Thiazolidinediones, an antidiabetic drug, promote bone loss and provoke fractures, suggesting a protective role of phytoestrogens rich herbal supplement along with rosiglitazone. The aqueous methanol extract (AME) of Trifolium resupinatum L. var. minus Boiss. (Fabaceae) and defatted AME (DAME) protective effects were assessed against rosiglitazone (Ros) inducing osteoporosis in type 2 diabetic male rats. The bioactive extract was investigated for its phenolics. Eighty male Albino rats (10/group) were used; G1: control, G2: diabetic rats + Ros (10 mg/kgbw/d for 30 days; Ros-rats), G3-G5 and G6-G8: Ros-rats + AME or DAME (24, 12, 6 mg/kgbw/d, each). OPG, RANKL and β2- microglobulin serum levels, femur BMD (Dexa) and OC, COL and ACP5 osteogenic genes mRNA (qRT-PCR) were evaluated. Phenolics were isolated and characterized by chromatographic, chemical and spectral techniques. Their HPLC profiling was also analyzed. Ros-rats + DAME (24 mg/kgbw) revealed the most significant effects on bone biomarkers. It modulated OPG (ng mL-1), RANKL (pg mL-1) and β2- microglobulin (μg mL-1) serum levels with 2.46 ± 0.44, 96.640 ± 06.12, 0.22 ± 0.04, compared to those of Ros--rats (1.62 ± 0.26, 162.42 ± 11.34, 0.46 ± 0.05), respectively. Also, it improved BMD (mg cm-2) of proximal, distal and total femur bones (102.8 ± 8.6, 104.6 ± 10.2, 106.0 ± 9.2), compared to Ros-rats (82.3± 6.4, 81.2 ± 7.2, 82.1 ± 4.8), respectively and up-regulated OC & COL and down-regulated ACP5 expressions. From DAME, 12 phenolics were identified and HPLC profiling showed major phenolics; chlorogenic acid > formononetin> quercetin> pseudobaptigenin. DAME in combination therapy with rosiglitazone provides protective effect on bone, possibly due to its phenolics.
Key words: Persian clover, Thiazolidinediones, diabetes, serum bone markers, bone mineral density, osteogenic genes, phytoestrogens.
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