Elsevier

Journal of Ethnopharmacology

Volume 131, Issue 2, 15 September 2010, Pages 282-289
Journal of Ethnopharmacology

In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment

https://doi.org/10.1016/j.jep.2010.06.039Get rights and content

Abstract

Aim of the study

A novel topical paste used for fracture healing (FH), consisting of the extracts of six herbs, Radix Dipsaci, Ramulus Sambucus Williamsii, Rhizoma Notoginseng, Flos Carthami, Rhizoma Rhei and Fructus Gardeniae, was developed according to the classical theory of traditional Chinese medicine. This study aimed to determine the effectiveness of this formula, and some of its important chemical components in the promotion of fracture healing. The transdermal transport of FH was also examined.

Materials and methods

The osteogenic, angiogenic and nitric oxide suppressing effects of FH and its important chemical marker components were assessed by using osteoblastosacroma UMR-106 cells, human umbilical vein endothelial cells (HUVEC) and murine macrophage RAW264.7 cells, respectively. The bone healing effects of the FH paste and its transdermal absorption were determined using a rabbit fracture model. The callus sizes, bone specific alkaline phosphatase levels and biomechanical properties of the healed bone were assessed.

Results

FH significantly increased the cell proliferation in UMR-106 and HUVEC cells and inhibited the nitric oxide production in murine macrophage in dose-dependent manner. Its important chemical components asperosaponin VI, ginsenoside Rg1 and emodin were shown to be acting positively in the respective in vitro studies. FH paste significantly improved the bone healing in the rabbit fracture model, as was indicated by the increases in callus size at weeks 2–5, and the elevations in bone specific alkaline phosphatase activities at weeks 5–6. The analysis using LC/MS/MS also showed the presence of important chemical marker components of the FH formula in the plasma after 8 weeks of topical treatment.

Conclusion

This study presents the first scientific evidence of the efficacy of a herbal paste in the promotion of fracture healing. There were evidences of transdermal transport of the chemical components, control the inflammation through nitric oxide inhibition, promotion of angiogenesis, and bone healing in the in vitro tests, as well as in the experimental animal.

Introduction

Musculoskeletal injury is becoming more and more common and the expenses involved pose significant burdens on public health planning. Although advances in the early care of injured patients helped improving survival rates, subsequent complications of musculoskeletal injuries remain difficult problems (Button et al., 2009). Hence effective treatment is crucial for the patients suffering from serious injuries. Diverse and sophisicated methods, for example, using mechanical stimulation (Palomares et al., 2009), electrical and electromagnetic devices (Ciombor and Aaron, 2005), low-intensity ultrasound (Busse and Bhandari, 2004) and local injection/implantation of bone morphogenetic protein (Aspenberg, 2005) have been developed as adjuvant therapies for fracture healing. In spite of increasing evidences showing that these interventions can positively promote bone healing (Childs, 2003). Their clinical uses are limited because of the high costs expensive, complicated instrumentations and lack of uniform results (Winn et al., 1999). Fracture healing is a complex physiological process, starting after the local bleeding and inflammation, followed by the complicated activities of mesenchymal precursor cells that lead to formation of soft extracellular matrix tissue, cartilage and the bone (Matsuyama et al., 2005). It consists of a well-orchestrated sequence of events which leads to the proliferation and differentiation of osteoprogenitor cells to endothelial cells which form vascular tissues, and to osteoblast which form bone tissues.

In the old practice of Traditional Chinese Medicine (TCM), bone setting and injury therapy have enjoyed a very long history of over 3000 years. The TCM practitioners usually relied on the use of topical herbal pastes, by which, the herbal essence is believed to go directly into the underlying tissues and initiates, for metabolic enhancements of tissue repairs (Ma, 1998). Compared with the conventional oral route of administration, topical herbal paste has a variety of advantages. It avoids the initial hepatic screening; It works continuously as absorption of the drug goes on; It reduces possible systemic side effect and it does not involve invasive manoeuvres (Ramchandi et al., 1997). Due to cultural reasons, traditional belief and clinical efficacy, many Asian people are still relying on TCM in the treatment of bone fractures and other injuries. This study aims to explore the scientific evidence of topical agents and to elucidate their biological effects on bone fracture healing. According to the TCM theory, the pathological syndrome of bone fracture include: the redness and pain in the fracture area, the swelling and stagnation of circulation; and the slow bone healing. The fracture healing could be therefore improved with those herbs that control inflammation, that promote blood circulation and that stimulate bone regeneration. In this study, three pairs of herbs were accordingly chosen from each of the three major groups known to be anti-inflammatory, circulation promoting and bone forming. They were: (i) Rhizoma Rhei and Fructus Gardeniae (anti-inflammatory), (ii) Rhizoma Notoginseng and Flos Carthami (circulation promotion) and (iii) Radix Dipsaci and Ramulus Sambucus Willamsii (bone forming).

Selection of the six herbs has been based on their popularity of use in traditional practice and the literature support for their bioactivities. We find it unadvisable to copy those complicated formulations found in the classical literature because of the large number of herbs used, making further studies unfavorable. The biological effects of the six herb mixture formula (FH) formula and its ingredients could then be assessed using appropriate cell lines and an animal model.

Section snippets

Herbal materials and extraction

The raw herbs of Radix Dipsaci (RD), Ramulus Sambucus Williamsii (RS), Rhizoma Notoginseng (NG), Flos Carthami (FC), Rhizoma Rhei (RR) and Fructus Gardeniae (FG) were purchased from a herbal supplier in AnHui Province, China. The identities of all herbs, except Ramulus Sambucus Williamsii, were authenticated using thin-layer chromatography with reference to methods recommended by the Chinese Pharmacopoeia (Chinese Pharmacopoeia, 2010). The identity of Ramulus Sambucus Williamsii was identified

Effects of FH extracts and chemical marker compounds on the osteoblastic proliferation of UMR-106 cells

As shown in Fig. 1A, both aqueous and ethanolic extracts significantly increased the cell proliferation of UMR-106 cells at all studied concentrations (15–120 μg/mL) from 10 to 31% and 24 to 60%, respectively, using thymidine incorporation assay. Similar dose-dependent responses were demonstrated in MTT assay, but significant result was not observed in the aqueous group at relatively low concentrations (15–30 μg/mL). At 60 μg/mL of aqueous and ethanolic extracts, the cell viabilities were

Discussion

In our present study, we showed that the aqueous and ethanolic FH extracts significantly promoted osteogenesis and angiogenesis as were indicated by the increased proliferation responses of the relevant cell cultures. In addition, these extracts were shown to be effective in inhibiting the NO production in murine macrophages, suggesting that the extracts were anti-inflammatory. We also demonstrated that the chemical marker compounds of the FH formula, asperosaponin VI, ginsenoside Rg1 and

Acknowledgements

The authors would like to thank the Ming Lai Foundation, the International Association of Lions Clubs District 303-HK & Macau Tam Wah Ching Chinese Medicine Resource Centre for their support to this work. Also thanks to Professor Cao Hui (National Engineering Research Centre for Modernization of Traditional Chinese Medicine, Guangdong, China) for the microscopic identification of Ramulus Sambucus Williamsii.

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