Osteoporosis (OP) is a disorder characterized by low bone mass and structural deterioration of the bone microarchitecture, and it leads to an increased risk of bone fragility and fracture. Over 75 million people worldwide suffer from OP, of which 80% are postmenopausal women [1
], and there is a direct relationship between the lack of estrogen and OP development [2
]. In women, the two major causes of bone loss are menopause-induced estrogen deficiency and aging. Postmenopausal OP generally develops when, after menopause, estrogen levels drop precipitously. Senile OP is related to aging and it results from deficiencies in dietary calcium and vitamin D, and increased activity of the parathyroid glands. Calcium and vitamin D are implicated in gynecologic and reproductive conditions [3
], and improve vasomotor disturbances, quality of life, and sexual function in menopausal women [5
]. Moreover, high intakes of dietary vitamin D and calcium lowers risk of early menopause compared to low intakes [6
Meyer, which has been used intensively as a traditional herbal medicine for thousands of years [7
], has become a most popular research subject in the field of natural medicine. The active components of P. ginseng
include ginsenosides, polysaccharides, and flavonoids. In particular, approximately 40 types of ginsenosides have been identified and studied for their anticancer, antidiabetic, antioxidant, and immune regulatory effects [7
]. The protective effect of ginseng on glucocorticoid-, inflammation-, radiation-, and ovariectomized (OVX)-induced OP has been reported [12
]. P. ginseng
prevented dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells by lowering caspase-3 and -9 expression and increasing antiapoptotic genes, such as Bcl2 and Inhibitor of Apoptosis Protein [13
]. Moreover, P. ginseng
increased level and activity of alkaline phosphatase, osteocalcin, Runt-related transcription factor, and bone morphogenic proteins, thereby preventing dexamethasone-induced trabecular bone loss. Preventive effect of P. ginseng
and P. notoginseng
on bone loss has been extensively studied in OVX-induced OP model in rats [14
]. P. ginseng
decreased osteoclast (OC) numbers and OP in OVX rats [14
], and saponins from P. notoginseng
prevented OVX-induced decrease in bone mineral density (BMD) and deterioration in bone microarchitecture, and also suppressed OC turnover [15
]. Moreover, ginsenoside Rg1 countervailed the decreased BMD in OVX rats [17
], ginsenoside Rb2 revealed anti-osteoporosis effect by reducing oxidative damage and bone resorbing cytokines in osteoblast cells [19
]. In contrary, ginsenoside Rb1 increased osteogenic differentiation and osteogenic proteins, however, did not halt osteoporotic bone loss in OVX rats [20
]. However, despite accumulating data on the effect of P. notoginseng
on OP in rats, the effect of P. ginseng
on postmenopausal OP is not clearly defined; thus, we investigated the effect of P. ginseng
on this disorder.
(cabbage) is highly nutritious and possess a wide range of bioactive compounds, such as glucosinolates, vitamins, phenols, anthocyanin, and carotenoids [21
]. Approximately 200 types of glucosinolates have been identified in plants [24
], and over 30 types are known to possess anti-cancer activities [25
]. In addition, B. oleracea
has anti-mutagenic, antioxidant, gastric and duodenal ulcer recovery, liver protection, and liver function improvement, and also inhibits microbial reproduction [28
]. Although there are very few reports showing an anti-osteoporotic effect of B. oleracea
, we found a paper showing that B. oleracea
stabilize bone mass after menopause [32
]. We also found protective effect on osteoporosis in rats in a preliminary study (data not shown); thus, we employed B. oleracea
in this study.
Anti-osteoporotic drugs lead to improvements in OP, but they also cause many side effects, including gastrointestinal intolerability, osteonecrosis, osteosarcoma, and cancers [33
]. Therefore, there is a great demand for safe and more efficacious anti-osteoporotic drugs. In this study, we evaluated the effect of P. ginseng
as a safe medicine for long-term use in postmenopausal OP treatment using OVX-induced mice. Our results suggest that co-treatment of P. ginseng
with B. oleracea
restores OVX-induced decreases in bone weight and BMD and inhibits OC formation in vivo and in vitro, and thus can be used for treatment for postmenopausal OP.
2. Materials and Methods
2.1. P. ginseng and B. oleracea Extract Powders and Reagents
Standardized P. ginseng extract powder (Lot No. H1312-6010, ginsenosides: Rg1 + Rb1 + Rg3 > 7.0 mg/g) and B. oleracea extract powder were provided by Korea Ginseng Corporation (Daejeon, Korea). Dried B. oleracea was extracted twice with a 17-fold volume of distilled water for 8 h at 80 °C, filtered with 1-µm pore-size filter, and the filtrate was concentrated. The 17β-estradiol was purchased from Dalim Biotech (Preda®; Seoul, Korea) and heparin was obtained from Kimble Chase (Vineland, NJ, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.
2.2. OVX Operation in Mice
Seven-week-old female C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea) and acclimated to the environment for 1 week prior to experiment initiation. The mice were maintained under pathogen-free conditions at a temperature of 23 ± 1 °C and humidity of 55 ± 5% under a 12-h light/dark cycle. The mice were provided a normal chow diet and water ad libitum during the experimental period. All procedures were conducted in accordance with the institutional guidelines approved by the Animal Care and Use Committee of Inha University (INHA 161214-465), and we followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [37
Sham and OVX operation were conducted on 8-week-old mice. The mice were operated under general anesthesia induced by intraperitoneal injection of 180 µL of ketamine–rompun cocktail containing 50 mg/kg ketamine (Yuhan, Seoul, Korea) and 20 mg/kg rompun (Bayer Korea, Ansan, Korea). The bilateral ovaries were exposed and removed from the mice, and the muscle wall and skin incisions were closed with 6-0 silk sutures (AILEE, Busan, Korea). In the sham-operated group, the ovaries were exposed using the same procedure, but were left intact.
2.3. Pg and Bo Treatment
A pilot study was conducted to determine the administration dose of Pg. OVX mice were treated with 100, 300, and 500 mg/kg Pg daily for 10 weeks. Although not significant, the mice treated with 500 mg/kg/day Pg showed higher recovery of OVX-induced bone loss compared those treated with 100 and 300 mg/kg/day Pg (data not shown). Thus, Pg and Bo were used at a concentration of 500 mg/kg/day in the subsequent experiments.
Mice were randomly assigned to experimental groups (10 mice in each group): Normal (naïve control group without incision), Sham (sham-operated control), OVX (OVX mice treated with vehicle), E2 (positive control treated with 1 mg/kg 17β-estradiol), Pg500 (500 mg/kg Pg), Bo500 (500 mg/kg Bo), Pg:Bo = 1:1 (250 mg/kg Pg + 250 mg/kg Bo), Pg:Bo = 1:3 (125 mg/kg Pg + 375 mg/kg Bo), and Pg:Bo = 3:1 (375 mg/kg Pg + 125 mg/kg Bo). E2, Pg, and Bo dissolved in water were administered orally at a volume of 0.2 mL/mouse once a day. From 1 week of operation, the mice were fed with the test substance daily for 10 weeks.
At the end of the administration period, the mice were anaesthetized by intraperitoneal injection of 180 µL of ketamine–rompun cocktail, and approximately 1 mL of blood samples were collected by cardiac puncture. The mice were sacrificed by cervical dislocation and bones were harvested. The animals were weighed weekly and administration dosages were adjusted according to weight.
2.4. Measurement of Bone Loss in OVX Mice
Animals were sacrificed 10 weeks following sample administration and femurs and tibias were dissected free of soft tissue. Bone weight and length were measured using a digital caliper (Mitutoyo, Kanagawa, Japan). OP is a disease in which the density and quality of bone are reduced, and density of bone is often evaluated by measuring BMD. Bone mineral content (BMC) and BMD of the right femur were analyzed using a Dual-energy X-ray absorptiometry (DXA, Norland Medical Systems, White Plains, GA, USA).
2.5. Histological Preparation and TRAP Staining
The femurs were fixed with 4% paraformaldehyde, decalcified in 10% ethylenediaminetetraacetic acid for 4 weeks, dehydrated in a graded series of 70–100% ethanol washes, and then embedded in paraffin. The paraffin blocks were cut with a microtome (SM 2010R; Leica, UK) to obtain 5-μm-thick tissue sections, which were then stained with hematoxylin and eosin (H&E). The sections were also stained with tartrate-resistant acid phosphatase (TRAP) by using a Leukocyte Acid Phosphatase kit according to the manufacturer’s instruction. Briefly, the sections were washed with phosphate-buffered saline (Hyclone, Logan, UT, USA), and fixed with a fixative solution containing citrate solution, acetone, and 37% formaldehyde. The fixed sections were subsequently incubated with TRAP staining solution for 60 min at 37 °C, and washed twice with water. TRAP-positive multinucleated cells with more than three nuclei were counted as OCs.
2.6. Blood Sampling and Analysis
After a 12-h fasting period, blood samples were collected by cardiac puncture in a heparinized tube, and plasma samples were obtained by centrifugation at 900× g for 5 min at room temperature. Next, plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, and cholesterol were determined using Hitachi 7080 Automatic Analyzer (Tokyo, Japan).
2.7. OC Differentiation In Vitro
RAW 264.7 cells (ATCC, Rockville, MD, USA) were cultured in alpha-minimum essential medium (Hyclone) with 10% fetal bovine serum and 50 ng/mL receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) for 4 days. The medium was replaced with fresh RANKL-containing differentiation medium at day 3. Cells were fixed and stained for TRAP, and formation of multinucleated functional OCs was assessed by counting the number of TRAP-positive cells containing more than three nuclei.
2.8. Statistical Analysis
All data were plotted as means ± standard error of mean (SEM), and group differences were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Statistical significance was defined at p < 0.05.
At menopause, estrogen deficiency impairs normal bone remodeling cycle by increasing osteoclastic resorption activity without a corresponding increase in osteoblastic activity; therefore, the amount of bone resorbed is greater than the amount of bone deposited, leading to a net loss of bone [33
]. OVX causes estrogen deficiency, and an OVX-induced osteoporotic mouse model has frequently been used. OP drugs and hormone replacement therapy lead to improvements, but they cause diverse unfavorable side-effects and need to be determined individual appropriate dose [33
]; therefore, many menopausal women avoid taking these treatments for a prolonged period. Thus, we interested in the efficacy of herbal medicines and functional foods that are expected to have fewer adverse effects than chemical drugs and hormone replacement therapy.
In this study, we investigated the effect of P. ginseng
and B. oleracea
on postmenopausal OP, which was accompanied by decreased estrogen level. After OVX surgery, decreased estrogen level led to increased body weight, and Pg and Bo did not decrease the OVX-induced increase in body weight. This result is contradictory to reports showing the significant body weight restoring effect of P. ginseng
in OVX mice [40
]. P. ginseng
restored the estrus cycle and estrogenic activity of OVX mice and inhibited OVX-induced obesity. Treatment with P. ginseng
extract at 18.0 g/kg/day (36 times higher than Pg500) for 4 weeks showed the greatest decrease in OVX-induced increase in weight gain, whereas ginseng extract at 12.0 g/kg (24 times higher than Pg500) showed no significant effect [40
]. Lee et al. [41
] showed that treatment with 5% P. ginseng
extract (50 g/kg, 100 times higher than Pg500) for 15 weeks resulted in 19% decrease in body weight, compared with that in OVX mice. From these results, we conclude that a certain amount of ginseng uptake is necessary to produce a noticeable effect against OVX-induced increase in weight. However, 36.0 g/kg is equivalent to 2160 g for a woman weighing 60 kg; thus, it is impossible to consume this amount of ginseng extract daily.
Bone mass and BMD are indicators of OP. Treatment with Pg:Bo combination restored OVX-induced bone loss (Figure 3
A), and Pg:Bo = 3:1 recovered BMC and BMD to the levels observed in Sham mice (Figure 3
B,C), indicating a synergistic effect of Pg and Bo in restoring BMC and BMD. There is much controversy on the effect of ginseng on BMD [15
]. Treatment with 300 mg/kg/day P. notoginseng
extract for 13 weeks restored decreased BMD in OVX rats [15
], but treatment with 300~1000 mg/kg/day P. ginseng
mixture for 7 weeks did not restore BMD in OVX rats [18
Proper functioning of OCs, which are responsible for bone resorption, is necessary to maintain bone mass and BMD. The anti-osteoporotic activities of ginseng mixtures and different ginsenosides have been studied using cells and animals [12
]. In this study, single treatment with Pg did not exert significant effect on OC formation; however, treatment with Pg:Bo = 3:1 inhibited OC formation in vivo and in vitro, suggesting that the administration dose of ginseng or its co-administration with a suitable material, in this case Bo, may be an important factor underlying the effect of ginseng on OC formation. Although we did not measure the effect of ginseng on osteoblast function and bone formation, the inhibitory effect of Pg:Bo combination on bone loss appeared to be somewhat related to bone production capacity. Further studies are required to clarify this relationship.
Estrogen deficiency alters glucose metabolism and body fat distribution [45
]; at the postmenopausal period, body weight gain, insulin resistance, hyperlipidemia, and visceral fat accumulation occur. Furthermore, bone metabolism disorder in OP can influence global glucose homeostasis and energy metabolism [46
]. P. ginseng
has been traditionally used to treat diabetes, and ginsenosides are considered as major bioactive components mediating the antidiabetic effect of P. ginseng
]. P. ginseng
reduced the circulating levels of free fatty acids and triglycerides, and normalized hyperinsulinemia and hyperglycemia [41
]. Consistent with these reports, Pg decreased blood glucose level to the levels observed in Sham and Normal mice. The antidiabetic actions of estrogen have been well recognized, and the incidence of diabetes rapidly increases at approximately the time of menopause [51
]; however, in this study, E2
did not lower OVX-induced glucose increase. Although further investigation is required, it is possible that a dominant negative estrogen receptor β2 might be induced and involved in responses to E2
]. Notable increases in ALT and AST levels are considered a sign of liver injury, but they can also indicate damage in another organs. AST and ALT levels increased at postmenopause [53
], and have been reported to be related to insulin resistance [54
]. Thus, we assumed that the increased levels of AST and ALT after operation contributed to the increased glucose level in OVX mice.
In this study, the most prominent effect of Pg on OVX-induced bone loss was shown by Pg:Bo = 3:1 combination. Pg:Bo combination protected OVX-induced bone loss by increasing BMD and inhibiting OC formation. Thus, we proposed that P. ginseng in combination with a suitable material, in this case Bo, might be beneficial in protecting against postmenopausal OP, and that finding the suitable material and determining the dose and ratio of the combination are critical in developing P. ginseng as an effective remedy for postmenopausal OP. However, the extracts of Pg and Bo contain various components whose effects are unknown, so it is not clear which substances are responsible for their action, and the characteristics of signaling mechanism by which these extracts inhibit OP remains to be determined. In addition, more studies, especially randomized controlled trials, are needed to apply the results of this study.