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Experimental Gerontology 42 (2007) 594–600

Antioxidative action of royal jelly in the yeast cell

Polona Jamnik, Dusan Goranovic, Peter Raspor *

University of Ljubljana, Biotechnical Faculty, Food Science and Technology Department, Chair of Biotechnology, Jamnikarjeva 101,

1000 Ljubljana, Slovenia

Received 19 October 2006; received in revised form 5 January 2007; accepted 8 February 2007Available online 20 February 2007

Abstract

Royal jelly is a bee product, secreted from the hypopharingeal and mandibular glands of worker bees. There are many reports onpharmacological activities of royal jelly in experimental animals, but there are few about its antioxidative properties connected to aging.The aim of the work was to investigate the antioxidative action of royal jelly in the cell of the yeast Saccharomyces cerevisiae as a modelorganism. Yeast was cultivated in YEPD medium enriched with different concentrations of royal jelly like 1, 2 and 5 g/L. Yeast growthwas monitored by measuring optical density. At different time points cell energy metabolic activity was measured using the cell energymetabolism indicator resazurin, and 2 0,7 0-dichlorofluorescein was applied to estimate intracellular oxidation. Additionally, protein pro-file of cell extract was analyzed by 2-D electrophoresis. Results showed that royal jelly decreased intracellular oxidation in a dose depen-dent manner. Additionally it affected growth and cell energy metabolic activity in a growth phase dependent manner. Protein profileanalysis showed that royal jelly in the cell does not act only as a scavenger of reactive oxygen species, but it also affects protein expres-sion. Differentially expressed proteins were identified.� 2007 Elsevier Inc. All rights reserved.

Keywords: Yeasts; Royal jelly; Antioxidants; Aging; Intracellular oxidation; Cell energy metabolism; Proteomics

1. Introduction

Royal jelly (RJ) is a glandular secretion produced byworker honeybees to feed honeybee larvae during their firstthree days of life. It is the sole food for the honeybee queenduring its life span (Schmidt, 1996) and it is known that thehoneybee queen lives for several years compared to a fewmonths for the worker bee (Krell, 1996). Chemical compo-sition shows that RJ is mainly composed of proteins, sugars,lipids, vitamins, free amino acids and a large number of bio-active substances (Howe et al., 1985; Koya-Miyata et al.,2004; Nagai and Inoue, 2004; Simuth et al., 2004; Stockeret al., 2005). Therefore, RJ is extensively used as a cosmeticor dietary supplement due to the belief that it exerts onhuman beings similar effects as it does on honeybees.

Several pharmacological effects in experimental animalshave already been demonstrated, including vasodilative

0531-5565/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2007.02.002

* Corresponding author. Tel.: +386 1 4231161; fax: +386 1 2574092.E-mail address: peter.raspor@bf.uni-lj.si (P. Raspor).

and hypotensive activities (Shimoda et al., 1978), antitu-moral activity (Townsend et al., 1959; Townsend et al.,1960; Townsend et al., 1961), antihypercholesterolemicactivity (Nakajin et al., 1982), antifatigue effect (Kamakuraet al., 2001), promotion of collagen production (Koya-Miyata et al., 2004), insulin-like activity (Okuda et al.,1998; Salazar-Olivo and Paz-Gonzales, 2005), estrogeniceffects (Mishima et al., 2005), hypoglycaemic activity (Dixitand Patel, 1964; Fujii et al., 1990; Kramer et al., 1977),anti-inflammatory activity and wound-healing properties(Fujii et al., 1990), but there are a few reports about itsantioxidative role connected to antiaging effects (Iannuzzi,1990; Rembold, 1965; Inoue et al., 2003). Although thefundamental mechanisms of aging are still poorly under-stood, a growing body of evidence points towards reactiveoxygen species (ROS) as one of the primary determinants(Kregel and Zhang, 2007). Nagai et al. (2001) examinedthe antioxidative effect of RJ and other bee products bymeasuring scavenging abilities of the superoxide radical.RJ showed high scavenging activity surpassed only by

P. Jamnik et al. / Experimental Gerontology 42 (2007) 594–600 595

propolis among the examined bee products. Additionally,Nagai and Inoue (2004) and Nagai et al. (2006) confirmedantioxidative properties of water and alkaline extracts ofRJ and enzymatic hydrolysates from RJ. Thus antioxidantproperties of RJ determined in vitro may show a high anti-aging effect. Inoue et al. (2003) have already investigatedthe effect of royal jelly on the oxidative DNA damageand life span of C3H/HeJ mice. In mice that were fed withRJ as a dietary supplement for 16 weeks, the level of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxida-tive stress, was significantly reduced in kidney and serum.Furthermore, the average life span was extended by about25% compared to the control group. The results indicatedthat RJ increased the average life span of C3H/HeJ mice,possibly through the mechanism of reduced oxidativedamage.

Most studies exploring the biological effects of RJ onorganisms other than honeybees have been done employ-ing whole-animal experimental systems (Townsend et al.,1959; Townsend et al., 1960; Dixit and Patel, 1964; Kra-mer et al., 1977; Fujii et al., 1990; Vittek, 1995; Sveret al., 1996; Kamakura et al., 2001; Inoue et al., 2003).We used yeast as a model organism to investigate theantioxidative action of RJ in the cell by measuring intra-cellular oxidation, cell energy metabolic activity, andanalysing protein profile of yeast cell extract. Microor-ganisms are useful models for study of different aspectsof oxidative stress on biochemical, molecular-biologicaland cellular level. Oxidative damages to proteins, lipids,nucleic acids and other cell components as well as defensesystems against oxidative stress are basically almost sim-ilar at all levels of cellular organization (Sigler et al.,1999).

2. Materials and methods

2.1. Microorganism and cultivation

The yeast Saccharomyces cerevisiae – ZIM 2155 wasobtained from the culture collection of industrial microor-ganisms (ZIM) at the Biotechnical Faculty, Chair of Bio-technology, Ljubljana.

Cells were cultivated in YEPD medium with differentconcentrations of RJ 0, 1, 2 and 5 g/L. Yeast growth wasmonitored by measuring OD650 and samples were takenafter 6, 9, 11 and 13 h to measure cell energy metabolicactivity, intracellular oxidation and to analyze protein pro-file. All experiments were performed in triplicates.

2.2. Estimation of intracellular oxidation

Intracellular oxidation was estimated by using 2 0,7 0-dichlorofluorescein (H2DCF), which is able to react withoxidants. It is given as 2 0,7 0-dichlorofluorescein diacetate(H2DCFDA), which easily penetrates the plasma mem-brane and is hydrolyzed inside the cells by non-specificesterases. Non-fluorescent 2 0,7 0-dichlorofluorescein

(H2DCF) is oxidized to fluorescent 2 0,7 0-dichlorofluoresce-in (DCF), which is determined fluorimetrically (Jakubow-ski and Bartosz, 1997). It has been established that DCFcan penetrate from the cell. Thus, measurement of extracel-lular concentrations of DCF is a valid measure of oxida-tion processes inside the cells (Jakubowski and Bartosz,1997).

After 6, 9, 11 and 13 h from RJ addition cells were sed-imented by centrifugation and resuspended in the 50 mMpotassium phosphate buffer (pH 7.8) in concentration of1 · 108 cells/mL. H2DCFDA (Sigma) was added as a stocksolution of 1 mM ethanol solution to final concentration of10 lM. After 1-h incubation at 28 �C and 200 rpm cell sus-pensions were centrifuged and fluorescence of the superna-tants was measured using Tecan microplate reader(excitation and emission wavelengths of 488 and 520 nm,respectively). The results were expressed as fluorescence(RFU) normalized on total cell number.

2.3. Determination of cell energy metabolic activity

Cell energy metabolic activity was determined by CellT-iter-Blue� Cell Viability Assay (Promega). It uses the dyeresazurin, which is reduced to resorufin inside living cells(O’Brien et al., 2000). Resazurin can penetrate cells, whereit becomes reduced to the fluorescent product, resorufin,which can diffuse from cells and back into the surroundingmedium. It is known that resazurin acts as an intermediateelectron acceptor in the electron transport chain betweenthe final reduction of oxygen and cytochrome oxidase bysubstituting for molecular oxygen as an electron acceptor(Page et al., 1993). Measurement of resazurin fluorescenceis therefore an indicator of cell energy metabolism (Mag-nani and Bettini, 2000).

After 6, 9, 11 and 13 h from royal jelly addition 100 lLof cell suspension in concentration of 1 · 107 cells/mL and20 lL CellTiter-Blue� reagent were placed in 96-wellmicroplate. After 75-min incubation with resazurin at28 �C fluorescence was measured using Tecan microplatereader (excitation and emission wavelengths of 550 and595 nm, respectively). The results were expressed as fluores-cence (RFU) normalized on total cell number.

2.4. Cell extract preparation

Cells were collected by centrifugation at 4000 rpm for5 min and washed once with 0.9% solution of NaCl. 0.5 gcells (wet weight) was suspended in 2.5 mL extraction buf-fer (40 mM Tris–HCl, pH 8.0; 2% (w/v) 3-[(3-cholamido-propyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS),65 mM dithiothreitol (DTT)) containing protease inhibitorcocktail (Complete, Mini; Roche) – 1 tablet per 10 mL ofbuffer. The cells were disrupted by vortexing with acidwashed glass beads (Sigma, diameter: 425–600 lm) fourtimes, 1 min each with 1-min intervals for cooling the mix-ture on ice. The cell homogenate was centrifuged at 20,000g

for 20 min at 4 �C.

596 P. Jamnik et al. / Experimental Gerontology 42 (2007) 594–600

2.5. Protein assay

The protein content in the cell extracts was determinedby the method of Bradford (1976) using bovine serum albu-min as standard.

2.6. Two-dimensional electrophoresis

Two-dimensional (2-D) electrophoresis was performedaccording to Gorg (1991) with minor modifications. Sam-ples (100 lg protein) were mixed with rehydration solution(9 M urea, 2% (w/v) CHAPS, 2% (v/v) immobilized pHgradient (IPG) buffer, 18 mM DTT, a trace of bromophe-nol blue) and applied on 13-cm immobilized pH 4–7 gradi-ent (IPG) strips (Amersham Pharmacia Biotech). Afterrehydration (13 h) isoelectric focusing (IEF) as first dimen-sion was carried out at 20 �C on a Multiphor II (AmershamPharmacia Biotech). The following voltage program wasapplied: 300 V (gradient over 1 min), 3500 V (gradient over1.5 h), 3500 V (fixed for 4.33 h). Prior to sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE),the IPG strips were equilibrated in SDS equilibration buffer(50 mM Tris–HCl, pH 8.8; 6 M urea, 30% (v/v) glycerol,2% (w/v) SDS, a trace of bromophenol blue) containing1% DTT for 15 min, and containing 4.8% iodoacetamidefor an additional 15 min. SDS–PAGE as the second dimen-sion was carried out with 12% running gel on the verticaldiscontinuing electrophoretic system SE 600 (Hoeffer Sci-entific Instruments) at constant 20 mA/gel 15 min and thenat constant 40 mA/gel until the bromophenol blue reachedthe bottom of the gel. 2-D gels were silver stained usingprotocol compatible with matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometry(MALDI-TOF MS) (Yan et al., 2000). For each sampletriplicate 2-D gels were run at the same conditions.

2.7. Protein visualization and image analysis

2-D gels were recorded using an Artixscan 1800f scanner(Microtek). Gel image analysis was done with 2-D Dymen-sion software version 2.02 (Syngene) and included spotdetection, spot quantification, pattern warping and match-ing. For all spot intensity calculations, normalized volumevalues were used. The results are expressed as a ratio of thenormalized volume of protein spot in RJ-treated cellsdivided by normalized volume of matched protein spot inuntreated control cells at the same time of exposure.

2.8. Protein identification

The protein spots of interest were excised from the gelsand analyzed by LC–MS/MS using ESI-TRAP instrumentat the Aberdeen Proteome Facility (University of Aber-deen, Aberdeen, Scotland). The Mascot software was usedto search MSDB 20060529x database. The following searchparameters were applied: S. cerevisiae as species; trypticdigest with a maximum number of one missed cleavage.

The peptide mass tolerance was set to ±1.5 Da and frag-ment mass tolerance to ±0.5 Da. Additionally, carbami-domethylation and oxidation of methionine wereconsidered as possible modifications.

3. Results and discussion

Since little is known about antioxidative action of RJ inthe cell connected to anti-aging effects (Rembold, 1965;Iannuzzi, 1990; Inoue et al., 2003), we investigated in ourstudy the effect of royal jelly on yeast growth, intracellularoxidation, cell energy metabolic activity and proteinexpression in model organism yeast S. cerevisiae.

3.1. The effect of RJ on yeast growth, cell energy metabolicactivity and intracellular oxidation

Yeast cells were cultivated with different concentrationsof RJ . During cultivation at different time points yeastgrowth, cell energy metabolic activity and intracellular oxi-dation were measured. As shown in Fig. 2 decreased resa-zurin fluorescence was observed in a dose dependentmanner at 6 and 9 h. It is known that resazurin acts asan intermediate electron acceptor in the electron transportchain between the final reduction of oxygen and cyto-chrome oxidase by substituting for molecular oxygen asan electron acceptor (Page et al., 1993) and measurementof resazurin fluorescence is therefore an indicator of cellenergy metabolism (Magnani and Bettini, 2000). There-fore, we can conclude that decreased fluorescence resultsfrom decreased reduction of resazurin indicating lower cellenergy metabolism and consequently lower ATP pool size.On the other hand, it has to be highlighted that reducedATP pool size did not result in slower growth, furthermore,maximal specific growth rate remained the same as in thecontrol (Fig. 1). It seems that optimal growth can occurat lower ATP pool size. Cells require energy all the time.Energy produced in the cell is used for making new mate-rials for growth and also for maintenance, which includesrepair of damages, maintaining internal conditions withina narrow range, and detecting and responding to environ-mental changes (Verduyn, 1991). In the case of RJ treatedcells lower ATP pool size did not cause growth decline, butit might result from decreased cell requirement for energy,since decreased intracellular oxidant level was observed in adose dependent manner (Fig. 3). That indicates lowerenergy consumption for induction of endogenous antioxi-dant defence systems and repair systems. This was alsoconfirmed from 2-D gels, where down-regulation of enzy-matic antioxidant system Cu–Zn superoxide dismutasewas observed at 6 h (2.2-fold). Decreased cell requirementfor energy in RJ treated cells was observed also from glu-cose consumption rate measured at 6 h, which was for14.5% lower compared to control.

At 11 h difference in resazurin fluorescence between con-trol and RJ treated cells was not observed any more, butafter 11 h slight increasing was observed at 5 g/L RJ indi-

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

0 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26Time (hours)

Opt

ical

den

sity

Control1.0 g RJ/L2.0 g RJ/L5.0 g RJ/L

9

Fig. 1. Monitoring of growth of the yeast S. cerevisiae – ZIM 2155 atdifferent concentrations of RJ by measuring optical density.

P. Jamnik et al. / Experimental Gerontology 42 (2007) 594–600 597

cating increased cell energy metabolic activity, which mightbe connected to shift into postdiuaxic phase (Fig. 2). Thuswas more significant at higher concentrations of RJ com-pared to control and was still observed at 24 h (Fig. 1).This was confirmed also by de novo induction or up-regu-lation of some proteins in cells treated with 5 g/L of RJ for13 hours (Table 1 and Fig. 4). On the other hand, at 2 g/LRJ resazurin fluorescence was maintaining at the samelevel, but at 1 g/L RJ and control decreasing in resazurinfluorescence was observed after 11 h (Fig. 2). Inspite ofslight increasing of cell energy metabolic activity at 5 g/LRJ, intracellular oxidation was still reduced compared tocontrol (approximately 25%) (Fig. 3). This was seen alsofrom down-regulation of antioxidant enzyme Cu–Znsuperoxide dismutase and vacuolar protease B (Table 1and Fig. 4).

The results suggest that RJ treatment provides preven-tion against intracellular ROS formation and consequently

0

10000

20000

30000

40000

50000

60000

5 6 7 8 9 10 11 12 13 14Time (hours)

Flu

ore

scen

ce (

RF

U)

Control

1.0 g RJ/L

2.0 g RJ/L

5.0 g RJ/L

Fig. 2. The effect of royal jelly on cell energy metabolic activity in theyeast S. cerevisiae – ZIM 2155. The results are expressed as fluorescencenormalized on total cell number.

oxidative damages of cell components, which could be con-nected to anti-aging effect. Similarly, Inoue et al. (2003)showed that in mice fed with RJ as a dietary supplementfor 16 weeks, the level of 8-OHdG, a marker of oxidativestress, was significantly reduced in kidney and serum andalso the average life span was extended by about 25% com-pared to control.

3.2. The effect of RJ on protein expression

Since it is known that antioxidants do not act only asscavengers of reactive oxygen species, but they affect geneexpression in cultured cells, in laboratory animals, and inhumans (Rimbach and De Pascual-Teresa, 2005), we inves-tigated the effect of RJ on protein expression by using 2-Delectrophoresis. Proteins were separated on immobilizedpH 4–7 IPG strips followed by 12% SDS–PAGE.

Protein profile was analyzed only in yeast cells treatedwith 5 g/L RJ for 13 h. Comparison of protein profile oftreated and non-treated yeast cells showed differences inprotein expression (Fig. 4), which were analyzed using soft-ware. The threshold, at least 1.5-fold up- or down-regula-tion, was chosen to exclude proteins that differ inintensity due to small random variations during the perfor-mance of experiments.

A total of 22 spots were found to be differentiallyexpressed compared to control. The densities of six spotswere decreased and five spots were found up-regulated,while eleven protein spots were detected only in RJ treatedcells. Some of them were successfully identified by LC–MS/MS (hits with Score > 31; individual ion scores > 31 indi-cated identity or extensive homology at p < 0.05). Informa-tion of these identified proteins together with fold ratio arelisted in Table 1. Additionally identified protein spots arenumbered and indicated by arrows as shown in Fig. 4.Two of six protein spots that were down-regulated in RJtreated cells were identified as cerevisin precursor (vacuolarprotease B) and Cu–Zn superoxide dismutase, that mightindicate decreased intracellular oxidation and consequentlyless oxidative damaged proteins. Namely, Cu–Zn superox-ide dismutase is responsible for superoxide anion removingfrom the cytosol (Fridovich, 1995), while vacuolar proteaseB is a subtilisin-like serine protease found in the vacuole ofthe yeast S. cerevisiae and is responsible like other prote-ases for hydrolysis of damaged proteins (Moehle et al.,1987). The yeast S. cerevisiae has evolved two different pro-teolytic systems: (i) vacuolar system, which contains a vari-ety of non-specific proteases including vacuolar protease Band (ii) non-vacuolar system with highly specific proteasesresiding at different cellular locations (Hilt and Wolf,1992). Five spots were detected as RJ induced and onewas identified as phosphoglycerate kinase suggestingincreasing of cell energy metabolism after 11 h from RJaddition. Namely, phosphoglycerate kinase is one of twoenzymes required for ATP generation in glycolysis. It catal-yses transfer of a phosphoryl group from the acyl phos-phate of 1,3-diphosphoglycerate to ADP thus forming

Table 1Identification of proteins by LC–MS/MS searching MSDB 20060529x database

SpotID

Protein AccessionNo.

Ratio change TheoreticalMr (Da)/pI

Score Sequence coverage(%)

1 H+-exporting ATPase (EC 3.6.3.6) chain B,vacuolar

S45996 New in RJ treatedcells

57770/4.95 146 13

2 H+-exporting ATPase (EC 3.6.3.6) chain B,vacuolar

S45996 New in RJ treatedcells

57770/4.95 449 34

3 Glycine cleavage T protein GCV1 S54642 New in RJ treatedcells

44612/8.91 325 37

4 Transcription modulator WTM1 S60957 New in RJ treatedcells

48467/5.18 783 46

5 Cerevisin (EC 3.4.21.48) precursor A29358 fl3.49 69807/5.94 398 136 Superoxide dismutase (EC 1.15.1.1) (Cu–Zn)

[validated]DSBYC fl5.37 15959/5.62 127 23

7 Phosphoglycerate kinase (EC 2.7.2.3) KIBYG ›2.12 44867/7.74 154 13

Individual ion scores > 31 indicate identity or extensive homology at p < 0.05.

0

10000

20000

30000

40000

50000

60000

70000

Time (hours)

Fluo

resc

ence

(RFU

)

Control1,0 g RJ/L2,0 g RJ/L5,0 g RJ/L

5 6 7 8 9 10 11 12 13 14

Fig. 3. The effect royal jelly on intracellular oxidation of the yeastS. cerevisiae – ZIM 2155. The results are expressed as fluorescencenormalized on total cell number.

598 P. Jamnik et al. / Experimental Gerontology 42 (2007) 594–600

ATP and 3-phosphoglycerate (Watson et al., 1982). Amongprotein spots that were detected only in royal jelly cells,four were identified. Two different spots (number 1 and 2)were both identified as H+-exporting ATPase chain B,vacuolar. Vacuolar H+-ATPase maintains vacuolar pH. Thehydrolysis of cytosolic ATP by the vacuolar H+-ATPase

7 pI 4

Mr (kD116

66.2

45.0

35.0

25.0

18.4

14.4

Fig. 4. Comparison of protein profile of control (left) and RJ-treated yeast ceDetails for each spot are listed in Table 1.

is required for the movement of protons from cytosol intothe interior of the vacuole. The proton gradient generatedin this way across the yeast vacuolar membrane is utilizedby other membrane-bound transporters to drive the accu-mulation of ions and small molecules, amino acids andmetabolites into the vacuole (Kane, 2006). Its inductionmight be related to increased cell energy metabolism inRJ-treated cells compared to control. The other two iden-tified spots include glycine cleavage T protein GCV1 andtranscription modulator WTM1. Glycine cleavage T pro-tein GCV1 belongs to a family of glycine cleavage T pro-teins, part of the glycine cleavage multienzyme complex(GCV) found in bacteria and the mitochondria of eukary-otes. It catalyses the catabolism of glycine in eukaryotes(McNeil et al., 1997). Transcription modulator WTM1belongs to Wtm proteins, a novel family of S. cerevisiae

transcriptional modulators, that have role in meiotic regu-lation and silencing. Wtm1p was identified as a proteinpresent in a large nuclear complex. This protein has twohomologs, Wtm2p and Wtm3p, which probably arose bygene duplications. All members of this family appear tobe synergistically involved in the repression of the meio-sis-specific gene IME2 (Pemberton and Blobel, 1997). Itis known that the expression of meiosis- and sporulation-specific genes is tightly repressed in haploid cells and cells

7 pI 4

a)

lls (right). Arrows and numbers indicate spots identified by LC–MS/MS.

P. Jamnik et al. / Experimental Gerontology 42 (2007) 594–600 599

growing with adequate nitrogen and a fermentable carbonsource. Only under the correct genetic and environmentalconditions are meiosis-specific genes expressed (Mitchell,1994).

In summary, RJ affected protein expression and proteinsidentified in this study include antioxidant enzyme (Cu–ZnSOD), a regulating protein (transcription modulatorWTM1) and proteins that play important role in proteoly-sis and several metabolic pathways (vacuolar protease B,glycine cleavage T protein GCV1, vacuolar H+-ATPase).Some of them were not found to be dependent of RJ anti-oxidant properties.

4. Conclusions

Using the yeast S. cerevisiae as a model organism weshowed decreased intracellular oxidation in RJ-treated cellscompared to control, which resulted in better vitality ofyeast cells. Additionally, this is the first approach to ourknowledge, to study the RJ action in the cell using proteo-mic approach.

Acknowledgements

We thank Mrs. Alesa Kandus and Mrs. Jana Potokarfrom Medex in Ljubljana for kindly providing royal jellysamples. This work was supported by the Ministry of Edu-cation, Science and Sport of the Republic of Slovenia (Re-search program: Microbiology and biotechnology of foodand environment: P4-0116).

The Aberdeen Proteome Facility is funded jointly bySHEFC, BBSRC and the University of Aberdeen.

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