Effect of Gamma Radiation on Essential Oil Production in Different Plant Parts of Lemongrass, Cymbopogon Citratus.

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1 Scientia Agriculturae E-ISSN: X / P-ISSN: DOI: /PSCP.SA Sci. Agri. 5 (3), 2014: PSCI Publications Effect of Gamma Radiation on Essential Oil Production in Different Plant Parts of Lemongrass, Cymbopogon Citratus. Sharifah-NR, SA 1, Hanina, MN 1, Mahir, AM 2, CW-Zanariah, CWN 1, Siti-Salhah, O 1, M-Noor, I 3 1. Faculty of Science and Technology, Universiti Sains Islam Malaysia (USIM), Bandar Baru Nilai, Nilai, Negeri Sembilan, Malaysia 2. Centre for Collaborative Innovation, c/o UKM Technology Sdn. Bhd., Universiti Kebangsaan Malaysia, UKM Bangi, Selangor 3. Malaysia Genome Institute, UKM-MTDC Technology Centre, Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor Corresponding Author emno@cgat.ukm.my Paper Information A B S T R A C T Unique production of flowers from lemongrass mutant that have been Received: 18 January 2014 successfully produced by previous study has given the opportunity to study its essential oil yield and composition. Thus, objective of this study is to Accepted: 2 March 2014 analyse the yield and chemical composition of essential oils extracted from different plant parts of lemongrass, C. citratus mutant as compared to its Published: 20 March 2014 control. Essential oils from different plant parts of lemongrass mutant and control were extracted by steam distillation and then analysed by Triple Quadruple Gas Chromatography-Mass Spectrometry (Agilent 7000A). Results have showed very significant differences (P ) between different plant parts in lemongrass mutant and control with flower part of lemongrass mutant contained the higher percentage of essential oil (0.3804%). Chemical compound analysis result have showed that myrcene, one of the main minor compounds in essential oil extracted from all part of lemongrass control was absent in essential oils extracted from stalk, leaves and flowers of lemongrass mutant. Radiation has triggered significant changes in yield and chemical composition of essential oil extracted in lemongrass mutant as compared to its control PSCI Publisher All rights reserved. Key words: Lemongrass, Cymbopogon citratus, mutation breeding, essential oil, gamma radiation Introduction Study on mutation breeding program of the genus Cymbopogon has started about 30 years ago. FAO/IAEA Mutant Varieties Database (MVD) has reported that only six mutant varieties cultivars of Cymbopogon genus (i.e. C. winterianus) officially been released for past two decades (Bhanumati, Bibhuti, Niranjan, Phullara, Sourar and Subir) (Malusznski et al., 2001). These varieties of C. winterianus have been developed to adapt at North Indian and South Indian conditions. Mutation breeding techniques using x-rays radiation to these citronella grasses have resulted in genetic divergence including changes in oil composition, but have not resulted in improved cultivars (Oyen, 1999, Maluszynski et al., 2001). X-rays radiation experiment on Cymbopogon flexuosus (Nees ex Steud) Wats had resulted in the isolation of a methyl-eugenol deficient mutant. The results had shown that once the methyl eugenol is absent from the oil, it resembles the oil of citronella (Java type) and could be good substitute for the same oil (Choudhary and Kaul, 1979). While other study using x-rays radiation had showed genetic divergence including changes in oil composition in citronella grass, C. winterianus (Oyen, 1999, Maluszynski et al., 2000). Meanwhile, study of radiation treatment on C. citratus had shown that short duration exposure of mild supplemental ultraviolet-b (suv-b) irradiation on its field grown had resulted an increased in essential oil yield to % compared to control. This does suggested that treatment of suv-b exposure could stimulate the production of oil cells in changing the quality and percentage of essential oil contents of lemongrass (Rima et al., 2009). Other study on gamma radiation treatment on seed of Palmarosa (Cymbopogon martini Stapf.) has showed an increase in yield and quality of the essential oil (Srivastava and Tyagi, 1986). In our previous study, we have successfully produced new mutant varieties of lemongrass, C. citratus using optimum doses 80γ of gamma radiation. Unique characteristic have been observed in the long above ground stem (not stalk) with obvious appearance of nodes and internodes together with unique production of flowers (Sharifah et al., 2012). Arena et. al (2014) have suggested that ionising radiation may have different effects on plant metabolism, growth and

2 reproduction, depending on radiation dose, plant species, developmental stage and physiological traits. Thus, objective of this study is to analyse the yield and chemical composition of essential oils extracted from different plant parts of lemongrass, C. citratus mutant as compared to its control. Materials And Methods Plant Material Lemongrass mutant and its control were harvested from plants that have reached the maturity age of 6 months and divided to four different plant parts (stalk, mid stem, leaves and flowers). All samples were dried at room temperature for 1 week and cut into smaller pieces (1-2 inch) prior to essential oil extraction. Essential oil extraction 100g of the samples were submitted to steam distillation for 2 hours. Each sample was made triplicates. The volatiles were collected until no oil drop out. Moisture was removed by sodium sulphate anhydrous. Extract were weight and diluted with isopropanol (AR grade, Sigma Aldrich) for chemical compound analysis. Chemical Compound Analysis Chemical compound analysis was analyzed by Triple Quadruple Gas Chromatography-Mass Spectrometry (Agilent 7000A). Analyses were performed in MS1 Scan Mode and column use was HP-5MS fused-silica column (30 m X 0.32 mm i.d., film thickness 0.25µm, Agilent) with helium as carrier gas. Oven temperature was 40 C for 2 min, and then programmed heating from 40 to 200 C at a rate of 5 C/min and hold at 200 C for 2 min resulting in the complete elution of all peaks analyzed. Injector and detector temperatures were 300 C. All samples were diluted in isopropanol. Spectra was recorded in electron impact (EI) ionisation mode at 70 ev. The quadrupole mass detector, ion source and transfer line temperatures were set, respectively, at 150, 230 and 280 C. Mass spectra was scanned in the range m/z amu at 1 s intervals. Identification of essential oil constituents Essential oil constituents were identified by comparing retention times of the chromatogram peaks with those of reference compounds run under identical conditions, by comparison of retention indices (Kovats, 1965). Retention indices (RI) were calculated for all components using homologous series of n-alkanes (C 8 -C 24 ) injected in conditions equal to the samples ones. The formula used to calculate the RI was; where; I = retention index, n = the number of carbon atoms in the smaller alkane, N = the number of carbon atoms in the larger alkane, z = the difference of the number of carbon atoms in the smaller and larger alkane, t r = the retention time. Kovat s retention indices that have been calculated and mass spectra that have been scanned were analysed based on comparison of corresponded with data (Adam, 1989) and mass spectra libraries (National Institute of Standards and Technology 98). Statistical analysis The yield of essential oil was determined in triplicate, and the results were expressed as mean values. An analysis of variation (ANOVA) of the results was performed using PROC GLM (Generalised Linear Model) and Duncan Multiple Ranges Test. Statistical analysis was performed with SAS statistical software. Results And Discussion Result analysed have showed that there are very significant differences (P ) between mutant and control of lemongrass and their different plant parts. Essential oil yielded from lemongrass mutant is much higher in stalk (0.0586%) and leaves (0.2591%) while in mid stem is lower (0.0501%) as compared to control (stalk; %, mid stem; % and leaves; %) (Figure 1, Table 1 and 2). Essential oil yielded from flower parts of mutant is the highest (0.3804%) compared to all part of lemongrass mutant and control. This is 46.82% higher in its leaves. Up to present, little is known about any study performed on essential oil extracted from lemongrass flower especially in C. citratus. However in Cymbopogon martini which is known as Palmarosa grass has showed that essential oil content in its flowers is higher than the stems and leaves (de Guzman and Reglos, 1999). Generally, essential oil is produced higher in flowers than leaves and other parts of plants due to its physiology structure. Most of essential oil bearing plants are unique in possessing specialised secretory type epidermal hairs or trichomes to synthesise and accumulate large quantities of these compounds 2

3 (Fahn 1988). These epidermal appendages (glandular trichomes, glandular hairs, resin ducts etc. occur in different plant parts from flowers to roots with special individual attributes which are responsible in giving variability in essential oil accumulation percentage and composition. Meanwhile, citral content in essential oil extracted from all parts of lemongrass control and mutant were presented in Figure 2. Most of the citral content were above 75 % (control stalk; 80.24%, control mid stem; 82.44% and control leaves; 77.23%, mutant leaves; 79.89% and mutant flower; 76.48%) which have been set by ISO standard for lemongrass essential oil (ISO 3217: 1974) except in stalk (51.39%) and mid stem (65.89%) of lemongrass mutant. 0/4500 0/4000 0/3804 0/3500 0/3000 0/2500 0/2000 0/1500 0/2291 0/2591 0/1000 0/0500 0/0000 0/0586 0/0501 0/0133 0/0112 Control Mutant Control Mutant Control Mutant Mutant Stalk Mid Stem Leaf Flower Figure 1. Comparison of essential oil yield percentage different plant parts of lemongrass mutant and control. 100/00 90/00 80/00 70/00 60/00 50/00 40/00 30/00 20/00 80/24 51/39 82/44 65/89 77/23 79/89 76/48 Other Components Citral 10/00 0/00 Control Mutant Control Mutant Control Mutant Mutant Stalk Mid Stem Leaf Flower Figure 2. Comparison of citral content percentage in essential oil extracted from different plant parts of lemongrass mutant and control. Table 1. ANOVA Test of essential oil yield from different plant parts of lemongrass mutant and control Source of Variation DF Essential Oil Yield Lemongrass Type *** Plant Parts *** Lemongrass Type X Plant Parts ** Error 14 3

4 Total 20 DF = Degree of Freedom, Probability; P 0.01*, P 0.001**, P *** Table 2. Duncan Multiple Range Test of essential oil yield from different plant parts of lemongrass mutant and control Factor Level N Essential Oil Yield Mean D Lemongrass Type Mutant A Control B Plant Parts Stalk C Mid Stem C Leaves B Flower A N = Total number of data; D = Duncan Classification, mean values sharing the same alphabet were not significantly different Table 3. Chemical composition of essential oil extracted from different plant parts of lemongrass mutant and control RT KI Compound Composition (%) Control Mutant Stalk Mid Mid Leaf Stalk Stem Stem Leaf Flower trans pinane pinane methyl-5-heptene-2-one myrcene d-limonene y-terpinene linalool isopentyl 2me-butanoate menthone trans chrysanthemal cis-verbenol isopinocamphone alpha thujenal b-citronellol oxinecarbox neral nerol geranial trans-carvone oxide tetraallyloxyethane epoxylinalooloxide neric acid pyridinone dihydroxysechellane oxiranecarboxaldehyde isopropyl -2-methyl geranic acid cis-carvone oxide trimethyl-bicyclo linalool isobutyrate trimethyl-bicyclo geranyl acetate alpha-trans-bergamotene cyclopropacarboxylic acid germacrene verbenyl propyl ether b-bisabolene methyl dodecanoate nona-2,3, dienoic acid citronellyl acetate caryophyllene oxide (z)-8-dodecenyl acetate globulol juniper camphor selinene beta-cyclocitral benzophenone (z,e)-5,7-dodecadienyl acetate (z)-3-hexenyl hexanoate isopropylphenol agarospirol juniper camphor benzimidaxol longiborneol

5 umbeliferone hexadecanoic acid methyl-2-bomene 0.70 A dash (-) indicate the absence of component in the oil. RT= Retention Time; RI= Relative retention indices to C 8-C 24 n-alkanes on HP-5MS Column. Figure 3. Chromatogram of chemical composition in essential oil extracted from stalk of lemongrass control. Figure 4. Chromatogram of chemical composition in essential oil extracted from stalk of lemongrass mutant. Figure 5. Chromatogram of chemical composition in essential oil extracted from mid stem of lemongrass control. 111

6 Figure 6. Chromatogram of chemical composition in essential oil extracted from mid stem of lemongrass mutant. Figure 7. Chromatogram of chemical composition in essential oil extracted from leaves of lemongrass control. Figure 8. Chromatogram of chemical composition in essential oil extracted from leaves of lemongrass mutant. 112

7 Figure 9. Chromatogram of chemical composition in essential oil extracted from flower of lemongrass mutant. Conclusion Radiation has triggered significant changes in yield and chemical composition of essential oil extracted in lemongrass mutant as compared to its control. Essential oil extracted from lemongrass mutant flowers gives the highest yield while myrcene were found absent in all parts of lemongrass mutant essential oil except in the mid stem. Further study need to be done to analyse the biological properties differences between them. Acknowledgement We would like to express our sincere thanks to Dr. Herman, S. from Universiti Kebangsaan Malaysia (UKM) for his contribution in the gamma irradiation procedure. This work was fully supported by USIM s Faculty of Science and Technology grant for postgraduate student. References Adams PR Identification of essential oil components by gas chromatography/mass spectrometry (4th ed.), Allured Publishing Corporation, Carol Stream, Illinois, USA. Arena C, De Micco V, De Maio A Growth alteration and leaf biochemical responses in Phaseolus vulgaris exposed to different doses of ionising radiation. Plant Biology, 16: Choudhary DK, Kaul BL Radiation induced methyl-eugenol deficient mutant of Cymbopogon flexuosus (Nees ex Steud) Wats. Proc. Indian Aad. Sci., 88B, Part II (3): de Guzman CC, RA Reglos. Cymbopogon martini (Roxb.) J.F. Watson. In Oyen LPA, Nguyen XD. (Eds). Plant Resources of South-East Asia. No 19: Essential-Oil Plants. Prosea Foundation: Bogor Indonesia: Fahn A Secretory tissues in vascular plants. New Physiologist, 108 (3): Kovats ES Gas chromatographic characterization of organic substances in the retention index system. Adv. Chromatogr, 1: Maluszynski M, Nichterlein K, Van Zanten L, Ahloowalia BS Officially Released Mutant Varieties The FAO/IAEA. Mutation Breeding Review. 65 (3), Oyen LPA Cymbopogon citratus (DC.) Stapf. In Oyen LPA and Nguyen XD. (Eds). Plant Resources of South-East Asia. No 19: Essential- Oil Plants. Prosea Foundation: Bogor Indonesia: Rima K, Agrawal SB, Abhijit S Evaluation of changes in oil cells and composition of essential oil in lemongrass (Cymbopogon citratus (D.C.) Stapf.) due to supplemental ultraviolet-b irradiation. Current Science, 97 (8): Sharifah NRSA, Mahir AM, Jusoff K Unique Flowers Produced from West Indian Lemongrass, Cymbopogon citratus (DC.) Stapf. Through Induced Mutation. World Applied Sciences Journal, 17 (Towards the Traceability of Halal and Thoyyiban Application): Srivastava HK, Tyagi BR Effects of seed irradiation on yield and quality of essential oil in palmarosa (Cymbopogon martini Stapf.). Euphytica. 35 (2):