Effects of long-term administration of royal jelly on pituitary weight and gene expression in middle-aged female rats.

To determine the effects of ingested royal jelly (RJ) on the pituitary in middle-aged female rats, we performed a long-term RJ administration test. Several animals showed age-related increases in pituitary weight, and RJ administration compensated for the increase. RJ tended to down-regulate prolactin mRNA and up-regulated thyroid-stimulating hormone beta mRNA in the pituitary. This suggests that RJ compensates for age-associated decline in pituitary functions.

Estrogenic activities of Fatty acids and a sterol isolated from royal jelly.

We have previously reported that royal jelly (RJ) from honeybees (Apis mellifera) has weak estrogenic activity mediated by interaction with estrogen receptors that leads to changes in gene expression and cell proliferation. In this study, we isolated four compounds from RJ that exhibit estrogenic activity as evaluated by a ligand-binding assay for the estrogen receptor (ER) beta. These compounds were identified as 10-hydroxy-trans-2-decenoic acid, 10-hydroxydecanoic acid, trans-2-decenoic acid and 24-methylenecholesterol. All these compounds inhibited binding of 17beta-estradiol to ERbeta, although more weakly than diethylstilbestrol or phytoestrogens. However, these compounds had little or no effect on the binding of 17beta-estradiol to ERalpha. Expression assays suggested that these compounds activated ER, as evidenced by enhanced transcription of a reporter gene containing an estrogen-responsive element. Treatment of MCF-7 cells with these compounds enhanced their proliferation, but concomitant treatment with tamoxifen blocked this effect. Exposure of immature rats to these compounds by subcutaneous injection induced mild hypertrophy of the luminal epithelium of the uterus, but was not associated with an increase in uterine weight. These findings provide evidence that these compounds contribute to the estrogenic effect of RJ.

A dipeptide YY derived from royal jelly proteins inhibits renin activity.

Renin is the rate limiting enzyme in the renin-angiotensin (RA) system that regulates blood pressure and electrolyte balance. In this study, we investigated the renin inhibitory effect of a royal jelly (RJ)-derived peptide. A dipeptide YY was isolated from the digested fraction of RJ proteins by proteases and was found to inhibit human renin activity. The inhibition constant (Ki) of YY was estimated to be 10 microM when the Km was 0.16 microM using sheep angiotensinogen as the substrate. The peptide was observed to lower blood pressure in spontaneously hypertensive rats.

Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines.

Royal jelly (RJ) has diverse physiological and pharmacological functions. We observed its weak estrogenic activity in the previous study. RJ stimulated the proliferation of mouse osteoblast-like MC3T3-E1 cells at 0.1 mg/ml, and the effect was blocked by the specific estrogen receptor antagonist ICI 182,780. The addition of 0.1-1.0 mg/ml RJ enhanced collagen production in culture medium. Oral administration of RJ to normal female mice for 9 weeks increased the ash content of their tibiae. DNA microarray analysis revealed significant changes in gene expression related to extracellular matrix formation when the femurs of mice fed RJ were analyzed. Quantitative reverse transcriptase-PCR (RT-PCR) confirmed up-regulation of procollagen I alpha1 gene expression. These data suggest that RJ as a whole or some of its individual components stimulates production of type I collagen and other activities for bone formation through action on osteoblasts.

Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells.

We have previously reported that Brazilian propolis extracts inhibited growth of HL-60 human myeloid leukemia cells, which is partly attributed to the induction of apoptosis associated with granulocytic differentiation. In this study, we isolated three compounds which induce granulocytic differentiation evaluated by nitroblue tetrazolium (NBT)-reducing assays from the water extract of propolis and identified as 4,5-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 3,4-di-O-caffeoylquinic acids by NMR analysis. Cell growth inhibitory activity of these caffeoylquinic acids was found in HL-60 cell, which was mainly attributed to the induction of apoptosis. Furthermore, the potency of caffeoylquinic acid derivatives to induce granulocytic differentiation was examined in HL-60 cells. Caffeic, quinic, and chlorogenic acids had no effects on the NBT-reducing activity, while 3,4,5-tri-O-caffeoylquinic acid induced more than 30% of NBT-positive cells. These results suggest that the number of the caffeoyl groups bound to quinic acid plays an important role in the potency of the caffeoylquinic acid derivatives to induce granulocytic differentiation. This is the first report demonstrating that the caffeoylquinic acid derivatives induce granulocytic differentiation of HL-60 cells.

Royal jelly has estrogenic effects in vitro and in vivo.

Royal jelly (RJ) from honeybees (Apis mellifera) is traditionally thought to improve menopausal symptoms. The potential estrogenic activities of RJ were investigated using various approaches. RJ competed for binding of 17beta-estradiol to the human estrogen receptor alpha and beta but its affinities were weak compared with diethylstilbestrol and phytoestrogens. The reporter gene expression assays suggested that 0.1-1 mg/ml RJ activated estrogen receptors, leading to enhanced transcription of a reporter gene through an estrogen-responsive element. 1 mg/ml RJ stimulated the mRNA expression of estrogen-responsive pS2 and vascular endothelial growth factor (VEGF) by increasing gene transcription in MCF-7 cells. Treatment with RJ at concentrations ranging from 0.5 to 1 mg/ml enhanced MCF-7 cell proliferation, but concomitant treatment with 1 microM tamoxifen blocked this effect. In vivo studies using ovariectomized rats showed that 17beta-estradiol (20 mg/kg, s.c.) treatment restored VEGF expression in both uterus and brain, whereas RJ (1 g/kg, s.c.) restored it in uterus but not in brain. These findings provide evidence that RJ has estrogenic activities through interaction with estrogen receptors followed by endogenous gene expressions.

Effects of propolis on cell growth and gene expression in HL-60 cells.

Brazilian propolis obtained from honeybee hives was extracted with water or ethanol. Cell growth-inhibitory activities of these propolis extracts were found in HL-60 human myeloid leukemia cells. The extracts-induced apoptosis in the cells, which was characterized by morphological and nucleosomal DNA fragmentation analysis. The apoptosis was mainly attributed to the induction of granulocytic differentiation, which was evaluated by nitro blue tetrazolium (NBT) reducing assays and cytofluorometric analysis for the expression of cell surface marker CD11b. DNA microarray analysis was performed to examine the gene expression profiles in the propolis-treated HL-60 cells accompanied with granulocytic differentiation, which were compared with those in all-trans retinoic acid-treated cells. Several genes were up- or down-regulated. Two genes encoding S100 calcium binding protein A9 and ferritin, heavy polypeptide 1 were up-regulated, which were also confirmed by semi-quantitative reverse transcriptase-PCR (RT-PCR). Propolis-induced growth inhibition in HL-60 cells was, at least in part, due to differentiation with gene expression profiles, which are similar to those induced by all-trans retinoic acid.

Antihypertensive effect of peptides from royal jelly in spontaneously hypertensive rats.

We have shown that Protease N treated Royal Jelly (ProRJ) and peptides from ProRJ (Ile-Tyr (IY), Val-Tyr (VY), Ile-Val-Tyr (IVY)) inhibited angiotensin I-converting enzyme (ACE) activity and they have an antihypertensive effect in repeated oral administration for 28 d on spontaneously hypertensive rats (SHR). We investigated the contributive ratio of these peptides in ProRJ for antihypertensive effect in single oral administration on SHR. In single oral administration of each peptide and peptides mixture (MIX; IY, VY and IVY) at doses of 0.5, 1 and 10 mg/kg, systolic blood pressure (SBP) of SHR was reduced dose-dependently. This antihypertensive effect was held for 8 h. These results suggest that peptides contributed to the antihypertensive effect of ProRJ. And the contributive ratio of MIX in ProRJ for antihypertensive effect was computed to be about 38%. Therefore it is considered that intake of peptides, as a functional food would be beneficial for improving blood pressure in people with hypertension.