Major royal jelly proteins accelerate onset of puberty and promote ovarian follicular development in immature female mice

The major royal jelly proteins(MRJPs)are the central constituents responsible for the specific activities of royal jelly.Here MRJPs via oral administration daily for 45 consecutive days were evaluated the effects on the reproductive parameters in immature female mice(FM).Neonatal FM were divided into four groups fed MRJPs with doses of 0,125,250 and 500 mg/kg/body weight(M125,M250 and M500).The results in M125,M250 and M500 showed that the times of estrus were accelerated by 10.7%,15.5%and 10.7%,the secondary follicles number were increased by 50.7%,78.8%and 38.6%,the Graafian follicles were increased by 600.0%and 774.0%and 150.0%,respectively.M500 induced multi-oocyte follicles.The serum estradiol levels of the three groups were increased by 47.1%,64.9%and 31.1%,the action of MRJPs raising hormone secretion level is mainly via upregulating expression of ERˇgene.Antioxidant parameters of ovarian tissue showed that the malondialdehyde levels in M125 and M250 were decreased,the superoxide dismutase activities and glutathione peroxidase activities in M125 and M250 were increased.In conclusion,MRJPs may accelerate onset of puberty and promote follicular development in FM.Our findings would facilitate better understanding of the benefit effect of MRJPs as the key ingredient in royal jelly on promoting fertility performance.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines

Royal jelly （R J） is a well-known bioactive substance. It contains large amounts of major royal jelly proteins （MRJPs）, which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum （FBS） in different types of human cell culture, the prolif- eration of the complex serum with different ratios of MRJPs/FBS （M/F） was evaluated on five cell lines： 293T, HFL-I, 231, HCT116, and Changliver using MTT （3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide） assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor （EGF） and insulin-transferrin-selenium （ITS）, pro- moted proliferations of Changliver, 293T, HCT116, and H FL-I by 18.73%-56.19% （P〈0.01）. Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein(RJP)at different degrees of hydrolysis(DH)by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34%, 11.65%, 15.19%, 21.38% and 23.91%, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20%. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Anti-senescence effect and molecular mechanism of the major royal jelly proteins on human embryonic lung fibroblast(HFL-I) cell line

Royal jelly (R J)from honeybee has been widely used as a health promotion supplement.The major royal jelly proteins (MRJPs)have been identified as the functional component of RJ.However,the question of whether MRJPs have anti-senescence activity for human cells remains.Human embryonic lung fibroblast (HFL-I)cells were cultured in media containing no MRJPs (A),MRJPs at 0.1mg/ml (B),0.2mg/ml (C),or 0.3mg/ml (D),or bovine serum albumin (BSA)at 0.2mg/ml (E).The mean population doubling levels of cells in media B,C,D,and E were increased by 12.4%,31.2%,24.0%,and 10.4%,respectively,compared with that in medium A.The cells in medium C also exhibited the highest relative proliferation activity,the lowest senescence,and the longest telomeres.Moreover, MRJPs up-regulated the expression of superoxide dismutase-1(SOD1)and down-regulated the expression of mammalian target of rapamycin (MTOR),catenin beta like-1(CTNNB1),and tumor protein p53(TP53).Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials,amide carbonyl group vibrations and aromatic hydrogens.These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line,and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1,major royal jelly protein 1 antibody

Major royal jelly protein 1（MRJP1）, designated apalbumin 1, has been regarded as a freshness marker of royal jelly（RJ）. A MRJP1-specific peptide（IKEALPHVPIFD） identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody（antiSP-MRJP1 antibody）. Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1（anti-R-MRJP1 antibody） reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay（ELISA） using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ（0 d）. Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage（P〈0.0001）. Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.