Background
Osteoporosis (OP) is one of the most serious diseases in the modern world, and OP patients frequently suffer from fragility fractures in the hip, spine and wrist, resulting in a limited quality of life. OP is often observed in elderly people, particularly postmenopausal women, and is caused by an imbalance between bone formation and bone resorption (reviewed in ref. [
1‐
3]). Bisphosphonates (BPs) are the most effective class of anti-resorptive drugs currently available and are frequently used in the clinical treatment of OP, owing to the great advantages. Despite showing clinically beneficial effects in the treatment of OP, serious side effects of BPs have been reported, including bisphosphonate-related osteonecrosis of the jaw (BRONJ) [
4]. Medical practitioners and basic researchers have therefore focused on the use of novel therapeutic materials such as anabolic and hormone-like agents that can replace and/or reduce the use of BPs.
In recent decades, Chinese medical herbal extracts have been extensively investigated for their effects on proliferation and differentiation of osteoclasts (OCs) and osteoblasts (OBs)
in vitro, and/or therapeutic potency in OP
in vivo (
e.g., Cistanche salsa[
5],
Anoectochilus formosanus[
6],
Acanthopanax senticosus[
7],
Herba Epimedii[
8] and
Curcuma longa[
9‐
12]). In order to identify more candidates of herbal extracts that have therapeutic effects not only in OP, but are also effective in the treatment of oral and skeletal diseases, an institutional collaborative project between Showa University and Tokyo University of Marine Science and Technology was launched in 2010 [
13]. Within this project, more than 400 bioactive herbal products were examined. After screening of the products by an osteoclast-formation-inhibition experiment utilizing RAW264.7 cells, 3 Chinese medical herbs, the root barks
Melia azedarach (
M. azedarach; commonly known as bead-tree or Cape lilac),
Corydalis turtschaninovii (
C. turtschaninovii; crested lark), and
Cynanchum atratum (
C. atratum; swallowwort) were chosen for further investigation. Although water or ethanol extracts of the roots were reported to contain biologically-active chemicals [
14‐
19], the main compounds and precise mechanisms for the pharmacological effects of the extracts are unknown. In the present study, we reveal that these herbal extracts not only induce apoptosis of mature OCs, but also increase differentiation of OBs and chondrocytes
in vitro. These findings suggest the feasibility of the use of these herbal extracts as novel therapeutics in OP.
Discussion
A number of basic and clinical studies investigating chemical treatment for OP, including parathyroid hormone (PTH), vitamin D
3 and selective estrogen receptor modulator (SERM), have been reported. Currently, BPs are widely used as a therapeutic medicine for OP, as well as bone-metastatic cancers, since they effectively inhibit bone resorption. Despite great pharmacological and clinical advantages of BPs, however, serious side effects, such as renal failure and BRONJ, have also been reported [
4]. This suggests an urgent need for the identification and development of novel medicines. Botanical therapeutics are traditional medicines such as Chinese herbal medicines. The therapeutic effects of a variety of herbal extracts have been studied and reported worldwide. Among these, one of the most investigated herbal extracts is curcumin (reviewed in ref. [
28]), which is isolated from the rhizome of
Curcuma longa (commonly known as turmeric). Curcumin has been reported to have anti-cancer, anti-viral, anti-arthritic, anti-amyloid, anti-oxidant, and anti-inflammatory properties, and is considered a potential therapeutic agent in the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer’s disease, inflammation and OP [
9‐
12]. This accumulation of evidence encouraged us to explore the use of herbs other than
C. longa for their potential therapeutic effects in OP, and hence an institutional collaborative project was commenced [
13].
During bone remodeling [
29], bone resorption by OCs occurs prior to bone formation by OBs. It has been suggested that suppression of proliferation and maturation of OCs prevents excess bone loss. To this end, more than 400 bioactive herbal products were subjected to a preliminary screening utilizing RAW264.7 cells, and we subsequently narrowed these candidates down to three:
M. azedarach, C. turtschaninovii and
C. atratum. The bark of
M. azedarach has been utilized as a therapeutic medicine for tinea imbricata in the Chinese pharmacopoeia [
18,
19]. Methanolic extract of
C. turtschaninovii has been reported to have anti-allergic effects [
15], and is used in traditional Chinese medicine in the treatment of gastric and duodenal ulcers, cardiac arrhythmic disease, rheumatism, and dysmenorrhea [
14]. Finally, the root bark extract of
C. atratum has been used as an anti-febrile and diuretic [
16], and has been reported to show anti-acetylcholinesterase and anti-amnesic activities
in vivo in mice [
17].
In addition to previous reports on pharmacological availability, we showed in the present study that these extracts are capable not only of suppressing proliferation and/or maturation of OCs (Figure
2), but also of inducing cell death (apoptosis) by increasing caspase activity (Figure
3). The results of biochemical assays showed inconsistencies with those of hystochemistry in part. However, it is supposed that the former represents more accurate results than the later does, since histochemical staining often detects a non-specific artifact, and thereby, shows inconsistent results. It is important to note that an increase in the mitochondrial pro-apoptotic/pro-survival protein ratio is required for apoptosis in various cells, including OCs [
30]. All of the extracts increased expression of Bax, Bad and Bak, whereas the effects on expression of Bcl-2 and Bcl-X
L differed for each extract (Figure
4). On the otherhand, AD increased p53 protein, as well as Bax, Bad and Bak. In any case, the Bcl-2 pathway and subsequent activation of caspase is involved in the apoptotic effects observed with these compounds, and interestingly, it is suggested thay the apoptotic signal pathway might be different from that by BPs.
Since anti-OP therapeutics are required to have minimal effects in reducing bone formation, we investigated the effects of the herbal extracts on proliferation and differentiation of OBs (Figures
5 and
6) and chondrocytes (Figures
7 and
8) cell lines. All of the extracts exhibited positive effects on partial, but not terminal, maturation of OBs and chondrocytes, suggesting that these compounds satisfy the requirements for therapeutics used in OP. Finally, the extracts demonstrated the ability to induce differentiation of OBs, but not OCs, from primary MBM cells, reinforcing the effects of Osteoblast-Inducer Reagent, albeit the effect was likely to be saturated at the high dosage (10 μg/ml) of those extracts (Figure
9). The results from the present study imply that we have successfully, at least in part, uncovered a novel potential activity of these extracts to be used as medicines for OP. We are of course aware that further investigation, such as isolation and analysis of bioactive chemicals, detailed molecular and cellular experiments
in vitro, and pre-clinical studies
in vivo, is required in order to ensure that there are not serious side effects associated with the use of the herbal extracts, as has been reported with chemically synthesized medicines. Indeed, several of these studies are currently underway, and our findings will be reported in the near future.
Conclusions
In the present study, we have successfully uncovered a novel potential activity of three Chinese medical herbal extracts from the root barks of M. azedarach, C. turtschaninovii, and C. atratum to be used as medicines for OP. All of the extracts showed capabilities of inducing OCs to undergo apoptosis, OBs and chondrocyte to differentiate, but not to grow. Moreover, the extracts induced osteoblastic, but not osteoclastic differentiation in primary MBM cells. In conclusion, these findings suggest the feasibility of the use of these herbal extracts as novel therapeutics in OP.
Acknowledgments
This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (KAKENHI C to YM and SK, and B to SS) and Nakatomi Foundation (to YM). The authors wish to thank Drs. Yasuto Yoshihama, Tatsuo Shirota, Hiroaki Kamatani, Yasumasa Yoshizawa, Tomohiko Kutsuna, Sayaka Yoshiba, Arisa Yasuda, Hikari Tsukamoto, Rika Nagasaki, Ryota Kishigami and Yuji and Sayaka Kurihara for their helpful suggestions and Ms. Miho Yoshihara for secretarial assistance.
Competing interests
The authors declare that they have no financial competing interests.
Authors’ contributions
YM significantly contributed to and performed the present study, applied for the grant supporting the study, prepared the figures, and wrote the manuscript. SK applied for the grant supporting the study, and helped to draft the figures and manuscript. CL, SB and AK performed histochemical and biochemical assays. TK prepared the materials and purified the herbal extracts. KY participated in the design and coordination of the study. SS conceived the idea, applied for the grant supporting the study, and proofread the figure and manuscript. All authors read and approved the final manuscript.