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Ethanol extract of Peperomia pellucida (Piperaceae) promotes fracture healing by an anabolic effect on osteoblasts
Journal of Ethnopharmacology 148 (2013) 62–68
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethanol extract of Peperomia pellucida (Piperaceae) promotes fracture healing by an anabolic effect on osteoblasts Florence Tsofack Ngueguim a,n, Mohd Parvez Khan b, Jean Hubert Donfack c, Deepshikha Tewari b, Theophile Dimo a, Pierre Kamtchouing a, Rakesh Maurya d, Naibedya Chattopadhyay b,nn a
Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon Division of Endocrinology and Center for Research on Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226021, India c Department of Biomedical Sciences, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon d Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226021, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 November 2012 Received in revised form 21 March 2013 Accepted 22 March 2013 Available online 8 April 2013
Ethnopharmacological relevance: The whole plant or some part of Peperomia pellucida (L.) HBK is used in some parts of Cameroon as a treatment for fracture healing. Aim of the study: To evaluate the effect of ethanolic extracts of Peperomia pellucida (L.), a Cameroonian medicinal plant on bone regeneration following bone and marrow injury, and determine the mode of action. Materials and methods: Ethanol extract of Peperomia pellucida was administered at 100 and 200 mg/kg doses orally to adult female Sprague-Dawley rats having a drill hole injury (0.8 mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of calcein labeling at the drill hole site was performed to evaluate bone regeneration. 3-D microarchitecture of drill hole site was analyzed by micorocomputed tomography. Osteogenic effects of the extract were evaluated by assessing mineralized nodule formation of bone marrow stromal cells and expression of osteogenic genes (mRNA level of type-1 collagen, bone morphogenetic protein-2 and osteocalcin genes) in the femur. Results: Ethanol extract from Peperomia Pellucida (L.) dose-dependently induced bone regeneration at the fracture site. At 200 mg/kg dose, the extract significantly increased mineral deposition compared to controls. The extract also improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. In addition, the extract increased the formation of mineralized nodules from the bone marrow stromal cells. Furthermore, the extract induced the expression of osteogenic genes in the femur including type 1 collagen, osteocalcin and BMP-2, compared to control. Conclusion: Ethanolic extract of P. pellucid (L.) accelerates fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying its traditional use. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Peperomia pellicuda (L.) Sprague-Dawley rats Bone regeneration Osteogenic Bone histomorphometry
1. Introduction Bone fracture can occur due to trauma (accident) or pathological reasons such as osteoporosis (Doblaré et al., 2004). When fracture occurs, there is an auto-activated healing involving many local and systemic growth factors and hormones, and extracellular matrix components. This interaction between the local and n
Corresponding author. Tel.: +237 99 64 25 10. Corresponding author. E-mail addresses: [email protected] (F.T. Ngueguim), [email protected] (N. Chattopadhyay). nn
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.03.063
systemic mediators causes mesenchymal stem cells to migrate, proliferate and differentiate at the fracture site. Metabolic bone diseases including primary and secondary osteoporosis are essentially caused by the deficiency in osteoblast function (Dutmanee et al., 2007; Liu et al., 2011). Pharmacological agents to reduce fracture risk in osteoporosis include bisphosphonates, selective estrogen receptor modulators (SERMs) and calcitonin (Gerstenfeld and Einhorn, 2003; Delaney, 2006; Gass and Dawson-Hughes, 2006). Parathyroid hormone (PTH 1-34) remains the only anabolic agent available for clinical use for postmenopausal osteoporosis that has recently been recommended by the US Food and Drug Administration to carry a black-box warning because it is
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associated with an increased risk of osteosarcoma in rats (John et al., 2002). In many African countries, due to the adverse effects of synthetic drugs and their higher cost; many people still use medicinal plants to treat diseases related to bone (Adjanohoun et al., 1996). The effectiveness of a few plant extracts in promoting bone fracture healing has been demonstrated including Ulmus wallichiana (Swarnkar et al., 2011), Elephantopus mollis and Spilanthes africana (Ngueguim et al., 2012). Peperomia pellucida (Linn.) Humb. Bonpl. & Kunth is a plant from a family of Piperaceae. It is a succulent herb of about 30 cm high, ruderal and occurring especially around dwelling areas. In some parts of Cameroon the seeds and the whole plant are used to treat fracture (Adjanohoun et al., 1996; Ngueguim et al., 2012). It is also used in the treatment of various ailments such as abdominal pain, headache and hypertension (Adjanohoun et al., 1996). To the best of our knowledge, fracture healing effect of Peperomia pellucida (L.) has not been scientifically demonstrated. Therefore, we investigated the effects of ethanol extract of Peperomia pellucida on fracture healing in rats using a drill hole injury model of bone and bone marrow. Fracture healing efficacy was investigated using (a) dynamic histomorphometry to determine the rate of new bone formation at the drill hole site, (b) static histomorphometry to assess the microarchitectural characteristics of the newly formed callus and (c) cellular and molecular studies to assess anabolic effect on osteoblasts.
2. Materials and methods 2.1. Reagents and chemicals Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). All fine chemicals were purchase from Sigma-Aldrich (St-Louis, MO). 2.2. Plant material and extraction Fresh plant of Peperomia pellucida (Linn.) was collected from Dschang region (Cameroon) in November 2010 precisely in damp areas. The plant was authenticated by Pr. Zafack Louis, a Botanist (Department of Vegetal Biology and Physiology, University of Yaounde 1). A voucher specimen (19555/SRFCam) was deposited in National Herbarium in Cameroon. The whole plant was cut into small pieces, air-dried, and powdered. The powder of plant material was extracted with ethanol following previously described method (Sharan et al., 2010). The mixture was filtered; the filtrate obtained was concentrated under reduced pressure. The yield (w/w), calculated from the weight of initial powder and the final powder, obtained after evaporation of solvent was 8.12%. 2.3. Animal study Animal studies were conducted in accordance with current legislation on animal experiments (Institutional animal Ethical committee) at CSIR-Central Drug Research Institute. Four-monthold Sprague-Dawley female rats with body weights of 200 720 g were taken for the study. Each group consisted of 6 rats. Control group received acacia gum in distilled water as vehicle; while others groups received individual plant extracts (dissolved with acacia gum in distilled water) at two empirically determined doses −100 and 200 mg/kg.
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et al., 2011).The front skin of the mid-femur in rats was incised straight and longitudinally at 1 cm in length under anesthesia. After splitting the muscle, we stripped the periosteum to expose the femoral bone surface. A drill-hole injury was made by inserting a drill bit with a diameter of 0.8 mm in the anterior portion of the diaphysis of one femur, 2 cm above the knee joint. The treatment began the very next day after creating the injury. Plant extract at the doses of 100 and 200 mg/kg were administered orally for 12 consecutive days. Twenty four hours before autopsy, all animals were given a fluorochrome, calcein (20 mg/kg) dissolved in normal saline via intraperitoneal route. The rats were sacrificed under anesthesia and the injured femur (right) was collected and fixed in 70% isopropanol for microarchitecture analysis. Left (uninjured femur) was used for harvesting bone marrow stromal cell (BMC) cultures. For the measurement of bone microarchitectural parameters in the drill-hole, bones were embedded in an acrylic material and 50 μm sections were made using Isomet Bone cutter. Photographs were taken under confocal microscope (Carl Zeiss LSM 510 Meta) aided with appropriate filters. The calcein binding intensity which is an indication of the amount of callus regeneration in the bony hole was calculated using Carl Ziess AM 4.2 image analysis software. 2.5. Mineralization of BMC For mineralization studies, bone marrow stromal cells (BMCs) from the left femora of treated or untreated groups was flushed out with medium. These cells were seeded in 6-well plates (2 106 cells/well) and cultured in osteoblast differentiation medium (10−7 M dexamethasone, 10 mM β-glycerophosphate and 50 mg/ml ascorbic acid). The medium was changed after every two days. The cultures were continued for 21 days after those cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. The fixed cells were stained with alizarin red-S (40 mM, pH 4.5) for 30 min followed by washing with H2O. Stained cells were first photographed under light microscope and alizarin stain was then extracted by 10% (v/v) acetic acid with shaking at room temperature for 30 min. Cells were scrapped out of the culture plates, centrifuged (20,000g for 15 min) and supernatant was collected. To the supernatant, 10% (v/v) ammonium hydroxide was added to bring the pH of the solution to 4.5. Absorbance of the solution was read at 405 nm as described before (Trivedi et al., 2008; Siddiqui et al., 2010; Dixit et al., 2012). 2.6. Micro-computed tomography (mCT) Assessment of internal microstructure (both 2-D and 3-D) of the mineralized tissue in the bony hole was analyzed by mCT, using Sky Scan 1076 CT scanner (Aartselaar, Belgium). Bones were cleaned of soft tissues and scanned using a X-ray source of 70 KV, 100 mA with a pixel size of 18 mm. The images were reconstructed using Sky Scan Nrecon software, which facilitates network-distributed reconstruction carried out on four personal computers running simultaneously. Callus bone was captured by drawing ellipsoid contour with CT analyzer software. Microarchitectural parameters including, bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) trabecular number (Tb.N) and structure model index (SMI) were quantified as described before (Uchida et al., 2000; Sharan et al., 2011; Ngueguim et al., 2012). 2.7. Quantitative real-time polymerase chain reaction (qPCR)
2.4. Drill-hole injury in femur A drill-hole injury was created in both vehicle and extract treated groups as described before (Tanaka et al., 2010; Sharan
Total RNA was isolated from left femur after flushing with medium. The femur of each animal was cleaned properly of any remaining soft tissue, then crushed with TRIzol (Invitrogen)
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according to the procedure describe by the manufacturer. The concentration and the purity of RNA were determined by measuring the absorbance at 260/280 nm using a spectrophotometer (NANO-Drop). cDNA was synthesized with revertAid kit (Fermentas, Austin, USA) from 2 μg of total RNA using a commercial first-strand cDNA synthesis kit. SYBR green chemistry was used to perform quantitative determination of, collagen type-1 (Col1), bone morphogenetic protein (BMP-2), osteocalcin and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript level following an optimized protocol (Sharan et al., 2011). Primer sequence of the gene of interest: col1, BMP-2 osteocalcin and GAPDH are shown in Table 1. 2.8. Statistical analysis Data are expressed as mean 7S.E.M. The data obtained from each treatment were subjected to one-way ANOVA followed by Table 1 Primer sequence of various genes used for qPCR. Gene name
Primer sequence
Accession number
Collagen1
F-CATGTTCAGCTTGTGGACCT R-CGAGCTGACTTCAGGGATGT
NM-053304
BMP-2
F-CGGACTGCGGTCTCCTAA R-GGGGAAGCAGCAACACTAGA
NM-007553.2
Osteocalcin
F-ATAGACTCCGGCGCTACCTC R-CCAGGGGATCTGGGTAGG
NM-013414
GAPDH
F-CAGCAAGGATACTGAGAGCAAGAG R-GGATGGAATTGTGAGGGAGATG
NM-017008
Newman–Keuls test of significance using Prism version 3.0 software. One way ANOVA was applied to test the variation between the groups and Newman–Keuls as post test to make the multiple comparisons between the doses of the extracts. Qualitative observations have been represented following assessments made by three individuals blinded to the experimental designs.
3. Results 3.1. Effect of the ethanol extract of Peperomia pellucida on bone regeneration Quantification of bone regeneration by calcein label (mineral deposition) at the site of drill hole injury was used to evaluate the effect of ethanol extract of Peperomia pellucida (Fig. 1). The extract administered for 2 weeks induced a significantly higher mineral deposition over the control (rats receiving vehicle). Fig. 1A showed representative confocal microscopy images of calcein deposition at the fracture site in the various experimental groups. The increase was 9.45% (p o0.05) at 100 mg/kg dose and 84.85% (p o0.01) at 200 mg/kg dose, compared to the control (Fig. 1B). The extract treatment exhibited dose-dependent increase in mineral deposition over the control. 3.2. Effect of the extract on microarchitecture of regenerated bone at the drill-hole site After 2 weeks administration of the extracts, mCT analysis at the drill hole site of the various groups was performed (representative images in Fig. 2A). Quantification of various mCT parameters
Fig. 1. Ethanol extract from Peperomia pellucida accelerates bone regeneration in drill-hole. (A) Representative confocal images (100 ) of calcein labeling in the callus of drill-hole of various groups after 12 days of treatments. (B) Quantification of the mean intensity of calcein label per pixel. All values are expressed as mean 7SEM (n ¼6); *p o0.05;**p o0.01.
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Fig. 2. Peperomia pellucida extract improves microarchitectural parameters in the drill-hole. (A) Representative mCT images from the center of the bony hole at different doses. (B) mCT analysis showing BV/TV, bone volume/tissue volume (%); Tb.N, strut number (1/mm); Tb.Th, strut thickness (mm); SMI, structure model index; Tb.Sp, strut spacing (mm). All values are expressed as mean7 SEM (n¼ 6 rats/group); npo 0.05; nnp o 0.01.
showed that, both doses of the extract improved the bone microarchitecture. Compared to the control; the extract at both doses increased bone volume (BV/TV, p o0.05), trabecular thickness (Tb.Th, p o0.01), trabecular number (Tb.N, p o0.05), and decreased trabecular separation (Tb.Sp, P o0.05) and structure model index (SMI, Po 0.05) (Fig. 2B).
levels of all three genes over the control. At 200 mg/kg dose, Peperomia pellucida extract had a robust induction of 25–30 folds on the mRNA levels of col1 and osteocalcin over the control. BMP-2 levels were significantly higher in the rats treated with 200 mg/kg dose of Peperomia pellucida extract over the control (po0.001).
3.3. Effect of the extract on mineralized nodule formation in BMC
4. Discussion
At the end of various treatments, BMC from femur of vehicle or extract treated rats were cultured to induce mineralized nodules formation. Fig. 3A represents the photomicrograph of alizarin red-S stained cells showing the formation of mineralized nodules. The quantification done by the percent change of alizarin red-S stained cells over control showed that Peperomia pellucida extract treatment exhibited dose-dependent increase in mineralized nodule formation of BMC over control (Fig. 3B). The percentage change of alizarin red-S stained cells was approximately 24% and 50% at the doses of 100 mg/kg and 200 mg/kg respectively compared to the control.
Peperomia pellucida is an herb used in Cameroonian traditional medicine to treat fracture (Adjanohoun et al., 1996). However, no systematic study is available to confirm the efficacy of this plant in accelerating healing of fracture. At present, there is no orally efficacious agent/compound available to treat fracture. In the present study, we firstly used dynamic histomorphometry to assess whether the plant extract could induce bone regeneration at the fracture site, then employed static histomorphometry (bone microarchitecture using μCT) to assess the quality of the callus that were being formed (Tanaka et al., 2010; Sharan et al., 2011; Ngueguim et al., 2012) and lastly assessed its osteogenic effect in vitro and ex vivo. The results showed that, the Peperomia pellucida extract dose-dependently increased mineral deposition with improved quality of callus formation at the fracture site, which was due likely to its stimulatory effect on osteoblastic cells. Paralleling in vivo bone formation, at the cellular level, the extract treatment stimulated new bone formation by increased differentiation of bone marrow osteoprogenitor cells in ex vivo
3.4. Effect of the extract on expression of osteogenic genes in femur We investigated the effect the extract on osteogenic genes that are expressed during bone formation including, type1 collagen (col1), bone morphogenetic protein-2 (BMP-2) and osteocalcin. As shown in Fig. 4, the results revealed that, daily administration of Peperomia pellucida extract exhibited a dose dependant increase in the mRNA
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Fig. 3. Peperomia pellucida extract stimulated the production of osteoprogenitor cells in bone marrow stromal cells. (A) Representative photomicrographs of mineralized nodules (MN) of all doses show increased intensity and larger-sized nodules by Peperomia pellucida treatment when compared with control. (B) Quantification of mineralization was done by extraction of alizarin red-S dye as described in Section 2. All values are expressed as mean 7 SEM (n¼ 6); *po 0.05, **po 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cultures as well as by increased expression of osteogenic genes of bones. BMP-2 is known to augment fracture healing in several animal models and clinical studies. Human recombinant BMP-2 is clinically used for open tibial and spinal surgeries for accelerating fracture by applying it directly to the site as this cytokine has no oral bioavailability (Govender et al., 2002). Because Peperomia pellucida extract significantly increased mRNA levels of BMP-2 in the femur along with other osteogenic genes, it appeared that the mechanism of accelerating fracture healing by the extract involved endogenous BMP-2 production. Together, these results demonstrate the ability of the extract to stimulate the recruitment of osteoblast at the injury side and to maintain the quality and the integrity of the bone. μCT measurements showed increased BV/TV, Tb.Th, Tb.N and decreased Tb.Sp of the callus by Peperomia pellucida treatment resulting in a more compact assembly of the newly formed bone over the control. SMI represents the relative amounts of rods and plates in a 3D structure, with high values indicating more rods over plates, and greater accumulation of micro-damage. Peperomia pellucida resulted in preferred plate-like structure (low SMI) of the newly formed bone suggesting superior surface geometry and curvature distribution in the newly formed callus compared to control. In folk medicine, Peperomia pellucida is used in the form of macerate or decoction (Adjanohoun et al., 1996). It is administered by oral route or as a paste at the fracture site after scarification. Extraction by ethanol used in this study and water used in folk medicine are both polar solvents. Thus the putative osteogenic compounds responsible for the fracture healing appear to be polar. Phytochemistry of the whole plant of Peperomia pellucida show the presence of alkaloids, saponins, tannins, flavanoids, steroids and glycosides (Pulak et al., 2011). Various flavonoids have osteogenic effect in vivo (Sharan et al., 2009). Notably, a few compounds
isolated from the whole plant have exhibited estrogen-‘like’ effect in CV-1 cells transfected with human estrogen receptor (Xu et al., 2006). It is plausible that flavonoids and phytoestrogens present in the plant extract contribute to its osteoblast stimulating effect and consequent rapid fracture healing effect in vivo. Further studies are required to demonstrate this hypothesis. Effective therapeutic dose for various herbal extracts in rodents have a wide range and often 1.0 g/kg dose is used (Shirke et al., 2008, Haihua et al., 2010). At 100 mg/kg dose of Peperomia pellucida extract, several osteogenic parameters were increased over the control. Maximum mineral deposition and osteogenic stimulation was observed with 200 mg/kg dose of Peperomia pellucida extract. A dose of 200 mg/kg in rats is translated to 32.4 mg/kg in humans by following the equation: human equivalent dose (mg/kg)¼rat dose (mg/kg) [200] X rat Km factor, [6/human Km factor, that is, 37] (Reagan-Shaw et al., 2007). For a 60-kg person, the daily requirement of the extract for fracture repair is calculated at 1.9 g/day, which is favorable for adherence to treatment. Collectively, our findings suggest that the Peperomia pellucida extract accelerates fracture healing and improves callus quality by increasing the production of osteoprogenitor cells and their differentiation to osteogenic lineage thereby enhancing the recruitment of osteoblast to the injury site as well as stimulating the ability of the cells to produce osteogenic cytokine, BMP-2 and deposition of matrix protein, col1, ultimately accelerate fracture healing.
5. Conclusion Our study in a preclinical set up clearly demonstrated that daily oral administration of an ethanol extract from Peperomia pellucida at a favorable dose of 200 mg/kg accelerates the healing of bone
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Fig. 4. qPCR determination of mRNA levels of osteogenic genes, col1, BMP-2 and osteocalcin in femora of various groups. Each assay was performed in triplicate and results are represented as mean 7 S.E.M. of percentage/fold change; nPo 0.05; nnnPo 0.001.
and marrow injury. This effect seems to be due to the osteogenic effect of the extract thereby promoting recruitment and differentiation of osteoblast at the fracture site. Our study justifies the traditional use of the plant in fracture healing and could therefore be considered as an alternative therapy for fracture repair. Future studies will determine the bio-active marker and its mechanism of action of the plant extract.
Acknowledgments The authors thank Federation of Indian Chamber and Commerce Industry (FICCI) through CV Raman fellowship awarded to Dr. T.F. Ngueguim. The authors also sincerely thank Mama Awatsa Marceline, Nankeng Helene and other traditional healers who agreed to disclose the plants they use for fracture healing. We also thank Pr. Louis Zapfack for the authentication of the plant. Authors acknowledge generous grant support from CSIR (ASTHI program).
References Adjanohoun, J.E., Aboukakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu, K.J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabu, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional medicine and pharmacopoeia. Contribution to Ethnobotanical and Floristic Studies in Cameroon. Centre de Production de Manuels Scolaires, Porto-Novo (Rep. Du Benin), p. 641.
Delaney, M.F., 2006. Strategies for the prevention and treatment of osteoporosis during early postmenopause. American Journal of Obstetrics Gynecology 194, S12–S23. Dixit, P., Chand, K., Khan, M.P., Siddiqui, J.A., Tewari, D., Ngueguim, F.T., Chattopadhyay, N., Maurya, R., 2012. Phytoceramides and acylated phytosterol glucosides from Pterospermum acerifolium Willd. seed coat and their osteogenic activity. Phytochemistry 81, 117–125. Doblaré, M., Garcia, J.M., Gomez, M.J., 2004. Modelling bone tissue fracture and healing: a review. Engineering Fracture Mechanics. 71, 1809–1840. Dutmanee, S., Kanogwun, T., Narattaphol, C., Jantarima, P., Kukiat, T., Jarinthorn, T., Tuangporn, S., Nateetip, K., 2007. Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor κB ligand/ osteoprotegerin ratio. Bone 42, 535–546. Gass, M., Dawson-Hughes, B., 2006. Preventing osteoporosis-related fractures: an overview. The American Journal of Medicine 119, 3S–11S. Gerstenfeld, L.C., Einhorn, T.A., 2003. Developmental aspects of fracture healing and the use of pharmacological agents to alter healing. Journal of Musculoskeletal and Neuronal Interactions 3, 297–303. Govender, S., Csimma, C., Genant, H.K., Valentin-Opran, A., Amit, Y., Arbel, R., Aro, H., Atar, D., Bishay, M., Börner, M.G., Chiron, P., Choong, P., Cinats, J., Courtenay, B., Feibel, R., Geulette, B., Gravel, C., Haas, N., Raschke, M., Hammacher, E., van der Velde, D., Hardy, P., Holt, M., Josten, C., Ketterl, R.L., Lindeque, B., Lob, G., Mathevon, H., McCoy, G., Marsh, D., Miller, R., Munting, E., Oevre, S., Nordsletten, L., Patel, A., Pohl, A., Rennie, W., Reynders, P., Rommens, P.M., Rondia, J., Rossouw, W.C., Daneel, P.J., Ruff, S., Ruter, A., Santavirta, S., Schildhauer, T.A., Gekle, C., Schnettler, R., Segal, D., Seiler, H., Snowdowne, R.B., Stapert, J., Taglang, G., Verdonk, R., Vogels, L., Weckbach, A., Wentzensen, A., Wisniewski, T., 2002. BMP-2 Evaluation in surgery for Tibial Trauma (BESTT) Study Group Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. Journal of Bone Joint Surgery 84A, 2123–2134. Haihua, S., Hideko, A., Masakazu, H., Liang, Z., Ke, A., Wenqi, H., Kazuo, H., 2010. Anti-hypersensitivity effects of Shu-jing-huo-xue-tang, a Chinese herbal medicine, in CCI-neuropathic rats. Journal of Ethnopharmacology 131, 464–470. John, L., Vahle, M.S., Gerald, G.L., Jamie, K.Y., Paul, C.F., Jeffery, A.E., Michael, S.W., Yanfei, L.M., James, B.N., 2002. Skeletal changes in rats given daily subcutaneous
68
F.T. Ngueguim et al. / Journal of Ethnopharmacology 148 (2013) 62–68
injections of recombinant human parathyroid hormone (1–34) for 2 years and relevance to human safety. Toxicologic Pathology 30, 312–321. Liu, M.J., Li, Y., Pan, J.H., Liu, H., Wang, S.J., Teng, J.R., Zhao, H.Y., Ju, D.H., 2011. Effects of zuogui pill (see text) on Wnt singal transduction in rats with glucocorticoidinduced osteoporosis. Journal of Traditional Chinese Medecine 31, 98–102. Ngueguim, F.T., Khan, M.P., Donfack, J.H., Siddiqui, J.A., Tewari, D., Nagar, G.K., Tiwari, S.C., Dimo, T., Maurya, R., Chattopadhyay, N., 2012. Evaluation of Cameroonian plants towards experimental bone regeneration. Journal of Ethnopharmacology 141, 331–337. Pulak, M., Priya, A., Satya, V., 2011. Ethno-medicinal, phytochemical and pharmacological review of an amazing medicinal herb Peperomia pellucida (L.) HBK. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2, 258–264. Reagan-Shaw, S., Nihal, M., Ahmad, N., 2007. Dose translation from animal to human studies revisited. Journal of the American Societies for Experimental Biology 22, 659–661. Sharan, K., Siddiqui, J.A., Swarnkar, G., Maurya, R., Chattopadhyay, N., 2009. Role of phytochemicals in the prevention of menopausal bone loss: evidence from in vitro and in vivo, human interventional and pharma-cokinetic studies. Current Medical Chemistry 16, 1138–1157. Sharan, K., Siddiqui, J.A., Swarnkar, G., Tyagi, A.M., Kumar, A., Rawat, P., Kumar, M., Nagar, G.K., Arya, K.R., Manickavasagam, L., Jain, G.K., Maurya, R., Chattopadhyay, N., 2010. Extract and fraction from Ulmus wallichiana Planchon promote peak bone achievement and have a nonestrogenic osteoprotective effect. Menopause 17, 393–402. Sharan, K., Sharan, J.M., Swarnkar, G., Siddiqui, J.A., Khan, K., Kumari, R., Rawat, P., Maurya, R., Sanyal, S., Chattopadhyay, N., 2011. A novel quercetin analogue from a medicinal plant promotes peak bone mass achievement and bone healing
after injury and exerts an anabolic effect on osteoporotic bone: the role of aryl hydrocarbon receptor as a mediator of osteogenic action. Journal of Bone and Mineral Research 26, 1–16. Shirke, S.S., Jadhav, S.R., Jagtap, A.G., 2008. Methanolic extract of Cuminum cyminum inhibits ovariectomy-induced bone loss in rats. Experimental Biology and Medicine. 233, 1403–1410. Siddiqui, J.A., Swarnkar, G., Sharan, K., Chakravarti, B., Sharma, G., Rawat, P., Kumar, M., Khan, F.M., Pierroz, D., Maurya, R., Chattopadhyay, N., 2010. 8″-Biapigeninyl osteoprotective action of 8,8-biapigeninyl in ovariectomized mice. Molecular and Cellular Endocrinology 323, 256–267. Swarnkar, G., Sharan, K., Siddiqui, J.A., Chakravarti, B., Rawat, P., Kumar, M., Kamal, R.A., Maurya, R., Chattopadhyay, N., 2011. A novel flavonoid isolated from the steambark of Ulmus wallichiana Planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation. European Journal of Pharmacology 658, 65–73. Tanaka, K., Tanaka, S., Sakai, A., Ninomiya, T., Arai, Y., Nakamura, T., 2010. Deficiency of vitamin A delays bone healing process in association with reduced BMP-2 expression after drill-hole injury in mice. Bone 47, 1006–1012. Trivedi, R., Kumar, S., Kumar, A., Siddiqui, J.A., Swarnkar, G., Gupta, V., Kendurker, A., Dwivedi, A.K., Romero, J.R., Chattopadhyay, N., 2008. Kaempferol has osteogenic effect in ovariectomized adult Sprague-Dawley rats. Molecular and Cellular Endocrinology 289, 85–93. Uchida, S., Doi, Y., Kudo, H., Furukawa, H., Nakamura, T., Fujimoto, S., 2000. Transient expression of activin betaA mRNA on osteoprogenitor cells in rat bone regeneration after drill-hole injury. Bone 27, 81–90. Xu, S., Li, N., Ning, M.M., Zhou, C.H., Yang, Q.R., Wang, M.W., 2006. Bioactive compounds from Peperomia pellucida. Journal of Natural Product 69, 247–250.
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Ethanol extract of Peperomia pellucida (Piperaceae) promotes fracture healing by an anabolic effect on osteoblasts Florence Tsofack Ngueguim a,n, Mohd Parvez Khan b, Jean Hubert Donfack c, Deepshikha Tewari b, Theophile Dimo a, Pierre Kamtchouing a, Rakesh Maurya d, Naibedya Chattopadhyay b,nn a
Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon Division of Endocrinology and Center for Research on Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226021, India c Department of Biomedical Sciences, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon d Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226021, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 November 2012 Received in revised form 21 March 2013 Accepted 22 March 2013 Available online 8 April 2013
Ethnopharmacological relevance: The whole plant or some part of Peperomia pellucida (L.) HBK is used in some parts of Cameroon as a treatment for fracture healing. Aim of the study: To evaluate the effect of ethanolic extracts of Peperomia pellucida (L.), a Cameroonian medicinal plant on bone regeneration following bone and marrow injury, and determine the mode of action. Materials and methods: Ethanol extract of Peperomia pellucida was administered at 100 and 200 mg/kg doses orally to adult female Sprague-Dawley rats having a drill hole injury (0.8 mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of calcein labeling at the drill hole site was performed to evaluate bone regeneration. 3-D microarchitecture of drill hole site was analyzed by micorocomputed tomography. Osteogenic effects of the extract were evaluated by assessing mineralized nodule formation of bone marrow stromal cells and expression of osteogenic genes (mRNA level of type-1 collagen, bone morphogenetic protein-2 and osteocalcin genes) in the femur. Results: Ethanol extract from Peperomia Pellucida (L.) dose-dependently induced bone regeneration at the fracture site. At 200 mg/kg dose, the extract significantly increased mineral deposition compared to controls. The extract also improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. In addition, the extract increased the formation of mineralized nodules from the bone marrow stromal cells. Furthermore, the extract induced the expression of osteogenic genes in the femur including type 1 collagen, osteocalcin and BMP-2, compared to control. Conclusion: Ethanolic extract of P. pellucid (L.) accelerates fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying its traditional use. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Peperomia pellicuda (L.) Sprague-Dawley rats Bone regeneration Osteogenic Bone histomorphometry
1. Introduction Bone fracture can occur due to trauma (accident) or pathological reasons such as osteoporosis (Doblaré et al., 2004). When fracture occurs, there is an auto-activated healing involving many local and systemic growth factors and hormones, and extracellular matrix components. This interaction between the local and n
Corresponding author. Tel.: +237 99 64 25 10. Corresponding author. E-mail addresses: [email protected] (F.T. Ngueguim), [email protected] (N. Chattopadhyay). nn
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.03.063
systemic mediators causes mesenchymal stem cells to migrate, proliferate and differentiate at the fracture site. Metabolic bone diseases including primary and secondary osteoporosis are essentially caused by the deficiency in osteoblast function (Dutmanee et al., 2007; Liu et al., 2011). Pharmacological agents to reduce fracture risk in osteoporosis include bisphosphonates, selective estrogen receptor modulators (SERMs) and calcitonin (Gerstenfeld and Einhorn, 2003; Delaney, 2006; Gass and Dawson-Hughes, 2006). Parathyroid hormone (PTH 1-34) remains the only anabolic agent available for clinical use for postmenopausal osteoporosis that has recently been recommended by the US Food and Drug Administration to carry a black-box warning because it is
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associated with an increased risk of osteosarcoma in rats (John et al., 2002). In many African countries, due to the adverse effects of synthetic drugs and their higher cost; many people still use medicinal plants to treat diseases related to bone (Adjanohoun et al., 1996). The effectiveness of a few plant extracts in promoting bone fracture healing has been demonstrated including Ulmus wallichiana (Swarnkar et al., 2011), Elephantopus mollis and Spilanthes africana (Ngueguim et al., 2012). Peperomia pellucida (Linn.) Humb. Bonpl. & Kunth is a plant from a family of Piperaceae. It is a succulent herb of about 30 cm high, ruderal and occurring especially around dwelling areas. In some parts of Cameroon the seeds and the whole plant are used to treat fracture (Adjanohoun et al., 1996; Ngueguim et al., 2012). It is also used in the treatment of various ailments such as abdominal pain, headache and hypertension (Adjanohoun et al., 1996). To the best of our knowledge, fracture healing effect of Peperomia pellucida (L.) has not been scientifically demonstrated. Therefore, we investigated the effects of ethanol extract of Peperomia pellucida on fracture healing in rats using a drill hole injury model of bone and bone marrow. Fracture healing efficacy was investigated using (a) dynamic histomorphometry to determine the rate of new bone formation at the drill hole site, (b) static histomorphometry to assess the microarchitectural characteristics of the newly formed callus and (c) cellular and molecular studies to assess anabolic effect on osteoblasts.
2. Materials and methods 2.1. Reagents and chemicals Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). All fine chemicals were purchase from Sigma-Aldrich (St-Louis, MO). 2.2. Plant material and extraction Fresh plant of Peperomia pellucida (Linn.) was collected from Dschang region (Cameroon) in November 2010 precisely in damp areas. The plant was authenticated by Pr. Zafack Louis, a Botanist (Department of Vegetal Biology and Physiology, University of Yaounde 1). A voucher specimen (19555/SRFCam) was deposited in National Herbarium in Cameroon. The whole plant was cut into small pieces, air-dried, and powdered. The powder of plant material was extracted with ethanol following previously described method (Sharan et al., 2010). The mixture was filtered; the filtrate obtained was concentrated under reduced pressure. The yield (w/w), calculated from the weight of initial powder and the final powder, obtained after evaporation of solvent was 8.12%. 2.3. Animal study Animal studies were conducted in accordance with current legislation on animal experiments (Institutional animal Ethical committee) at CSIR-Central Drug Research Institute. Four-monthold Sprague-Dawley female rats with body weights of 200 720 g were taken for the study. Each group consisted of 6 rats. Control group received acacia gum in distilled water as vehicle; while others groups received individual plant extracts (dissolved with acacia gum in distilled water) at two empirically determined doses −100 and 200 mg/kg.
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et al., 2011).The front skin of the mid-femur in rats was incised straight and longitudinally at 1 cm in length under anesthesia. After splitting the muscle, we stripped the periosteum to expose the femoral bone surface. A drill-hole injury was made by inserting a drill bit with a diameter of 0.8 mm in the anterior portion of the diaphysis of one femur, 2 cm above the knee joint. The treatment began the very next day after creating the injury. Plant extract at the doses of 100 and 200 mg/kg were administered orally for 12 consecutive days. Twenty four hours before autopsy, all animals were given a fluorochrome, calcein (20 mg/kg) dissolved in normal saline via intraperitoneal route. The rats were sacrificed under anesthesia and the injured femur (right) was collected and fixed in 70% isopropanol for microarchitecture analysis. Left (uninjured femur) was used for harvesting bone marrow stromal cell (BMC) cultures. For the measurement of bone microarchitectural parameters in the drill-hole, bones were embedded in an acrylic material and 50 μm sections were made using Isomet Bone cutter. Photographs were taken under confocal microscope (Carl Zeiss LSM 510 Meta) aided with appropriate filters. The calcein binding intensity which is an indication of the amount of callus regeneration in the bony hole was calculated using Carl Ziess AM 4.2 image analysis software. 2.5. Mineralization of BMC For mineralization studies, bone marrow stromal cells (BMCs) from the left femora of treated or untreated groups was flushed out with medium. These cells were seeded in 6-well plates (2 106 cells/well) and cultured in osteoblast differentiation medium (10−7 M dexamethasone, 10 mM β-glycerophosphate and 50 mg/ml ascorbic acid). The medium was changed after every two days. The cultures were continued for 21 days after those cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. The fixed cells were stained with alizarin red-S (40 mM, pH 4.5) for 30 min followed by washing with H2O. Stained cells were first photographed under light microscope and alizarin stain was then extracted by 10% (v/v) acetic acid with shaking at room temperature for 30 min. Cells were scrapped out of the culture plates, centrifuged (20,000g for 15 min) and supernatant was collected. To the supernatant, 10% (v/v) ammonium hydroxide was added to bring the pH of the solution to 4.5. Absorbance of the solution was read at 405 nm as described before (Trivedi et al., 2008; Siddiqui et al., 2010; Dixit et al., 2012). 2.6. Micro-computed tomography (mCT) Assessment of internal microstructure (both 2-D and 3-D) of the mineralized tissue in the bony hole was analyzed by mCT, using Sky Scan 1076 CT scanner (Aartselaar, Belgium). Bones were cleaned of soft tissues and scanned using a X-ray source of 70 KV, 100 mA with a pixel size of 18 mm. The images were reconstructed using Sky Scan Nrecon software, which facilitates network-distributed reconstruction carried out on four personal computers running simultaneously. Callus bone was captured by drawing ellipsoid contour with CT analyzer software. Microarchitectural parameters including, bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) trabecular number (Tb.N) and structure model index (SMI) were quantified as described before (Uchida et al., 2000; Sharan et al., 2011; Ngueguim et al., 2012). 2.7. Quantitative real-time polymerase chain reaction (qPCR)
2.4. Drill-hole injury in femur A drill-hole injury was created in both vehicle and extract treated groups as described before (Tanaka et al., 2010; Sharan
Total RNA was isolated from left femur after flushing with medium. The femur of each animal was cleaned properly of any remaining soft tissue, then crushed with TRIzol (Invitrogen)
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according to the procedure describe by the manufacturer. The concentration and the purity of RNA were determined by measuring the absorbance at 260/280 nm using a spectrophotometer (NANO-Drop). cDNA was synthesized with revertAid kit (Fermentas, Austin, USA) from 2 μg of total RNA using a commercial first-strand cDNA synthesis kit. SYBR green chemistry was used to perform quantitative determination of, collagen type-1 (Col1), bone morphogenetic protein (BMP-2), osteocalcin and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript level following an optimized protocol (Sharan et al., 2011). Primer sequence of the gene of interest: col1, BMP-2 osteocalcin and GAPDH are shown in Table 1. 2.8. Statistical analysis Data are expressed as mean 7S.E.M. The data obtained from each treatment were subjected to one-way ANOVA followed by Table 1 Primer sequence of various genes used for qPCR. Gene name
Primer sequence
Accession number
Collagen1
F-CATGTTCAGCTTGTGGACCT R-CGAGCTGACTTCAGGGATGT
NM-053304
BMP-2
F-CGGACTGCGGTCTCCTAA R-GGGGAAGCAGCAACACTAGA
NM-007553.2
Osteocalcin
F-ATAGACTCCGGCGCTACCTC R-CCAGGGGATCTGGGTAGG
NM-013414
GAPDH
F-CAGCAAGGATACTGAGAGCAAGAG R-GGATGGAATTGTGAGGGAGATG
NM-017008
Newman–Keuls test of significance using Prism version 3.0 software. One way ANOVA was applied to test the variation between the groups and Newman–Keuls as post test to make the multiple comparisons between the doses of the extracts. Qualitative observations have been represented following assessments made by three individuals blinded to the experimental designs.
3. Results 3.1. Effect of the ethanol extract of Peperomia pellucida on bone regeneration Quantification of bone regeneration by calcein label (mineral deposition) at the site of drill hole injury was used to evaluate the effect of ethanol extract of Peperomia pellucida (Fig. 1). The extract administered for 2 weeks induced a significantly higher mineral deposition over the control (rats receiving vehicle). Fig. 1A showed representative confocal microscopy images of calcein deposition at the fracture site in the various experimental groups. The increase was 9.45% (p o0.05) at 100 mg/kg dose and 84.85% (p o0.01) at 200 mg/kg dose, compared to the control (Fig. 1B). The extract treatment exhibited dose-dependent increase in mineral deposition over the control. 3.2. Effect of the extract on microarchitecture of regenerated bone at the drill-hole site After 2 weeks administration of the extracts, mCT analysis at the drill hole site of the various groups was performed (representative images in Fig. 2A). Quantification of various mCT parameters
Fig. 1. Ethanol extract from Peperomia pellucida accelerates bone regeneration in drill-hole. (A) Representative confocal images (100 ) of calcein labeling in the callus of drill-hole of various groups after 12 days of treatments. (B) Quantification of the mean intensity of calcein label per pixel. All values are expressed as mean 7SEM (n ¼6); *p o0.05;**p o0.01.
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Fig. 2. Peperomia pellucida extract improves microarchitectural parameters in the drill-hole. (A) Representative mCT images from the center of the bony hole at different doses. (B) mCT analysis showing BV/TV, bone volume/tissue volume (%); Tb.N, strut number (1/mm); Tb.Th, strut thickness (mm); SMI, structure model index; Tb.Sp, strut spacing (mm). All values are expressed as mean7 SEM (n¼ 6 rats/group); npo 0.05; nnp o 0.01.
showed that, both doses of the extract improved the bone microarchitecture. Compared to the control; the extract at both doses increased bone volume (BV/TV, p o0.05), trabecular thickness (Tb.Th, p o0.01), trabecular number (Tb.N, p o0.05), and decreased trabecular separation (Tb.Sp, P o0.05) and structure model index (SMI, Po 0.05) (Fig. 2B).
levels of all three genes over the control. At 200 mg/kg dose, Peperomia pellucida extract had a robust induction of 25–30 folds on the mRNA levels of col1 and osteocalcin over the control. BMP-2 levels were significantly higher in the rats treated with 200 mg/kg dose of Peperomia pellucida extract over the control (po0.001).
3.3. Effect of the extract on mineralized nodule formation in BMC
4. Discussion
At the end of various treatments, BMC from femur of vehicle or extract treated rats were cultured to induce mineralized nodules formation. Fig. 3A represents the photomicrograph of alizarin red-S stained cells showing the formation of mineralized nodules. The quantification done by the percent change of alizarin red-S stained cells over control showed that Peperomia pellucida extract treatment exhibited dose-dependent increase in mineralized nodule formation of BMC over control (Fig. 3B). The percentage change of alizarin red-S stained cells was approximately 24% and 50% at the doses of 100 mg/kg and 200 mg/kg respectively compared to the control.
Peperomia pellucida is an herb used in Cameroonian traditional medicine to treat fracture (Adjanohoun et al., 1996). However, no systematic study is available to confirm the efficacy of this plant in accelerating healing of fracture. At present, there is no orally efficacious agent/compound available to treat fracture. In the present study, we firstly used dynamic histomorphometry to assess whether the plant extract could induce bone regeneration at the fracture site, then employed static histomorphometry (bone microarchitecture using μCT) to assess the quality of the callus that were being formed (Tanaka et al., 2010; Sharan et al., 2011; Ngueguim et al., 2012) and lastly assessed its osteogenic effect in vitro and ex vivo. The results showed that, the Peperomia pellucida extract dose-dependently increased mineral deposition with improved quality of callus formation at the fracture site, which was due likely to its stimulatory effect on osteoblastic cells. Paralleling in vivo bone formation, at the cellular level, the extract treatment stimulated new bone formation by increased differentiation of bone marrow osteoprogenitor cells in ex vivo
3.4. Effect of the extract on expression of osteogenic genes in femur We investigated the effect the extract on osteogenic genes that are expressed during bone formation including, type1 collagen (col1), bone morphogenetic protein-2 (BMP-2) and osteocalcin. As shown in Fig. 4, the results revealed that, daily administration of Peperomia pellucida extract exhibited a dose dependant increase in the mRNA
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Fig. 3. Peperomia pellucida extract stimulated the production of osteoprogenitor cells in bone marrow stromal cells. (A) Representative photomicrographs of mineralized nodules (MN) of all doses show increased intensity and larger-sized nodules by Peperomia pellucida treatment when compared with control. (B) Quantification of mineralization was done by extraction of alizarin red-S dye as described in Section 2. All values are expressed as mean 7 SEM (n¼ 6); *po 0.05, **po 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cultures as well as by increased expression of osteogenic genes of bones. BMP-2 is known to augment fracture healing in several animal models and clinical studies. Human recombinant BMP-2 is clinically used for open tibial and spinal surgeries for accelerating fracture by applying it directly to the site as this cytokine has no oral bioavailability (Govender et al., 2002). Because Peperomia pellucida extract significantly increased mRNA levels of BMP-2 in the femur along with other osteogenic genes, it appeared that the mechanism of accelerating fracture healing by the extract involved endogenous BMP-2 production. Together, these results demonstrate the ability of the extract to stimulate the recruitment of osteoblast at the injury side and to maintain the quality and the integrity of the bone. μCT measurements showed increased BV/TV, Tb.Th, Tb.N and decreased Tb.Sp of the callus by Peperomia pellucida treatment resulting in a more compact assembly of the newly formed bone over the control. SMI represents the relative amounts of rods and plates in a 3D structure, with high values indicating more rods over plates, and greater accumulation of micro-damage. Peperomia pellucida resulted in preferred plate-like structure (low SMI) of the newly formed bone suggesting superior surface geometry and curvature distribution in the newly formed callus compared to control. In folk medicine, Peperomia pellucida is used in the form of macerate or decoction (Adjanohoun et al., 1996). It is administered by oral route or as a paste at the fracture site after scarification. Extraction by ethanol used in this study and water used in folk medicine are both polar solvents. Thus the putative osteogenic compounds responsible for the fracture healing appear to be polar. Phytochemistry of the whole plant of Peperomia pellucida show the presence of alkaloids, saponins, tannins, flavanoids, steroids and glycosides (Pulak et al., 2011). Various flavonoids have osteogenic effect in vivo (Sharan et al., 2009). Notably, a few compounds
isolated from the whole plant have exhibited estrogen-‘like’ effect in CV-1 cells transfected with human estrogen receptor (Xu et al., 2006). It is plausible that flavonoids and phytoestrogens present in the plant extract contribute to its osteoblast stimulating effect and consequent rapid fracture healing effect in vivo. Further studies are required to demonstrate this hypothesis. Effective therapeutic dose for various herbal extracts in rodents have a wide range and often 1.0 g/kg dose is used (Shirke et al., 2008, Haihua et al., 2010). At 100 mg/kg dose of Peperomia pellucida extract, several osteogenic parameters were increased over the control. Maximum mineral deposition and osteogenic stimulation was observed with 200 mg/kg dose of Peperomia pellucida extract. A dose of 200 mg/kg in rats is translated to 32.4 mg/kg in humans by following the equation: human equivalent dose (mg/kg)¼rat dose (mg/kg) [200] X rat Km factor, [6/human Km factor, that is, 37] (Reagan-Shaw et al., 2007). For a 60-kg person, the daily requirement of the extract for fracture repair is calculated at 1.9 g/day, which is favorable for adherence to treatment. Collectively, our findings suggest that the Peperomia pellucida extract accelerates fracture healing and improves callus quality by increasing the production of osteoprogenitor cells and their differentiation to osteogenic lineage thereby enhancing the recruitment of osteoblast to the injury site as well as stimulating the ability of the cells to produce osteogenic cytokine, BMP-2 and deposition of matrix protein, col1, ultimately accelerate fracture healing.
5. Conclusion Our study in a preclinical set up clearly demonstrated that daily oral administration of an ethanol extract from Peperomia pellucida at a favorable dose of 200 mg/kg accelerates the healing of bone
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Fig. 4. qPCR determination of mRNA levels of osteogenic genes, col1, BMP-2 and osteocalcin in femora of various groups. Each assay was performed in triplicate and results are represented as mean 7 S.E.M. of percentage/fold change; nPo 0.05; nnnPo 0.001.
and marrow injury. This effect seems to be due to the osteogenic effect of the extract thereby promoting recruitment and differentiation of osteoblast at the fracture site. Our study justifies the traditional use of the plant in fracture healing and could therefore be considered as an alternative therapy for fracture repair. Future studies will determine the bio-active marker and its mechanism of action of the plant extract.
Acknowledgments The authors thank Federation of Indian Chamber and Commerce Industry (FICCI) through CV Raman fellowship awarded to Dr. T.F. Ngueguim. The authors also sincerely thank Mama Awatsa Marceline, Nankeng Helene and other traditional healers who agreed to disclose the plants they use for fracture healing. We also thank Pr. Louis Zapfack for the authentication of the plant. Authors acknowledge generous grant support from CSIR (ASTHI program).
References Adjanohoun, J.E., Aboukakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu, K.J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabu, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional medicine and pharmacopoeia. Contribution to Ethnobotanical and Floristic Studies in Cameroon. Centre de Production de Manuels Scolaires, Porto-Novo (Rep. Du Benin), p. 641.
Delaney, M.F., 2006. Strategies for the prevention and treatment of osteoporosis during early postmenopause. American Journal of Obstetrics Gynecology 194, S12–S23. Dixit, P., Chand, K., Khan, M.P., Siddiqui, J.A., Tewari, D., Ngueguim, F.T., Chattopadhyay, N., Maurya, R., 2012. Phytoceramides and acylated phytosterol glucosides from Pterospermum acerifolium Willd. seed coat and their osteogenic activity. Phytochemistry 81, 117–125. Doblaré, M., Garcia, J.M., Gomez, M.J., 2004. Modelling bone tissue fracture and healing: a review. Engineering Fracture Mechanics. 71, 1809–1840. Dutmanee, S., Kanogwun, T., Narattaphol, C., Jantarima, P., Kukiat, T., Jarinthorn, T., Tuangporn, S., Nateetip, K., 2007. Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor κB ligand/ osteoprotegerin ratio. Bone 42, 535–546. Gass, M., Dawson-Hughes, B., 2006. Preventing osteoporosis-related fractures: an overview. The American Journal of Medicine 119, 3S–11S. Gerstenfeld, L.C., Einhorn, T.A., 2003. Developmental aspects of fracture healing and the use of pharmacological agents to alter healing. Journal of Musculoskeletal and Neuronal Interactions 3, 297–303. Govender, S., Csimma, C., Genant, H.K., Valentin-Opran, A., Amit, Y., Arbel, R., Aro, H., Atar, D., Bishay, M., Börner, M.G., Chiron, P., Choong, P., Cinats, J., Courtenay, B., Feibel, R., Geulette, B., Gravel, C., Haas, N., Raschke, M., Hammacher, E., van der Velde, D., Hardy, P., Holt, M., Josten, C., Ketterl, R.L., Lindeque, B., Lob, G., Mathevon, H., McCoy, G., Marsh, D., Miller, R., Munting, E., Oevre, S., Nordsletten, L., Patel, A., Pohl, A., Rennie, W., Reynders, P., Rommens, P.M., Rondia, J., Rossouw, W.C., Daneel, P.J., Ruff, S., Ruter, A., Santavirta, S., Schildhauer, T.A., Gekle, C., Schnettler, R., Segal, D., Seiler, H., Snowdowne, R.B., Stapert, J., Taglang, G., Verdonk, R., Vogels, L., Weckbach, A., Wentzensen, A., Wisniewski, T., 2002. BMP-2 Evaluation in surgery for Tibial Trauma (BESTT) Study Group Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. Journal of Bone Joint Surgery 84A, 2123–2134. Haihua, S., Hideko, A., Masakazu, H., Liang, Z., Ke, A., Wenqi, H., Kazuo, H., 2010. Anti-hypersensitivity effects of Shu-jing-huo-xue-tang, a Chinese herbal medicine, in CCI-neuropathic rats. Journal of Ethnopharmacology 131, 464–470. John, L., Vahle, M.S., Gerald, G.L., Jamie, K.Y., Paul, C.F., Jeffery, A.E., Michael, S.W., Yanfei, L.M., James, B.N., 2002. Skeletal changes in rats given daily subcutaneous
68
F.T. Ngueguim et al. / Journal of Ethnopharmacology 148 (2013) 62–68
injections of recombinant human parathyroid hormone (1–34) for 2 years and relevance to human safety. Toxicologic Pathology 30, 312–321. Liu, M.J., Li, Y., Pan, J.H., Liu, H., Wang, S.J., Teng, J.R., Zhao, H.Y., Ju, D.H., 2011. Effects of zuogui pill (see text) on Wnt singal transduction in rats with glucocorticoidinduced osteoporosis. Journal of Traditional Chinese Medecine 31, 98–102. Ngueguim, F.T., Khan, M.P., Donfack, J.H., Siddiqui, J.A., Tewari, D., Nagar, G.K., Tiwari, S.C., Dimo, T., Maurya, R., Chattopadhyay, N., 2012. Evaluation of Cameroonian plants towards experimental bone regeneration. Journal of Ethnopharmacology 141, 331–337. Pulak, M., Priya, A., Satya, V., 2011. Ethno-medicinal, phytochemical and pharmacological review of an amazing medicinal herb Peperomia pellucida (L.) HBK. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2, 258–264. Reagan-Shaw, S., Nihal, M., Ahmad, N., 2007. Dose translation from animal to human studies revisited. Journal of the American Societies for Experimental Biology 22, 659–661. Sharan, K., Siddiqui, J.A., Swarnkar, G., Maurya, R., Chattopadhyay, N., 2009. Role of phytochemicals in the prevention of menopausal bone loss: evidence from in vitro and in vivo, human interventional and pharma-cokinetic studies. Current Medical Chemistry 16, 1138–1157. Sharan, K., Siddiqui, J.A., Swarnkar, G., Tyagi, A.M., Kumar, A., Rawat, P., Kumar, M., Nagar, G.K., Arya, K.R., Manickavasagam, L., Jain, G.K., Maurya, R., Chattopadhyay, N., 2010. Extract and fraction from Ulmus wallichiana Planchon promote peak bone achievement and have a nonestrogenic osteoprotective effect. Menopause 17, 393–402. Sharan, K., Sharan, J.M., Swarnkar, G., Siddiqui, J.A., Khan, K., Kumari, R., Rawat, P., Maurya, R., Sanyal, S., Chattopadhyay, N., 2011. A novel quercetin analogue from a medicinal plant promotes peak bone mass achievement and bone healing
after injury and exerts an anabolic effect on osteoporotic bone: the role of aryl hydrocarbon receptor as a mediator of osteogenic action. Journal of Bone and Mineral Research 26, 1–16. Shirke, S.S., Jadhav, S.R., Jagtap, A.G., 2008. Methanolic extract of Cuminum cyminum inhibits ovariectomy-induced bone loss in rats. Experimental Biology and Medicine. 233, 1403–1410. Siddiqui, J.A., Swarnkar, G., Sharan, K., Chakravarti, B., Sharma, G., Rawat, P., Kumar, M., Khan, F.M., Pierroz, D., Maurya, R., Chattopadhyay, N., 2010. 8″-Biapigeninyl osteoprotective action of 8,8-biapigeninyl in ovariectomized mice. Molecular and Cellular Endocrinology 323, 256–267. Swarnkar, G., Sharan, K., Siddiqui, J.A., Chakravarti, B., Rawat, P., Kumar, M., Kamal, R.A., Maurya, R., Chattopadhyay, N., 2011. A novel flavonoid isolated from the steambark of Ulmus wallichiana Planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation. European Journal of Pharmacology 658, 65–73. Tanaka, K., Tanaka, S., Sakai, A., Ninomiya, T., Arai, Y., Nakamura, T., 2010. Deficiency of vitamin A delays bone healing process in association with reduced BMP-2 expression after drill-hole injury in mice. Bone 47, 1006–1012. Trivedi, R., Kumar, S., Kumar, A., Siddiqui, J.A., Swarnkar, G., Gupta, V., Kendurker, A., Dwivedi, A.K., Romero, J.R., Chattopadhyay, N., 2008. Kaempferol has osteogenic effect in ovariectomized adult Sprague-Dawley rats. Molecular and Cellular Endocrinology 289, 85–93. Uchida, S., Doi, Y., Kudo, H., Furukawa, H., Nakamura, T., Fujimoto, S., 2000. Transient expression of activin betaA mRNA on osteoprogenitor cells in rat bone regeneration after drill-hole injury. Bone 27, 81–90. Xu, S., Li, N., Ning, M.M., Zhou, C.H., Yang, Q.R., Wang, M.W., 2006. Bioactive compounds from Peperomia pellucida. Journal of Natural Product 69, 247–250.