Analysis of constituents of the eastern Nigeria mistletoe, Loranthus micranthus linn revealed presence of new classes of osteogenic compounds.

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE Mistletoe extracts (decoctions) are used traditionally in eastern Nigeria for the management of bone pain, post menopausal syndrome and diabetes amongst several other ailments. While scientific evidence supporting its folkloric use as an antidiabetic agent has been documented, the age-long practice of its use in treatment of post menopausal syndrome has not been scientifically validated. Postmenopausal osteoporosis accounts for one of the prevalent disease conditions in aging population globally. This situation is exacerbated by the lack of osteogenic therapy. In search for plants of Nigerian origin with osteogenic potential, we evaluated eastern Nigerian mistletoe, having ethnotraditional claims of anti-diabetic, anti-hypertensive and anti-cancer activities as well as preventive effect in various post-menopausal syndromes. MATERIALS AND METHODS: Methanolic extracts of mistletoe leaves harvested from three host tress - Kola acuminata (KM), Citrus spp (CM) and Garcinia kola (GKM) - were evaluated for osteoblast viability and osteogenic activities using primary rat calvaria culture. Lupeol (1) was isolated from the stem bark of Bombax ciba and its congener, dihydoxylupeol palmitate (2) in addition to three other compounds; 3-methoxy quercetin (3), 3,4,5-trimethoxy gallate (4), and friedelin (5) were isolated from the leaves of mistletoes species. Following their chemical characterization, the compounds were evaluated for osteogenic potential using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes - bone morphogenetic protein-2 (BMP2) and osteoblast transcription factor (RUNX2) - in primary calvarial cultures harvested from neonatal rats. Uterine estrogenicity of the extracts was tested in adult female Sprague Dawley rats. RESULTS: Methanol extracts of mistletoe from three hosts exhibited increase in ALP activity (a marker of osteoblast differentiation) at lower concentrations (0.2-0.8 µg/ml) and either no or inhibitory effect at higher concentrations (1.6 and 3.2 µg/ml). None of the extract had cytotoxicity to osteoblasts at the concentrations tested. Five compounds viz. 1 from Bombax ciba, and 2-5 were isolated from the mistletoe leaves. Out of these, 5 exhibited significant loss of osteoblast viability and hence it was not considered further. All four compounds exhibited stimulatory effects on osteoblast differentiation as assessed by ALP assay and determination of osteogenic gene expression. Compound 2 was relatively more potent than its precursor, compound 1 in stimulating BMP2 upregulation. KM did not show uterine estrogenicity. CONCLUSION: Methanolic extracts from the three mistletoes species possess in vitro osteogenic activity, and from these extracts three new classes of compounds have been found to promote osteoblast differentiation in vitro. In light of these findings, we propose that mistletoe species may be developed as safer alternative(s) in the management of diseases where lack of bone formation is the pathology.