Quercetin is one of the most important and widely studied dietary bioflavonoids. It is ubiquitously found in fruits and vegetables (66). For several years in China, Quercetin and its derivatives have been used in the treatment of osteoporosis because of their natural anti-oxidant property (67). Quercetin regulates various pathways involved in maintaining bone homeostasis such as the RANK/RANKL/OPG System, MAPK signalling, apoptotic pathway, canonical Wnt/β Catenin signalling, BMP and transforming growth factor (TGF-β) signalling ( Figures 1A, B ). Further quercetin exhibits anti-oxidative, anti-inflammatory and angiogenic properties through which it maintains a balance between osteoblastogenesis and osteoclastogenesis (66). Prouillet et al., showed that, in MG-63 human osteoblasts, quercetin had a stimulatory effect on alkaline phosphatase (ALP) activity in the range of 1-50 mM without any significant cytotoxic effects. Quercetin-induced ALP activation requires the ERK pathway and rapidly stimulates it, because inhibition of this pathway by the MEK inhibitor PD 98059 reduced the enhancing actions of quercetin. Moreover, the direct role of ER involved in the effect of quercetin was shown by the fact that ER antagonist ICI 182780 prevented quercetin-induced increase in ALP activity (68). While the previous study showed involvement of ER, another study on the effects of quercetin pretreatment on osteogenic differentiation and proliferation of Human Adipose Tissue Derived Stromal Cells (hADSC) indicated an ER-independent mechanism (69, 70). In mouse monocyte/macrophage cell line RAW264.7, quercetin and quercetin-3-O-glucoside (Q3G) were found to decrease the number of RANK-L-induced TRAP positive multi-nucleated osteoclast cells significantly in dose dependent manner. Treatment with quercetin suppressed the expression of osteoclast related genes such as the calcitonin receptor (CTR), CTSK, MMP-9 and NFATc1. NFATc1 is a master regulatory transcription factor of osteoclast differentiation regulated by RANK-L via activator protein-1 (AP-1) and NF-kB (71, 72). Actin-ring formation, which is important for bone resorption in osteoclast-like mononucleated cells (OCLs) was disrupted by quercetin. This suggests a possible role of quercetin in regulating the signal transducing molecules involved in actin-ring formation: p60 c-src tyrosine kinase, phosphoinositide-3-kinase (PI3K), GTP-binding proteins (GTP-BP) and protein kinase A (PKA) (73). IL-17 is an osteoclastogenic inflammatory cytokine promoting the production of other destructive cytokines such as the macrophage migration inhibitory factor (MIF), tumour necrosis factor-alpha (TNF-α) and RANK-L which in turn increase reactive oxygen species (ROS) and osteoclastic differentiation in rheumatoid arthritis (RA). IL-17-stimulated RA-fibroblasts-like synoviocytes (RA-FLS), when treated with quercetin decreased the production of RANK-L, TNF-α, IL-6 and IL-8. Quercetin decreased the IL-17-induced phosphorylation of mammalian target of rapamycin (mTOR), ERK and NF-kB in RA-FLS, whereas it increased the IL-17-induced phosphorylation of AMP-activated protein kinase (AMPK). Since AMPK is known to counteract and inhibit mTOR signalling, the effect of quercetin on AMPK activation suppresses mTOR and induces apoptosis in osteoclasts (74). MC3T3-E1 cells, treated with Lipopolysaccharide (LPS), a pro-inflammatory glycolipid suppressed the m-RNA and protein expression levels of ALP, RUNX2, OSX and OCN, thus inducing apoptosis and inhibiting the differentiation of osteoblasts via the JNK pathway. Quercetin reversed this condition by increasing the phosphorylation of ERK-1/2, which inhibited the induction of apoptosis by p38 MAPK and JNK. Further, quercetin upregulated the expression of anti-apoptotic proteins B-cell lymphoma-2 (BCL-2) and BCL-XL, while it downregulated the apoptotic proteins caspase-3, BCL-2 associated X apoptosis regulator (BAX) and cytochrome c (75). In osteoblasts isolated from foetal rat calvaria quercetin aglycone upregulated the m-RNA and protein levels of three anti-oxidant genes heme oxygenase-1 (HO-1), γ-glutamate cysteine ligase catalytic subunit (GCLC) and catalase. However, it did not upregulate Nuclear factor erythroid 2–related factor 2 (Nrf-2), the transcription factor of these three genes. Quercetin also downregulated the phosphorylated levels of ERK1/2 and NF-kB, which suggests an anti-inflammatory response associated with the activation of anti-oxidant genes (76). This is in contrast to the studies on MC3T3-L1 osteoblasts and MG-63 osteosarcoma cells (68, 77). Further studies are required to confirm the exact role of ERK1/2, NF-kB p65 and Nrf-2 in mediating the anti-oxidative responses (76). Zhou et al., investigated the effect of quercetin on angiogenesis and found that it increased the expression of angiogenic factors VEGF, angiopoietin 1 (ANG-1), basic fibroblast growth factor (bFGF) and TGF-b, ultimately leading to bone regeneration (66). Another pathway regulating bone homeostasis is the Wnt/b-catenin pathway. Pre-treatment of MC3T3-E1 cells with quercetin increased the protein levels of Wnt3 and β-catenin, which is responsible for osteoblast differentiation (75). One of the mechanisms by which TNF-α suppresses osteoblastogenesis is by inhibiting the activation of SMAD signal transduction by TGF-β and BMP-2. The effect of quercetin in this case, only added to the inhibitory effect of TNF-α, rather than suppressing it. Thus, the overall effect of quercetin on bone formation involves complex competing pathways which may depend on the dose and the concentrations of cytokines and growth factors prevailing in the micro-environment (72).

Kaempferol and its derivatives are natural bioflavonoids enriched in fruits and vegetables and are used as nutraceuticals. Kaempferol possesses various medicinal properties some of which are directly associated with bone-sparing effects (78). Both adipocytes and osteoblasts are differentiated from multipotential mesenchymal stem cells in bone marrow. During the process of ageing, there is a reciprocal increase in adipogenesis and decrease in osteogenesis in the bone marrow, which has to be inhibited and reversed to treat bone diseases such as osteoporosis. The in vitro studies of Ritu et al., showed that kaempferol inhibited the differentiation of bone marrow mesenchymal stem cells(BMSCs) to adipocytes, whereas it stimulated increased osteoblast differentiation (79). This is supported by the fact that kaempferol downregulated the LPS-induced expression of lipid-anabolism genes (sterol regulatory element binding protein-1c [SREBP-1c], fatty acid synthase [FAS] and peroxisome proliferated activated receptor-gamma [PPAR-g]) in BMSCs ( Figure 2 ). On the contrary, it promoted the expression of genes involved in lipid catabolism (carnityl palmitoyl transferase [CPT-1], PPAR-α and acetyl CoA carboxylase [ACC]), thus preventing adipogenesis (80). Kaempferol treatment of BMSCs increased the expression of important downstream regulatory proteins in the mTOR pathway, which suggests its involvement in the differentiation of osteoblasts. The role of mTOR in osteogenesis was validated by Zhao et al., where treatment BMSCs with a specific inhibitor of mTOR called rapa, resulted in decreased levels of osteogenic activity. However, several other studies exhibit controversies over the role of mTOR in bone formation (81). In mouse calvarial osteoblast cell line MC3T3, kaempferol inhibited the TNF a-induced signalling in osteoblasts and thereby reduced the secretion of osteoclastogenic cytokines interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). It also blocked the TNFa-induced nuclear translocation of NF-kB, a transcriptional regulator of MCP-1. Further, kaempferol antagonised the RANKL induced differentiation of RAW264.7 cells to osteoclasts by inhibiting c-Fos expression, an immediate early oncogene, which is indispensable for osteoclastogenesis (82). In LPS treated BMSCs, kaempferol reversed the downregulation of expression of chondrogenic markers SRY-Box Transcription Factor 9 (SOX-9), COL-2 and Aggrecan and strongly elevated their levels. Besides, kaempferol caused a significant decrease in the levels of matrix metallo-proteinase-3 (MMP-3), MMP-13, ADAM metallopeptidase with thrombospondin Type 1 Motif-4 (ADAMTS-4), ADAMTS-5. The inflation of pro-inflammatory cytokines IL-6, IL-1β, inducible nitric oxide synthase (iNOS) and TNF-α induced by lipopolysaccharide (LPS) was reduced by kaempferol, while it increased the level of anti-inflammatory cytokine IL-10 (80, 83). The LPS-induced activation of NF-kB was also inhibited by kaempferol, as was shown by the reduced nuclear staining of p-65 (80). Treatment with kaempferol of ATDC5 cells, led to a marked increase in the mRNA levels of genes encoding COL-2 and COL-10, which are markers of fully differentiated chondrocytes. Also, kaempferol induced the activation of ERK and p38 MAP kinase pathway. Further, it promoted the expression of BMP-2 and BMP-4, thereby suggesting that stimulation of chondrogenesis occurs via BMP-2 signalling pathway in ATDC-5 cells (84). Treatment with 8-prenyl kaempferol, a prenyl flavonoid on MC3T3-E1 cell line, regulated osteoblast differentiation via BMP-2 signalling pathway, which subsequently triggered SMAD1/5/8. This led to the activation of the transcription factor RUNX2 which promoted bone mineralization by regulating the expression of COL-1, OPN and ON (85). Kaempferol induced luciferase activity in rat primary osteoblasts transfected with pERE-Luc and also triggered phosphorylation of ER-a, which suggested that kaempferol acts via ER activation. This was confirmed when pre-treatment with ICI 182,780 completely blocked the kaempferol-induced pERE-Luc activity. Additionally, kaempferol upregulated ALP activities and the transcription of several bone differentiation marker genes such as the COL1A1, ON, OCN, RUNX2 and OSX (86). A study by prouillet et al., on MG-63 human osteoblastic cell strain demonstrated that kaempferol induced increase in ALP activation involves the ERK pathway. This was shown by incubating the cells with PD 98059, an inhibitor of ERK pathway, which reduced the stimulatory effects of kaempferol on ALP. ICI 182780, a pure anti-estrogen, inhibited ERK activation and reduced the levels of ALP in kaempferol treated cells which shows that kaempferol activates ERK pathway via the ERs (69, 85). MAP kinase activation via a non-genomic action of ER can lead to downstream modulation of the transcription factor AP-1 which has been predicted to have a binding site on the promoter of the ALP gene. This transcription factor can act as a possible link between rapid ERK activation and increased ALP activity (69). Pretreatment with kaempferol of MC3T3-E1 cells exhibited a marked reduction in antimitochondrial antibody (AMA) induced-cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, [Ca²⁺]_i elevation, and ROS production. Kaempferol induced the activation of AKT, PI3K and cAMP response binding element protein (CREB) inhibited by AMA, which are known to be involved in osteoblast-like cell proliferation and differentiation (87). RANK-L induced differentiation of RAW 264.7 cells to osteoclasts was shown to be inhibited by kaempferol by suppressing the expression of osteoclastogenic factors TRAF6, NFAT-c1, and c-Fos. Osteoclastogenesis was also suppressed by inhibiting autophagy related factors beclin-1 and sequestosome 1 (p62/SQSTM1) (83, 88). Furthermore, in dexamethasone-induced rat calvarial osteoblasts, kaempferol decreased osteoblast apoptosis by inducing expression of the anti-apoptotic gene BCL-2 and suppressing BAX, a pro-apoptotic gene (78).

Icariin is the main active prenylated flavonol glycoside isolated from the herb Epimedium pubescens. It has been widely used for several centuries in Chinese herbal medicine and is known to possess “bone strengthening” properties (89, 90). Naturally isolated icariin is becoming an interesting alternative in the prevention and treatment of bone diseases (90). It has been known to enhance osteoblastic differentiation and proliferation, inhibit bone resorption, and induce apoptosis of osteoclasts (89). Pre-osteoblastic MC3T3-E1 cells treated with icariin upregulated the levels of osteogenic markers RUNX2, OCN, BSP and ALP in a dose dependant manner. Besides, Icariin was effective in upregulating RUNX2, BSP and OCN levels in mouse primary osteoblasts as well. mRNA expression levels of inhibitor of DNA binding-I (Id-1), a transcriptional target of BMP/SMAD signalling was increased in MC3T3-E1 cells when treated with icariin, whereas expression of RUNX2 m-RNA was upregulated in both MC3T3- E1 cells and POBs. This suggests the involvement of BMPs and RUNX2 signalling in osteogenesis induced by icariin (91, 92). In adult female osteoblast-like cells, icariin caused a significant increase in ALP activity and nitric oxide (NO) levels followed by increased proliferation and mineralisation of osteoblasts ( Figure 3A ). NO is known to exhibit inhibitory effects on bone resorption by suppressing osteoclasts activity and precursor recruitment connected to iNOS activity. Moreover, icariin treatment increased BMP-2/SMAD protein expression as well. Both NO and BMP-2/SMAD activate the transcription of RUNX2 gene, thereby regulating bone homeostasis. Icariin also attenuated caspase-3 activity in the osteoblast-like cells on the 28^th day of treatment with icariin, thereby exhibiting its anti-apoptosis effect (93). Sheng et al. demonstrated that treatment with icariin upregulated OCN synthesis, ALP activity, calcium deposition and collagen synthesis in BMSCs, thus promoting osteogenic differentiation. Additionally, icariin increased the expression levels of marker genes and proteins namely RUNX2, OSX, BMP-2 and IGF in osteogenic cultures. Ming et al. and Huang et al. found that icariin inhibited osteoclastogenesis induced by RANKL and M-CSF in mouse bone marrow culture and inhibited bone resorption by stimulating apoptosis of mature osteoclasts (89). In the study of Wu et al., on BMSCs, it was found that ERK, p38 and JNK signalling pathways were all phosphorylated indicating their participation in osteoblast proliferation, differentiation and mineralisation. Blocking these three pathways significantly inhibited ALP activity and expression of COL1, OPN and OCN. Besides, icariin treatment has also been reported to instigate osteogenic differentiation of BMSCs through the activation of PI3K–AKT–eNOS–NO–sGC–cGMP–PKG signalling pathway (94). Icariin caused significant inhibition of NF-kB activation in RANKL-induced RAW264.7 cells by degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkB-a) ( Figure 3B ). RANKL-induced expression of downstream regulatory factors c-Fos and NFATc1 were decreased after treatment with icariin, which in turn reduced the levels of target osteoclastogenic proteins such as CTSK and TRAP (95). Treatment with icariin on bone mesenchymal stem cells upregulated the expression of osteogenic genes RUNX2, ALP, and COL1 and decreased the expression levels of adipogenic genes —PPARγ, fatty acid binding protein-4 (Fabp4), and adipsin, thus inhibiting the differentiation of BMSCs into adipocytes. Icariin promoted the phosphorylation of Glycogen synthase kinase-3β (GSK-3b) and elevated the levels of active b-catenin in the nucleus of BMSCs. Inhibition of the Wnt signalling pathway brought down the phosphorylation of GSK-3b, caused degradation of b-catenin and upregulated the expression of adipogenic genes, thus confirming the intervention of the Wnt pathway in the differentiation of BMMSCs (92, 96). Recently it was found that cyclin D1, a mitogenic signal sensor that pushed cells from G0 phase into the proliferative cycle, was significantly increased in icariin treated BMSCs (92). Iron overload and accumulation in post-menopausal women and elderly men has been found to be linked to bone metabolism abnormalities like osteopenia, osteomalacia and osteoporosis. Treatment with icariin reversed the iron-overload-induced elevation of ROS and mitochondrial dysfunction caused by the collapse of mitochondrial membrane potential. Thus, icariin attenuated the increase in osteoclasts differentiation and promoted osteoblasts proliferation and differentiation in iron-overloaded MC3T3-E1 osteoblasts (97). Icariin inhibited hypoxia induced apoptosis in neonatal rat calvarial osteoblasts. It reduced the expression levels of caspase-3 and upregulated the mRNA expression levels of BCL-2, thereby inhibiting apoptosis. Also, icariin supplementation diminished the intracellular malondialdehyde (MDA) levels and ROS production, while increasing the activity of SOD, anti-oxidant enzyme to ameliorate the hypoxia induced stress (98). In the LPS-induced osteoclastogenesis model, icariin treatment reduced the LPS-induced activities of osteoclast differentiation marker protein TRAP and regulator of bone resorption- acid phosphatase (ACP). Moreover, icariin suppressed the LPS-induced RANKL expression, whereas it elevated the LPS-inhibited expression of OPG, an osteogenic marker. In addition, icariin could inhibit the synthesis of osteoclastogenic pro-inflammatory cytokines such as IL-6 and TNF-a. Alongside this, icariin reduced prosteoglandin-E2 (PGE-2) production by obstructing synthesis of cyclo-oxygenase -2 (COX-2), therefore inhibiting bone resorption (99). Considering the limitations of animal models in determining the therapeutic efficacy and pharmacological properties of icariin and derivatives, further verification using mammalian models, primates and human clinical trials is required (90).

Myricetin belongs to a subclass of bio-flavonoids called flavonols. It is majorly found in berries, fruits, vegetables, medicinal herbs and tea plants (100, 101). Myricetin is known to possess antioxidant, anti-inflammatory, antimicrobial, anti-viral, antioxidative, anti-tumorigenic and antiallergic properties. Recent studies also provide evidence for myricetin exhibiting osteo-protective properties and inhibiting osteo-clastogenesis (102, 103). Huang et al., demonstrated that myricetin treatment suppressed RANKL-induced differentiation of mouse macrophage RAW264.7 cells into characteristic TRAP-positive multinucleated osteoclast-like cells (OCL). The hypothesis that impairment of osteoclast differentiation would also result in the inhibition of osteoclast bone resorption was confirmed by bone resorption assay, which showed complete bone resorption activity at myricetin concentrations ≥50 mm (100). In the Titanium particle-induced mouse calvarial osteolysis model, myricetin disrupted the RANKL-induced F-actin ring formation, a characteristic feature of mature osteoclasts responsible for bone resorption. It also decreased the RANKL-induced expression of osteoclastogenic markers TRAP, CTR, CTSK, V-ATPase-d2, c-Fos, and NFATc1 ( Figure 4 ). Further, Myricetin inhibited the production of pro-inflammatory cytokines TNF-a and IL-1b, thereby suppressing the NF-kB pathway and MAPK pathways (p38, JNK1/2, and ERK1/2) responsible for osteoclast formation and bone resorption (104). Ying et al., showed that treatment with myricetin elevated the serum OCN and ALP levels in rats with streptozotocin-induced diabetic osteoporosis. The levels of serum anti-oxidants SOD and catalase were also increased in response to addition of myricetin (105). In human chondrocytes, myricetin reduced the levels of IL-1b stimulated inflammatory mediators and cytokines such as PEG-2, COX-2, iNOS, IL-6 and TNF-a as well as the elevated levels of MMPs, thereby inhibiting extracellular matrix (ECM) degradation and promoting generation of COL-2. Regulation of these mediators was associated with the repression of NF-kB pathway by the activation of Nrf2/HO-1 with a possible mediation of the PI3K/AKT pathway (106). Pre-treatment of human gingival fibroblasts with myricetin suppressed the LPS-induced expression of MMP-1, MMP-2 and MMP-8. RANKL-stimulated RAW264.7 cells when pre-treated with myricetin, exhibited reduced phosphorylation of p38 and ERK pathways, inhibited phosphorylation of c-Src and impeded the degradation of IkB-a. Moreover myricetin showed inhibitory effects on the m-RNA expression of osteoclastogenic markers such as TRAP, c-FOS, CTSK and NFATc-1 (107). Myricetin exhibits protective effects against 2-deoxy-D-ribose induced oxidative damage in MC3T3-E1 cells by decreasing the levels of protein carbonyl, advanced oxidation protein products, and MDA. Besides it elevated the levels of ALP activity, collagen content, calcium deposition, OCN and OPG in the presence of 2-deoxy-D-ribose (108). In human bone marrow stromal cells (hBMSCs), myricetin upregulated the levels of m-RNA expressions of osteogenic markers OCN, COL-1, ALP and RUNX2. Apart from that, myricetin triggered the Wnt/b-catenin pathway and upregulated the expression of several downstream genes such as T-cell factor-1(TCF-1) and lymphoid enhancer factor-1 (LEF-1) (109). Hsu et al., showed that treatment with myricetin on the conditionally immortalized human fetal osteoblastic cell line (hFOB) and the human osteosarcoma cell line MG-63, caused a significant upregulation of BMP-2, which in turn increased the phosphorylated levels of SMAD 1/5/8 and p38, one of the MAPK pathways (110). The effects of myricetin on Dexamethasone(DEX) treated MC3T3 cells revealed that, it ameliorated the DEX-induced inhibition of bone formation markers namely RUNX2, BSP, OPN, OCN, COL1A1 and ALP. Besides myricetin promoted matrix mineralisation via the ERK signalling pathway and downregulated TRAP activity and C-terminal telopeptide of type I collagen (CTx) in DEX treated cells (111). Pre-treatment with myricetin on MG-63 cells, reduced the synergistic effect of IL-1b and TNF-a on anti-Fos immunoglobulin-M (IgM) mediated apoptosis of osteoblasts, thereby attenuating the activation of apoptotic proteins caspase-8 and caspase-3, and upregulating the levels of the anti-apoptotic protein FLICE inhibitory protein (FLIP) (112). Overall, myricetin has proven to exhibit osteogenic properties and further studies are required to use it as a therapeutic agent against bone diseases.

Naringin, a polymethoxylated flavonoid glycoside, is an active ingredient of citrus fruits and Chinese herbal medicine. It possesses several pharmacological effects, including bone-protective properties. Zhu et al. demonstrated that naringin exhibits anti-osteoporosis property in a fashion similar to estrogen, by binding to the estrogen receptors. This might replace estrogen-replacement therapy in clinical use (113, 114). In the study of Li et al., naringin promoted the osteogenic proliferation and differentiation of BMSCs and also exhibited a 5-7 day delay between the start of naringin treatment and the burst of ALP expression. This suggested a delayed differentiation pattern of the BMSCs in response to naringin treatment (114). In human amniotic fluid stem cells (hAFSCs), naringin was shown to upregulate ALP activity and calcium deposition in a dose dependent manner. Naringin significantly promoted the expression of osteogenic marker genes including ALP, OPN and COL-1 as well as the osteoclastogenesis-inhibition marker gene OPG, thus enhancing the osteogenic differentiation of hAFSCs ( Figure 5 ). This differentiation was shown to be regulated via the BMP and Wnt/b-catenin pathways involving BMP-4, RUNX2, b-catenin and cyclin D1 (115). Further, naringin induces the apoptosis of osteoclasts via inhibition of activation of the death receptor pathway (Fas, TNF) or mitochondrial apoptosis pathway. In the study conducted by Li et al., it was confirmed that naringin could downregulate the mRNA expression levels of the pro-apoptotic marker gene BCL-2 and downregulate the expression levels of the anti-apoptotic marker gene BAX (116). Treatment of RAW627.4 cells with naringin abrogated RANKL induced formation of TRAP positive osteoclast cells. Additionally, naringin attenuated the gene expression levels of osteogenic markers such as CTSK, CTR and TRAP as well as osteoclastogenic fusion genes including dendritic cell-specific transmembrane protein (DC-STAMP), and V-ATPase d2 (d2). Further, naringin suppresses the RANKL induced activation of NF-kB via inhibition of degradation of IkB and suppresses the activation of ERK pathway as well (117). Recent studies have shown naringin being an HMG-CoA reductase inhibitor, might possibly promote BMP-2 expression and induce bone formation, suggesting the possible involvement of mevalonate pathway. In co-cultures of osteoblasts and bone marrow cells, naringin suppressed the IL-1 induced osteoclastogenesis (118). Naringin may also possess the ability to downregulate the expression of PPARγ in BMSCs, thus reducing adipogenesis and promoting bone formation. In addition, naringin inhibited the mRNA expression of osteoclastogenic markers including RANK, TRAP, MMP-9 and NFATc1, whereas it upregulated c-Fos expression in RAW627.4 cells (119). Li et al., showed that increased levels of SOD, catalase and MDA in dexamethasone (DEX)-treated-inflammatory bowel disease (IBD) rats were significantly reduced by the intervention of naringin (120). The experiments of Wu et al., revealed that naringin induced osteoblast proliferation, differentiation and maturation in cultured osteoblasts. Besides, in MC3T3-E1 osteoblastic cells, the stimulatory effects of naringin on the expression of BMP-2 was found to involve the activations of PI3K, AKT, c-Fos/c-Jun and AP-1 pathways. Furthermore, it was found that the osteo-protective effects of naringin on UMR-106 cells were attributed to its positive effect on the Wnt/b-catenin pathway via AMPK and AKT signalling (121, 122). Kanno et al., demonstrated that naringin inhibited the LPS-induced production of NO and the expression of inflammatory gene products such as TNF-a, IL-6, iNOS, COX-2 and the transcriptional activity of NF-kB. Suppression of these pro-inflammatory cytokines which are the positive regulators of osteoclastogenesis via the inhibition of NF-kB might result in the inhibition of osteoclastogenesis and bone resorption (123). Further, naringin promoted angiogenesis and neovascularization during fracture callus formation in murine osteoporotic models, likely by regulating the expression of VEGF in osteocytes (119). Naringin’s diverse effects on bone indicate its potential in the treatment and prevention of many common orthopaedic conditions. Naringin strongly reduces osteoclastogenesis, inflammation, and adipogenesis and promotes osteoblastic differentiation from progenitor cells for the maintenance and preservation of both cartilage and bone. However additional research is required to assess the ways in which the pharmacokinetic properties of naringin can be improved, in order to optimize its therapeutic effects.

Daidzein is a phytoestrogen belonging to the iso-flavonoid group and abundantly found in soy products. Considering the fact that daidzein can bind to estrogen receptors a and b and have estrogenic effects, they can be used as an alternative to estrogen replacement therapy (124). Osteoblast cell cultures treated with exhibited enhanced osteoblast viability and induced their differentiation from osteoprogenitors to terminally differentiated osteoblasts. Moreover, daidzein increased the ALP activity, OCN synthesis and the mRNA expression levels of BMP-2 in primary osteoblast cell cultures (125). Exposure of porcine osteoblasts to daidzein increased the nuclear levels of the osteogenic transcription factor RUNX2 that was blocked by ICI 182,780. Daidzein also caused a heightened secretion of OPG in the medium of porcine control OB, while it decreased the membrane content of RANKL (126). Picherit et al. demonstrated that in ovariectomised rat model of postmenopausal osteoporosis, oral administration of daidzein arrested both cancellous and cortical bone loss or only cortical bone loss, while manifesting no estrogenic activity on the uterus. Therefore, this proves that daidzein has no adverse effects on the uterus (127). Treatment with daidzein on ovariectomized mice significantly reduced the production of ROS and TNF-a by activated T-cells, both of which are involved in the stimulation of osteoclastogenesis (128). In osteoblast like MG-63 cells, administration of daidzein caused a remarkable elevation in the levels of ALP and COL-1 and also protected against cisplatin induced apoptosis via an ER-dependent MEK/ERK and PI3K/AKT activation (129). Daidzein promoted osteoblast proliferation and differentiation via the BMP pathway, which upregulated the phosphorylated levels of SMAD 1/5/8. This in turn, led to an increase in the expression of osteogenic marker genes, including ALP, RUN-X2, COL-1 and OSX (130). Furthermore, daidzein demonstrated anti-osteoclastic activity in RAW264.7 cells by downregulating the expression levels of TNF-a induced c-Fos and NFAT-c1 (both of which are important regulators of osteoclast differentiation) in an ER dependent manner. In addition, daidzein inhibited nuclear translocation of NFAT-c1 and also reduced the levels of NF-kB and DC-STAMP levels (131). However, high levels of daidzein cannot not always be beneficial. A correct balance is always required for optimum activity. A study by Dang et al., using mouse bone marrow cells and mouse osteoprogenitor KS483 cells has shown that at concentrations below 20 μM, they inhibit osteogenesis and at concentrations higher than 30 μM, it stimulates adipogenesis (124). This proves that a proper amount of daidzein should be taken, and high or lower levels may not tend to be beneficial to the human body.

Luteolin is a flavonoid found in many herbal extracts and has been a part of the traditional culture in Asian countries through medicines and supplements. Exposure of mouse bone marrow derived macrophages (BMMs) to luteolin inhibited osteoclast differentiation induced by RANKL and also downregulated the expression of osteoclast related genes such as NFATc1, c-Src, DC-STAMP, MMP-9, CTSK and TRAP. Moreover, luteolin suppressed bone resorption in a dose-dependent manner in mature osteoclasts incubated with RANKL and M-CSF (132). In RAW264.7 cells, luteolin inhibited the formation of mature TRAP-positive osteoclasts induced by RANKL via the suppression of activating transcription factor (ATF2) downstream of p38 MAPK and NFATc1, thus inhibiting bone resorption. This was accompanied by the disruption of actin rings of the osteoclasts (133). The effects of luteolin on the prevention of bone loss in experimental periodontitis in Wistar rats were assessed and it was found that treatment with luteolin remarkably decreased the alveolar bone loss by attenuating osteoclastogenic activity and production of osteoclastogenic markers including MMP-9 and RANKL. Besides, it upregulated osteoblastic activity via the increased expression of osteogenic markers such as tissue inhibitor of metalloproteinase (TIMP-1), BMP-2, and OPG expressions (134). Nash et al. demonstrated that Luteolin-treated mouse osteoblasts exhibited elevated ALP activity and collagen formation via interactions with estrogen receptors (135). Luteolin treatment of MC3T3-E1 osteoblasts abrogated the 3-morpholinosydnonimine (SIN-1)-induced production of oxidative stress markers which included NO, PGE2, TNF-a and IL-6, thus preventing osteoclastogenesis and bone resorption in diseases linked with the overproduction of inflammatory mediators such as arthritis (136). In cultured human periodontal ligament cells (HPDLCs), administration of 1µmol of luteolin strongly enhanced cell viability, ALP activity and increased calcified nodules content. Additionally luteolin significantly upregulated the mRNA and protein expression levels of osteoblast specific markers such as ALP, BMP2, OSX and OCN and the relative expression levels of β-catenin and cyclin D1 (137). Yang et al., demonstrated that in murine calvarial osteoblasts administration of luteolin suppressed the IL-1b-induced expressions of MMP-9 and MMP-13 via a possible inhibition of the ERK pathway, thus preventing excessive degradation of bone matrix (138). Luteolin dose dependently suppressed the mRNA and protein expression levels of pro-inflammatory cytokines and mediators including TNF-a, IL-6, COX-2 and iNOS in LPS-stimulated mouse alveolar macrophage MH-S and peripheral macrophage RAW 264.7 cell lines via inhibition of phosphorylated NF-kB and AP-1 mediated through blockage of Akt and IkB kinase (IKK) phosphorylation. Further, luteolin inhibited the production of ROS as well (139). In a study by Abasi et al., it was found that luteolin at lower concentrations conferred protection against high-glucose-induced cell death compared to its cytotoxic effects at high doses. Thus, in order to utilise the protective cations of luteolin, it is safest to avoid consuming high doses of luteolin in food supplements (140).

Genistein, a phytoestrogen, is a non-steroidal compound, that shows structural similarity to estradiol-17β. This enables genistein to bind to sex hormone binding proteins and estrogen receptors, thus exhibiting anti-estrogenic and estrogenic properties, the former being done by competing with estradiol with estrogen receptors (141, 142). Anderson et al. discovered a tendency in OVX rats treated with genistein to maintain a better bone mass when compared to the untreated control rats and conjugated estrogen-treated rats, with the low-dose genistein treated groups exhibiting the highest numerical effect on bone retention. Several studies have implied that at low doses genistein acts through estrogen receptors, thus rendering bone-preserving effects. However, it has also been shown that genistein at high doses might induce multiple cellular effects and may not necessarily cause estrogen receptor activation. Thus, further studies are required to determine the effects of non-pharmacological doses of genistein (143). In a study conducted by Li et al. on Sprague Dawley rats, it was found that genistein at both high and low doses, caused a remarkable increase in the BMD, bone volume and also resulted in denser subchondral trabecular bone in vivo. At low doses, genistein upregulated the mRNA expression levels of osteogenic markers including ALP, OCN, OPG, ERα and ERβ, whereas it downregulated the osteoclastogenic marker RANKL both in vivo and invitro. High dose genistein decreased the mRNA levels of bone homeostasis related markers such as ALP, OCN, OPG, RANKL and ERα, while it increased ERβ expression levels invitro and in vivo, thus not only inhibiting bone resorption but also bone formation at higher doses- (144). Fanti et al., demonstrated that treatment with genistein of OVX rats lead to an approximate 50% percent reduction in distal femur cancellous bone loss and loss of whole tibia BMD. Highest genistein dose (25 mg/g/day) resulted in larger uterine size compared to the intermediate dose which provided maximum bone-sparing effects but lesser uterine size, thus suggesting a possible non-estrogen mediated mechanism of genistein such as direct interaction with cellular enzymes including via direct interaction with cellular enzymes as diverse as 5-LOX, COX, cyclic AMP phosphodiesterase, protein kinases, DNA topoisomerase II and 11b-hydroxysteroid dehydrogenase ( Figure 6 ). Moreover, genistein treatment suppressed the elevated levels of pro-inflammatory cytokine TNF-a, an inhibitor of osteogenesis (145). In MC3T3 pre-osteoblastic cells, treatment with genistein altered the expression levels of genes associated with cell proliferation, cell migration, cell differentiation, and inflammatory responses. Successive knockdown analyses showed that two upregulated genes (Ereg and Efcab2) and three downregulated genes (Hrc, Gli1, and Iftm5) play crucial roles in the differentiation of osteoblasts via increasing the expressions of osteoblast-associated markers such as RUNX2, ALP and BMP-2 (146). Administration of genistein to human bone marrow stromal cells suppressed its differentiation into adipocytes by inhibiting the mRNA levels of PPARg and CCAAT/enhancer binding proteins (C/EBPs), while it enhanced osteoblastogenesis, thus preventing bone loss associated with excessive adipogenesis (147). Besides, genistein was also found to increase the expression levels of b-catenin and reduced the levels of IL-6 in Sprague Dawley rats (148). Liao et al. have shown that genistein promotes osteoblastic differentiation by the activation of p38 MAPK-RUNX2 pathway. Moreover, several other studies have revealed a possible cross talk between this pathway and other pathways mediated by BMP and protein kinase C (PKC) (149). Genistein also has shown to induce osteoblast proliferation and differentiation from BMSCs through the involvement of ER–NO–cGMP pathway (150). The expressions of two main osteoclastogenic markers c-Fos and NFATc1, were found to be inhibited by genistein. Furthermore, genistein inhibited RANKL-induced degradation of IkB and nuclear translocation of NF-kB and also suppressed the expressions of IL-1 and CTSK mediated by tyrosine kinase-NF-kB pathway. These effects led to the inhibition of differentiation of osteoclasts and subsequent bone resorption (89).

Hesperidin, also called Hesperetin-7-O-glucuronide is a flavonoid abundantly found in citrus fruits and belongs to the flavonoid subgroup called flavonones. Hesperidin is a glycoside flavonoid which when absorbed gets hydrolysed into the aglycone form by gut microbiota and undergoes further metabolic changes-1 (151). Several studies have reported hesperidin to act as a potential bioactive compound in maintaining bone health in OVX rat models (152). In primary osteoblasts obtained from wistar rats, hesperidin, was found to upregulate the mRNA levels of ALP and OCN via upregulation of RUNX2 and OSX, the two important transcription factors in relation to osteoblasts, which are a part of the MAPK and BMP signalling pathways ( Figure 7 ). Phosphorylation of SMAD1/5/8 complex also seemed to be increased, thus suggesting the participation of the BMP pathway through activation of SMAD1/5/8. Moreover, noggin, a protein secreted by osteoblasts and known to hinder the BMP pathway was found to be downregulated by hesperidin (151). Besides, treatment with hesperidin showed slight modulation in the levels of c-Jun and c-Fos, which form a part of the transcription factor AP-1 responsible for the activation of osteoblast-related genes. This indicates the possible intervention of hesperidin through the MAPK signalling pathways (152). In periodontal ligament stem cells (PDLSCs), administration of hesperitin, increased the mRNA level of the osteogenic transcription factor Fos-related antigen-1 (FRA-1) and also the protein levels of OPN and COL-1A. Under conditions of high glucose, the ROS produced by PDLSCs were scavenged by hesperitin. Furthermore, hesperitin also stimulated the activation of Wnt/b-catenin pathway mediated by the activation of PI3K/AKT signalling (153). A study by Kim et al., demonstrated a possible antiresorptive effect of hesperitin through the inhibition of four pathways namely NIK/IKK, ERK, p38, and JNK, which in turn suppressed the NF-kB signalling responsible for osteoclastogenesis and also showed effects on the redox regulating transcription factors Trx/Ref-1 (154). Additionally, exposure to hesperidin of male gonad-intact senescent rats, attenuated the production of the pro-inflammatory cytokine IL-6 (155). Although the exact mechanism of action of hesperidin hasn’t been elucidated, the above-mentioned pathways have been discovered as of yet to be regulated by hesperidin.

Apigenin is a flavonoid belonging to the subgroup flavone and is widely present in several fruits and vegetables such as olives, apples and parsley. Although only minimum information is present on the role of apigenin in bone metabolism, a few studies indicate the role of apigenin in preventing bone loss (156). Pre-treatment of H2O2 induced MC3T3-E1 cells with apigenin, caused an upregulation of anti-oxidant enzymes SOD1, SOD2 and glutathione peroxidase (GPx), thus counteracting the ROS produced. Further, apigenin remarkably increased the expression levels of genes responsible for osteoblast differentiation such as ALP, OPN, OPG, BSP, OSX, OCN and BMPs (BMP2, BMP4 and BMP7). Other anti-oxidant properties of apigenin include activating H2O2-induced reduced expression of AKT2, PI3K and ERK, all of which are key-regulators of pathways involved in survival, thus inhibiting apoptosis osteoblasts ( Figure 8 ). These findings suggest the role of apigenin in the treatment of bone diseases associated with oxidative stress (157). Apigenin treatment of TNF-a-induced MC3T3-E1 osteoblasts, reduced its production of IL-6 and NO involved in bone resorption, suggesting apigenin’s intervention in treating bone disorders such as osteoporosis characterised by excessive production of inflammatory cytokines (158). In a study by Lee et al., it was demonstrated that apigenin supressed the activity of collagenase in RA and also showed that apigenin inhibited LPS-induced production of NO and COX-2 by RAW 264.7 macrophage cells. In addition, apigenin significantly attenuated the TNFa-induced adhesion of monocytes to human umbilical vein endothelial cell (HUVEC) monolayer and TNF-a-stimulated elevation of vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and E-selectin-mRNA, all of which are involved in RA (159). Treating LPS-induced macrophages with apigenin, profoundly suppressed the production of IL-6, IL-1β, and TNF-α via regulating various signalling pathways. Apigenin suppressed LPS-induced production of IL-1β by disrupting caspase-1 activation via hampering the inflammasome assembly. Also, apigenin arrested the LPS-stimulated production of IL-6 and IL-1β by decreasing the mRNA stability through inhibition of ERK1/2 activation. Additionally, apigenin inhibited the activation of NF-kB via induced by TNF-α and IL-1β thus providing evidence to use apigenin for potentially treating inflammatory bone diseases (160). Furthermore, Zhang et al., have shown the involvement of JNK and p38 MAPK signalling pathways in stimulating osteoblast differentiation via upregulation of osteoblast-specific genes (161). In conclusion, the pathways discussed above provide evidence to use apigenin as a possible intervention in the treatment of bone-related diseases. Figure 9 depicts an overview of the flavonoids that regulate molecular mechanism in bone remodelling.

Puerarin, a natural isoflavone isolated from the Chinese herb Pueraria lobata, exhibits osteogenic effects similar to 17-β-estradiol, suggesting a therapeutic role in the treatment of osteoporosis in the future. Puerarin treatment on rat osteoblasts increased the levels of ALP and stimulated osteoblastic proliferation via a possible mediation of the PI3K/Akt pathway (162). Puerarin alleviated pathological bone graft defects and apoptosis of BMSCs and increased their proliferation and differentiation. Further, it decreased the levels of proinflammatory cytokines and promoted the levels of anti-inflammatory cytokines, thus ameliorating bone loss via inflammation (163). In human osteoblasts (hOBs), treatment with puerarin was shown to inhibit serum-free-induced apoptosis by upregulating the expression of BCL-2 and downregulating the expression of BAX through the activation ERK-1/2 signalling pathway (164). Besides puerarin is well accepted as an autophagy regulator and osteoclastogenesis inhibitor, with the exact role of autophagy in puerarin-regulated osteoclastogenesis still being unclear (165). In RANKL-induced BMMs, osteoclastogenesis was alleviated with puerarin treatment, which inhibited the expression of osteoclastogenic genes and the TRAF6/ROS-dependent MAPK/NF-κB signalling pathway (166). Petunidin, a compound belonging to the flavonoid family anthocyanin has been shown to be a promising natural agent in inhibiting osteoclastogenesis and promoting bone formation. Treating RAW264.7 cells with petunidin significantly inhibited osteoclastogenesis by suppressing the mRNA expression of osteoclastogenic markers c-Fos, NFATc1, MMP9, CTSK, and DC-STAMP. Petunidin stimulated the gene expression of osteogenic markers BMP-2 and OCN, whereas it inhibited mRNA expression of MMP-2, MMP-9, MMP-13 and the proteolytic activities of MMP-9 and MMP-13 in MC3T3-E1 cells (167). The isoflavonoid formononetin has been suggested to be a natural selective estrogen receptor modulator (SERM), and exhibit estrogenic activity on bone cells, thus inhibiting the development of osteoporosis in post-menopausal women (168). A study by Singh et al. revealed that treatment with formononetin on overiectomised (OVx) osteopenic mice repaired the cortical bone defect and promoted bone regeneration accompanied by elevated expression of osteogenic markers BMP-2, RUNX2 and OCN (169). Formononetin treatment on C2C12 progenitor cells remarkably enhanced ALP activity, calcium deposition, and the expression of osteogenesis specific markers including ALP, RUNX2, OCN and BMP isoforms. It was also demonstrated that osteogenic differentiation in these cells treated with formononetin was enhanced by p38 MAPK dependent SMAD 1/5/8 signalling pathways (170). In BMMs, treatment with formononetin regulated OPG and RANKL expression levels, and inhibited RANKL induced TNF-α, IL-1β, IL-6, MCP-1 and macrophage inflammatory protein-1α (MIP-1α). These were accompanied by a reduction in RANKL induced activation of the NF-κB p65 subunit, degradation of IκBα, activation of AKT, ERK, JNK and p38 MAPK (171). In addition, formononetin inhibited classic osteoclastogenic markers significantly. Furthermore, it possesses angiogenic properties required for bone fracture healing and upregulates expression of VEGF and VEGF-R2 (172). Naringenin a dihydro flavonoid compound abundantly found in fruits such as orange, pomelo and drynaria has shown to possess osteogenic effects. In BMSCs, treatment with naringenin upregulated the gene and protein expression levels of ALP, RUNX2, C-X-C chemokine receptor type 4 (CXCR4) and stromal cell-derived factor 1 (SDF-1) via the SDF-1/CXCR4 signalling pathway (173). Calycosin, an isoflavonoid phytoestrogen, significantly suppressed osteoclast formation from BMMs and inhibited the expression of osteoclastogenic markers, including CTSK, TRAP and MMP-9. Moreover, calycosin attenuated the expression levels of NFATc1 and c-Fos via inhibition of activation of NF-kB and MAPKs thereby preventing bone resorption (174). Curcuma longa, a member of the family Zingiberaceae commonly referred to as turmeric, contains an important flavonoid named curcumin. A study by Folwarczna et al. exhibited that curcumin reduced serum estradiol and mineralization, and increased bone formation and histomorphometric properties of the bone (175–178). Curcumin was found to downregulate the Wnt/β-catenin pathway, AKT pathway, BCL-2, NF-kB, COX-2 and activated GSK-3β, thus preventing oxidative stress and inflammatory responses induced by these pathways (177, 179–187). A study by Notoya et al., utilising rat calvarial osteoblast-like cells, showed that curcumin inhibited the proliferation of osteoblasts without induction of apoptosis. This occurred due to the expression of p21 protein, which resulted in the arrest of cell cycle (188). In another study by Yamaguchi et al. with an analogue of curcumin UBS109, it was found to increase SMAD activity, BMP-induced SMAD activation and TGF-β-induced SMAD activation. It was also found to inhibit TNF-α-induced SMAD suppression. This might be crucial to enhance the differentiation of osteoblasts (189). Also, curcumin slightly inhibited the enhancement of RANKL by IL-1α in human bone marrow stromal cells (190). Epigallocatechin-3-gallate is a flavonoid found abundantly in green tea. Epigallocatechin (EGCG) was found to have promote differentiation of osteoblasts in murine BMSCs. In a study by Lin et al., EGCG showed upregulation of osteogenic-related genes including osteocalcin, RUNX2, OCN, ALP and BMP2, resulting in increased mineralization in a cultured mesenchymal stem cell line derived from bone marrow (191). The expression of RUNX2 and OSX, which are important for mesenchymal stromal cells to differentiate to osteoblasts, was increased by EGCG, and thereby resulted in increased osteogenesis (192). Oleuropein, a flavonoid found in green olives and the olive tree, has recently been deeply researched for its multiple health benefits (193). In a study by Santiago-Mora et al. using the periodontitis model in rats, Oleuropein downregulated the genes linked with adipogenesis such as lipoprotein lipase and PPAR‐γ and upregulated the factors promoting osteogenesis such as OCN, RUNX2 and ALP and eventually enhanced osteoblastic differentiation (194). Moreover, oleuropein reduced JNK, p38 MAPK and ERK1/2, and prevented the translocation of NF‐KB from cytosol to nucleus which is important for the activation of NF‐KB (195). Castejon et al. also demonstrated a downregulation of MAPK and NF‐KB pathway, reduced MMP‐3, COX‐2, TNF‐α, MMP‐1 and IL‐6 levels in IL‐1β-induced synovial fibroblast cells by oleuropein (196, 197). OCN and BMP4 are augmented by this flavonoid, and TRAP osteoclasts are inhibited (194, 198). BMP4 is linked with high OPG production, and this leads to a higher rate of osteoblastogenesis (199).