In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment
Graphical abstract
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.
References (32)
- et al.
Therapeutic ultrasound and fracture healing: a survey of beliefs and practices
Archives of Physical Medicine and Rehabilitation
(2004) - et al.
The role of electrical stimulation in bone repair
Foot and Ankle Clinics
(2005) - et al.
Current concepts of bone healing
Clinics in Podiatric Medicine and Surgery
(2001) - et al.
Changes in total alkaline phosphatase level after hip fracture: comparison between femoral neck and trochanter fractures
Journal of Orthopaedic Science
(2006) Overview of the fracture healing cascade
Injury
(2005)- et al.
Improvement of fracture healing by systemic administration of growth hormone and local application of insulin-like growth factor-1 and transforming growth factor-β1
Bone
(2002) - et al.
Do serological tissue turnover markers represent callus formation during fracture healing?
Bone
(2005) - et al.
Are herb-pairs of traditional Chinese medicine distinguishable from others? Pattern analysis and artificial intelligence classification study of traditionally defined herbal properties
Journal of Ethnopharmacology
(2007) Drugs and fracture repair
Acta Orthopaedica
(2005)- et al.
Refining animal models in fracture research: seeking consensus in optimising both animal welfare and scientific validity for appropriate biomedical use
BMC Musculoskeletal Disorders
(2007)