A standardized extract of Coleus forskohlii root protects rats from ovariectomy-induced loss of bone mass and strength, and impaired bone material by osteogenic and anti-resorptive mechanisms

Abstract

Introduction: In obese humans, Coleus forskohlii root extract (CF) protects against weight gain owing to the presence of forskolin, an adenylate cyclase (AC) activator. As AC increases intracellular cyclic adenosine monophosphate (cAMP) levels in osteoblasts that has an osteogenic effect, we thus tested the skeletal effects of a standardized CF (CFE) in rats.

Methods: Concentrations of forskolin and isoforskolin were measured in CFE by HPLC. CFE and forskolin (the most abundant compound present in CFE) were studied for their osteogenic efficacy in vitro by alkaline phosphatase (ALP), cAMP and cyclic guanosine monophosphate (cGMP) assays. Femur osteotomy model was used to determine the osteogenic dose of CFE. In growing rats, CFE was tested for its osteogenic effect in intact bone. In adult ovariectomized (OVX) rats, we assessed the effect of CFE on bone mass, strength and material. The effect of forskolin was assessed in vivo by measuring the expression of osteogenic genes in the calvarium of rat pups.

Results: Forskolin content in CFE was 20.969%. CFE increased osteoblast differentiation and intracellular cAMP and cGMP levels in rat calvarial osteoblasts. At 25 mg/kg (half of human equivalent dose), CFE significantly enhanced calcein deposition at the osteotomy site. In growing rats, CFE promoted modeling-directed bone formation. In OVX rats, CFE maintained bone mass and microarchitecture to the level of sham-operated rats. Moreover, surface-referent bone formation in CFE treated rats was significantly increased over the OVX group and was comparable with the sham group. CFE also increased the pro-collagen type-I N-terminal propeptide: cross-linked C-telopeptide of type-I collagen (PINP : CTX-1) ratio over the OVX rats, and maintained it to the sham level. CFE treatment decreased the OVX-induced increases in the carbonate-to-phosphate, and carbonate-to-amide-I ratios. CFE also prevented the OVX-mediated decrease in mineral crystallinity. Nanoindentation parameters, including modulus and hardness, were decreased by OVX but CFE maintained these to the sham levels. Forskolin stimulated ALP, cAMP and cGMP in vitro and upregulated osteogenic genes in vivo.

Conclusion: CFE, likely due to the presence of forskolin displayed a bone-conserving effect via osteogenic and anti-resorptive mechanisms resulting in the maintenance of bone mass, microarchitecture, material, and strength.

Keywords: Coleus forskohlii; bone material and quality; histomorphometry; micro-computed tomography; new bone formation; osteotomy; post-menopausal osteoporosis.

Figure 1

CFE stimulated osteoblast differentiation, cAMP and cGMP in vitro and promoted bone regeneration at the femur osteotomy site. (A) RCO were treated with CFE, and differentiation was assessed by ALP assay. (B) RCO were treated with CFE at the indicated time points and cAMP and (C) cGMP production were measured. (D) Adult female rats were treated with vehicle and CFE at indicated doses after femur osteotomy for 12 days, and representative images (10X) of calcein deposition at the osteotomy site are shown. (E) Quantification of the calcein deposition data are presented (n = 6 bones/group). All data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p<0.001 vs. vehicle.

Figure 2

CFE promoted new bone formation in growing rats. (A) Femur length. (B) Representative μCT images (left panel) and quantitative μCT parameters of the tibia metaphysis (right panel). %BV/TV, percent bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness. (C) Representative μCT images (upper panel) and quantitative μCT parameters of the femur and tibia diaphysis (lower panel). Ct.Th, Cortical thickness and B.Ar, bone area. (D) Left panel showing the representative images of double calcein labeling (scale bar, 100 µm) and histomorphometry parameters (right panel) of the indicated groups. (E) 3-point bending strength of femur was determined by a bone-strength tester. All data are expressed as mean ± SEM (n = 6 bones/group); *p <0.05, **p <0.01, and ***p<0.001 vs. vehicle-treated group.

Figure 3

CFE prevented bone loss in osteopenic rats. (A) Representative images of tibia metaphysis, and L5 vertebrae are shown. (B) Shown are the quantitative μCT parameters of the tibia metaphyses, and L5. BMD, bone mineral density; Tb.Sp, trabecular spacing; Conn.D., connectivity density; and SMI, structure model index. (C) The L5 compression strength was determined by a bone-strength tester. All data are expressed as mean ± SEM (n = 6 bones/group); *p <0.05, **p <0.01, and ***p<0.001 vs. sham.

Figure 4

CFE has an osteoanabolic effect in osteopenic rats. (A) Upper panel showing representative images (scale bar, 100 µm) of single and double calcein labeled bone surfaces at tibia metaphysis; white arrows- single label and yellow arrows- double label surfaces; and the lower panel showing the histomorphometry parameters in the indicated groups. (B) Ex vivo mineralization assay was performed in bone marrow stromal cells obtained from the indicated groups. (C) Serum procollagen type I N-propeptide (PINP), cross-linked C-telopeptide of type I collagen (CTX1) levels and their ratio were determined by ELISA from the serum of rats with indicated treatments. For ELISA, serum samples of n = 6 rats from each group were taken. For ex vivo mineralization femurs and for histomorphometry tibia sections of n = 3 rats from each group were used. All data are expressed as mean ± SEM; *p <0.05, and ***p<0.001 vs. sham.

Figure 5

Forskolin has osteogenic effect in vitro and in vivo. (A) RCO were treated with forskolin at the indicated concentrations and differentiation was assessed by ALP assay. (B) RCO were treated with forskolin (10nM) for the indicated time points and intracellular cAMP and (C) cGMP production were measured. (D) Rat pups (1-day old) were injected with forskolin at the indicated doses for 5 consecutive days and the relative expression of osteogenic genes in the calvarial tissue were measured. All values are expressed as mean ± SEM; *p <0.05, **p<0.01 and ***p<0.001 vs. sham.