Osteoporosis represents an imbalance between bone formation and
bone resorption. As a result of low
estrogen levels, it is markedly prevalent during menopause, thus making such patients susceptible to fractures. Both bone formation and resorption are modulated by
nitric oxide (NO). Currently, there are no risk-free
pharmaceutical prevention
therapies for
osteoporosis. COMB-4, a nutraceutical combination of Paullinia cupana, Muira puama, ginger, and L-
citrulline, known to activate the NO-cGMP pathway, was reported to accelerate fracture healing in the rat. To determine whether COMB-4 could be effective in preventing menopausal
osteoporosis, it was compared to
estradiol (E2) in an ovariectomized (OVX) rat
osteoporosis model. Nine-month-old female Sprague Dawley rats were divided into
SHAM, OVX, OVX+E2, and OVX+COMB-4. After 100 days of treatment, bone mineral density (BMD) and bone mineral content (BMC) were measured by DXA scan. TRAP staining was performed in the femur and lumbar vertebrae.
TRACP 5b and
osteocalcin levels were assayed in the serum. MC3T3-E1 cells were differentiated into osteoblasts and treated with COMB-4 for one week in order to evaluate
calcium deposition by
Alizarin staining, cGMP production by ELISA, and upregulation of the
nitric oxide synthase (NOS)
enzymes by RT-PCR. OVX resulted in a decrease in BMD, BMC, and serum
osteocalcin and an increase in serum
TRACP 5b. Except for an increase in BMC with COMB-4, both E2 and COMB-4 reverted all bone and
serum markers, as well as the number of osteoclasts in the vertebrae, to
SHAM levels. Incubation of MC3T3-E1 cells with COMB-4 demonstrated an increase in the three NOS
isoforms, cGMP, and
calcium deposition. COMB-4 increased BMD in OVX rats by inhibiting
bone resorption and increasing
calcium deposition presumably via activation of the NO-cGMP pathway. It remains to be determined whether COMB-4 could be a potential nutraceutical
therapy for the prevention of premenopausal bone loss.