The effects of bee pollen extract on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vitro and in vivo were investigated. Bone tissues were cultured for 48 hr in serum-free Dulbecco's modified Eagle's medium containing either vehicle or water-or ethanol-solubilized extracts (10, 100, or 1000 µg/ml of medium) obtained from the bee pollen of Cistus ladaniferus. Calcium content in the femoral-diaphyseal or -metaphyseal tissues was significantly increased in the presence of water-solubilized extract (100 or 1000 µg/ml) and ethanol-solubilized extract (1000 µg/ml). An increase was also observed in the presence of water-solubilized extract (100 µg/ml) obtained from Fagopyrum esculentum, Camellia sinesis, or Brassica napus L. Alkaline phosphatase activity and DNA content in the femoral-diaphyseal or -metaphyseal tissues in vitro were significantly increased in the presence of water-solubilized extract (100 or 1000 µg/ml) obtained from the bee pollen. The effects of the bee pollen extract (100 µg/ml) in increasing bone components were completely inhibited in the presence of cycloheximide (10 -6 M), an inhibitor of protein synthesis, in vitro. Moreover, the calcium content and alkaline phosphatase activity in the femoral-diaphyseal or -metaphyseal tissues were significantly increased by the oral administration of water-solubilized extracts (5 or 10 mg/100 g body weight) obtained from the bee pollen of Cistus ladaniferus once daily for 7 days. The DNA content in the diaphyseal or metaphyseal tissues was significantly increased by the oral administration of water-solubilized extract (10 mg/100 g) of bee pollen cistus. The dose of 1.0 mg/100 g caused a significant increase in the diaphyseal and metaphyseal alkaline phosphatase activity or the metaphyseal DNA content in vivo. This study demonstrates that the extract of bee pollen has an anabolic effect on bone components in rats in vitro and in vivo.

The effect of various flavonoids, which are present in food and plants, on bone calcium content and osteoclastogenesis were investigated to compare action of flavonoid on bone formation and bone resorption in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose) supplemented with antibiotics and bovine serum albumin. Amoung quercetin, myricetin, kaempferol, isorhamnetin, curcumin, hesperidin, or astaxanthin in the range of 10(-7)-10(-5)M, culture with quercetin (10(-6) or 10(-5)M) caused a significant increase in diaphyseal calcium content. Such an effect was not seen in other compounds. Mouse bone marrow cells were cultured for 7 days in the presence of parathyroid hormone (PTH; 10(-7)M), a bone-resorbing factor, in vitro. Culture with PTH caused a significant increase in osteoclast-like cell formation. This increase was significantly inhibited in the presence of quercetin, myricetin, kaempferol, isorhamnetin, or curcumin in the range of 10(-8)-10(-6)M. Such an effect was not seen in the case of hesperidin or astaxanthin. In addition, culture with PTH (10(-7)M) caused a significant decrease in diaphyseal calcium content. This decrease was completely prevented in the presence of quercetin, myricetin, kaempferal, or isorhamnetin of 10(-6)M. This study demonstrates that various flavonoids have a potent inhibitory effect on osteoclastogenesis and bone resorption rather than bone formation in vitro. Among various flavonoids, quercetin had a stimulatory effect on bone formation and an inhibitory effect on bone resorption in vitro.

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The effect of bee pollen cistus extract on serum and bone biochemical components in streptozotocin (STZ)-diabetic rats was investigated. The water-solubilized extracts were obtained from the bee pollen of Cistus ladaniferus. Rats received a single subcutaneous administration of STZ (6.0 mg/100 g body weight), and then the animals were orally administered water-solubilized extract (5, 10, or 20 mg/100 g body weight) of bee pollen cistus once daily for 14 days. The administration of STZ caused a significant decrease in body weight and a significant increase in serum glucose, triglyceride, and calcium levels, indicating a diabetic state. These alterations were significantly prevented by the administration of the extract (5, 10, or 20 mg/100 g). Serum inorganic phosphorus concentration was significantly decreased in STZ-diabetic rats, and the decrease was significantly prevented after administration of the extract of 10 or 20 mg/100 g. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues were significantly decreased in STZ-diabetic rats. These decrease were significantly prevented after administration of the extract of 10 or 20 mg/100 g. The disphyseal DNA content was also significantly decreased in STZ-diabetic rats. This decrease was significantly prevented after the administration of the extract of 10 or 20 mg/100 g. This study demonstrates that the intake of bee pollen cistus extract has preventive effects on bone loss in STZ-diabetic rats, and that the intake has partial restorative effects on serum biochemical findings with the diabetic state.

The bee pollen Cistus ladaniferus (C. ladaniferus) extract has an anabolic effect on bone metabolism. The effects of the fractionated extracts obtained from bee pollen on bone calcium content and osteoclast-like cell formation in vitro were investigated. Rat femoral-diaphyseal and -metaphyseal tissues were cultured for 48 hr in a medium containing either vehicle or a water-solubilized extract with the membrane fractions obtained from bee pollen. The active component of bee pollen in increasing calcium content in diaphyseal tissues was seen in the fraction of molecular weight (MW) of less than 1000, and it was not observed in fractions of greater than MW 1000. Culture with the fractionated bee pollen extract (25 or 50 µg/ml of medium) of less than MW 1000 caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. The parathyroid hormone (PTH; 10 -6 M)-induced decrease in diaphyseal calcium content was significantly prevented in the presence of the fractionated bee pollen extracts (10 µg/ml) of less than MW 1000 or greater than MW 1000. Mouse marrow cells were cultured for 7 days in a medium containing PTH (10 -6 M) in the presence or absence of the fractionated bee pollen extract (10 or 50 µg/ml). The PTH-induced increase in osteoclast-like cell formation was markedly suppressed in the presence of extracts of less than MW 1000 as compared with that in the presence of fractions of greater than MW 1000. The effects of the fractionated bee pollen extracts of less than MW 1000 in increasing diaphyseal calcium content and in inhibiting PTH-induced osteoclastic cell formation were significantly decreased upon heat treatment for 20 and 60 min at 80°C. This study demonstrates that the active component of bee pollen C. ladaniferus extract, which stimulates bone formation and inhibits osteoclastic bone resorption, is the fraction with MW less than 1000.