Li et al. [21] analyzed the effect of puerarin (5–20 $\mu$M) on the proliferation of osteoblasts from female mice using the MTT assay. They noted that the proliferation of osteoblasts increased after 24 hours of culture. It should be noted that after 48 h and 72 h even higher proliferation was observed but this effect differed depending on the dose (5 $\mu$M, p 0.05; 10 and 20 $\mu$M, p 0.01). Additionally, the authors of the experiment found a statistically significant decrease in RANKL (receptor activator of nuclear factor kappa-B ligand) protein expression in osteoblasts after treatment with puerarin. At the same time, they noted an increase in OPG (osteoprotegerin) protein expression. These results showed that puerarin may inhibit osteoclastogenesis-related processes and thus prevent bone resorption [21]. Similar results were obtained by Yuan et al. [22] who showed that puerarin increased mRNA expression of OPG/GAPD in M3T3-E1 cells and simultaneously decreased RANKL mRNA expression. Wang et al. [23] confirmed that puerarin stimulates osteogenesis in osteoblast-like MC3T3-E1 cells. The observed increase in the viability of MC3T3-E1 cells indicates a significant effect of puerarin on proliferation, while the increase in alkaline phosphatase (ALP) activity (which is a marker of osteoblast activity) shows a stimulating effect of osteoblastic differentiation. However, the most beneficial effect was observed in the case of puerarin administration at 20 $\mu$M. In addition, it has been noted that puerarin may stimulate osteocalcin (a marker of bone formation) secretion in MC3T3-E1 cells. The authors of the experiment suggest that puerarin may stimulate osteogenesis by up-regulating the expression of OPN (osteopontin) and OPG, which constitute bone turnover markers. The results of an experiment by Shan et al. [24] confirmed that puerarin (10–40 $\mu$M) stimulates cell differentiation in MC3TC-E1 cells. As a result of treatment with puerarin, the researchers observed an increase in ALP activity, collagen type 1 secretion as well as osteocalcin secretion. Furthermore, the experiment showed an increase in bone mineralization of MC3T3-E1 cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed an increase in expression of miR-106b after treatment of MC3T3-E1 cells with puerarin. This may indicate the anti-osteoporotic properties of puerarin, as miR-106b can have an inhibitory effect on osteoclastogenesis and osteolysis. A similar effect was observed by Feng et al. [25] who confirmed that puerarin stimulates the viability and differentiation of MC3T3-E1 cells by downregulation of miR-204. An in vitro experiment published in 2012 showed that puerarin does not affect rat osteoblast proliferation and cell viability. On the other hand, it has been found that puerarin (0.01–0.1 $\mu$M) may stimulate osteoblast differentiation. It was observed that cells treated with puerarin had higher ALP activity and Col I (type I collagen) secretion. In addition, this study showed that treatment with puerarin caused a significant increase in phospho-p38 MAPK (mitogen-activated protein kinase) and $\beta$-catenin proteins, indicating that puerarin could increase osteoblast differentiation via p38 MAPK and Wnt/b-catenin pathways [26]. Another experiment proved that puerarin promoted the proliferation and differentiation of human osteoblastic MG-63 cells. The highest puerarin activity was found at a dose of 1 $\mu$M. Additionally, it was observed that puerarin increased ALP activity as well as stimulated collagen synthesis in osteoblastic cells. It was also noted that puerarin inhibited cisplatin-induced apoptosis in MG-63 cells via ER-dependent MEK/ERK and PI3K (phosphatidylinositol 3-kinase-protein kinase B)/Akt activation [27]. Similarly, Zhang et al. [28] concluded and proved that puerarin (2.5–100 $\mu$M) positively affects rat calvaria osteoblastic bone formation through activation of the PI3K/AKt signaling pathway. An experiment by Liu et al. [29] has provided evidence that puerarin may prevent bone loss. The authors of the research noted that puerarin protected human osteoblasts from serum-free-induced apoptosis. The highest effectiveness was found for puerarin concentration of 10${}^{-8}$ M. Additionally, treatment with puerarin caused an increase in Bcl-2 (B-cell lymphoma 2) proteins while decreasing the expression of Bax in osteoblasts. It was also found that the anti-apoptotic effect was mediated by the ERK signaling pathway. Wang et al. [30] proved that puerarin (0.01–1 $\mu$M) stimulated the production of osteoprotegerin by human osteoblastic MG-63 cells. It should be noted that the increase in OPG expression caused by puerarin was comparable to the effect obtained with the use of 17$\beta$-estradiol. Simultaneously, puerarin inhibits the production of RANKL and IL-6 (interleukin 6), which may indicate the inhibition of bone resorption. The researchers also showed that puerarin stimulates ER-alpha, ER-beta, and steroid hormone receptor coactivator expression, which proves that puerarin acts via the ERE (estrogen response elements) pathway. Tiyasatkulkovit et al. [31] further proved that both puerarin (1000 nM) and Pueraria mirifica (PM) extract (100 $\mu$g/mL may increase the expression of ALP over type I collagen in primary baboon osteoblasts. At the same time, they did not observe the effect of puerarin and PM on other markers of bone formation: Runx2 (run-related transcription factor 2), osteocalcin and osterix. Additionally, the authors of the experiment noted that both puerarin and PM extract lowered the levels of RANKL mRNA expression (bone resorption marker). However, they did not cause any changes in OPG expression. An increase in ALP activity after treatment with puerarin was also observed in a study performed by Wang et al. [32] The highest ALP activity was found after the application of puerarin at 10${}^{-6}$ mol/L. At the indicated dose of puerarin, the most significant positive effect on the number of mineralized modules (5.64) was also noted. It should also be noted that too high doses of puerarin (10${}^{-3}$ mol/L) may inhibit ALP activity, osteoblast differentiation, reduce the formation of mineralized osteoblast nodules and, as a result, adversely affect bone formation. In turn, Wang et al. [32] proved that puerarin can stimulate proliferation and differentiation of osteoblasts in a high-glucose environment. This is an important fact because high glucose levels can disturb the balance between osteoclastic/osteogenic activity. Consistent with the obtained results, scientists suggest that puerarin may be a promising agent in the treatment of diabetic osteoporosis (DOP) [33].

An experiment conducted by Yuan et al. [22] has proven that puerarin (10${}^{-7}$–10${}^{-6}$ mol/L) may inhibit osteoclast formation induced by RANKL. The researchers observed that RAW264.7 cells treated with puerarin formed a lower number of TRAP-positive cells (tartrate resistant acid phosphatase). One study showed that puerarin (10–50 $\mu$M) inhibited LPS-induced (lipopolysaccharide) differentiation from osteoclast precursor RAW264.7 cells. According to the researchers, puerarin was significantly capable of inhibiting the production of pro-inflammatory cytokines: PGE2 (prostaglandin E2), IL-1$\beta$ (interleukin 1$\beta$) and TNF-$\alpha$ (tumor necrosis factor alpha), which may stimulate osteoclast differentiation. In consequence, a statistically significant decrease in mRNA expression of TRAP, cathepsin K and MMP-9 (metalloproteinase 9) occurred. At the same time, it was observed that puerarin inhibited the activation of Akt in RAW264.7 cells, thus preventing osteoclastogenesis [34]. The studies conducted by Yang et al. [35] provided conclusions about the beneficial effect of puerarin (1–25 $\mu$M) on the inhibition of RANKL-induced osteoclast activation in bone marrow-derived macrophages. The researchers observed that the cells treated with puerarin formed a lower number of TRAP-positive cells. Additionally, it was observed that puerarin decreased the expression of osteoclast-related genes including cathepsin K, a nuclear factor of activated T cell cytoplasmic 1 and calcitonin receptor. The observed effects differed depending on the dose, and the most beneficial results were obtained after treatment with puerarin at a dose of 25 $\mu$M. Based on these data, puerarin was found to be able to inhibit osteoclast formation [35]. The effect of puerarin on the inhibition of osteoclastogenesis was also confirmed by Lin et al. [36]. They observed that puerarin caused the inhibition of migration of osteoclast precursors (OCP) by blocking the production of monocyte chemotactic protein-1 (MCP-1). It should be mentioned here that MCP-1 is a key mediator of osteoclastogenesis and plays a role in bone resorption.

Li and Peng proved that puerarin (10–100 $\mu$M) stimulated the proliferation of human periodontal ligament stem cells. This effect differed depending on the dose and the increase in puerarin concentration (100 $\mu$M) was correlated with greater proliferation. Additionally, they observed that puerarin contributed to ALP activity. This research also showed that puerarin increased the osteogenic differentiation of periodontal ligament stem cells [37]. Wang et al. [38] noted that the osteocalcin level and ALP activity in bone marrow cells treated with ethanol + puerarin (0.01 mg/mL) were higher compared to cells treated with ethanol only. The results obtained show that puerarin may protect bone marrow stromal cells from alcohol-induced osteonecrosis [38]. The essence of the presented results of in vitro studies on puerarin is presented in Table 1 (Ref. [21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38]).

Li et al. [21] proved the anti-osteoporotic activity of puerarin 6-O-xyloside. They showed that ovariectomized rats (OVX) treated with puerarin (intraperitoneal injection) (20, 40, 60 mg/kg/d) had significantly higher blood calcium levels compared to the control OVX group. Additionally, higher blood phosphorus levels were observed in animals treated with puerarin. However, this effect was differed depending on the dose. The highest level of the indicated components was found with the administration of puerarin at 60 mg/kg/d. This research also assessed ALP activity. It was observed that the use of puerarin at doses of 40 or 60 mg/kg/d increased serum levels of ALP. At the same time, an increase in osteoprotegerin levels was observed [21]. Another study that proved the anti-osteoporotic effect of puerarin was conducted by Michihara et al. [39]. They showed that in ovariectomized mice during 8-week oral (per os) administration of puerarin (5 mg/kg/d) urinary DPD (doxypyridinoline) levels were lower compared to mice that did not consume this isoflavonoid. In the conducted experiment it was observed that the control group had a significantly lower number of femoral trabeculae than the OVX-puerarin group, which indicates that puerarin may protect the trabecular structure from destruction. In the group fed with puerarin a significantly lower activity of TRAP, which is a bone absorption marker, was also found. It should be noted, however, that the administration of puerarin did not affect the level of osteocalcin. Based on the observed lack of effect of puerarin on morphology and weight of oviducts and uterus, the researchers indicated that the observed properties are independent of the estrogen receptor-mediated pathway. They also proved this effect by observing that puerarin did not stimulate the growth of MCF-7 cells [39]. Similarly, Tanaka et al. [40] noted that puerarin may inhibit ovariectomy-induced bone loss. In the experiment, the researchers administered kudzu vine ethanol extract (PVEE) per os to female mice. Of isoflavones present in it, puerarin was found in the highest amounts (52%). This extract was administered at 20 mg/kg/d for 8 weeks. The authors of this research showed that animals which consumed PVEE had lower levels of bone resorption markers such as DPD and TRAP compared to OVX mice. At the same time, it was noted that the use of PVEE inhibited the reduction in femoral bone mineral density. A lower number of matured osteoclasts in the distal femur was also observed in the group treated with PVEE. On the other hand, administration of PVEE did not affect the serum bone-specific alkaline phosphatase activity. At the same time, Liang et al. [41] noted that puerarin may have a beneficial effect on bones in rats with diabetes. It was observed that daily intraperitoneal administration of puerarin (60 mg/kg/d) caused a decrease in caspase-3 expression in osteoblasts. The authors of the experiment suggest that high blood glucose levels are associated with an increase in the expression of caspase-3, which in turn may contribute to osteoblast apoptosis and lead to the development of osteoporosis. Moreover, Luo et al. [42] proved that oral administration of kudzu root extract (0.45, 0.9 and 1.8 g/kg/d) to ovariectomized rats may have a beneficial effect on bone health. In the conducted studies it was observed that the use of kudzu root extract resulted in lowering the serum levels of C-terminal telopeptide of collagen type I (CTx). This indicates that this extract may inhibit bone resorption. Additionally, it has been shown that the administration of P. lobata extract (100 mg/kg/d) results in a statistically significant reduction in the levels of osteocalcin as well as ALP and CTx, which are markers of bone resorption and remodeling [43]. It is also worth mentioning that studies conducted by Wang et al. [38] on an animal model showed that puerarin may inhibit alcohol-induced osteonecrosis. Marrow and bone necrosis were found in mice belonging to the model group treated with ethanol (intragastric). An increase in the number of empty osteocyte lacunae in the femur was also observed. Such adverse lesions were not found in mice treated with intramuscular injection of puerarin (0.5 g/kg/d). Additionally, higher expression of osteocalcin mRNA was found in animals treated with puerarin [38]. Research results published in 2020 also indicated that puerarin (15.4 and 30.8 mg/kg/d) administered i.p. inhibits titanium particle-induced osteolysis [44]. Zhang et al. [34], on the other hand, proved that calvarial injection of puerarin (1 mg/kg/d) is capable of inhibiting LPS-induced bone loss. The authors found that after the injection of LPS there was a significant increase in the number of osteoclasts and an increase in the area of osteolysis. At the same time, significant reductions in calvaria weight were observed. Mice additionally treated with puerarin had a significantly lower number of osteoclasts as well as decreased osteolysis area. Lower bone mass loss, compared to the puerarin-free group (68.5 vs. 47.9 mg of calvaria weight), was observed as well [45]. The beneficial effect of Pueraria lobata extract (PE) was also confirmed by Lee et al. [46]. They observed that the administration of PE (25–1600 mg/kg) to ovariectomized (OVX) rats resulted in a reduction in the level of bone turnover markers, such as osteocalcin, C-terminal telopeptide fragment of type I collagen, deoxypyridinoline, and pyridinoline, the concentration of which was elevated in OVX rats. The highest effect was seen in animals receiving 1600 mg PE/kg. Moreover, researchers confirmed that puerarin caused an increase in the plasma level of estradiol, which indicates that PE has estrogenic activity and its activity is similar to that of phytoestrogens. Therefore, it seems that puerarin may slow down the osteoporotic changes associated with estrogen deficiency, which is characteristic in postmenopausal women [46].

An experiment by Tanaka et al. [40] showed that ovariectomized mice fed with kudzu vine ethanol extracts (PVEE) had a lower degree of trabecular destruction than mice belonging to the control OVX-group. The beneficial effect of puerarin on bones was also noted in a research conducted by Yuan et al. [22]. Ovariectomized mice received a diet containing puerarin at different doses: 2, 4 or 8 mg/d. The authors observed that mice receiving food enriched with puerarin (2 mg/d) had a higher mineral density of various regions of the femur – proximal femur, middle femur, distal femur compared to animals fed without this additive [22]. In an experiment conducted by Liang et al. [41], femur X-rays showed that animals treated with puerarin had a lower loss of bone mass compared to those not treated with this additive. It was shown that femoral BMD (bone mineral density) in rats treated with puerarin was 9.7% higher than in the control group. Moreover, rats with diabetes had a higher number of osteoclasts clumped together as well as higher cortical bone reduction. In rats treated with puerarin, these lesions were milder [41]. Similar results were also obtained by Cho et al. [47] who administered isoflavones from Pueraria lobata per os to ovariectomized mice. Of isoflavones found in P. lobata extract, puerarin was present at the highest concentration (7.5%). Daidzein (4.2%) and genistein (1.9%) were present in smaller amounts. The researchers found that mice fed with P. lobata extract (200 and 500 mg/kg/d) had significantly higher femoral BMD than those fed without this additive [47]. Similarly, in another study, researchers showed that rats consuming the kudzu root had higher BMD of the middle femur [42]. In turn, Wang et al. [26] proved that puerarin stimulates bone formation. They showed that ovariectomized rats treated with puerarin (i.g.; 20 mg/kg/d) had a higher femur BMD and BMC (bone mineral content). The positive effect of Pueraria mirifica administration on bone health was also noted by Suthon et al. [48]. The researchers showed that rats treated with P. mirifica (50 mg/kg/d) by gavage had a higher total BMD of L4 (fourth lumbar vertebra), as well as trabecular BMD of L4 and tibial metaphysis, compared to rats not treated with isoflavones. The effect of dietary isoflavones from Puerariae Radix on bone metabolism in ovariectomized rats was also studied by Lim et al. [43]. The researchers administered orally P. lobata extract, containing high amounts of isoflavones, to rats. Puerarin constituted 57.6% of all isoflavones. The remaining isoflavones were daidzein (30.4%) and genistein (12.0%). They found that rats treated with the extract at 100 mg/kg/d had a significantly higher femoral bone mineral density compared to the control OVX-group. Such an effect was not observed in the group treated with P. lobata extract at 30 mg/kg/d. Yang et al. [49] showed that rats with ligature-induced periodontitis and treated with puerarin at 200 mg/kg/d by gavage had a lower volume of bone loss compared to those not treated with isoflavonoids. In this research, it was noted that the consumption of puerarin inhibited alveolar bone loss by inhibiting RANKL production and reducing the number of active osteoclasts [49]. The experiment performed on healthy male C57BL/6J mice showed that the intraperitoneal administration of puerarin (10 and 50 mg/kg/d) caused inhibition of osteolysis induced by titanium particles. The authors of the research noted that mice treated with puerarin had higher BMD and BV/TV (bone surface/total volume). However, this effect was dose-dependent, and a more beneficial effect was observed when a higher dose of puerarin was used. In addition, micro-CT scanning showed a lower number of pores in mice treated with puerarin. The researchers also noted a statistically significant reduction in eroded surface area, in the number of TRAP-positive cells, as well as in the osteoclast surface to bone surface ratio [49]. Interesting research results were observed by Liu et al. [50], who noted that administration of puerarin combined with zinc (50 mg/kg/d + 0.25 mg/kg/d) by gavage could prevent mandibular bone loss in OVX rats more effectively than administration of these compounds separately. The experiment showed that rats treated with puerarin + zinc had statistically significantly higher bone mineral density compared to rats fed without these additives, as well as compared to those consuming puerarin + zinc separately. In contrast, studies performed on adult ovariectomized rats showed that rats treated with puerarin (administered i.p.) (50 mg/kg/d) from Pueraria lobata had a lower BMD of the proximal tibia compared to animals not treated with this isoflavonoid. This means that puerarin was not capable of reducing loss of BMD induced by ovariectomy [47]. In 2020, the results of a meta-analysis on the effect of puerarin administration on bone mass for OVX-induced postmenopausal osteoporosis in a murine model were published. Based on eight randomized studies, the authors showed that the use of puerarin can have a beneficial effect on the increase in bone mineral density [51]. The beneficial effect of puerarin on bones was also noted by Li et al. [52]. They showed that administration of puerarin at 100 mg/kg/d resulted in a statistically significant increase in cortex and trabeculae BMD. Simultaneously, the authors of the experiment noticed that in the tested rats there was an improvement in the integrity of the intestinal mucosa. Moreover, positive changes in gut microbiota were noted. For example, Lactobacillaceae and Bifidobacteriaceae bacteria were found to grow. Additionally, puerarin increased the content of SCFAs. The authors suggested that puerarin may prevent osteoporotic changes by modulating the community of gut microbiota and repairing intestinal mucosal integrity [52]. The essence of the presented results of puerarin studies in animal models is presented in Table 2 (Ref. [21, 22, 26, 34, 35, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56]).

Li et al. [21] observed that the administration of puerarin 6-O-xyloside to rats had a beneficial effect on bone microarchitecture. Compared to the control OVX group, in animals treated with puerarin, the cortical and trabecular bone were thickened. It should be noted that the level of pathological lesions in femoral bone tissue was dependent on the dose of puerarin administered. The least adverse lesions occurred after administration of 60 mg puerarin/kg/d [21]. Urasopon et al. [53] conducted research in which they administered Pueraria mirifica to rats after orchidectomy. They found that the enrichment of food with P. mirifica inhibited bone loss in trabecular and cortical bones. It should be stressed that this effect was dependent on the dose of P. mirifica. The most positive results were achieved in the group of rats treated with the highest amounts of P. mirifica (1000 mg/kg/d) [53]. Wang et al. [26] showed that the administration of puerarin to rats resulted in the improvement of trabecular bone structure. They noted that both trabecular number and trabecular thickness were significantly higher in the OVX + puerarin group compared to the OVX group [26]. The beneficial effect on bone histomorphometry was also confirmed by Suthon et al. [48]. They observed that rats treated with P. mirificashowed an improvement in such parameters as bone volume (BV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and osteoblast surface (Ob.S) [48]. Similarly, Lim et al. [43] observed that puerarin can effectively improve the histomorphometry parameters. It was noted that the proximal tibia had significantly higher trabecular number (Tb.N) as well as BV/TV. However, no difference was found for such parameters as bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th) and Tb.Sp. This research also showed an increase in plasma osteocalcin level in the group treated with puerarin, with a simultaneous slight decrease in alkaline phosphatase activity [43]. Additionally, the bone morphometry of the trabeculae of mandibles proved that animals treated with puerarin + zinc showed higher values for such parameters as BV/TV and Tb/Tb.N. At the same time, a significant reduction in Tb.Sp compared to OVX + puerarin and OVX + zinc group was noted. The authors also compared the levels of serum bone biochemical markers. They showed significantly higher calcium levels and simultaneously lower levels of TRAP and RANKL. The results of this study showed that the combined use of puerarin and zinc may be an effective way to inhibit osteoclastogenesis [50]. Xiao et al. [54] confirmed that puerarin protects against bone mass loss in ovariectomy-induced osteoporosis model mice. They reported that the animals that received puerarin had improved the bone structural features. The following parameters were improved: BV/TV, BS/BV, BS/TV, Tb.N. The authors of the experiment proved that puerarin has a bone protective effect by suppressing osteoclastogenesis via inhibition of the TRAF6/ROS-dependent MAPK/NF-$\kappa$B signaling pathway [54]. Interesting observations were made by Guo et al. [55], who proved that puerarin inhibits the osteoporotic changes associated with streptozotocin (STZ)-induced diabetes. The authors observed that puerarin improves bone microstructure. Higher BV/TV and Tb.N values were noted in rats receiving puerarin. At the same time, the reduction in Tb.Sp was noted. Moreover, lower levels of OPG and CTX were found. The scientists indicated that this effect may be related to suppressed inflammation [55]. Anti-osteoporotic activity was also demonstrated by Pueraria lobata fermented (FPE) with Lactobacillus paracasei JS1. Researchers observed that ovariectomized mice treated with FPE were characterized by improved bone architecture. BV/TV, Tb.Th, and Tb.N were higher compared to OVX group. At the same time, a lower value of Tb.Sp was noted. Additionally, an increase in bone mineral density was noted in mice receiving FBE [56].