Aroma profile of leaf and stem of lemon balm (Melissa Officinalis L.) grown under greenhouse conditions.
Lemon Balm (Melissa officinalis L.) a member of Lamiaceae is a perennial herb native to southern climates of Europe and North America. It is presently found in both wild and cultivated states. The highest levels of essential oil have been extracted in late summer from the lower parts of the plants [1,9]. The essential oil contains geraniol, citronellol, cintronellal, linalool, eugenol acetate and nerol. Oil of balm has also been shown to have antiviral, antibacterial and antispasmodic activity. Balm has been reported to be an insect repellent. As a medicinal plant, lemon balm has traditionally been employed against catarrh, fever , flatulence  and headaches . Polyphenolic compounds are commonly found in medicinal plants and they have been reported to have multiple biological effects, including antioxidant activity . Oil yield of lemon balm in 17 regions of europe was reported from 0.06% to 0.16% . Tinmaz et al.  reported that oil yield is 0.14% at the begining of blooming. The highest level of oil yield reported by Adzet et al.  was 0.4%. Askari and Sefidkon  used aerial parts of lemon balm cultivated in three states of Iran for isolation of oil and reported oil yield 0.14-0.26%. Major components of oil were citronellal and [beta]-caryophyllene. Carvacrol was dominant in Tehran sample. Sadraei et al.  isolated the oil from dried aerial parts and identified 17 components.
Geranial (35.3%) and neral (24.5%) were the major components. Carnat et al.  found 14 constituents in leaf oil of lemon balm which citral (48.0%) and citronellal (40.0%) were the majors. The composition and quantity of essential oil from a particular species could be markedly affected by harvesting season , geographical environment and other agronomical factors [7,11].
This study focuses on identification of essential oil components in leaf and stem of lemon balm when the plants grown under greenhouse conditions.
Material and Methods
This study was conducted in experimental glasshouse of Islamic Azad University, Firoozabad Branch (28[degrees]35' N, 52[degrees]40' E; 1327 m above sea level). Breeded seeds were sown and the seedlings were transplanted in pots containing 1/3 soil, 1/3 sand and 1/3 peat (v/v) at 4-6 leaf stage and kept at 27[+ or -]3/17[+ or -]3[degrees]C day/night temperatures. The soil of pots were tested before transplanting and soil texture was sandy-loam with pH=7.73, organic C=2.27%, total N=0.22%, available P=20.19 mg/kg, available K=176.7 mg/kg and EC=1.94 dS/m. After six months plants were cut to 10 cm above the pot soil surface and were dried at room temperature. Leaves were separated from the stem by hand.
Isolation of essential oils was performed using hydrodistillation of 50 g sample of leaves and stems separately by using a Clevenger-type apparatus over 4 hours. The oils were dried over sodium sulphate and the yields of the essential oils (w/w) were calculated.
Gas Chromatography analysis was performed on an Agilent technologist model (6890 USA) series II gas chromatograph equipped with flame ionization detector and capillary column HP-5 (30 m ' 0.25 mm, 0.25 [micro]m film thicknesses). The chromatographic conditions were as follows: The oven temperature increased from 60 to 240[degrees]C at a rate of 3[degrees]C/min. The injector and detector temperatures were 240 and 250[degrees]C, respectively. Helium used as the carrier gas was adjusted to a linear velocity of 32 cm/s. The samples were injected using split sampling technique by a ratio of 1:20. Quantitative data was obtained from electronic integration of peck areas without the use of correction factors. Essential oil was also analyzed by Hewlett- Packard GC-MS (model 6890 series II) operating at 70e V ionization energy. Equipped with a HP-5 capillary column (phenyl methyl siloxane (30 m '0.25 mm, 0.25 Lim film thickness) with He as the carrier gas and a split ratio of 1:20. The retention indices for all the components were determined according to the Van Den Doll method using n-alkanes as standard. The compounds were identified by comparison of retention indices (RRI- AP-5) with those reported in the literature and by comparison of their mass spectra with the Wiley and mass finder 3 libraries or with the published mass spectra.
The data was collected from three replications and standard deviation was calculated by excel software. Comparison of the leaf and stem oil components was performed by t-test.
Results and Discussion
The yield of essential oils of leaves and stem were 0.36% and 0.014% respectively. Oil yield of lemon balm in 17 regions of Europe was reported from 0.06% to 0.16%  that is lower than our investigation. Genotype and geographical characteristics can produce such differences. Tinmaz et al.  reported that oil yield is 0.14% at the begining of blooming but we harvest the plants at vegetative stage of growth. The highest level of oil yield reported by Adzet et al.  was 0.4% that is almost similar to our study. We applied drug leaves and stem separately to isolate oil but whole aerial parts are used in some of study.
Results obtained from qualitative and quantitative analysis of essential oils have been shown in Table 1. Twenty compounds representing 94.5% of the oil of leaves and 19 components representing 87.1% of the oil of stems were identified. Geranial was the major component of both leaf (43.1%) and stem (34.9%) oils. According to our results, citral (geranial+neral) was 76.5% in leaf oil that is higher than report of Chin et al. . They used vapour distillation for isolation of oil but we applied hydrodistillation. Our findings about geranial is similar to Sari and Ceylon  reports about 11 population of balm. Nerol and geraniol are oxidative form of neral and geranial that have been reported by Patora  as major components of balm oil. High level of geranial and neral and absence of geraniol and nerol was reported in peppermint . Our results revealed that there is 82% oxygenated monoterpenes (rosefuran, linalool, citronellal, isogeranial, rosefuran epoxide, neral, geranial, methyl geranate, geranyl acetate), 3.8% hydrocarbon sesquiterpenes (a-caryophyllene, [beta]-caryophyllene, [alpha]-humulene), 1.4% oxygenated sesquiterpenes (caryophyllene oxide, geranyl linalool) and 7.3% miscellaneous (6-methyl-5-hepten-2-one, E-[beta]-ionone, pentadecanal, heptadecane, 6,10,14-trimethyl-2- pentadecanone, hexadecanoic acid) in leaf oil. Comparison of leaf and stem oil components by t-test shown significant differences in percentages of several components (Table 1). Citronellal, linalool, rosefuran, [alpha]-caryophyllene, [alpha]-humulene, pentadecanal, heptadecane, and geranyl linalool was not identified in stem oil.
In this study, citronellal was 0.5% in leaf oil and was not detected in stem oil.
Other researchers [3,5,10,12] showed high level of citronellal in lemon balm oil. The percentage and composition of essential oil could be markedly affected by the geographical environment, places that plants is grown, physical and chemical characteristics of soil, seed source and genotype, plant age, parts of plant which is used for oil isolation and oil isolation method.
This research was supported by Islamic Azad University, Firoozabad Branch, Iran and Medicinal and Natural Products Chemistry Research Center of Shiraz Medical Sciences, Shiraz, Iran. We would like to thank Professor Ramin Miri for his help and the use of laboratory facilities.
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(1) Shahram Sharafzadeh, (2) Morteza Khosh-Khui, (3) Katayoon Javidnia
(1) Islamic Azad University, Firoozabad Branch, Iran.
(2) Department of Horticultural science, Shiraz University, Shiraz, Iran.
(3) Medicinal Plant Research Center, Medical University of Shiraz, Shiraz, Iran.
Shahram Sharafzadeh; Department of Agriculture, Islamic Azad University, Firoozabad Branch, Iran.
Table 1: Amounts of the different chemical components in leaf and stem oil of lemon balm. RI Component name % in leaf Oil 981 1-octen-3-ol Nd 986 6-methyl-5-hepten-2-one 1.0 [+ or -] 0.2 989 2-pentylfuran Nd 1013 (2E,4E)-heptadienal Nd 1094 Rosefuran T 1101 Linalool 0.4 [+ or -] 0.2 1151 Citronellal 0.5 [+ or -] 0.1 1164 Isogeranial 1.2 [+ or -] 0.2 1175 Rosefuran epoxide 0.2 [+ or -] 0.1 1215 (2E,4E)-nonadienal Nd 1240 Neral 33.4 [+ or -] 2.3 1269 Geranial 43.1 [+ or -] 3.0 1318 (2E,4E)-decadienal Nd 1325 Methyl geranate 0.3 [+ or -] 0.1 1384 Geranyl acetate 2.9 [+ or -] 0.3 1406 [alpha]-caryophyllene 0.2 [+ or -] 0.1 1418 [beta]-caryophyllene 2.4 [+ or -] 0.4 1450 [alpha]-humulene 1.2 [+ or -] 0.8 1486 (E)-[beta]-ionone 0.1 [+ or -] 0.1 1581 Caryophyllene oxide 1.2 [+ or -] 0.5 1641 Caryophylla-4(14),8(15)-dien-5-ol Nd 1697 Pentadecanal 4.7 [+ or -] 2.0 1700 Heptadecane 0.8 [+ or -] 0.4 1780 Tetradecanoic acid Nd 1845 6,10,14-trimethyl-2-pentadecanone 0.1 [+ or -] 0.1 1976 Hexadecanoic acid 0.6 [+ or -] 0.2 2026 Geranyl linalool 0.2 [+ or -] 0.1 Total (%) 94.5 RI Component name % in stem Oil t-test 981 1-octen-3-ol 0.9 [+ or -] 0.2 986 6-methyl-5-hepten-2-one 1.1 [+ or -] 0.3 ns 989 2-pentylfuran 1.4 [+ or -] 0.4 1013 (2E,4E)-heptadienal 0.9 [+ or -] 0.2 1094 Rosefuran Nd 1101 Linalool Nd 1151 Citronellal Nd 1164 Isogeranial 0.6 [+ or -] 0.1 ** 1175 Rosefuran epoxide 0.4 [+ or -] 0.1 ** 1215 (2E,4E)-nonadienal 0.4 [+ or -] 0.1 1240 Neral 23.5 [+ or -] 2.4 ** 1269 Geranial 34.9 [+ or -] 2.3 0 1318 (2E,4E)-decadienal 1.8 [+ or -] 0.1 1325 Methyl geranate 0.6 [+ or -] 0.1 0 1384 Geranyl acetate 7.5 [+ or -] 0.4 ** 1406 [alpha]-caryophyllene Nd 1418 [beta]-caryophyllene 3.5 [+ or -] 0.5 0 1450 [alpha]-humulene Nd 1486 (E)-[beta]-ionone 0.4 [+ or -] 0.1 ** 1581 Caryophyllene oxide 4.0 [+ or -] 0.4 ** 1641 Caryophylla-4(14),8(15)-dien-5-ol 0.4 [+ or -] 0.1 1697 Pentadecanal Nd 1700 Heptadecane Nd 1780 Tetradecanoic acid 0.1 [+ or -] 0.1 1845 6,10,14-trimethyl-2-pentadecanone 0.4 [+ or -] 0.1 ** 1976 Hexadecanoic acid 4.3 [+ or -] 3.0 ns 2026 Geranyl linalool Nd Total (%) 87.1 RI, retention index Means of three replications [+ or -] SD (standard deviation) ns, not significant **, significant (P<0.01) *, significant (P<0.05) N.d., not detected T, trace=less than 0.05%
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|Title Annotation:||Original Article|
|Author:||Sharafzadeh, Shahram; Khosh-Khui, Morteza; Javidnia, Katayoon|
|Publication:||Advances in Environmental Biology|
|Date:||Mar 1, 2011|
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