Background
A bone fracture is a break in the continuity of the bone [
1]. Bone fractures can either be of traumatic origin (motor accidents or falls) or of pathological origin (mainly osteoporosis) in most cases, as a result of bone fragility [
2].
A Bone as an organ has the ability to regenerate naturally when a fracture occurs [
3]. Generally in modern medicine, treatment of bone fractures involve the use of a cast or plaster to immobilise the fractured limb, followed by the administration of analgesics (pain killers) and anti-inflammatory drugs to patients. The broken bone is then allowed to regenerate naturally on its own [
3], since there is no orally active pharmacological agent available for rapid fracture repair [
4]. Even though fractures heal naturally, it would be of great interest to discover alternatives to accelerate a bone healing process, thereby reducing the healing period, reducing the cost of treatment and helping patients to quickly regain their good health.
Peperomia pellucida (L.)HBK, belongs to the family Piperaceae. It is a herbaceous plant found mainly in America, Africa and Asia. The species develops during rainy periods and thrives in damp, humid soils and under the shade of trees [
5].
Traditional uses include treatment of abdominal pain, abscesses, acne, boils, colic, fatigue, gout and rheumatic joint pain [
6]. Literature data confirms the hypoglycemic, anti-inflammatory and analgesic effects of
P. pellucida [
7] and equally its antidiabetic [
8] properties. Several animal models are used to show bone regeneration among which is drill-hole injury model [
4,
9,
10]. We recently demonstrated that, the ethanolic extract of
P. pellucida promotes fracture healing in rat by anabolic effects on osteoblasts using drill-hole injury model [
10]. In addition, this plant contains calcium [
11] and metabolites as flavonoids [
12] which are known respectively to contribute in matrix deposition during osteogenesis [
13] and promote bone formation [
12,
14]. The aqueous extract is commonly used in Cameroonian traditional medicine for fracture management. Therefore, we investigated the effects of the aqueous whole plant extract of
P. pellucida on fracture healing in female rats.
Discussion
Peperomia pellucida is a herb used in Cameroonian traditional medicine to treat fracture. However, no systematic study is available to confirm the efficacy of the aqueous extract of this plant in accelerating fracture healing. Till date, there is no orally available agent/compound used to treat fractures. In the present study, we firstly evaluated the mineral content of the aqueous whole plant extract of this plant. Minerals and most especially calcium and phosphorus (hydroxyapatite) build up the inorganic content of bones [
18]. In this study, results of the analyses of the mineral content of the aqueous extract of
P. pellucida reveal the presence of some minerals like potassium, phosphorus, magnesium, calcium and sodium. These results are in accordance with those of Ooi
et al. [
11] who found out that
P. pellucida harvested in Malaysia, contained these similar minerals. The presence of these minerals in
P. pellucida therefore makes it an appropriate dietary source of bone minerals making us to suggest that the administration of this plant extract to both unfractured and fractured rats could help to build up strong bones by providing them with essential minerals important in mineralization of osteoid, during bone formation [
13].
The results of body weight of animals during the experimental period reveal that, the administration of the plant extract at the dose of 400 mg/kg induces an increase in body weight of fractured and unfractured rats. These results suggest that,
P. pellucida aqueous extract could enhance appetite at higher doses. The red marrow in the femur is the principal site of hematopoiesis in the body [
19]. Thus, an injury to this organ may affect the haematopoietic process. Bone haematology revealed that the RBCs and platelets counts remained within the normal range after the 14 days period of treatment. This indicates that the plant extract did not have any relevant effect on these blood cells production, and suggesting maintenance of the bone marrow integrity. However, the administration of the plant extract at the doses of 100 and 200 mg/kg led to a significant increase in WBCs levels suggesting an inflammatory response [
19] of the extract. To confirm this suggestion, further studies need to be performed. On the other hand, it is known that thrombocytes promote bone regeneration and their activation and function can be measured as mean platelet volume (MPV) [
20]. The plant extract at high dose tripled the MPV in fractured and unfractured rats. This result suggests the ability of the plant extract to promote bone regeneration by accelerating migration and proliferation of osteogenic cell in fractured rat. However in unfractured rat the increase in MPV confirms the capability of the plant extract to act on thrombocyte activation and function which could be beneficial when fracture occurs. On the contrary some authors have shown that, the increase in MPV could be related to problems in the union of fracture in human [
21].
P. pellucida plant has been shown to possess osteogenic compounds like flavonoids and phytoestrogens [
2], which have the ability to stimulate the recruitment of osteoblasts at the injury site [
9] and to increase the amount and activities of osteoblasts [
22] respectively. The increased activity of osteoblasts leads to the rapid deposition of osteoid or soft callus (which fills the fracture gap) which is later on mineralised, thereby adding weight to bone. Calcium level is an important marker of bone healing. The creation of the fracture resulted in a reduced serum calcium levels and a high bone calcium level. These results could indicate a mobilisation of calcium ions from serum to bone. It has been shown that, calcitonin is a hormone secreted as a negative feedback mechanism in response to an increase in plasma calcium concentration [
23], an increase which could be due to the high calcium content of the plant extract. The results of mineral composition of the extract confirm this assertion. In bones, the results show that the plant extract at the doses of 100 and 200 mg/kg favoured calcium mineralisation. This result is in consonance with a previous report [
10] which showed that
P. pellucida ethanolic extract dose-dependently increased mineral deposition. This could be attributed not only to the presence of calcium in the extract but equally to the presence of phytoestrogens and flavonoids in the extract [
2] which stimulate osteoblastic activities. These active osteoblasts in turn secrete high amounts of alkaline phosphatase and osteocalcin which are important in bone mineralisation [
19] hence, calcium deposition. Conversely to the doses of 100 and 200 mg/kg, the plant extract at the dose of 400 mg/kg in both fractured and normal rats, induced a non-significant decrease in levels of calcium when compared to the normal control. This may suggest bone healing during which, osteoblasts activities and secretions return to their normal levels [
24]. This view correlates with the reduction in AP activities and complete closure of fracture gap (X-rays examination) at this dose as observed in these groups.
Phosphorus is an essential bone-forming element because it is required for the appropriate mineralisation of the skeleton [
25]. Evaluation of phosphorus levels in animals revealed that, bone phosphorus concentrations are higher than serum levels. This could be due to an accumulation of phosphorus in bones which is a reservoir for this mineral [
18]. In bones, the extract induced an increase in the deposition of this mineral at the doses of 100 and 200 mg/kg. This could possibly be as a result of the high phosphorus content of our extract. In fact, during a bone healing process, calcium and phosphorus crystals derived from nutritional sources are deposited in bones in the form of hydroxyapatite [
13] in the phase of bone mineralisation. This is in accordance with the results of calcium levels which have also increased in these groups. The increase mineralisation could equally be attributed to the presence of copper [
11] in the extract which enhances bone formation and skeletal mineralization [
25]. In the groups receiving the plant extract at the dose of 400 mg/kg, there was a decrease in the bone mineral content to baseline values which could be due to the reduction in the activity of osteoblasts after fracture healing [
24], leading to a decrease in mineralisation an hence in phosphorus deposition. This goes in line with the results of alkaline phosphatase activity and calcium content which equally reduced after administration of the plant extract at the dose of 400 mg/kg during 14 days.
The detection of specific serum biomarkers of bone formation, such as alkaline phosphatase can be clinically useful in evaluating the progress of a healing process. Alkaline phosphatase is secreted by osteoblasts during the maturation phase of bone formation [
13]. Fracture induction provokes a decrease in bone alkaline phosphatase as observed in this study. When the plant extract was administered, there was a non-significant increase in bone alkaline phosphatase activity. This result suggests that the aqueous extract of
P. pellucida could accelerate the maturation phase of osteoblasts. In addition, the presence of zinc in this extract [
11] could enhance osteoblastic activity and alkaline phosphatase activity [
25]. It is important to note that, at the dose of 400 mg/kg in fractured rats, the increase in alkaline phosphatase activity was lower than that observed at lower doses. This could indicate bone healing during which the osteoblastic activities and secretions decrease to reach the normal value. Some studies confirm a decrease in bone specific alkaline phosphatase to baseline values during natural normal fracture healing [
24,
26]. X-rays examination helps to assess a bone healing process [
27]. The results obtained after x-rays examination at the site of the fracture reveal that, 14 days after the fracture, the callus formed is already observable. In the fracture control group, the diameter of the hole has slightly reduced. This indicates that the natural process of bone healing has occurred [
3]. The administration of the extract accelerates a fracture healing process which is dose dependent. There is a decrease in the diameters of the fracture gaps in the treated groups with complete closure in the rats treated with the highest dose (400 mg/kg). These results suggest the powerful effect of the plant extract to fill up the gap. This was made evident with the histology realised at the fracture sites. In fact after 14 days of fracture induction, animals presented a loose bone structure which confirms once again a natural bone regeneration. When administered, the plant extract led to more packed and dense cells suggesting an acceleration of a bone repair process. Previous works done by Ngueguim
et al. [
10] showed that ethanol extract of
P. pellucida significantly increases the expression of BMP-2 genes. Thus, the rapid healing process observed, could be due to an increase in the expression of BMPs which are potent inducers of osteogenesis leading to fracture healing [
13].
Acknowledgments
Authors are grateful for technical assistance received from Mrs. Catherine CANNET through the “Association pathologie cytologie developpement” program, Novartis, Bâle. We would like to thank the International Institute for Tropical Agriculture (IITA), Yaounde (Cameroon) and the “Centre d’Imagerie médicale de Yaoundé” for their technical help respectively for mineral content of the plant extract and bone radiography analysis.