Potential anti-osteoporotic effects of herbal extracts on osteoclasts, osteoblasts and chondrocytes in vitro
© Mukudai et al.; licensee BioMed Central Ltd. 2014
Received: 10 May 2013
Accepted: 10 January 2014
Published: 17 January 2014
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. Although bisphosphonates (BPs) are the most effective class of anti-bone-resorptive drugs currently available and the most commonly prescribed for the clinical treatment of OP, they are known to cause serious side effects such as bisphosphonate-related osteonecrosis of the jaw. Novel therapeutic materials that can replace the use of BPs have therefore been developed.
We commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only in OP, but also in oral and skeletal diseases. In the present study, we report on 3 Chinese medical herbal extracts from the root barks of Melia azedarach, Corydalis turtschaninovii, and Cynanchum atratum.
All of these extracts inhibited osteoclast proliferation and induced apoptosis by up-regulation of caspase activity and increase of mitochondrial pro-apoptotic proteins expression. Furthermore, the extracts enhanced differentiation, but did not affect proliferation of both osteoblasts and chondrocytes. The osteo-inducible effect was also observed in cultured primary bone marrow cells.
Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects in OP. In this study, we elucidate the potency of these herbal extracts as novel candidates for OP therapy.
KeywordsHerbal extract Osteoporosis Osteoclast Osteoblast Shondrocyte Traditional Chinese medicine
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) . 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, Anoectochilus formosanus, Acanthopanax senticosus, Herba Epimedii 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 . 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.
Preparation of the root bark and BP
Approximately 400 kinds of dry herbal roots, including M. azedarach, C. turtschaninovii and C. atratum, were imported from China. The plant materials were formally surveyed and identified by Laboratory of Nutraceuticals and Functional Foods Science, Graduate School of Marine Science and Technology. The dry powdered roots (100 g) were extracted and concentrated to 1 mg/ml under reduced pressure as described previously . Alendronate (AD; Wako Pure Chemical Industries, Osaka, Japan) was used as a BP control, and added at a final concentration of 0.01 to 100 μM into the culture medium.
RAW264.7 cells (mouse monocytes) were cultured and allowed to differentiate into OCs, as described previously . MC3T3E1 cells (mouse osteoblastic cells) were cultured in α-minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and Osteoblast-Inducer Reagent, a cocktail of L-ascorbic acid, dexamethasone and β-glycerophosphoric acid, (Takara Bio, Shiga, Japan), and ATDC5 cells (mouse chondrosarcoma cells) were cultured in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 Ham (DMEM/F-12) supplemented with 10% FBS and Insulin-Transferrin-Sodium selenite Supplement (Roche Diagnostics, Indianapolis, IN). Normal mouse bone marrow (MBM) cells from 8- to 9-wk-old female ICR mice were purchased from Takara Bio, and grown in RPMI 1640 medium supplemented with 10% FBS, according to the manufacturer’s protocol. All cells were grown at 37°C, 5% CO2 and 100% humidity.
Cells were seeded at a density of 3 × 103 cells/ well in 48-well cell culture plates and permitted to grow to maturation for 3 (for RAW264.7 and MBM cells) or 7 days (for MC3T3 and ATDC5 cells) as described above. Thereafter, the herbal extracts (1, 10 and 100 μg/ml) or AD were added to the medium (0.5 ml/ well). After 3 days, the cells were stained for tartrate-resistant acid phosphatase (TRAP)  and alkaline phosphatase (ALPase) activity  and also stained with crystal violet , toluidine blue  and alizarine red , as described previously, with slight modification.
Cell viability and apoptosis assays
For 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, the cells were seeded at a density of 1 × 103 cells/ well in 96-well cell culture plates (100 μl medium in each well). Once the cells were differentiated, herbal extracts or BP were added as described above, and the MTT assay was performed as described previously . The activities of caspase 3/7, 8 and 9 were measured by Caspase-Glo (Promega, Madison, WI) and GloMax-Multi + Detection System (Promega), according to the manufacturer’s protocol. Genomic DNA fragmentation was investigated using a commercial kit (Apopladder EX, Takara), according to the manufacturer’s protocol.
Western blotting analysis
Total cellular protein was prepared as described previously , protein concentration was measured by Quick Start Bradford Reagent (Bio-rad, Hercules, CA) using bovine serum albumin as a standard, and aliquots were stored at -80°C until use. Twenty micrograms of protein was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) in 4- 20% gradient gel (Bio-Rad), and the blot was transferred onto polyvinylidene difluoride membrane (Life Technologies, Carlsbad, CA). Blocking, incubation with primary and horseradish peroxidase-conjugated secondary antibodies and washing of the blots were carried out as previously described . Subsequently, the signal was visualized using Amersham ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd., Buckinghamshire, UK) and ChimiDoc XRS Plus ImageLab System (Bio-Rad). The primary and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA) and GE Healthcare, respectively.
Cells were seeded at a density of 3 × 103 cells/ well in 24-well cell culture plates and cultured as described above in the presence of herbal extracts or AD (1 ml medium/ well). Thereafter, cells were lysed with 0.3 ml of 0.02% Triton X-100 (Sigma Aldrich, St. Louis, MO) in physiological saline, sonicated, and stored at -80°C until use. DNA and sulfated glycosaminoglycan (GAG) content were measured as described previously , and ALPase activity was assayed with a commercial kit (pNPP Phospatase Assay Kit, BioAssay Systems, Hayward, CA). Calcium and PO4 content were also measured with commercial kits (Calcium C-test and Phosphor C-test, Wako). Meanwhile, the conditioned medium (CM) of cultured cells was collected, centrifuged at 1 × 104 g for 5 min at 4°C, concentrated with Amicon Ultra-0.5 ml 3 k (Merck Millipore, Billerica, MA), and the resulting aliquot (25 μl) was subjected to enzyme-linked immunosorbent assay (ELISA) for mouse osteocalcin using a commercial kit (Mouse Osteocalcin EIA Kit, Biomedical Technologies, Stoughton, MA). Osteocalcin content and activity were normalized to DNA content in the cell layer lysate.
RNA isolation and real-time PCR analysis
The genes, primer sequence for sense (upper row) and anti-sense (lower row) strands references and GenBank accession numbers utilized for real-time PCR are shown
GenBank accession No
Col II α1
Unless otherwise specified, all experiments were repeated at least three times, and similar results were obtained in the repeated experiments. The two-tailed, unpaired Student’s t-test was used for analysis. Data are expressed as means ± standard deviation of triplicate data. A P-value < 0.05 was considered significant.
Assessment of optimal dosage of alendronate used as an apoptosis-inducible control
In vitro screening of herbal extracts utilizing TRAP staining in OCs
The herbal extracts induce apoptosis in OCs via activation of caspases
The herbal extracts do not enhance proliferation but induce differentiation of OBs
The herbal extracts increase ALPase activity of chondrocytes, but do not affect synthesis of cartilage-specific ECM
The herbal extracts induce osteoblastic, but not osteoclastic differentiation in primary bone marrow cells
A number of basic and clinical studies investigating chemical treatment for OP, including parathyroid hormone (PTH), vitamin D3 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 . 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. ), 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 .
During bone remodeling , 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 , and is used in traditional Chinese medicine in the treatment of gastric and duodenal ulcers, cardiac arrhythmic disease, rheumatism, and dysmenorrhea . Finally, the root bark extract of C. atratum has been used as an anti-febrile and diuretic , and has been reported to show anti-acetylcholinesterase and anti-amnesic activities in vivo in mice .
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 . All of the extracts increased expression of Bax, Bad and Bak, whereas the effects on expression of Bcl-2 and Bcl-XL 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.
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.
Bisphosphonate-related osteonecrosis of the jaw
- M. azedarach:
- C. turtschaninovii:
- C. atratum:
Minimum essential medium
Fetal bovine serum
Dulbecco’s modified Eagle’s medium nutrient mixture F-12 Ham
Mouse bone marrow
Tartrate-resistant acid phosphatase
3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide
Sodium dodecyl sulfate polyacrylamide electrophoresis
Glycosaminoglycan CM, conditioned medium
Enzyme-linked immunosorbent assay
Selective estrogen receptor modulator.
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.
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