) bone repair process with no or minimal adverse side effects [16].

Moreover, given their affordability and accessibility, they may play an important role in reducing family economic pressure. In low-income settings, they hold a very important place in the healthcare systems as up to 80 percent of people rely on them for their primary health care. Lannea acida A. Rich (Anacardiaceae) is distributed in Africa and used to treat various ailments including musculoskeletal disorders and gynaecological complains [17,18], to enhance fertility and facilitate parturition [19]. In vitro, the aqueous and methanolic extracts of stem bark induced uterotonic effects through oxytocin receptors [19]. Chronic, non-resolving inflammation is detrimental to fracture healing [20]. Upon fracture inflammatory and ischemic conditions can cause excessive oxidative stress resulting in irreversible damage to cells associated with bone repair [21]. This situation is aggravated by conditions of intrinsic oxidative stress such diabetes and postmenopausal osteoporosis [22-23]. In vivo studies reported inflammatory, analgesic [24], and antioxydative [25] properties of L. acida stem bark extracts. Moreover, our previous study showed that an ethanolic stem bark extract of L. acida exhibited estrogenic properties and prevented bone loss in an ovariectomized rat model of osteoporosis [26]. All these results suggest that L. acida might promote the osteoporotic fracture healing. Therefore, the present study has been designed to evaluate the potential of the ethanolic stem bark extract of the L. acida to promote bone fracture healing in drill-hole injury in estrogen-deficient Wistar rat model of osteoporosis.

Chemicals and Reagents

The antibiotic penicillin (xtapen®) and diclofenac (Dicloecnu®) were obtained from CSPC Zhongnuo pharmaceutical (Shijiazhuang City, China) and ECNU pharmaceutical (Yanzhou City, China), respectively. The 17β-estradiol valerate (Progynova®) were provided by DELPHARM (Lille, France).

Plant Collection and Authentication

The stem barks of L. acida were collected in Moutourwa (Far- North, Cameroon) in July 2014. The plant sample was authenticated at the national Herbarium of Cameroon (HNC-IRA) with voucher number 40942 HNC.

Animals

Healthy, nulliparous and non-pregnant female Wistar rats (2.5- 3 months), from the Animal house of the Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé 1, Cameroon. They were housed in groups of five in plastic cages and kept at room temperature, under natural illumination. They were given soy free diet and water ad libitum. Animals were handled in accordance to the European Union on Animal Care (CEE Council 86/609) guidelines approved by the Institutional National Ethics Committee of the Cameroonian Ministry of Scientific Research and Technology Innovation.

Plant Extraction

The air-dried and powdered plant material (2kg) was soaked 3 times in 6L of 95% ethanol at room temperature for 48h. After filtering, the combined solution was concentrated under reduced pressure (40°C, 337 mbar) using a rotary evaporator to obtain 272 mg of extract.

Determination of the Mineral Composition of the Extract

To determine the content of calcium, magnesium, potassium, sodium and phosphorus in the extract, 0.5 g of L. acida extract was diluted in 10 mL of supra pure HNO3 and placed in the MARS 6 microwave digestion system (CEM Matthew, USA) for 30 minutes. Thereafter, demineralized water was added to the tubes up to a total volume of 50 mL. One mL of this digested sample was diluted in 9 mL of 1% HNO3 in falcon tubes. In reference to calibration curves of standards, the mineral contents of sample were determined using a Spectro Arcos inductively coupled plasma – optical emission spectrometer ICPOES (SPECTRO Analytical Instruments, Germany).

Fracture Healing Assessment

Animal Randomization, Drill-Hole Femur Injury and Treatment: Before the bilateral ovariectomy, vaginal smears of each animal were daily performed for 12 consecutive days to be sure that they are normocyclic. Thereafter, twenty-five of them were ovariectomized (OVX) under anaesthesia (10 mg/kg diazepam and 50 mg/kg ketamine, i.p.), while 5 were sham-operated (SHAM). To assure the situation of estrogen depletion and constant presence in diestrus step, the vaginal smears of the ovariectomized animals were daily checked for five consecutive days from the day 14 after ovariectomy. Thereafter, animals were divided into 6 groups of five animals each (n = 5). Fractures of the proximal femur are the osteoporotic fractures with the highest morbidity and mortality [12]. Yousefzadeh, et al. [27] reported that 30 days is the time needed for inducing osteoporosis in rat femur after bilateral ovariectomy. Accordingly, thirty days after the ovariectomy a femoral drill-hole injury was created in all ovariectomized rats as described by Ngueguim, et al. [28]. Briefly, following a 1-cm long skin and musculature incision, and the exposure of the surface of the femur under anaesthesia (diazepam and ketamine), a 1-mm diameter hole was created by inserting a drill bit into the anterior portion of the diaphysis. Thereafter, animals were sutured and submitted from the very next day to a 60-day (once daily at 4 - 5 p.m.) oral treatment. The animals were weighed once weekly. Group I (SHAM) and Group II (OVX: ovariectomized rats serving as negative control) received distilled water; Group III (E2V: OVX rats serving as positive control) received 1 mg/kg estradiol valerate; Group IV, V and VI (LA 50, LA 100 and LA 200) were OVX animals treated with L. acida extract at the doses of 50, 100 and 200 mg/kg, respectively. At the end, the rats were sacrificed under anaesthesia (10 mg/kg of diazepam and 50 mg/kg of ketamine, i.p.) after an overnight fast and the blood samples collected. The uterus, vagina and femurs were peeled off to remove the surrounded tissues. After weighing uterus and femurs, all these tissues were fixed in 10% formaldehyde.

Preparation of Bone Homogenate

After washing in NaCl 0.9%, the proximal portion of the left femur was cut and weighed (0.5 g). The tissue was then homogenized in ice-cold medium using a glass Teflon-Potter and 3 mL of deionized water. After centrifuging at 3000 rpm at 5°C for 15 min, the supernatant was collected for estimating the antioxidant activity. Catalase (CAT) and glutathione peroxidase (GPx) activities as well as the ferric reducing antioxidant power (FRAP) were determined according to the methods of Wilbur, et al. [29], Ellman [30], and Benzie and Strain [31], respectively. Calcium and inorganic phosphorus contents as well as alkaline phosphatase activity were also measured using Biolabo (Maizy, France) reagent kits.

Blood Analyses

The blood samples collected in EDTA tubes were analysed using a Mindray BC-2800 Auto Haematology Analyser. The samples in dry tubes were centrifuged at 3500 rpm at 5°C for 10 min, and the serum analysed to measure the serum calcium and inorganic phosphorus contents as well as the alkaline phosphatase (ALP) activity using reagent kits from Biolabo (Maizy, France).

Bone Radiography

The bone callus formation was evaluated as described by Ngueguim, et al. [28]. Briefly, the femur was exposed for 5 milliseconds to X-rays emitted by an Allengers X-ray machine (maximum voltage 125 Kv, main voltage 55 Kv, filtration 0.9 Al/75 (permanent) at a distance of 1 m). An AGFA CR 30-Xm detector (CRAGFA, Belgium) was used for obtaining and analysing the images.

Histological Analysis

A Zeiss Axioskop 40 microscope equipped with an AxioCam MR digital camera connected to a computer allowed the analysis of 5 μm sections of paraffin-embedded uterus and femur using MRGrab1.1 and Axio Vision 3.1 softwares (Carl Zeiss, Hallbergmoos, Germany). The sections were stained with haematoxylin and eosin.

Statistical Analysis

All results were expressed as mean ± standard error of mean (SEM) and analysis Graphpad Prism 5.03. One-way analysis of variance (ANOVA) and the Dunnett’s post hoc test were used. p value < 0.05 was considered as statistically significant.

Mineral Composition of the Extract

Table 1 summarizes the contents of potassium (K), phosphorus (P), magnesium (Mg), calcium (Ca) and sodium (Na) found in L. acida extract. Potassium was found to be the most abundant mineral (7387.91 ± 80.19 μg/g) followed by calcium (1657.20 ± 52.65 μg/g). The abundances of sodium, phosphorus and magnesium were 979.95 ± 4.45, 680.11 ± 3.38 and 343.99 ± 2.05 μg/g, respectively.

Table 1: Mineral composition of the ethanolic extract of L. acida.

Effects of L. Acida Ethanol Extract

Effects on Uterus and Vagina: Table 2 depicts the effects of the ethanolic extract of L. acida on uterus and vagina. A significant (p < 0.001) reduction of uterine relative wet weight as well as uterine and vagina epithelial heights was observed 90 days after ovariectomy in OVX animals compared with sham-operated (SHAM). Compared with OVX group, a 60-day treatment with the extract of L. acida from day 31 significantly increased (p < 0.05) the uterine (at 50 and 100 mg/kg) and vaginal (at 100 and 200 mg/kg) epithelial heights.

Table 2: Mineral composition of the ethanolic extract of L. acida.

Note: SHAM = normal animals receiving distilled water; OVX = Ovariectomized rats receiving distilled water; E2V = ovariectomized rats treated with estradiol valerate (1 mg/kg); L. acida = Ovariectomized rats treated with L. acida extract at the doses of 50, 100 and 200 mg/kg, respectively. Data are expressed as mean ± ESM (n = 5); *p< 0.05, **p< 0.01 and. ***p < 0.001 as compared with SHAM group. ###p < 0.001 and #p< 0.05 as compared with OVX group.

Effects on Femur Weight: A 90-day of estrogen depletion led to a significant reduction (p < 0.01) of the femur weight as compared with SHAM group (Table 2). At 50 and 100 mg/kg of L. acida extract, the femur wet and dry weights were significantly higher (p < 0.5) than that in OVX group following a 60 days’ oral treatment from the day 31.

Effects on Some Biomarkers of Serum and Bone: Table 3 shows an increased (p < 0.05) alkaline phosphatase (ALP) activity in both serum and bone 90 days after ovariectomy as compared with normal rats. The 60-day treatment with L. acida from day 31 after ovariectomy led to a lower ALP activity (p< 0.001) in serum (at 50 and 100 mg/kg) and bone (at 100 and 200 mg/kg) as compared with OVX group. Calcium levels in bone and serum were significantly lower (p< 0.001) in OVX animals as compared with SHAM group 90 days after ovariectomy. Compared to the OVX group, the serum calcium levels of the L. acida extract treated groups significantly increased (p< 0.001). The values were even higher than that observed in SHAM group. In bone, a significant increase was observed at the doses of 100 and 200 mg/kg group (p < 0.05). On the other hand, E2V-treated rats exhibited a higher (𝑝< 0.001) serum level of calcium, while no significant variation was observed in the femur. Regarding inorganic phosphorus, 90 days of endogenous estrogen depletion did not affect the bone and serum levels of inorganic phosphorus as compared with the SHAM group. Treatment with E2V (1 mg/kg) and L. acida (100 mg/kg) from the day 31 exhibited a higher level of inorganic phosphorus in serum as compared with OVX group (p < 0.01), while only 200 mg/kg of L. acida extract increased (p < 0.001) this parameter in bone.

Table 3: Effects of the ethanol extract of L. acida on some biomarkers of bone and serum after 60 days of treatment from day 31.

Note: SHAM = normal animals receiving distilled water (vehicle); OVX = Ovariectomized rats receiving distilled water; E2V = ovariectomized rats treated with estradiol valerate (1 mg/kg); L. acida = Ovariectomized rats treated with L. acida extract at the doses of 50, 100 and 200 mg/kg BW, respectively. All animals were fractured. Data are expressed as mean ± ESM (n = 5); *p< 0.05, **p< 0.01 and. *** p < 0.001 as compared to SHAM group. ###p < 0.001 and #p< 0.05 as compared to OVX group.

Effects on Some Oxidative Stress Parameters in Bone

Figure 1:

(A) Effects L. acida ethanol extract on the catalase,

(B) Glutathione peroxidase and

(C) FRAP activities in bone.

GPx = glutathione peroxidase; FRAP = Ferric Reducing Antioxidant Power; SHAM = normal control group receiving the vehicle (distilled Water), OVX = negative control group receiving the vehicle (distilled Water); E2V = OVX rats that received estradiol valerate (1 mg/kg BW); L. acida = OVX animals treated with de L. acida at the doses of 50, 100 and 200 mg/kg BW, respectively. Data expressed as mean ± SEM (n= 5); *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to OVX group. #p < 0.05, ##p < 0.01, and ### p < 0.001 as compared to SHAM group.

Ninety days after ovariectomy, a non-significant reduction (13.6%) of catalase (CAT) activity was observed in OVX rats as compared with the SHAM animals (Figure 1A). Treatment with E2V and L. acida (50 and 200 mg/kg) from the day 31 led to a lower CAT activity than that observed in OVX group (p< 0.001). However, the extract at 100 mg/kg significantly (p< 0.001) augmented the CAT activity at a value higher than that in the SHAM group. Compared with SHAM group, glutathione peroxidase (GPx) activity and the Ferric Reducing Antioxidant Power (FRAP) considerably diminished (p< 0.01) in bone of OVX rats (Figures 1B & 1C, respectively). The oral administration of E2V and L. acida for 60 consecutive days at all tested doses significantly (p < 0.001) increased GPx activity and FRAP as compared with the OVX group. The values of FRAP and GPx in all groups receiving the extract were close and higher than that of the SHAM group, respectively.

Effects on Bone Callus Formation

Figure 2: Effects L. acida ethanolic extract in the bone radiographies and histology. SHAM = normal control group receiving the vehicle (distilled Water); OVX = negative control group receiving the vehicle (distilled Water); E2V = OVX rats that received estradiol valerate (1 mg/kg BW); TB = trabecular bone; CB = cortical bone; BM = bone marrow; L. acida = OVX animals treated with de L. acida at the doses of 50, 100 and 200 mg/kg BW, respectively. Data expressed as mean ± SEM (n= 5); *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to OVX group. #p < 0.05, ##p < 0.01, and ### p < 0.001 as compared to SHAM group.

Radiographies of injured femurs showed a partial healing of bones in untreated OVX animals compared to the SHAM group. Ovariectomized and fractured rats that received E2V (1 mg/ kg) and L. acida at all the doses had total bone healing (Figure 2) characterized by a total callus formation and a complete bone union. Femurs in OVX group showed a deterioration of the microarchitecture as compared with the SHAM group. This was evidenced by the larger resorption lacunas (Figure 2). However, L. acida extract at all the tested doses improved the bone microstructure after fracture compared to OVX group. In fact, the histological sections of bones of these animals showed a quasinormal trabecular bone network. Furthermore, measurement of the trabecular bone thickness showed a significant (p< 0.05) reduction of trabecular bone thickness in OVX animals compared to the SHAM group. E2V and L. acida extract at all the tested doses for 60 days significantly (p< 0.001) increased the thickness of the trabecular bone as compared with the OVX rats.

Effects on Some Haematology Parameters

As far as haematological parameters are concerned, no significant variation was found in all parameters between the different groups (Table 4). Exception made for the white blood cell count, which was significantly decreased in the negative control group compared to the normal group.

Table 4: Effects of the ethanolic extract of L. acida extract on the hematological parameters.

Note: SHAM = normal animals receiving distilled water (vehicle); OVX = Ovariectomized rats receiving distilled water; E2V = ovariectomized rats treated with estradiol valerate (1 mg/kg); WBC = white blood cell count; RBC = red blood cell count; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; L. acida = Ovariectomized rats treated with L. acida extract at the doses of 50, 100 and 200 mg/kg BW, respectively. All animals were fractured. Data are expressed as mean ± ESM (n = 5); *p< 0.05, **p< 0.01 and. ***p < 0.001 as compared to SHAM group. ###p < 0.001 and #p< 0.05 as compared to OVX group

The bone-fracture healing capacity of the ethanolic stem bark extract of L. acida was investigated using a drill-hole femur fracture in postmenopausal Wistar rat model of osteoporosis. Bilateral ovariectomy is a widely accepted model for evaluating postmenopausal complications as well as the effects of substances on these complications. The lack of ovarian estrogen production for 90 days induced a significant reduction in uterine wet weight as well as uterine and vaginal epithelial heights in OVX group compared to SHAM group. This result is in accordance with many reports [26,32,33] and validates the bilateral ovariectomy. The 60-day treatment with L. acida extract had no effect on uterine wet weight but significantly increased the uterine and vaginal epithelial heights. This result suggests the presence of estrogenlike phytoconstituents in the extract and confirm the estrogenic potential of L. acida observed in our previous report [26].

Patient-related comorbidities such as osteoporosis contribute to bone fracture-healing complications [8]. By managing them bone healing can be improved. The significant reduction in femur mass and calcium content as well as the increase in bone alkaline phosphatase activity observed in OVX group as compared with SHAM group indicate the osteoporosis status in these animals. The process of consolidation and bone remodelling of a fracture is strictly dependent on bone turnover and the calcium and phosphate metabolism [10]. L. acida ethanolic extract significantly increased calcium and inorganic phosphorus levels in bone. Osteoblasts are responsible for the osteoid matrix formation and, through the production of non-collagenous proteins, initiate and regulate its mineralization. Therefore, the increase of bone calcium and inorganic phosphorus contents following treatment with L. acida is an indicator of bone formation. The higher the calcium and phosphorus contents, the greater the bone density and strength. L. acida extract also induced a significant increase of serum calcium and inorganic phosphorus levels suggesting an elevated intake of that minerals.

The analysis of this extract showed high amounts potassium, calcium, inorganic phosphorus and magnesium suggesting that the extract provides, at least in part, the minerals (calcium, inorganic phosphorus) necessary for the bone mineralization. Studies indicated that in addition to activate vitamin D, magnesium promotes osteoblast proliferation [34,35], while a high dietary intake of potassium is beneficial to bone health even in people with low dietary calcium intake [36]. Alkaline phosphatase (ALP), a specific biomarker of bone formation secreted by osteoblast during bone maturation [37], increased both in serum and femur of OVX-fractured rats compared to sham-operated animals. L. acida extract at the tested doses significantly reduced the ALP activity in bone and serum compared to OVX group. Several studies correlated the reduction of bone and serum ALP activity with the process of fracture healing [28,38,39]. Normal fracture healing is generated by increased osteoblastic activity, that secretes large quantities of ALP involved in the bone formation and mineralization [38].

Following a fracture, there is an acute increase in ALP activity, then it decreases with the formation of bone callus to return to normal when it ceases [38]. The faster bone healing occurs, the faster the ALP activity decreases. In line with this, it appears that compared to the OVX-fractured control animals, there is an acceleration of bone healing in OVX-fractured animals treated with L. acida extract. The radiographies of femurs from animals in this study showed a healing of fractures, except for the femur of OVXfractured animals, where the healing was not complete. According to our results, microarchitectures of bone of OVX-fractured rats were disorganized, with decrease in trabecular bone thickness. E2V and L. acida extract treatment reversed this bone disorganization and increased trabecular bone thickness compared to that observed in OVX-fractured control group. According to Potu, et al. [40], osteoporosis is characterized by reduction in trabecular and cortical bone thicknesses. L. acida ethanol extract inhibited bone lost and microarchitecture alteration by the same way improved bone fracture healing.

Oxidative stress has been suggested to be a mediator and indicator of osteoporosis [41-44]. It also been involved in the ischemia-reperfusion processes, especially during the callus formation, that occur after a fracture [45,46]. Sandukji, et al. [47] showed that antioxidant treatment improves bone parameters and oxidative stress related markers in patients with long-bone fractures, and thus might be beneficial in the healing. A decrease in GPx activity and FRAP was observed in OVX-fractured rats compared to SHAM group, while bone catalase (CAT) did not change. This result indicates the occurring of the oxidative stress in femur of OVX-fractured rats. Treatment of these animals with L. acida ethanolic extract induced a significant increase in CAT (at 100 mg/kg) and GPx (at all tested doses) activities suggesting an antioxidant activity in bone. On the other hand, the extract at all tested doses significantly increased bone FRAP in OVX-fractured animals. The FRAP measures the total antioxidant capacity of the antioxidants present in the sample and able to reduce ferric ion (Fe3+) involved in the generation the most potent hydroxyl radical. Accordingly, our results suggest that the ethanolic extract of L. acida increased non-enzymatic antioxidants and reduced lipid peroxides in the bone tissue.

The aim of this study was to evaluate the capacity of that extract to improve bone-fracture healing in estrogen-deficient Wistar rat model of osteoporosis. The extract reduced the alkaline phosphatase activity, increased calcium and inorganic phosphorus contents and improved the oxidative stress status in bone. In addition, a quasi-normal trabecular bone network at the fracture site accompanied the positive effects of the L. acida extract on callus formation and fracture healing. All these results suggest that the ethanol extract of stem bark of L. acida improves bone fracture healing in ovariectomized Wistar rat.

None declared.

All the authors accepted the responsibility of the manuscript’s content and approved the submission.

The authors declare no conflicts of interest.

1. Sànchez-Riera L, Wilson N (2017) Fragility fractures and their impact on older people. Best Practice & Research: Clinical Rheumatology 31(2): 169-191.
2. Hazavehei SM, Taghdisi MH, Saidi M (2007) Application of the health belief model for osteoporosis prevention among middle school girl students, Garmsar, Iran. Education for Health (Abingdon) 20(1): 23.
3. Pisani P, Renna MD, Consersano F, Casciaro E, Di Paolo M, et al. (2016) Major osteoporotic fragility fractures. Risk factor updates and societal impact. World Journal of Orthopedics 7(3): 171-181.
4. Hernlund E, Svedbom A, Ivergard M, Compston J, Cooper C, et al. (2013) Osteoporosis in the European Union: medical management, epidemiology and economic buerden. A report in collaboration with the international Osteoporosis Foundation (OIF) and the European Federation of Pharmaceutical Industry Associations (EFPIA). Archives of Osteoporosis 8(1): 136.
5. Williamson S, Landeiro F, McConnell T, Fulford-Smith L, Javaid MK, et al. (2017) Costs of fragility hip fractures globally: a systematic review and meta-regression analysis. Osteoporosis International 28(10): 2791-2800.
6. Williams SA, Chastek B, Sundquist K, Barrera-Sierra S, Leader DJr, et al. (20220) Economic burden of osteoporotic fractures in US managed care enrolees. The American Journal of Managed Care 26(5): e142-e149.
7. Leal J, Gray AM, Prieto-Alhambra D, Arden NK, Cooper C, et al. (2016) Impact of hip fracture on hospital care costs: a population-based study. Osteoporosis International 27(2): 549-558.
8. Haffler-Luntzer M, Hankenson KD, Ignatius A, Pfeifer R, Khader BA, et al. (2019) Review of animal model of comorbidities in fracture-healing research. Journal of Orthopaedic Research 37(12): 2491-2498.
9. Namkung-Matthai H, Appleyard R, Jansen J, Hao Lin J, Maastricht S, et al. (2001) Osteoporosis influences the early period of fracture healing in a rat osteoporotic model. Bone 28(1): 80-86.
10. Marongiu G, Dolci A, Verona M, Capone A (2020) The biology and treatment of acute long-bones diaphyseal fractures: overview of the current options for bone healing enhancement. Bone Reports 12: 100249.
11. Degli Esposti L, Girardi A, Saragoni S, Sella S, Andretta M, et al. (2019) Use of antiosteoporotic drugs and calcium/vitamin D in patients with fragility fractures: impact on re-fracture and mortality risk. Endocrine 64(2): 367-377.
12. Khan AZ, Rames RD, Miller AN (2018) Clinical management of osteoporotic fractures. Current Osteoporosis Reports 16: 299-311.
13. Shin YH, Shin WC, Kim JW (2020) Effect of osteoporosis medication on fracture healing: an evidence based review. Journal of Bone Metabolism 27(1): 15-26.
14. Black DM, Eastell R, Vittinghoff E, Li BH, Ryan DS, et al. (2020) Atypical femur fracture risk versus fragility fracture prevention with bisphosphates. New England Journal of Medicine 383(8): 743-753.
15. Grenn J, Czanner G, Reeves G, Watson J, Wise L, et al. (2010) Oral bisphosphates and risk of cancer of oesophagus, stomach and colorectum: case-control analysis within a UK primary cohort. British Medical Journal 341: c4444.
16. Barbalho SM, Araújo AC, Penteado Detregiachi CR, Buchaim DV, Guiguer EL (2019) The potential role of medicinal plants in bone regeneration. Alternative Therapies in Health and Medicine 25(4): 32-39.
17. Arbonnier M (2009) Arbres, arbustes et lianes des zones sèches d’Afrique de l’Ouest. Coed. In : MNHN-Quae (Edt.)., p. 396-416.
18. Gill LS (1992) Ethnomedicinal uses of plants in Nigeria. University of Benin Press, Benin City, Nigeria.
19. Ngadjui E, Kouam JY, Fozin GRB, Momo ACT, Deeh PDB, et al. (2021) Uterotrophic effects of aqueous and methanolic extracts of Lannea acida in Wistar rats: A in vitro Reproductive Sciences 28(9): 2448-2457.
20. Bahney CS, Zondervan RL, Allison P, Theologis A, Ashley JW, et al. (2019) Cellular biology of fracture healing. Journal of Orthopaedic Research 37(1): 35-50.
21. Kubo Y, Wruck CJ, Fragoulis A, Drescher W, Pape HC, et al. (2019) Role of Nrf2 in fracture healing: clinical aspects of oxidative stress. Calcified Tissue International 105(4): 341-352.
22. Dai K, Hao Y (2007) Quality of healing compared between osteoporotic fracture and normal traumatic fracture. In: Quin L, Genant HK, Griffith JF, Leung KS (Eds.)., Advanced bioimaging technologies in assessment of the quality of bone and scaffold materials techniques and applications. Springer, Berlin Heidelberg, pp. 531-541.
23. Hamada Y, Fujii H, Fukagawa M (2009) Role of oxidative stress in diabetic bone disorder. Bone 45(Suppl 1): S35-S38.
24. Owusu G, Ofori-Amoah J (2017) Anti-inflammatory and analgesic effects of an aqueous extract of Lannea acida stem bark. Journal of Pharmaceutical Research International 16: 1-8.
25. Sarwar M, Idress HA, Abdollahi MA (2011) Review on the recent advances in pharmacological studies on medicinal plants; animal studies are done but clinical studies need completing. Asian Journal of Animal and Veterinary Advances 6: 867-883.
26. Riepouo Mouchili O, Zingue S, Bakam BY, Ateba SB, Foyet SH, et al. (2017) Lannea acida Rich (Anacardiaceae) ethanol extract exhibits estrogenic effects and prevents bone loss in an ovariectomized rat model of osteoporosis. Evidence-Based Complementary and Alternative Medicine (2017): 7829059.
27. Yousefzadeh N, Kashfi K, Jeddi S, Ghasemi A (2020) Ovariectomized rat model of osteoporosis: a practical guide. EXCLI Journal 19: 89-107.
28. Ngueguim Tsofack F, Sakouong Talle SH, Donfack JH, Gounoue Kamkumo R, Dzeufiet Djomeni PD, et al. (2017) Aqueous extract of Peperomia pellucida (L.) HBK accelerates fracture healing Wistar rats. BMC Complementary and Alternative Medicine 2017(1): 188.
29. Wilbur Km, Bernheim F, Shapiro OW (1949) Determination of lipid peroxidation. Archives of Biochemistry and Biophysics 24: 305-310.
30. Ellman GL (1959) Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82: 70-77.
31. Benzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power, the FRAP assay. Analytical Biochemistry 239: 70-76.
32. Ateba SB, Njamen D, Medjakovic S, Hobiger S, Mbanya JC, et al. (2013) Eriosema laurentii De Wild (Leguminosae) methanol extract has estrogenic properties and prevents menopausal symptoms in ovariectomized Wistar rats. Journal of Ethnopharmacology 150(1): 298-307.
33. Zingue S, Njamen D, Mvondo MA, Magne Nde CB (2014) Preventive effects of the methanol soluble fraction of Milletia macrophylla Benth (Fabaceae) on an osteoporosis-like model on ovariectomized Wistar rats. Journal of Complement and Integrative Medicine 11(2): 83-92.
34. De Baaij JHF, Hoenderop JGJ, Bindels RJM (2015) Magnesium in man: Implications for health and disease. Physiological Reviews 95: 1-46.
35. Uwitonze AM, Razzaque MS (2018) Role of magnesium in vitamin D activation and function. Journal of the American Osteopathic Association 118: 181-189.
36. Kong SH, Kim JH, Hong AR, Lee JH, Kim SW, et al. (2017) Dietary potassium intake is beneficial to bone health in a low calcium intake population: The Korean National Health and Nutrition Examination Survey (KNHANES) (2008–2011). Osteoporosis International 28: 1577-1585.
37. Kini U, Nandeesh BN (2012) Physiology of bone formation, remodelling, and metabolism. In: Fogelman I, Gnanasegaran G, Van der Wall H (Eds.)., Radionuclide and hybrid bone imaging. Berlin Springer, p. 29-57.
38. Komnenou A, Karayannopoulou M, Polizopoulou ZS, Constantinidis TC, Dessiris A (2005) Correlation of serum alkaline phosphatase activity with the healing process of long bone fractures in dogs. Veterinary Clinical Pathology 34(1): 35-38.
39. Sousa C, Abreu H, Viegas C, Azevedo J, Reis R, et al. (2011) Serum total and bone alkaline phosphatase and tartrate-resistant acid phosphatase activities for the assessment of bone fracture healing in dogs. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 63: 1007-1111.
40. Potu BK, Rao MS, Nampurath GK, Chamallamudi MR, Nayak SR, et al. (2010) Anti-osteoporotic activity of the petroleum ether extract of Cissus quadrangularis in ovariectomized Wistar rats. Chang Gung Medical Journal 33(3): 252-257.
41. Carvellati C, Bonaccorsi G, Cremonini E, Bergamini CM, Patella A, et al. (2013) Bone mass density selectivity correlates with serum markers of oxidative damage in post-menopausal women. Clinical Chemistry and Laboratory Medicine 51(2): 333-338.
42. Cervellati C, Bonaccorsi G, Cremonini E, Romani A, Fila E, et al. (2014) Oxidative stress and bone resorption interplay as a possible trigger for postmenopausal osteoporosis. BioMed Research Internatioanl 2014: 569563.
43. Maggio D, Barabani M, Pierandrei M, Polidori MC, Catani M, et al. (2003) Marked decrease in plasma antioxidants in aged osteoporotic women. Results of a cross-sectional study. Journal of Clinical Endocrinology & Metabolism 88: 1523-1527.
44. Muthusami S, Ramachandran I, Muthusamy B, Vasudevan G, Prabhu V, et al. (2005) Ovariectomy induces oxidative stress and impairs bone antioxidant system in adult rats. Clinica Chimica Acta 360: 81-86.
45. Cetinus E, Kilinç M, Uzel M, Inanç F, Kututaş EB, et al. (2005) Does long-term ischemia affect the antioxidant status during fracture healing. Archives of Orthopaedic and Trauma Surgery 125(6): 376-380.
46. Sheweita SA, Koshhal K (2007) Calcium metabolism and oxidative stress in bone fracture: role of antioxidants. Current Drug Metabolism 8: 519-525.
47. Sandukji A, Al-Sawaf H, Mohamadin A, Alrashidi Y, Sheweita SA (2011) Oxidative stress and bone markers in plasma of patients with long-bone fixative surgery: role of antioxidants. Human & Expiental Toxicology 30(6): 435-442.