This article was downloaded by: [New York University] On: 01 December 2013, At: 19:31 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Essential Oil Bearing Plants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/teop20 Study of the Essential Oil of Limnophila Rugosa (Roth.) Merr in the South of Vietnam a Nguyen Truc Linh & Le Ngoc Thach b a Faculty of Chemical Technology, University of Cantho , 3/2 Street, Ninhkieu Dist., Cantho City , Vietnam b Department of Organic Chemistry, Faculty of Chemistry , University of Science, Vietnam National University-HCM City , 227 Nguyen Van Cu Street, Dist 5., Hochiminh City , Vietnam Published online: 12 Mar 2013 To cite this article: Nguyen Truc Linh & Le Ngoc Thach (2011) Study of the Essential Oil of Limnophila Rugosa (Roth.) Merr in the South of Vietnam, Journal of Essential Oil Bearing Plants, 14:3, 366-372, DOI: 10.1080/0972060X.2011.10643947 To link to this article: http://dx.doi.org/10.1080/0972060X.2011.10643947 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content This article may be used for research, teaching, and private study purposes Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions Jeobp 14 (3) 2011 pp 366 - 372 366 Journal of Essential Oil Bearing Plants ISSN Print: 0972-060X Online: 0976-5026 www.jeobp.com Study of the Essential Oil of Limnophila Rugosa (Roth.) Merr in the South of Vietnam Nguyen Truc Linh 1, Le Ngoc Thach 2* Downloaded by [New York University] at 19:31 01 December 2013 Faculty of Chemical Technology, University of Cantho, 3/2 Street, Ninhkieu Dist., Cantho City, Vietnam Department of Organic Chemistry, Faculty of Chemistry, University of Science, Vietnam National University-HCM City, 227 Nguyen Van Cu Street, Dist 5., Hochiminh City, Vietnam Received 14 February 2010; accepted in revised form 10 December 2010 Abstract: The essential oils of Limnophila rugosa (Roth.) Merr (Scrophulariaceae) in the south of Vietnam obtained by hydrodistillation under conventional heating or microwave irradiation showed a slight difference in chemical compositions The main constituents of these oils are trans-anethole (24.96-27.12 %) and methyl chavicol (70.79-71.00 %) In addition, microwave method gave a higher extraction yield in a considerably shorter time than conventional heating protocol The physical, chemical and antimicrobial properties of the oils were also determined Key words: Limnophila rugosa, Scrophulariaceae, chemical composition, trans-anethole, methyl chavicol, microwave hydrodistillation Introduction: Limnophila rugosa (Roth.) Merr., an annual herb native to wetlands in Vietnam, belongs to the Scrophulariaceae family 1,2 It is widely cultivated and used as food flavoring, seasoning or culinary herb In addition, the plant is also employed as a traditional medicine for colds, cough, laxative, diarrhoea, gonorrhea and anaprodisia 3,18 The chemical composition of Limnophila rugosa (Roth.) Merr essential oil, obtained by hydrodistillation (steam distillation) with conventional heating, from several regions including the north of Vietnam, 4,5 China, 6,7 Hungary, and India 9,10,11,18 had been studied However, only some of these reports mention the antimicrobial activity of the oils Moreover, the essential oil of Limnophila rugosa (Roth.) Merr from the south of Vietnam has not been studied yet Therefore, we decided to investigate this oil in term of both chemical and antimicrobial properties Recently, microwave-assisted extraction technique has been applied as a new and efficient method to isolate essential oils from plant materials 12 In 1996, we reported that a modified domestic microwave oven could be used as a heating source for fast hydrodistillation of essential oils from plant materials.13 Unfortunately, this system was subsequently proved to be not applicable for long hydrodistillation because water is quickly evaporated out of the distillation flask after several minutes In this paper, we report the first systematic study of essential oils from the aerial parts of Limnophila rugosa in the south of Vietnam obtained by hydrodistillation using both conventional *Corresponding author (Le Ngoc Thach) E-mail: © 2011, Har Krishan Bhalla & Sons Le Ngoc Thach et al / Jeobp 14 (3) 2011 366 - 372 367 heating (HDCH) and microwave irradiation (HDMW) We also introduce a newly-modified Clevenger type apparatus 14 designed for long hydrodistillation Oil contents in different parts of the plant and versus age of material were also studied The chemical compositions of oil products were analyzed by GC-MS The physical, chemical and antimicrobial properties of oils were also investigated Downloaded by [New York University] at 19:31 01 December 2013 Experimental Materials: All Limnophila rugosa samples were collected in the morning in Trang Bang Dist., Tay Ninh Province The plant was identified by the Department of Botany, School of Biology, University of Science, Vietnam National University-Ho Chi Minh City Gas chromatography: GC analyses were performed on an Aglient 6890N GC equipped with an FID and DB-5 capillary column (30 m x 0.32 mm x 0.25 μm) Nitrogen was used as the carrier at a flow rate of 1.74 ml/min The constant pressure mode at 9.32 psi was chosen on GC program The oven temperature was programmed from 60°C to 240°C at 3°C/min The injector and detector temperature was 250°C The injection volume was 1.0 μl The split ratio was 10:1 The percentage composition of the constituents of the oils was determined by area normalization Gas chromatography-mass spectrometry (GC/MS): An Agilent 7890 GC coupled to a 5973A Network Mass Selective Detector using HP-5MS capillary non-polar column (30.0 m x 0.25 mm x 0.25μm) Helium was employed as the carrier at a flow rate 1.20 mL/min The constant pressure mode at 10.604 psi was chosen on GC program The injection temperature was 250°C; the injection volume was 1.0 μl; the split ratio was 10:1, and the ionization voltage was 70 eV The oven temperature was programmed from 60°C to 240°C at 3°C/min The retention indices (arithemetic indices) of the oil components were calculated relative to the homologous series of C9-C17 n-alkanes by using the equation propounded by van den Dool and Kratz in 1963 Identification of constituents was based on comparing their mass spectra with those of standard compounds registered in the NIST 2008 library Moreover, the identification was also confirmed by the retention indices of each individual constituent relative to those reported in the literature compiled by Adams 15 Density of oil products was determined by using glass densitometer, and their [α] 27 were meaD sured by A Krüss Optronic polarimeter (Germany).16 Typical hydrodistillation procedure: Fresh aerial parts of the plant (300 g) were cut into small pieces, loaded onto a Clevenger type apparatus with distilled water (600 ml), and hydrodistilled using HDCH or HDMW The oil yield was measured as a function of time 14 Anti microorganism test: The oil is diluted in dimethyl sulphoxide (DMSO) The concentration can be calculated as following equations: C0 = 100 μl-1: concentration of original oil C1 = 1μl C0 + 9μl DMSO = 10-1 μl-1 C2 = 1μl C1 + 9μl DMSO = 10-2 μl-1 C3 = 1μl C2 + 9μl DMSO = 10-3 μl-1 C4 = 1μl C3 + 9μl DMSO = 10-4 μl-1 The antimicrobial activity of Limnophila rugosa aerial part oil was tested by paper disc method (standard diameter of mm) on Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans Le Ngoc Thach et al / Jeobp 14 (3) 2011 366 - 372 368 Downloaded by [New York University] at 19:31 01 December 2013 Results and discussion: The extraction yields in correspondence with extraction methods are shown in Figure The conventional method requires longer time (5 hours) but provides lower yield (0.1347 %) in comparison with the microwave method (0.1560 % yield in minutes) This result is consistent with the fact that microwave heating accelerates the evaporation of volatile compounds from plant materials Therefore, microwave-assisted extraction has been recently considered as a “green” technique due to its time and energy efficiency 17 It should be noted that our newly-designed apparatus for microwave-assisted hydrodistillation allows water to return to the distillation flask, thus, providing enough water for long distillation processes 13 The distribution of essential oils in different parts of plant materials was also investigated As illustrated in Figure 2, the leaf of the plant contains the highest amount of essential oil (0.2740 %), followed by the aerial part after flowering (AF) (0.1994 %), aerial part before flowering (BF) (0.1347 %) and stem (0.0151 %) It was also found that the oil content in the plant is dependent on the age of plant (nearly peak value observed at four months and slightly leveled off afterwards) Figure Oil yield vs the hydrodistillation time of HDMW (left) and HDCH (right) Figure The oil yields vs the plant part (left), and changes in oil yield vs the age of the plant (right) investigated by HDCH method Downloaded by [New York University] at 19:31 01 December 2013 Le Ngoc Thach et al / Jeobp 14 (3) 2011 366 - 372 369 The chemical compositions of the aerial part oil were identified by both GC-FID and GC-MSD technique as shown in Table Generally, oil products isolated by the two techniques show a slight difference in oil compositions For example, the oil obtained by HDCH method indicates the presence of limonene, coumarin, pentadecane… while these minor components were not found in the oil produced by HDMW method In addition, the percentages of major components, trans-anethole and methyl chavicol, are also variable with the heating methods On the other hand, these values are considerably different with that of the oil from China and the north of Vietnam For example, only 24.96 % of transanethole was found in the oil from the south of Vietnam while the figures for the oil from the north of Vietnam and China are 89.40 % and 76.39 %, respectively In contrast, methyl chavicol, the major component in the oil from the south of Vietnam (70.79 %), occurs as the minor one in the oil from the north of Vietnam (0.41%) (Table 2) The chemical constituents of the aerial part after flowering oil (AF) were also identified by GCMS technique The result indicates a transformation between two major components after flowering This can be easily observed by an increasing of trans-anethole (from 24.96 to 30.35 %) and decreasing of methyl chavicol (from 70.79 to 64.20 %) while the total percentage of these two components remains unchanged (Table 1) In general, oils isolated by using different techniques show a slight difference in compositions and abundance Besides, the oil constituents of the aerial part of Limnophila rugosa (Roth.) Merr vary with the geographic position of the herbs The percentage of each component is variable with the periods of plant growth Physical and chemical properties of aerial part oils isolated by both HDCH and HDMW methods were also determined (Table 3) 15 In general, the difference between two oils is negligible However, if compared with the oils from China and India, the difference becomes significant (Table 4) The anti-microbial activity of Limnophila rugosa aerial part oil was tested by paper disc method (standard diameter of mm) on Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans In addition, a typical fungi (Candida albicans) was also used to test the antifungal activity of this essential oil Interestingly, oil obtained from the microwave method exhibits stronger antimicrobial activity than the one from conventional method (Table 5) The antibacterial activity of the oil arises from the two main components The higher antimicrobial activity of the oil obtained from HDMW method can be attributed to the larger amounts of the two main components Previous studies also demonstrated significant anti-bacterial and anti-fungal activities of this oil 18,19 Infusion of leaves is used as diuretic and stomachic treatment in the Philippines and India 18 Moreover, L rugosa’s oil has antifungal activity toward Candida albicans, which cause genital infection in human 20 Further in vitro and in vivo tests are needed to confirm this finding The antifungal activity promises a broader usage of L rugosa’s oil Conclusions: Two different techniques (HDMW and HDCH) were used to extract essential oil from Limnophila rugosa (Roth.) Merr HDMW method is more efficient to extract main components (trans-anethole and methyl chavicol) Therefore, HDMW is a suitable technique for extracting essential oil from plant material containing a large amount of water HDCH method was applied to investigate the change in composition of oil versus period of growth The aerial parts can provide optimal oil yield after months Interestingly, a transformation between trans-anethole and methyl chavicol was observed in the oil of aerial part after flowering In addition, the leaves were found to give the highest oil yield Besides, the essential oil from Limnophila rugosa (Roth.) Merr indicates significant antibacterial and fungal activities Although further testing is needed, it is believed that these results would lead to a wider application for Limnophila rugosa oil References: Pham, H.H (2000) Plants in Vietnam Youth Ed HCM City pp 480 Le Ngoc Thach et al / Jeobp 14 (3) 2011 366 - 372 Downloaded by [New York University] at 19:31 01 December 2013 10 11 12 13 14 370 Ministry of Science, Technology and Enviroment (1996) The Red Book of Vietnam, Botanical Part Science and Technology Hanoi pp 170 Do, H.B (2003) Medicinal Plants and Animals in Vietnam Science and Technology Hanoi pp 993-994 Vu, N.L., Nguyen, D T and Nguyen, T T (1988) Limnophila rugosa (Roth.) Merr - A rich sources of anethole Journal of Pharmacy (4,5) 12-20 Tran, H.T (2003) Study of the essential oil of aerial part of Limnophila rugosa (Roth.) 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McGraw Hill New York Table Chemical composition (%) of the aerial parts (BF and AF) of Limnophila rugosa (Roth.) Merr No Constituents 10 11 12 13 14 15 16 17 18 19 20 1-Octen-3-ol 3-Octanol Limonene Linalool Methyl chavicol cis-Anethole p-Anisaldehyde 4-(1-Methylpropyl)phenol trans-Anethole 1-(4-Methoxyphenyl)-2-propanol Caryophylene Coumarin α-Caryophylene Pentadecane α-Farnesene Nerolidol 2-(1-Methoxypropyl)phenol Caryophylene oxide γ-Gurzunene Heptadecane Total % Oil composition Before flowering After flowering HDCH HDMW HDCH 0.18 0.17 70.79 0.26 0.37 0.08 24.96 0.18 0.96 0.11 1.09 0.04 0.13 0.12 99.44 0.10 71.00 0.31 0.46 27.12 0.14 0.22 0.30 0.08 0.08 0.07 99.88 0.73 0.27 0.05 0.28 64.20 0.39 0.03 30.35 1.32 0.21 1.39 0.08 0.03 0.16 0.08 99.57 Le Ngoc Thach et al / Jeobp 14 (3) 2011 366 - 372 372 Table Comparison of the main constituents of aerial part oils in the south of Vietnam and other regions % Oil composition Southern Vietnam Northern Vietnam Main constituents Downloaded by [New York University] at 19:31 01 December 2013 Linalool Methyl chavicol p-Anisaldehyde trans-Anethole Caryophylene α-Caryophylene 0.17 70.79 0.37 24.96 0.96 1.09 China 0.10 0.41 0.09 89.4 0.74 0.85 0.08 21.94 0.05 76.39 0.08 0.15 Table Physical and chemical properties of oil products obtained by HDCH and HDMW methods nD26 , [α α] 25 D IA IE 1.5226 1.5237 + 0.307 + 0.289 0.5745 0.5236 2.6552 2.5441 Method HDCH HDMW 0.9579 0.9585 IA : Index of Acide ; IE : Index of Ester Table Comparison of physical and chemical properties of aerial part oils in the south of Vietnam and other regions Properties China7 Southern Vietnam d nD 0.9579 30 1.5226 26,4 [α] 25 D + 0.30725 - 0.4524 IA IE 0.57 2.08 0.38 5.32 0.9756-0.9985 1.5283 22 India9 24 India10 0.986 10 1.530-1.53110 0.995922 1.522121 - - 3.02 31.78 1.15 12.58 Table Anti microorganism properties of Limnophila rugosa (Roth.) Merr volatile oil Microorganism C* = 10 Bacillus subtilis Escherichia coli, ATCC 25922 Staphylococcus aureus, ATCC 25923 Pseudomonas aeruginosa, ATCC 27853 Candida albicans *C : oil’s concentration (μl-1) 22 16.5 27 13 37.5 Zone of inhibition (mm) HDCH HDMW 10 -1 10-2 10 -3 10 10 -1 10 -2 12 6 6 6 6 6 23 22 15 11 >60 11 6 6 6 6 ... Journal of Essential Oil Bearing Plants ISSN Print: 0972-060X Online: 0976-5026 www.jeobp.com Study of the Essential Oil of Limnophila Rugosa (Roth.) Merr in the South of Vietnam Nguyen Truc Linh... several minutes In this paper, we report the first systematic study of essential oils from the aerial parts of Limnophila rugosa in the south of Vietnam obtained by hydrodistillation using both... China and the north of Vietnam For example, only 24.96 % of transanethole was found in the oil from the south of Vietnam while the figures for the oil from the north of Vietnam and China are 89.40