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.

Differential volatile organic compounds in royal jelly associated with different nectar plants

The aim of this work was to distinguish volatile organic compound（VOC） profiles of royal jelly（RJ） from different nectar plants. Headspace solid-phase microextraction（HS-SPME） was used to extract VOCs from raw RJ harvested from 10 nectar plants in flowering seasons. Qualitative and semi-quantitative analysis of VOCs extracts were performed by gas chromatography-mass spectrometry（GC-MS）. Results showed that VOC profiles of RJ from the samples were rich in acid, ester and aldehyde compound classes, however, contents of them were differential, exemplified by the data from acetic acid, benzoic acid methyl ester, hexanoic acid and octanoic acid. As a conclusion, these four VOCs can be used for distinguishing RJ harvested in the seasons of different nectar plants.

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.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellifera L.)

The protein composition of the egg development in the high royal jelly producing bees （Apis mellifera L.） was investigated. This pioneer study was to separate and quantify the proteins in the egg of the high royal jelly producing worker bees （Apis mellifera L.） by using two-dimensional gel electrophoresis along with their three-day development. The results showed that 160, 195, and 176 proteins, with a wide range of molecular weight （17-80 KDa） and relatively narrow scope of pI （4. 00-8.40） could be detected on day 1, day 2, and day 3, respectively, during the developmental process of the egg. Meanwhile 44 protein spots were constantly detected along with the egg development. Among them 36% were in the uptrend along with the egg development, 14% were in the downtrend, and 39% were of the largest expressed volume on day 2. In addition, the specific proteins were expressed on day 1, day 2, and day 3 （89, 77, and 80, respectively）. Besides the coexistent and specific proteins, 24 proteins were expressed on day 1 and day 2, but silenced on day 3, 49 proteins were expressed on day 2 and day 3, but silenced on day 1, only 3 proteins were expressed on day 1 and day 3, but silenced on day 2. The result indicates that egg development is a sequential and complex gene controlled process, where the eggs of day 2 express the most active proteins. The coexistent proteins suggest that it is conservative and indispensable for this event. These specific proteins suggest that the different developmental stage needs specific proteins to regulate it.

Water Soluble Propolis and Royal Jelly Enhance the Antimicrobial Activity of Honeys and Promote the Growth of Human Macrophage Cell Line

Due to the overuse and misuse of antibiotic, an increase in antibiotic resistance of pathogenic bacteria is evolving. Attention should be focused on natural alternatives to antibiotics, like propolis, royal jelly （R J） and honeys. They all have strong antibacterial properties due to the active substances they contain. This study investigated the effect of combination of water soluble propolis （WSP） Greitl20 or fresh royal jelly （F-RJ） （MiZigoj） and Forest honeys as antibacterial against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter baumanii, Staphylococcus aureus, methicillin resistant Staphylococcus aureus （MRSA）, Streptococcus pyogenes, Streptococcus agalactiae and Candida albicans. These substances are also cell growth promoters for human macrophage （TLT） cell line. WSP Greitl20, F-RJ （M） and different Forest honeys were prepared in saline as 10% solutions. The antimicrobial activity was expressed as the minimal inhibitory concentration （MIC） in mg/mL. The growth promotion activity was measured at optical density （OD） 595 nm. The combination ofWSP Greitl20 with different Forest honeys is better than F-RJ （M） in same combination with different Forest honeys. The best antibacterial/antifungal activity was found with the combination of 10% WSP Greit 120 in the Forest honey （1：10） from Italy or Spain. When measuring the growth promoting activity of TLT cell line, the best activity was detected at the combination of 10% WSP Greitl20 in the Forest honey from Italy （GI3 = 0.796 ± 0.014 and GI5 = 1.133± 0.022）. Antimicrobial and growth promoting activities are correlated and WSP-dependent.

Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin α3 and Integrin β1 mRNA Expression, via Akt/mTOR/HIF-1α Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.

Effect of royal jelly on in vitro fertilization and early embryo development following nicotine treatment in adult female rats

Objective:To scrutinize the protective role of royal jelly as an antioxidant on nicotine-induced changes in malondialdehyde(MDA)level,p53 expression,in vitro fertilization(IVF)rate,and early embryo development in adult female rats.Methods:A total of 56 adult female Wistar rats were divided into 8 groups(n=7 in each group).Group 1 served as an untreated control group,group 2,3 and 4 received nicotine at a dose of 0.50,1.00 and 2.00 mg/kg respectively,group 5 received royal jelly at a dose of 100.00 mg/kg,and group 6,7 and 8 received 0.50,1.00 and 2.00 mg/kg nicotine,respectively,with 100.00 mg/kg body weight royal jelly.Nicotine and royal jelly were administered daily for 49 days in the experimental groups intra-peritoneally and orally,respectively.At the end of the experimental period,p53 expression,IVF rate and early embryo development as well as MDA concentration were measured.Results:The IVF rate,number of cumulus oocytes,two-cell embryos and blastocysts decreased in the nicotine-treated groups in a dose-dependent manner.In addition,p53 mRNA expression and MDA levels increased in the nicotine-treated groups.Royal jelly co-administration led to partial improvement in the aforementioned parameters.Conclusions:Royal jelly may have a repro-protective effect in nicotine-administered female rats in terms of its anti-oxidant and anti-apoptotic properties.

Thymoquinone anticancer activity is enhanced when combined with royal jelly in human breast cancer

BACKGROUND Breast cancer is the most common cause of the majority of cancer-related deaths in women,among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options.Thymoquinone(TQ),the main bioactive constituent of Nigella sativa,has been extensively studied as a potent anticancer molecule against various types of cancers.Honeybee products such as the royal jelly(RJ),the nutritive secretion fed to honeybee queens,exhibit a variety of biological activities besides its anticancer effect.However,the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown.AIM To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ,RJ,and their combinations in the MDA-MB-231 cell line.METHODS Cells were treated with TQ,RJ,and their combinations for 24 h.Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,we determined the half-maximal inhibitory concentration of TQ.Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions.Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ,RJ,and their combination.Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation.RESULTS TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration.RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5μg/mL.High doses of RJ(200μg/mL)had greater toxicity against MDA-MB-231 cells.Interestingly,the inhibition of cell viability was most pronounced in response to 15μmol/L TQ and 5μg/mL RJ.A dose of 15μmol/L TQ caused a significant increase in the PreG1 population,while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations.TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ.In contrast,no significant regulation of Ki67 expression was observed,indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation.CONCLUSION This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells,which could confer an advantage for cancer therapy.

The endogenous antioxidant ability of royal jelly in Drosophila is independent of Keap1/Nrf2 by activating oxidoreductase activity

Royal jelly(RJ)is a biologically active substance secreted by the hypopharyngeal and mandibular glands of worker honeybees.It is widely claimed that RJ reduces oxidative stress.However,the antioxidant activity of RJ has mostly been determined by in vitro chemical detection methods or by external administration drugs that cause oxidative stress.Whether RJ can clear the endogenous production of reactive oxygen species(ROS)in cells remains largely unknown.Here,we systematically investigated the antioxidant properties of RJ using several endogenous oxidative stress models of Drosophila.We found that RJ enhanced sleep quality of aging Drosophila,which is decreased due to an increase of oxidative damage with age.RJ supplementation improved survival and suppressed ROS levels in gut cells of flies upon exposure to hydrogen peroxide or to the neurotoxic agent paraquat.Moreover,RJ supplementation moderated levels of ROS in endogenous gut cells and extended lifespan after exposure of flies to heat stress.Sleep deprivation leads to accumulation of ROS in the gut cells,and RJ attenuated the consequences of oxidative stress caused by sleep loss and prolonged lifespan.Mechanistically,RJ prevented cell oxidative damage caused by heat stress or sleep deprivation,with the antioxidant activity in vivo independent of Keap1/Nrf2 signaling.RJ supplementation activated oxidoreductase activity in the guts of flies,suggesting its ability to inhibit endogenous oxidative stress and maintain health,possibly in humans.

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.

Effects of Royal Jelly Supplementation on Glycemic Control and Oxidative Stress Factors in Type 2 Diabetic Female:A Randomized Clinical Trial

Objective： It has been proposed that royal jelly has antioxidant properties and may improve oxidative stress and glycemic control. Therefore, we investigated the effects of royal jelly supplementation in diabetic females. Methods： In this pilot, parallel design randomized clinical trial, 50 female volunteers with type 2 diabetes were randomly allocated to the supplemented （25, cases） and placebo （25, cases） groups, based on random block procedure produced by Random Allocation Software, given a daily dose of 1,000 mg royal jelly soft gel or placebo, respectively, for 8 weeks. Before and after intervention, glycemic control indices, antioxidant and oxidative stress factors were measured. Results： After royal jelly supplementation, the mean fasting blood glucose decreased remarkably （163.05_± 42.51 mg/dL vs. 149.68 ± 42.7 mg/dL）. Royal jelly supplementation resulted in significant reduction in the mean serum glycosylated hemoglobin levels （8.67%± 2.24% vs. 7.05% ± 1.45%, P=0.001） and significant elevation in the mean insulin concentration （70.28 ± 29.16 pmol/L vs. 86.46± 27.50 pmol/L, P=0.01）. Supplementation significantly increased erythrocyte superoxidase dismutase and glutathione peroxidase activities and decreased malondialdehyde levels （P〈0.05）. At the end of study, the mean total antioxidant capacity elevated insignificantly in both groups. Conclusions： On the basis of our findings, it seems that royal jelly supplementation may be beneficial in controlling diabetes outcomes. Further studies with larger sample size are warranted.

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.

Royal-jelly-based apitherapy can attenuate damages to male reproductive parameter following nicotine administration

Background:Nicotine administration can generate severe oxidative stress and lipid peroxidation.Royal jelly,with its antioxidant properties,acts as a scavenger of reactive oxygen species.This study describes the apitherapy effects of royal jelly on testicular damage following nicotine administration.Methods:Forty-eight male BALB/c mice were divided into 8 groups(n=6):saline,3 different doses of royal jelly(100,150,and 200 mg/kg body weight(BW)per day),nicotine(1.5 mg/kg),and 3 different groups of Nic+Roy(1.5 mg/kg of Nic+100,150,and 200 mg/kg BW per day of royal jelly).Nicotine was administrated intraperitoneally,and royal jelly was prescribed orally for 10 consecutive days.Serum levels of hormones(testosterone,luteinizing hormone,and follicle-stimulating hormone),total antioxidant capacity,nitric oxide(NO)status,malondialdehyde levels,sperm DNA fragmentation,sperm parameters,histopathological changes(H&E staining),immunohistochemistry against apoptotic proteins,and gene expression of Bcl-2,p53,Caspase-3,and Nrf2(real-time PCR)were assessed to evaluate the molecular and histological changes.Results:Hormone levels,sperm parameters,and status of antioxidants were decreased significantly(p<.05)following nicotine administration.Moreover,royal jelly treatment normalized hormonal and antioxidant characteristics,decreased apoptotic gene expression,increased Nfr2 gene expression,and restored histopathological alteration to the physiological status significantly(p<.05).Conclusion:Royal jelly upregulates the antioxidant status,inhibits the mitochondrialdependent apoptosis pathway,and increases the rate of proliferation.This therapeutic agent effectively protected the testis against nicotine-associated damages by antioxidant and anti-apoptotic effects.

Occupational Asthma Caused by Inhalable Royal Jelly ancl Its Cross-reactivity with Honeybee Venom

Royal jelly is a honeybee nutriment secreted from the glands in the hypopharynx of worker bees essential in the development of queen bees. Ingestion of royal jelly has been reporied to trigger rhinitis, asthma, and anaphylaxis, but occupational asthrna occurring after inhalation of volatile royal jelly is rare.

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.

Royal jelly for genitourinary syndrome of menopause:A randomized controlled trial

Objective:Genitourinary syndrome of menopause is a progressive condition characterized by a decrease in estrogen,which causes bothersome genital symptoms.The purpose of the study was to determine the effect of royal jelly on the genitourinary syndrome of menopause.Materials and methods:A randomized controlled trial was carried out from November 2018 to June 2019 in Bandar Abbas,Iran.The trial was registered in the Iranian Registry of Clinical Trials(1RCT20181107041585N1)with the main objective of determining if royal jelly could reduce the genitourinary syndrome of menopausal women.Eligible women were randomly assigned to receive either daily 1g of oral royal jelly or placebo for 8 weeks.The urogenital subscale of the Menopausal Rating Scale was used to determine genitourinary syndrome.Independent samples t-test was used for inter-group comparisons and paired samples t-test for pre-and post-treatment comparisons.Results:There were no differences in the severity of sexual problems,bladder complications,or vaginal dryness between groups before intervention.Although the intervention group's bladder complications improved slightly after eight weeks of royal jelly treatment compared to the control group(p?0.04),there were no significant changes in vaginal dryness,sexual problems,or total urogenital score.The within-group changes(before and after treatment)also showed no differences in urogenital symptoms.Conclusions:A daily dose of 1g royal jelly taken orally for 8 weeks did not alleviate menopausal genitourinary syndrome.No serious side effects were observed.To make more reliable decisions about the use and safety of royal jelly in the future,different doses of royal jelly and longer trials are required.