Effect of Harvesting Treatments and Distillation Methods on the Essential Oil of Lemon Balm and Apple Geranium Plants

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1 Jeobp 12 (2) 2009 pp ISSN X Effect of Harvesting Treatments and Distillation Methods on the Essential Oil of Lemon Balm and Apple Geranium Plants Khalid A. Khalid 1 *, Weiming Cai 2 and Aisha M.A. Ahmed 3 1 Department of Cultivation and Production of Medicinal and Aromatic Plants, National Research Centre, Dokki, Giza, Egypt 2 Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, 300 Fenglin Road, Shanghai , China 3 Botany Department, National Research Centre, Dokki, Giza, Egypt Received 06 May 2008; accepted in revised form 17 September 2008 Abstract: The effects of harvesting treatments and / or distillation methods (hydrodistillation, hydro-steam distillation and steam distillation) on the essential oil content and composition of Pelargonium odoratissimum L. and Melissa officinalis L. plants were carried out in a greenhouse at Shanghai Institute of Plant Physiology and Ecology (SIPPE), Shanghai, China, during the years of 2007 and The highest oil yield of P. odoratissimum L. and M. officinalis L. was obtained by hydrodistillation and the lowest by steam distillation. The essential oil was significantly decreased towards the second harvesting. The main component of P. odoratissimum essential oil extracted by hydrodistillation was methyl eugenol (25.9 % %), while the main component of hydro-steam distilled or steam distilled oil was limonene (30.5 % % for hydro-steam distilled oil and 38.8 % % for steam distilled oil). The most abundant compound of M. officinalis essential oil extracted by hydrodistillation or hydro- steam distillation was citronellal (36.1 % % for hydrodistillation and 27.3 % % for hydro- steam distillation ), whereas α-terpinene (20.2 % %) was the main component with steam distillation. The oxygenated compounds and hydrocarbon compounds were changed according to the distillation methods and / or harvesting number for both P. odoratissimum and M. officinalis. plants. Key words: Harvesting treatment, Essential oil, Lemon Balm (Melissa officinalis L.), Apple Geranium (Pelargonium odoratissimum L.), hydrodistillation, hydro-steam distillation and steam distillation. Introduction: Pelargonium odoratissimum L., Family Geraniacea, is commonly named Apple Geranium. The whole plant is an aromatic herb with astringent, tonic and *Corresponding author (Khalid A. Khalid ) E- mail: < ahmed490@gmail.com > 20

2 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp antiseptic effects 1,2. It is used internally in the treatment of debility, gastroenteritis and haemorrhage 2. Externally, it is used to treat skin complaints, injuries, neuralgia and throat infections 2. The essential oil obtained from Apple Geranium is applied in aromatherapy, perfumery and as an insect repellent 2,3. Lemon Balm (Melissa officinalis L.) of the family Lamiaceae, is a well known herb used to give fragrance to different food and beverage products. It has also been used as a medicinal plant for treatment of headaches, gastrointestinal disorders, nervousness, and rheumatism 4,5. The essential oil is a well-known antibacterial and antifungal agent, and it is also responsible for the mild depressive and spasmolytic properties of the plant 6. There are three main distillation methods for obtaining essential oil, namely, (i) waterdistillation (or hydro-distillation), (ii) steam-distillation and (iii) water- steam distillation. In the manufacture of essential oils using the method of water-distillation, the botanical material is completely immersed in water and the whole is brought to the boil. This method protects the oils so extracted to a certain degree since the surrounding water acts as a barrier to prevent it from overheating. When the condensed material cools down, the water and essential oil is separated and the oil decanted, to be used as essential oil. When steamdistillation is used in the manufacture and extraction of essential oils, the botanical material is placed in a still and steam is forced over the material. The hot steam helps to release the aromatic molecules from the plant material since the steam forces open the pockets in which the oils are held in the plant material. The molecules of these volatile oils then escape from the plant material and evaporate into the steam. The temperature of the steam needs to be carefully controlled, just enough to force the plant material to release the essential oil, yet not too hot as to burn the plant material or the essential oil. The steam which then contains the essential oil is passed through a cooling system (to condense the steam), which forms a liquid from which the essential oil and water is then separated. The water- steamdistillation method is basically a marriage between normal water-distillation and that of steam-distillation. The botanical material is immersed in water in a still, which has a heat source, and live steam is fed into the water and plant material mixture 7. The effects of different distillation methods on essential oil content and composition of aromatic plants has been previously reported. The highest essential oil yields of Thymus kotschyanus and rose-scented geranium (Pelargonium sp.) were obtained by the hydrodistillation method and the lowest by steam distillation The distillation methods also had effects on the essential oil components of rose-scented geranium, Nigella sativa and Satureja hortensis 7, 10,11. The essential oil of Salvia officinalis was significantly decreased towards the second harvesting 12. The minimum Ocimum americanum essential oil content was produced from the second harvesting also some components were changed compared with the first harvesting 13. First harvest gave the highest values of eugenol and linalool isolated form Ocimum basilicum 14. The aim of this study was to test the effect of harvesting treatments and / or distillation methods (hydrodistillation, hydro-steam distillation and steam distillation) on the essential oil content and composition of Pelargonium odoratissimum L. and Melissa officinalis L. plants. 21

3 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp Experimental Plant material: Experiments were carried out in a greenhouse at Shanghai Institute of Plant Physiology and Ecology (SIPPE), Shanghai, China, during the years of 2007 and The soil used in this study was sand: clay: peat moss (1:1:1). The seedlings of P. odoratissimum L. and M. officinalis L. were obtained from the Ministry of Agriculture of China through Shanghai Institute of Plant Physiology and Ecology. The seedlings were transplanted into plastic pots (30 cm diameter and 50 cm height). In the first week of November (2007), the pots were transferred to a greenhouse adjusted to 35/ 24 C, 90/60 % RH day/night and light intensity approximately 3700 Lux. Each pot was filled with 10 kg of air-dried soil. Three weeks after transplanting, the seedlings were thinned to three plants per pot. A randomized factorial design with five replications was used. Each replication contained six treatments (2 harvesting time of P. odoratissimum or M. officinalis x 3 distillation treatments). Each treatment had ten pots (3 plants per each). All agricultural practices were done according to the recommendation of the Chinese Ministry of Agriculture. Essential oil isolation: At full blooming, the plants were harvested two times (first, and second harvesting) during the growing seasons by cutting the plants 5 cm above the soil surface for collect the herb (first harvesting), then leave the plants to regrow till full blooming and cut again (second harvesting), similar to commercial production. Total fresh weights of the herbs (g/plant) were recorded. Fresh plants were collected from each treatment during the first and second harvesting. They were weighed to extract the essential oil. Fresh plant material (500 g) from each replicate of all treatments was subjected to hydrodistillation (HD), hydro-steam distillation (HD-SD) and steam distillation (SD) for 3 h using a Clevenger type apparatus 15. The essential oil content was calculated in percentage amount. In addition, total essential oil as g per plant was calculated by using the fresh weight of the herb. The essential oils extracted from P. odoratissimum and M. officinalis were collected from the first and second harvesting through hydrodistillation, hydro-steam distillation and steam distillation in order to identify the chemical constituents. GC-MS analysis: The ADELSIGLC MS system, equipped with a BPX5 capillary column (0.22 mm id x 25 m, film thickness 0.25 μm) was used. Analysis was carried out using helium as the carrier gas, with the flow rate at 1.0 ml/min. The column temperature was programmed from 60 C to 240 C at 3 C/min. The sample size was 2 μ1, the split ratio 1:20. The injector temperature was 250 C. The ionization voltage applied was 70 ev, mass range m/z amu. The Kovat s indices were determined by co-injection of the sample with a solution containing a homologous series of n-hydrocarbons in a temperature run identical to that described above. The separated components of the essential oil were identified by matching with the National Institute of Standards and Technology (NIST) mass spectral library data, comparison of the Kovat s indices with those of authentic components and with published data 16. The quantitative determination was carried out based on peak area integration. 22

4 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp Statistical analysis: The averages of data from each harvesting were statistically analyzed using analysis of variance (ANOVA) and values of least significant difference (LSD) at 5 % according to Snedecor and Cochran 17. Results and Discussion: A qualitative and quantitative comparison of the essential oil constituents based on the different distillation methods applied to P. odoratissimum and M. officinalis oil is presented in Tables 1, 2 and 3. The highest oil yield was obtained by hydrodistillation and the lowest by steam distillation during the first and second harvesting. This may be due to the fact that parameters such as type of plant material, mode of combination, mode of charging and grade of insulation play a more significant role in the steam distillation method compared to the other isolation techniques 7. These results are in agreement with previous work about the effect of distillation methods on essential oil content and composition of other essential oil-bearing plants such as Satureja rechingeri, Thymus kotschyanus and Pelargonium sp 7,9,10. The essential oil (percentage and g per plant) of P. odoratissimum was less than that of M. officinalis, which may be due to genetic differences between the two species from two families 18. Also these results may be due to the increment in herb weight per plant of M. officinalis compared with P. odoratissimum L. The essential oil contents of P. odoratissimum and M. officinalis were significantly decreased towards the second harvesting, which is in accordance with results obtained by Hendawy and Khalid 12. Forty constituents were found in the P. odoratissimum herb essential oil extracted by the three distillation methods. The main components of essential oil extracted by hydrodistillation were methyl eugenol (30.4 % and 25.9 %) and isomenthone (26.4 % and 26.9 %) during both first and second harvesting, while the main components of essential oil obtained by hydro-steam distillation were limonene (30.5 % and 32.4 %), isomenthone (20.8 % and 19.2 %) and methyl eugenol (21.3 % and 16.2 %) at both harvesting.main components obtained by steam distillation were limonene (49.4 % and 38.8 %), isomenthone (15.1 % and 19.3 %), and Methyl eugenol (12.2 % and 14.4 %) through the first and second harvesting, respectively. The highest amount of oxygenated compounds resulted from hydrodistillation and the lowest by hydro- and steam distillation at the first harvesting, while in the second harvesting the highest levels of oxygenated compounds were found in steam distilled and the lowest in hydro- steam distilled samples. Hydrocarbon levels were highest when applying hydro- steam distillation at the first harvesting, while in the second harvesting highest amounts of hydrocarbon were obtained by steam distillation. Forty six constituents were found in the M. officinalis herb essential oil extracted by the three distillation methods. The main components of essential oil extracted by hydrodistillation were citronellal (36.1 % and 37.8 %) and citronellol (18.3 % and 16.9 %) during both first and second harvesting, while the main components obtained by hydrosteam distillation were citronellal (29.8 % and 27.3 %), citronellol (18.4 % and 17.2 %) and linalool (13.0 % and 11.8 %) at both harvesting. On the other hand, α-terpinene (20.2 % and 21.9 %), linalool (19.5 % and 18.6 %), and citronellol (12.0 % and 13.1 %) were the most abundant essential oil constituents from first and second harvesting, respectively. The 23

5 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp highest amounts of oxygenated compounds resulted from hydrodistillation and the lowest by hydro- steam distillation at the first harvesting, while in the second harvesting highest levels of oxygenated compounds were obtained by steam distillation and lowest by hydro- and steam distillation. The highest hydrocarbons compounds were resulted from steam distillation during the first and second harvesting. These results are in accordance with those obtained by Sefidkon and co-workers 7 and other reports 11,19. We conclude from this study, that the distillation methods and / or harvesting time influenced the quantity and chemical composition of P. odoratissimum and M. officinalis oils The highest oil yield was obtained by hydrodistillation and the lowest by steam distillation, so hydrodistillation was recommended for the highest quantity of essential oil of both P. odoratissimum and M. officinalis. Hydrodistillation conditions is most suitable for a the highest percentage of methyl eugenol but Hydro-steam distillation or steam distillation are most suitable for the highest percentage of limonene isolated from P. odoratissimum. Hydrodistillation or hydro- steam distillation were recommended for the highest percentage of citronellal while steam distillation was recommended for the highest percentage of α- terpinene isolated from M. officinalis. The essential oil contents of P. odoratissimum and M. officinalis were significantly decreased towards the second harvesting and the amounts of essential oil components were changed. The oxygenated compounds and hydrocarbon compounds were changed according to the distillation methods and / or harvesting number for both P. odoratissimum and M. officinalis. plants. Acknowledgements: The authors would to thank the Third World Academy of Sciences (TWAS) and Chinese Academy of Sciences (CAS) for their support of this work. References 1. Grieve, M. (1984). A Modern Herbal. Penguin. London, UK 2. Bown, D. (1995). Encyclopaedia of Herbs and their Uses. Dorling Kindersley. London, UK. 3. Westwood, C. (1993). Aromatherapy - A guide for home use. Amberwood Publishing Ltd. Rochester, UK. 4. Bisset, N.G. (2001). Herbal Drugs and Phytopharmaceuticals. CRC Press. Boca Raton, London, New York, Washington DC. 5. Neda, M.D., Biljana, B., Marina, S. and Natasa, S. (2004). Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential oil. J. Agric. Food Chem. 52: Masakova, N.S., Tseevatuy, B.S., Trofimenko, S.L., and Remmer, G.S. (1979). The chemical composition of volatile oil in lemon-balm as an indicator of therapeutic use. Planta Med. 36: Sefidkon, F., Abbasi, K., Jamzad Z., and Ahmadi, S. (2007). The effect of distillation methods and stage of plant growth on the essential oil content and composition of Satureja rechingeri Jamzad. Food Chemistry, 100: Sefidkon, F., Abbasi, K. and Bakhshi Khaniki, G. (2006). Influence of drying and extraction methods on yield and chemical composition of the essential oil of 24

6 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp Satureja hortensis. Food Chem. 99: Sefidkon, F., Dabiri, M., and Rahimi-Bidgoly, A. (1999). The effect of distillation methods and stage of plant growth on the essential oil content and composition of Thymus kotschyanus Boiss & Hohen. Flavour Fragr. J. 14: Kiran, G.D., Babu, V., and Kaul, K. (2005). Variation in essential oil composition of rose-scented geranium (Pelargonium sp.) distilled by different distillation techniques. Flavour Fragr. J. 20(2): Stoyanova, A., Georgiev, E., Wajs, A. and Kalemba, D. (2003). A comparative investigation of the volatiles from seeds of Nigella sativa L. from Bulgaria. J. Essent. Oil Bearing Plants 3: Hendawy, S.F., and Khalid, K.A. (2005). Response of sage (Salvia officinalis L.) plants to zinc application under different salinity levels. J. Appl. Sci. Res. 1(2): Sabra, M.S.S. (2002). Response of Ocimum americanum L. plants to nitrogen fertilization. M.Sc. Thesis, Fac. Agric., Zagazig Univ., Egypt. 14. Abdelraouf, R.S. (2001). Production of sweet basil (Ocimum basilicum) in new reclaimed lands under different levels of bio fertilizers and plant densities. M.Sc. Thesis, Fac. Agric., Ain Shams Univ., Egypt. 15. Clevenger, J.F. (1928). Apparatus for determination of essential oil. J. Amer. Pharm. Assoc. 17: Adams, R.P. (1995). Identification of essential oil components by gas chromatography/mass spectroscopy. Allured. Carol Stream, Illinois, USA. 17. Snedecor, G.W. and Cochran, W.G. (1990). Statistical Methods. Iowa State College Press. Ames, Iowa, USA. 18. El-Beltagy, A.S. and Soliman, M.M. (1993). Effect of growth regulators on rates of recovery water logged tomato plants. Egypt. J. Hort. Sci. 12(2): Zrira, S., Elamrani, A., Pellerin, P., Bessière, J.M., Menut, C. and Benjilali, B. (2008). Isolation of Moroccan Ammi visnaga oil: Comparison between hydrodistillation, steam distillation and supercritical fluid extraction. J. Essent. Oil Bearing Plants 11(1):

7 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp Table 1. Effect of harvesting treatments, distillation methods and their interactions on the essential oil (Percentage and g per plant) of Pelargonium odoratissimum L. and Melissa officinalis L. plants Harvesting Distillation methods P. odoratissimum L. Essential oil M. officinalis L. Essential oil treatments Percentage g per plant Percentage g per plant First harvesting HD HD-SD SD Over all (Mean ) of First harvesting Second harvesting HD HD-SD SD Over all (Mean ) of Second harvesting Over all (Mean) of HD Distillation methods HD-SD SD L. S. D. at 0.05 Distillation methods Harvesting treatments Distillation methods *Harvesting treatments

8 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp Table 2. Effect of harvesting treatments and distillation methods on the essential oil components (Percentage) of Pelargonium odoratissimum L. No. Components KI* Identification method Distillation methods treatments First harvesting Second harvesting HD HD-SD SD HD HD-SD SD 1 α-pinene 939 KI & MS benzaldehyde 961 KI & MS Sabinene 976 KI & MS β-pinene 980 KI & MS Myrcene 991 KI & MS p-cymene 1026 KI & MS Limonene 1031 KI & MS ,8-Cineole 1033 KI & MS (Z)-β-Ocimene 1046 KI & MS (E)-β-Ocimene 1050 KI & MS γ-terpinene 1062 KI & MS Fenchone 1075 KI & MS Linalool 1098 KI & MS Undecane 1100 KI & MS Camphor 1143 KI & MS Isomenthone 1154 KI & MS Borneol 1180 KI & MS α-terpineol 1189 KI & MS Dodecane 1199 KI & MS Carveol 1212 KI & MS Fenchyl acetate 1226 KI & MS Pipritone 1252 KI & MS

9 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp table 2. (continued ). No. Components KI* Identification method Distillation methods treatments First harvesting Second harvesting HD HD-SD SD HD HD-SD SD 23 Tridecane 1299 KI & MS α-cubebene 1351 KI & MS α-copaene 1376 KI & MS β-cubebene 1390 KI & MS Tetradecane 1399 KI & MS Methyl eugenol 1401 KI & MS β-caryophyllene 1418 KI & MS α-caryophyllene 1454 KI & MS Germacrene D 1480 KI & MS β-selinene 1488 KI & MS Farnesene 1518 KI & MS γ-cadinene 1524 KI & MS Germacrene B 1556 KI & MS Caryophyllene oxide 1581 KI & MS Citronyllyl tiglate 1667 KI & MS Octadecane 1800 KI & MS Nonadecane 1900 KI & MS Eicosane 2000 KI & MS Total identified KI* = Confirmed by comparison with Kovats index on DB5 column

10 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp Table 3. Effect of harvesting treatments and distillation methods on the essential oil components (Percentage) of Melissa officinalis L. No. Components KI* Identification method Distillation methods treatments First harvesting Second harvesting HD HD-SD SD HD HD-SD SD 1 α-pinene 938 KI & MS Camphene 952 KI & MS Sabinene 976 KI & MS β-pinene 980 KI & MS Myrcene 991 KI & MS Octenal 995 KI & MS α-phellandrene 1005 KI & MS α-terpinene 1018 KI & MS Linalool 1098 KI & MS α-thujone 1102 KI & MS β-thujone 1116 KI & MS Fenchyl acetate 1126 KI & MS (E)-Rose oxide 1127 KI & MS Limonene 1131 KI & MS (Z)-β-Ocimene 1140 KI & MS Camphor 1143 KI & MS Citronellal 1153 KI & MS Isomenthone 1154 KI & MS Borneol 1165 KI & MS Menthol 1173 KI & MS Estragole 1195 KI & MS Carveol 1212 KI & MS Nerol 1228 KI & MS

11 Khalid A. Khalid et al. / Jeobp 12 (2) 2009 pp table 3. (continued ). No. Components KI* Identification method Distillation methods treatments First harvesting Second harvesting HD HD-SD SD HD HD-SD SD 24 Citronellol 1229 KI & MS Neral 1240 KI & MS Geraniol 1255 KI & MS Geranial 1271 KI & MS Limonene oxide 1304 KI & MS α-cubebene 1351 KI & MS Carvyl acetate 1362 KI & MS α-copaene 1376 KI & MS Geranyl acetate 1380 KI & MS (Z)-β-Damascenone 1382 KI & MS β-cubebene 1390 KI & MS Dodecanal 1397 KI & MS β-caryophyllene 1419 KI & MS α-humulene 1452 KI & MS β-selinene 1488 KI & MS Germacrene D 1491 KI & MS γ-cadinene 1524 KI & MS δ-cadinene 1540 KI & MS (Z)-β-Farnesene 1546 KI & MS Ledol 1565 KI & MS Caryophyllene oxide 1581 KI & MS Nonadecane 1903 KI & MS Eicosane 2000 KI & MS Total identified KI* = Confirmed by comparison with Kovats index on DB5 column