Food Chemistry 129 (2011) 1851–1859

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Food Chemistry

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Analytical Methods

Traditional Rosa damascena flower harvesting practices evaluatedthrough GC/MS metabolite profiling of flower volatiles

Krasimir Rusanov a,⇑, Natasha Kovacheva b, Mila Rusanova a, Ivan Atanassov a

a AgroBioInstitute, Sofia 1164, 8 Dragan Tsankov Blvd., Bulgariab Institute of Roses, Essential and Medical Cultures, Kazanlak 6100, 49 Osvobozhdenie Blvd., Bulgaria

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 March 2011Received in revised form 3 May 2011Accepted 26 May 2011Available online 2 June 2011

Keywords:Rosa damascenaTraditional flower harvestingFlower volatilesGC/MSMultivariate analysisMethyl eugenol

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.05.132

⇑ Corresponding author. Tel.: +359 2 9635411; fax:E-mail address: krusanov@abv.bg (K. Rusanov).

Industrial cultivation of Rosa damascena and rose oil production is a centuries-old industry based on well-established traditional hand-picking of rose flowers. Here the traditional rose harvesting practices wereevaluated through GC/MS analysis of the volatile compounds accumulation in R. damascena flowers ateight different developmental stages, flower buds with arrested development and flowers collected atsix daytime points related to flower picking. The accumulation of 80 out of 100 volatile compoundswas significantly influenced by the stage of flower development while 62 out of 103 compounds weresignificantly influenced by the daytime period of flower collection. Principal component analysis showedclose clustering of the flower developmental stages and daytime periods preferable for flower harvestingand production of high quality and quantity of rose oil. Taken together the obtained results provide fur-ther support to the currently applied traditional rose flower harvesting practices. The possible applica-tions of more precise flower harvesting for fine modulation of rose oil composition and reductionlevels of the unwanted compound methyl eugenol are discussed.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction R. damascena var. ‘‘Trigintipetala’’ happens once a year at the

Damask roses have been long time recognised for their strong fra-grance (Beales et al., 1998). Rosa damascena var. ‘‘Trigintipetala’’, alsoknown as the oil-bearing rose, is one of the most important Damaskroses industrially cultivated for production of rose oil and rose waterafter water steam distillation, or rose concrete and rose absoluteafter solvent extraction, (Widrlechner, 1981). Of these, the produc-tion of rose oil has largest economic impact (Kovacheva, Rusanov,& Atanassov, 2010). Rose oil consists of over 300 compounds (Kovats,1987; Ohloff, 1994) and its quality is evaluated by implementation ofan international standard, (ISO 9842, 2003). Rose oil is primarilyused as a fragrance ingredient in the perfumery and cosmetics indus-try and included in a large number of perfumes, creams, soaps, lo-tions and other cosmetic products. Beside this, the rose water andother products derived from R. damascena flowers are widely usedas flavour ingredients in various food products, like cakes, cookies,sweets, ice creams, beverages, etc. The main producers of rose oiland rose water are Bulgaria, Turkey and Iran. Smaller amounts ofmainly rose water are also produced in the countries of NorthernAfrica, India and China (Kovacheva et al., 2010).

The harvesting of rose flowers is the most labour-intense partof the industrial cultivation of oil-bearing roses and productionof rose oil (Gunes, 2005; Kovacheva et al., 2010). The blooming of

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beginning of the summer. The period of blooming is very shortand in Bulgaria it usually continues for 2–3 weeks, starting duringthe second half of May. The blooming period is extremely inten-sive. The rose bushes produce daily a large number of bloomingflower buds and full-blown flowers, which are picked by handand subjected to steam distillation within the same day. The roseharvesting practices are based on century-old traditions and areconsidered as essential for achieving high quality rose oil and rosewater. Traditionally the flower harvesting starts around 6:00 a.m.and ends around 12:00 p.m., since the content of rose oil drops dra-matically for rose flowers collected after noon (Dobreva & Kova-cheva, 2010). The majority of the rose buds which start bloomingearly in the morning hours reach full-blown before noon and haveto be collected on the same day. Faded flowers fully blown withinthe previous day are not harvested.

The quality and composition of the distilled rose oil are moni-tored and analysed in depth through GC or GC�MS analyses of vol-atile compounds (ISO 9842, 2003; Jalali-Heravi, Parastar, &Sereshti, 2008; Kovats, 1987). Comparative GC and GC�MS analy-ses of rose oil composition have also been used for characterisationof rose oils distilled from flowers of R. damascena cultivated indifferent geographic regions and/or representing differentgenotypes (Anac, 1984; Baser, 1992; Bayrak & Akgul, 1994;Dobreva & Kovacheva, 2010; Loghmani-Khouzani, Fini, & Safari,2007; Naqvi & Mandal, 1997; Nikolov et al., 1977; Rezaei, Jaimand,Tabaei-Aghdaei, Barazandeh, & Meshkinzadeh, 2003; Sood, Singh,

1852 K. Rusanov et al. / Food Chemistry 129 (2011) 1851–1859

& Singh, 1992), as well as for assessment of the effect of stage of flow-er development (Staikov, Decheva, & Balinova, 1975), date and timeof harvest (Baydar & Baydar, 2005; Dobreva & Kovacheva, 2010),flower storage and processing (Baydar & Baydar, 2005; Kazaz, Erbas,& Baydar, 2009; Kazaz, Erbas, Baydar, Dilmacunal, & Koyuncu, 2010;Staikov, Balinova-Tsvetkova, Decheva, & Kalaidjiev, 1975), and dis-tillation procedure (Baydar & Baydar, 2005). In addition GC–MSanalysis was applied for characterisation of the other rose flowerproducts, including rose concrete, rose absolute, rose water (Ayci,Aydinli, Bozdemir, & Tutas, 2005; Kurkcuoglu & Baser, 2003) and vol-atile emissions of the flower headspace (Picone, Clery, Watanabe,MacTavish, & Turnbull, 2004). However, the studies above have fo-cused on the characterisation of the end products from rose flowerprocessing and oil distillation, which makes difficult the more pre-cise study of the changes of levels of volatiles accumulation in roseflowers, in relation to other factors, like rose genotype, flower devel-opment, diurnal rhythm, etc. Recently, we proposed a simple andefficient procedure for GC�MS analysis of volatile accumulation inR. damascena flowers, based on direct hexane extraction (Rusanov,Kovacheva, & Atanassov, 2011). Here we apply this procedure toevaluate the traditional R. damascena flower harvesting practices,through comparative GC�MS analysis of volatiles accumulation inrose flowers at different developmental stages and daytime periodsrelated to flower collection. The possible impact of increased preci-sion of rose harvesting on the rose oil composition, and productionof rose oil with reduced methyl eugenol levels are discussed.

2. Materials and methods

2.1. Plant material

Samples of R. damascena flowers at eight stages of flower devel-opment and flower buds with arrested development were collectedfrom the experimental field of the Institute of Roses, Essential andMedical Cultures, Kazanlak within a short period around 8 a.m. onthe 21st of May 2010. For analysis of the daytime dynamics of vola-tile compounds accumulation, rose flowers at stage 6 were collectedat six time points: 6:00, 8:00, 10:00 a.m. and 12:00, 2:00, 4:00 p.m.on the 22nd of May 2010. All samples were collected in three biolog-ical replicates and immediately frozen in liquid nitrogen. Each repli-cate sample set was collected from a separate bush.

2.2. GC�MS analysis

Flower metabolite extraction and sample preparation forGC�MS analysis were carried out as described by Rusanov et al.

Fig. 1. Stages of R. damascena flower development used in the present study. Stages 1–8White arrow shows the necrosis of the petal abscission zone. Scale bar equals 1 cm and

(2011). All GC�MS analyses of flower extracts were carried outon an Agilent 7890A/5975C GC�MS system (Agilent Technologies,Santa Clara, CA). All compounds except nerol were analysed on anAgilent HP-5MS non-polar column with helium 5.0 as carrier gas ata septum purge flow of 3 mL/min, splitless injection of 1 lL of sam-ple, purge flow to split vent 50 mL/min at 2 min, 0.8 mL/min carriergas flow, 4.9 psi pressure and the following acquisition parame-ters: injector temperature 250 �C; oven program: 40 �C for 3 minthen 5 �C/min to 300 �C for 5 min; run time 60 min. On the HP-5MS non-polar column nerol and citronellol have similar retentiontimes and could not be chromatographically separated. Hence, ner-ol was also analysed on an Agilent DB-WAXetr polar column withhelium 5.0 as a carrier gas at a septum purge flow of 3 mL/min,splitless injection of 1 lL of sample, purge flow to split vent50 mL/min at 2 min, 0.8 mL/min carrier gas flow, 12.9 psi pressureand the following acquisition parameters: injector temperature200 �C; oven program: 40 �C for 0 min then 3 �C/min to 220 �Cfor 10 min; run time 70 min.

2.3. Data analysis

Individual compounds were detected and identified using AM-DIS Ver. 2.66 software (National Institute of Standards and Tech-nology (NIST), Gaithersburg, MD) and a custom-built MS libraryconsisting of compounds identified previously in rose oils. All datafiles of identified compounds were imported into the Mass ProfilerProfessional Ver. 2.1 software (Agilent Technologies), normalisedto the internal standard 2-nonadecanone (Sigma–Aldrich, St. Louis,MO) and used to produce heat maps, perform ANOVA statisticalanalysis for significance and principal component analysis (PCA).PCA was performed with the normalisation option of the Mass Pro-filer Professional software, where means are normalised to 0 andstandard deviations normalised to 1.

3. Results and discussion

3.1. Accumulation of volatiles during R. damascena flowerdevelopment

In order to investigate the metabolite changes during R. damasc-ena flower development, the process of flower development wasdivided in eight consecutive developmental stages (Fig. 1). The tra-ditional R. damascena flower harvesting for rose oil production in-volves collection of flower buds at stages 5, 6 and 7. The flowersfrom the last two stages produce a greater amount of rose oiland are preferable for harvesting (Staikov et al., 1975). The flowers

indicate normal flower development. AFD stands for arrested flower development.is valid for stages 1–8 shown in the figure.

K. Rusanov et al. / Food Chemistry 129 (2011) 1851–1859 1853

at stage 8 are faded, produce low amount of rose oil (Staikov et al.,1975) and are not harvested. Their anthers are brown and the pet-als start to discolour and fall (Staikov & Zolotovich, 1955). Stage 8flowers have become fully open and reached stage 7 within theprevious day. We included in the study also another rose flowermorphotype designated as AFD (arrested flower development,Fig. 1). Morphologically AFD buds are similar to rose flower budsat stage 4, but their petals start to discolour, fade and necrosis ap-pears in the petal abscission zone. The AFD flower buds do not con-tinue further their development. They form a separation layer atthe pedicel base and drop off within the next few days. The fre-quency of formation of AFD buds depends on the environmentalconditions and physiological state of the plant and may reach upto 50% of the flower buds within some years (Kosseva, Zolotovich,& Decheva, 1968).

Flower samples from the selected eight stages of flower devel-opment and AFD buds were collected in three replicates from theexperimental field of the Institute of Roses, Essential and MedicalCultures (IREMC) within a short period around 8 a.m. on the 21stMay, 2010. Each replica sample set was collected from a separateR. damascena bush. Hexane flower extracts from the sampleswere prepared and analysed on a GC�MS system as describedearlier (Rusanov et al., 2011). Typical GC chromatograms of hex-ane flower extracts corresponding to 3rd and 5th flower develop-mental stages are shown in Fig. 2a. The applied GC�MSmetabolite profiling resulted in the identification of a total of100 compounds, based on comparison with a custom build AM-DIS MS library consisting of compounds from rose essential oils.The number of compounds identified in each developmentalstage is presented in Fig. 3a. The metabolite profiling data wasnormalised and assessed via multivariate data analysis usingMass Profiler Professional software (Agilent Technologies). ANO-VA statistical analysis, applying a cut-off p-value of 60.01 re-vealed 80 compounds which are significantly influenced by thestage of flower development. Fig. 3c shows a heat map demon-strating the abundance changes of these 80 compounds duringthe different flower developmental stages. Multivariate analysiswas further conducted by means of principal component analysis(PCA) for the set of 80 compounds significantly influenced by thestages of flower development. The resulted 2D PCA plot is shownin Fig. 4a. Taken together the first two principal components, PC1and PC2, explain 83.67% of the total variation. The results fromthe PCA analysis show close clustering of the samples, corre-sponding to one and the same stage of flower development butcollected from different R. damascena bushes. At the same timethe PCA analysis demonstrates distribution of the studied sam-ples from different flower developmental stages into four majorclusters (Fig. 4a). Samples corresponding to flower stages 1 and2 and AFD flower buds are clustered together (cluster cl:1–2-AFD) and separated along PC1 from the rest of the samples. Clus-ters, cl:3–4 corresponding to flower stages 3 and 4 and cl:8corresponding to flower stage 8 are located at both ends ofPC2. The three stages of flower development, stages 5, 6 and 7,which are traditionally harvested for rose oil production are clus-tered together in cluster cl:5–7 located in the middle betweenthe clusters cl:3–4 and cl:8. Stages 6 and 7, which are the twomost preferable developmental stages for flower harvesting areclosely situated inside the cl:5–7 cluster. Stage 5 marks the ear-liest flower developmental stage used for flower harvesting. Thetraditional rose flower harvesting practices are based on collec-tion of rose flowers at stages 5, 6 and 7, producing distilled roseoil with typical R. damascena odour. Stages 6 and 7 re preferablefor harvesting due to the higher rose oil content, (Staikov et al.,1975). As can be seen from Fig. 3a stages 5, 6 and 7 also repre-sent the three developmental stages with the highest number ofidentified compounds which are present both in the rose flower

and in the rose oil. The results from the PCA analysis of the roseflower volatiles accumulated during different flower developmen-tal stages clearly demonstrate that the samples at flower devel-opmental stages related to the traditional rose flowerharvesting practices are located in one cluster and are separatedfrom the rest of the analysed samples. The results from the AN-OVA statistical analysis show that the rose flower development isrelated to significant changes of 80 out of the 100 analysed roseflower volatiles, which are present both in the rose flowers andthe distilled rose oil. This extends the earlier results reportedby Staikov et al. (1975), showing that the rose oil distilled fromflowers at these developmental stages contains highest amountsof the three major monoterpenoids (i.e., geraniol, citronellol, ner-ol) which are highlighted in the International Rose Oil Standard(ISO 9842, 2003). These results also demonstrate that traditionalrose harvesting is related not only to collection of flowers withhigher essential oil content and possessing high amount and‘right’ proportions of a few typical rose oil constituents, but italso accounts for a broader metabolite complexity of the roseflower volatile pool.

To assess the impact of the flower development on the accumu-lation of individual flower volatiles, we further analysed thechanges of the normalised intensity values of seven major com-pounds, which are main constituents of the distilled rose oil anda key part of ISO 9842, 2003 (Fig. 5a–d). The GC–MS analysis ofhexane flower extracts showed that the three monoterpenoidspossess similar flower developmental patterns. All of them wereabsent or were very weakly detected during the first three stagesof flower development and their accumulation rapidly increasedduring the transition from stage 3 to stage 5. The levels of geranioland citronellol accumulation reached their maximum at stage 5and nerol at stage 6. The next stages of flower development wererelated to a rapid decrease of citronellol levels and a more gradualdecline of geraniol and nerol. Thus, during the three developmentalstages 5, 6 and 7, which are traditionally used for rose flower har-vesting, the two major monoterpenoids geraniol and nerol are inthe range of 80–100% of their maximum levels of accumulation,whereas the levels of citronellol are reduced from 100% (stage 5)to 40% (stage 7). The major hydrocarbons nonadecane, heptadec-ane and heneicosane show similar developmental patterns to themonoterpenoids, but reach their maximum accumulation earlierat stages 3 and 4 and decrease during the next stages. The levelsof these hydrocarbons further decline at different rates. Accord-ingly, nonadecane was reduced from 80% to 60%, heptadecane from80% to 20% and heneicosane from 90% to 80% of their maximalintensities during the transition from stage 5 to stage 7. The lastmajor compound, phenylethyl alcohol, reaches its maximal levelof accumulation at stage 5 and remained generally unchangedwithin stages 5–7. The heat map presented in Fig. 3c demonstratesthat the harvested rose flowers contain a number of additional vol-atiles with significant changes in their levels of accumulation dur-ing the flower development from stage 5 to stage 7. The describedresults further suggest that the application of more precise har-vesting of rose flowers restricted to a narrower period of develop-ment, within the range of flower development stages traditionallyused for harvesting, will result in substantial changes in the flowervolatile spectra and therefore in the volatile composition of the dis-tilled rose oil and other flower products. At present such opportu-nities remain unexplored and unutilised. The application of moreprecise developmental guided flower harvesting will allow produc-tion of rose oil and other rose flower products with reducedcontent of methyl eugenol (MEG). MEG is a relatively minorconstituent of rose oil, which has recently caused increasingconcern, due to its reported toxicity/carcinogenicity (Opinion ofthe Scientific Committee on Food on Methyl Eugenol (4-Allyl-1,2-Dimethoxybenzene), 2001). MEG is not detected in hexane flower

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Fig. 2. Typical GC�MS chromatograms of R. damascena flower hexane extracts corresponding to (a) 3rd and 5th stages of flower development and (b) flower samples taken at10:00 a.m. and 4:00 p.m. Major flower volatiles designated: (1) b-Citronellol, (2) trans-geraniol, (3) phenylethyl alcohol, (4) heneicosane, (5) nonadecane, (6) heptadecane.

1854 K. Rusanov et al. / Food Chemistry 129 (2011) 1851–1859

extracts until stage 5 (Fig. 5d). During the transition from stage 5 tostage 7 the level of MEG increased to its maximum and after thatgradually decreased. Thus, the restriction of flower harvesting todevelopmental stages 5 and 6 will result in reduction of MEG inthe distilled rose oil.

3.2. Daily dynamics of R. damascena flower volatiles

Traditionally R. damascena flower collection starts shortly aftersunrise around 6:00 a.m. and ends around noon. Usually the larg-est volumes of rose flowers are harvested after 8:00 a.m. when

Fig. 3. Flower volatile compounds identified after GC�MS analysis. (a�b) Number of identified compounds in: (a) different flower developmental stages (b) different daytimepoints of flower collection. (c–d) Heat map showing the changes of abundance values normalised to the internal standard 2-nonadecanone of flower volatile compoundswhich are significantly influenced by (c) flower developmental stages (columns represent the analysed developmental stages) and (d) time of flower sampling during theharvesting day (columns represent the analysed daytime points of flower sampling). The left end of the heat map legend scale indicates absence of the compound. Changes ofthe colour from the left to the right end of the heat map scale indicate increase of the compound abundance. All compound abundances have been estimated on a non-polarcolumn (Agilent HP-5MS), except nerol which was analysed on a polar column (Agilent DB-WAXetr); (1) butanoic acid, 3,7-dimethyl-6-octenyl ester, (2) a-methyl-a-[4-methyl-3-pentenyl]oxiranemethanol, (3) 4-(2,6,6-trimethylcyclohex-1-enyl)butan-2-ol’.

K. Rusanov et al. / Food Chemistry 129 (2011) 1851–1859 1855

Fig. 4. Principal component analysis of normalised GC/MS metabolite profiling data of (a) different morphological stages of R. damascena flower development and (b)different times of flower sampling during the same harvesting day.

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most of the workers have already entered the rose fields. The mainreason for such a harvesting schedule is related to the daily varia-tions of the relative rose oil content distilled from the collectedrose flowers, which is highest for the flowers harvested between8:00 a.m. and noon (Dobreva & Kovacheva, 2010). Besides the dailyvariation of the rose oil content of the harvested flowers, Dobrevaand Kovacheva (2010) also reported substantial variation of the rel-ative abundances of some of the main rose oil constituents. In orderto investigate the daytime dynamics of volatile accumulation in R.damascena flowers, we conducted GC–MS analysis of hexane ex-tracts of flower samples collected at six daytime points: 6:00 a.m.,8:00 a.m., 10:00 a.m., 12:00 p.m., 2:00 p.m. and 4:00 p.m. Flowersfrom three separate rose bushes were collected at each time pointwithin the same day. All of the collected flowers were at stage 6 offlower development (Fig. 1). Typical GC chromatograms of hexaneflower extracts of samples taken at 10:00 a.m. and 4:00 p.m. areshown in Fig. 2b. The applied GC�MS metabolite profiling resultedin the identification of 103 compounds, based on comparison witha custom-built AMDIS MS library consisting of compounds from roseessential oils. The number of compounds identified at each daytimepoint is presented in Fig. 3b. ANOVA applying a cut-off p-value of60.01 revealed that 62 compounds were significantly influencedby the time of flower sampling. Principal component analysis wasconducted using the set of 62 compounds. The resulting PCA plot isshown in Fig. 4b. The first two principal components PC1 and PC2 ex-plain 87.54% of the total variation. As can be seen from Fig. 4b, theanalysed samples form three distinct clusters corresponding to flow-ers collected at different time points. Thus, the traditional time forthe end of the daily flower harvesting around 12:00 p.m. separateswell the before- and after- noon samples along PC1. The clustercl:14–16 h, corresponding to samples harvested at 2:00 p.m. and4:00 p.m., is situated at one of the ends of the PCA plot, whereasthe rest of the samples, which are traditionally harvested, are located

at the other end of the plot. The results from the PCA analysis showalso distinct separation into two clusters of the samples related tothe traditional daytime harvesting from 6:00 a.m. to 12:00 p.m.The cluster of the samples collected at the beginning of the tradi-tional daytime harvesting at 6:00 a.m. (cluster cl:6 h, Fig. 4b) is sep-arated along the PC2 from the cluster cl:8–12 h, containing the restof the samples related to more intensive harvesting. The 6:00 a.m.time point marks the beginning of a rapid increase of the light inten-sity and air temperature and therefore marks the beginning of thediurnal cycle of R. damascena. The last is related to complex changesin the rate of biosynthesis, accumulation and emission of flower vol-atiles, as part of their circadian diurnal rhythmicity (Dudareva,Pichersky, & Gershenzon, 2004). Correspondingly, strong diurnaloscillation of flower volatile emissions was reported also for R.damascena semperflorens cv. ‘Quatre Saisons’ (Picone et al., 2004),R. hybrida cv. Fragrant Cloud (Hendel-Rahmanim, Masci, Vainstein,& Weiss, 2007), and R. hybrida cv. Honesty (Helsper, Davies,Bouwmeester, Aafke, & Kampen, 1998).

The dynamics and complexity of the changes of volatile accu-mulation in the rose flowers are illustrated in Fig. 3d. Fig. 3dshows a heat map of the 62 compounds which are significantlyinfluenced by the time of flower sampling. The studied flowervolatiles show diverse patterns of daytime accumulation reflect-ing the proposed complexity of the regulation of their biosyn-thesis and accumulation (Dudareva et al., 2004; Hendel-Rahmanim et al., 2007; Picone et al., 2004). The changes ofthe intensities of the seven individual rose flower volatiles,which are part of the international rose oil standard, are shownin Fig. 5e–g. Whereas the accumulation of the two monoterpe-noids geraniol and nerol shows a similar pattern of reachingmaximum at 10:00 a.m., the maximal abundance of the thirdmonoterpenoid citronellol was detected at the beginning of thediurnal period at 6:00 a.m. and steadily decreased after that.

Fig. 5. Profile plots of the abundance values (as percentage from the maximum abundance value for each particular compound) of the major compounds which are part of theRose Oil International Standard (ISO, 2003). Plots (a–d) correspond to different morphological stages of the R. damascena flower development. Plots (e–g) correspond todifferent times of flower sampling during the harvesting day.

K. Rusanov et al. / Food Chemistry 129 (2011) 1851–1859 1857

The three major hydrocarbons also show diverse accumulationpatterns. While, the maximal level of heptadecane accumulationwas detected at the beginning of the diurnal period, the levels ofnonadecane was elevated during the 8:00 a.m. to 10:00 a.m. per-iod and the accumulation of heneicosane remained largely un-changed. The flower volatile accumulation levels for the timepoints 2:00 p.m. and 4:00 p.m., which are out of the traditionaldaytime flower harvesting period are lower than those of the6:00 a.m. to 12:00 p.m. period supporting the rationality of thetraditional rose harvesting. At the same time it has to be pointedout that the abundances of some volatiles, including major roseoil constituents like nerol and geraniol, are quite low at the

beginning of the daytime harvesting at 6:00 a.m. and compara-ble with those from the afternoon samples. Consequently, thevery early start of the daytime harvesting has to be reconsideredand if possible not applied at industrial scale. The described dataalso show that a number of the rose flower volatiles display sub-stantial variations of their flower abundances during the entireperiod of traditional daytime flower harvesting. Thus, the distil-lation of rose oil from flowers collected during a shorter timeperiod within the traditional daytime harvesting will allowdirectional changes of the rose oil composition accounting forselected sets of flower volatiles. Taken together the variations,described in the present study, in the levels of volatiles accumu-

1858 K. Rusanov et al. / Food Chemistry 129 (2011) 1851–1859

lated in the R. damascena flowers, collected according to the tra-ditional rose harvesting practices, make it possible to proposeapplication of a more precise rose flower harvesting, based ona well selected narrow stage of flower development and a day-time period of flower collection. It is expected that the proposedprecision of the flower harvesting parameters will allow finetuning of the rose oil composition, including avoiding the pres-ence of methyl eugenol in the distilled rose oil and other roseflower products. Although very attractive, the impact from suchprecise flower harvesting remains to be evaluated at industrialscale; it is also necessary to account for the effect of the post-harvesting flower storage prior to rose oil distillation.

R. Damascena flowers develop consecutively and normally asingle bush possesses flowers at different developmental stages.Traditional harvesting involves collection of flower buds at stages5, 6 and 7, as the last two stages are preferable. It is not clearhow the proposed harvesting of R. damascena flowers at precisestage of development, and specially harvesting of stage 5 flowers,will affect the overall flower and rose oil yield. We are currentlyplanning to test the effect of the practical application of preciserose flower harvesting during the forthcoming R. damascena har-vesting campaign. Additionally, the results of the present studypoint out the importance of the application of correct flower sam-pling procedure, in relation to volatile profiling for the character-isation of oil-bearing rose genetic resources and breeding lines. R.damascena flowers contain around thirty petals, of which the epi-dermal cells are the main location for biosynthesis and accumula-tion of flower volatiles (Staikov & Zolotovich, 1957; Bergougnouxet al., 2007). The flowers of a number of other oil-bearing rosesand seed progenies of R. damascena have more simple structurewith a lower number of petals. Thus, the analysis of volatile accu-mulation could be significantly disturbed by the reduction of therelative weight of the petals to the entire flower and by theincreasing rate of volatile emission once the flower with fewer pet-als is open. Considering the results of the present study it is possi-ble to suggest that the volatile profiling of rose flowers forevaluation of their potential for essential oil production shouldbe based on analysis of rose petals only collected around8:00 a.m. from flowers at stage 5, according to the proposed stagesof R. damascena flower development.

4. Conclusion

The GC�MS, ANOVA and PCA analyses of R. damascena flower vol-atiles described in the present study support the traditional roseflower harvesting practices, including collection of rose flowers atstages 5–7 starting at 6:00 a.m. and ending around 12:00 p.m. Theyfurther demonstrate that traditional rose flower harvesting is basedon the wide complexity of the flower volatile pool, as the accumula-tion levels of 80 out of 100 volatiles were significantly influenced bythe flower developmental stages and 62 out of 103 volatiles wereinfluenced by the time of flower collection. At the same time, sub-stantial variations of the accumulation of volatile compounds inthe rose flowers at developmental stages within the range whichis traditionally used for rose oil production were observed. Thismakes possible to propose a fine tuning of the composition of thedistilled rose oil by implementation of precise flower harvestingpractices, including harvesting of flowers at a narrower range offlower developmental stages and daytime period. An attractive im-pact from the proposed application of precise rose flower harvestingcould be the substantial reduction in the rose oil of the carcinogenicconstituent methyl eugenol. This could be directly achieved byrestriction of the industrial flower harvesting to collection ofrose flower buds at stage 5. The results of the study also suggestapplication of a common flower sampling procedure for proper com-parative analysis of essential oil composition from rose flowers.

Acknowledgements

The authors would like to thank Ivaila Dincheva (ABI) for excel-lent technical assistance and the National Science Fund at the Min-istry of Education, Youth and Science, Bulgaria for supporting thepresented research via project No.: DO02-105.

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