NPC Natural Product Communications Composition and Biological Activity of the Essential Oil of Litsea laevigata Nees. (Lauracea) 2007 Vol. 0 No. 0 1-3 Muhammed Arif Ma, Subbu Raj Ma, Leopold Jirovetzb and Mohamed Shafi Pa* a Department of Chemistry, University of Calicut, Kerala, India 673635 Department of Clinical Pharmacy and diagnostics, Althanstrasse 14,University of Vienna, A1090 Vienna, Austria b shafimuham@rediffmail.com Received: January XX, 2007; Accepted: January XX, 2007 The essential oil of berries of Litsea laevigata Nees., growing wild in Western Ghats, Kerala, India was obtained by hydrodistillation and was fractionated in a column using n-pentane and diethyl ether as eluents. The essential oil and its fractions were analysed by GC and GC-MS. Twenty six compounds representing 99.2% of the original oil were identified. The major components belong to the class of terpene hydrocarbons trans-α-Bergamotene(26.7%), α-Pinene (25%) and β-Pinene (8.2%). The anti microbial activity of essential oil of L.laevigata and its fractions against four Gram-positive and four Gramnegative bacteria (Staphylococcus aureus, Bacillus subtilis, Streptococcus faecalis, Staphylococcus albus, Escherichia coli, Pseudomonas aeruginosa , Protieus vulgaris, Klebsiella aerogenes) as well as two fungi (Candida albicans, Aspergillus niger) was studied. The bioassay showed that the oils exhibited moderate to high antimicrobial activity. Keywords: Litsea laevigata, Essential oil, Bergamotene, α-Pinene, β-Pinene, antimicrobial activity . The genus Litsea laevigata Nees. belongs to the Lauracea family. It is found in semi evergreen forests of the hilly regions of Kerala [1] and is endemic to south Western Ghats. It is a tree up to 10 m tall; Berries are 0.5 cm long, ellipsoid and violet when ripe. Flowering season is between December and January [2]. Literature survey showed that no phytochemical studies were reported on the species L.laevigata. Previous investigations of the chemical constituents of different species of Litsea have been reported [35]. Byung Sun Min et al. [3] isolated two lactones, litsealactone-A and litsealactone-B from the leaves of Litsea japonica, together with three known lactones, hamabiwalactone-A, hamabiwalactone-B, and akolactone-B. Hsing-I Cheng [4], et al. isolated six compounds from the leaves of Litsea acutivena, including one nor-neolignan, dehydroxymethylailanthoidol, litseakolide-D, litseakolide-E, litseakolide-F, litseakolide-G, and isolincomolide-D. Hong-Jie Zhang et al. [5] identified seven sesquiterpenes, named litseagermacrane, 7-epi-eudesm-4(15)-ene-1R,6R1α,6α-diol, 7-epi-eudesm-4(15)-ene-1β,6β-diol, 5- epi-eudesm-4(15)-ene-1β,6β-diol, eudesm-4(15)-ene1 β,6β-diol and litseahumulanes-A and B , from leaves and twigs. The objective of the present work was to characterise the volatiles present in the fruits of Litsea laevigata and to evaluate its antimicrobial properties. Hydrodistillation of L. Laevigata fruits yielded a colourless oil (LL) in 0.34% (w/w) yield, based on fresh weight of plant. In order to assess the antimicrobial property of different fractions, the essential oil was separated into two fractions by column chromatography using n-pentane (LLP) and diethyl ether (LLD). The identified constituents in different fractions are presented in table 1. The essential oil of Litsea laevigata (LL) represented 0.34% of the fresh weight of the berries and 99.2 % of the oil was identified by GC and GC-MS. Twenty six compounds were identified (table 1) in the essential oil sample LL of which monoterpenes were the major class of compounds (59.3%). The major compounds were α-pinene (25%), β-pinene (8.2%),αterpineol (5%), fenchol (3.5%), limonene (4.3%) and 1,8-cineole (4.5%). The percentage of sesquitertpenes 2 Natural Product Communications Vol. 0 (0) 2007 was 37.4 % whereas nonterpenoid compounds constituted only 2.5%. The important sesquiterpenes present were trans-α-bergamotene,(26.7%), αcopaene(4.1%) and β-santalene(1.7%). The essential oil fraction LLP was represented by non polar fraction of the original oil, thirty compounds were identified in this fraction, of which monoterpenes were the major class of compounds (54.5%). The percentage of sesquiterpene was about 42.5% whereas nonterpenoid compounds constitute only 1.2 %. The essential oil fraction LLD is represented by polar fraction of the original oil, thirty one compounds were identified in this fraction. The important compounds in this fraction were 1,8cineole(26.9%), α-terpineol (13.1%), fenchol (11.5%) and borneol (8.5%).. Both LLP and LLD contained compounds that could not be detected in the original oil. This can be due to the higher concentration of them during fractionation. Table 1: Constituents of Litsea laevigata essential oil and its fractions Compound trans-α-Bergamotene α-Pinene β-Pinene α-Terpineol 1,8-Cineole Limonene α-Copaene Camphene Fenchol Decanol Borneol β-Santalene α-Farnesene β-Farnesene α-Santalol β-Elemene α-Fenchene α-Bulnesene p-Cymene Cis-β-Ocimene β-Bisabolene β-Myrcene Pinocarveol Caryophyllene Terpinen-4-ol Epi-β-Santalene Junipene Myrtenol Verbenol Myrtenal Decanoic acid Camphene hydrate Hexadecanol Nonanol Total LL (%) 26.7 25 8.2 5 4.6 4.3 4.1 4 3.5 2.5 2.2 1.7 0.9 0.8 0.7 0.7 0.7 0.7 0.6 0.4 0.4 0.3 0.3 0.3 0.2 0.2 0.2 99.2 LLP (%) 16.5 25.3 2.1 0.7 8.4 3.1 6.2 7.5 0.8 0.7 0.4 5.1 1.3 5.1 1.8 2.1 1.4 0.4 0.4 0.2 0.3 0.7 1 1.1 0.1 2.4 0.2 1.6 0.9 0.5 98.2 LLD (%) 3.5 0.5 0.2 13.1 26.9 0.5 0.9 0.2 11.5 3.4 8.5 0.8 0.8 0.7 0.4 0.9 0.3 0.4 0.9 1.4 0.6 0.2 2.1 1.2 1.7 0.4 0.1 4.5 2.6 1.9 1.8 92.9 The composition of essential oil was determined by comparison of the ma spectrum of each component with Wiley GC/ MS library data and also from its retention index (RI). Author A et al. The essential oil of Litsea laevigata was colourless and possessed soft piney-woody, diffuse earthy, reminding of patchoulene, dry-herbal odour whereas the essential oil fraction LLP was colourless and had fresh, harsh-terpeny, diffuse citrus-metallic (myrcene-note) and soft herbal odour. The essential oil fraction LLD had light yellow colour and had fresh-terpeny, earthy (root-like), mild woodyaldehydic, later patchouli-bulnesene-like, fatty-sour in the background. The piney-woody, earthy and herbal odours can be attributed to the pinenes, terpineol, cineole, α -copaene and pinocarviol [6a,6b]. The odour impression of LLP and LLD are comparable but for the harsh-terpeny odour of LLP. This can be attributed to the high α- pinene content of LLP in comparison to LLD. Apart from these aroma compounds this essential oil contains small quantities of santalene and santalone which are sandalwood oil constituents. All these factors make this essential oil valuable in fine perfumery were piney –woody, earthy and herbal odour notes are desirable eg. in shower gels, deodorants etc., moreover the yield of this oil (0.34% of fresh weight) makes it a commercially viable product The result of the anti-microbial screening of the essential oil (LL) is given in table 2. All microorganisms exhibited concentration dependent activity. The oil is very active against gram-positive bacteria such as Streptococcus albus and fungus Aspergillus niger. The essential oil fractions LLP and LLD were less active against all of the above micro organisms compared to the original essential oil (LL). This shows a synergic action of molecules in antimicrobial activity. The polar fraction which contains more oxygenated compounds showed slightly higher anti-microbial activity than the nonpolar fraction. This observation is well known. The order of activity is phenols > aldehydes > alcohols > ketones > ethers > hydrocarbons [7]. The minimum inhibitory concentration (MIC) of the essential oil (LL) against different micro-organisms is given in table 2. The minimum inhibitory concentration is low for gram positive bacteria such as Staphylococcus albus and gram negative bacteria such as Escherichia coli. The two fungi Candida albicans and Aspergillus niger also showed lower MIC. The antimicrobial activity exhibited by the oil is fairly good even though it does not contain phenolics. Running title here Natural Product Communications Vol. 0 (0) 2007 3 Table 2: Antimicrobial activity of Litsea laevigata essential oil and its fractions Diameter of zone of inhibition(mm) Test Organism LLP LLD 24 38 33 60 37 9 11 26 13 20 16 15 25 20 17 ST D 27 27 30 34 30 21 40 26 14 37 19 12 13 11 15 19 17 17 12 15 35 34 30 12 25 LL Staphylococcus aureus Bacillus subtilis Streptococcus faecalis Staphylococcus albus Escherichia coli Pseudomonas aeruginosa Protieus vulgaris Klebsiella aerogenes Candida albicans Aspergillus niger MIC LL mg/mL 187.5 187.5 NT 93.75 93.75 375 NT 375 93.75 93.75 NT. Not tested Experimental Plant material: The fresh berries of Litsea laevigata were collected in the month of May 2007, from the Mathikettan forests, Kerala state, India at an altitude of 750m. The plant material was identified by Dr. A.K. Pradeep, Department of Botany, Calicut University. A voucher specimen (T-75) has been deposited in the specially maintained herbarium of the Department of Chemistry , Calicut University, Kerala, India. Isolation of the essential oil: The fresh fruits (3.5 kg) of Litsea laevigata were ground into a paste by means of an electric grinder and steam distilled for 3 h. The distillate was extracted with diethyl ether (2 x 100 mL) and dried over anhydrous sodium sulphate. After evaporation of the solvent, 12g (0.34% of the fresh weight) of colourless essential oil (LL) was obtained. Fractionation of essential oil: In order to assess the anti-microbial property of different components, the essential oils were separated into two fractions. A column was packed with 50g silica gel (100-200 mesh) using distilled n-pentane in a column of dimension 3 cm x 50 cm. About 5g of the essential oil (LL) was added on the column and eluted with 100 mL each of n-pentane, and diethyl ether successively and each 100 mL fractions were collected separately. The pentane fraction on evaporation yielded about 3g of oil (LLP) and the diethyl ether fraction on evaporation yielded about 1g of oil (LLD). Olfactoric Evaluations: Olfactometric study enabled the identification of the compounds responsible for different odour exhibited by it. The essential oil was diluted with dichloromethane, 10 μL placed on a commercial odour strip (Dragoco Co.) and its odour characterised by professional perfumers. Essential oil analysis: The GC-MS analyses were carried out by using a Shimadzu GC-17A with QP 5050 and the data system compaq-proLinea (class 5ksoftware), Hewlett-Packard GC-HP 5890 with HP5970 MSD and PC-Pentium (Böhm co; ChemstationSoftware) and Finnigan MAT GCQ with data system Gateway-2000-PS75 (Siemens Co., GCQ-software). An apolar 30 m OV-1-type column (0.32 mm i.d. and 0.25 μm film thickness) and helium as carrier-gas was used. Injector temperature: 250oC; interface heating: 300oC; ion source heating: 200oC, EI-mode; scan range: 41-450 amu. For compound identification Wiley - NBS- and NIST- library spectra (on line) as well as reference MS-spectral data were used. GC-FID analyses were carried out using a Shimadzu GC-14A with FID and the integrator CR6A-Chromatopac and a varian GC-3700 with FID and the integrator C-R1B-Chromatopac (Shimadzu Co.). The same column used for GC-MS was also used for GC-FID. Carrier gas: hydrogen; injector temperature was at 250oC and detector temperature at 320oC; temperature – program: 40oC/5 min to 280oC/5 min with a heating rate of 6oC/min. Quantifications were made by relative % peak-area calculations. Microbiological analysis Microbial strains and Antimicrobial method: Staphylococcus aureus (ATCC 25923), Bacillus subtilis(ATCC 6633), Streptococcus faecalis(ATCC 29212), Staphylococcus albus (ATCC 12228), Escherichia coli (ATCC 25992), Aspergillus niger (ATCC 1015), Protieus vulgaris(ATCC 13315), Klebsiella aerogenes (ATCC 9621), Candida albicans ( ATCC 10261), Pseudomonas aeruginosa (ATCC 27853) were used in this study. The antimicrobial activity test was carried out according to disc diffusion assay described by Rondon et. al. [8a]. The strains were maintained in agar conservation at room temperature.Every bacterial inoculum (2.5 mL) was incubated in Mueller-Hinton broth at 37 ˚C for 18 h. The bacterial inocolum was diluted in sterile 0.85% saline to obtain turbidity visually comparable to a McFraland N˚ 0.5 standard (106-8 CFU/mL). Every inoculum was spread over plates containing Muller-Hinton agar and a paper filter disc (6 mm) saturated with 25 μL essential oil. 4 Natural Product Communications Vol. 0 (0) 2007 The plates were left for 30min at room temperature and then incubated at 37 ˚C for 24 h. for bacteria and 96 h. for fungi. The inhibitory zone around the disc was measured and expressed in mm. A positive control was also assayed to check the sensitivity of tested organisms using Ciprofloxacin® (5μg/disc) for bacteria, Clotrimazole® (5μg/disc) for fungi. The minimal inhibitory concentration (MIC) was determined only with the original essential oil (LL). MIC was determined by dilution of the essential oil in dimethylsulfoxide (DMSO) and pipetting 10 μL of each dilution onto the filter paper disc. Dilutions of Author A et al. the oil within the concentration range of 93.75-750 mg/mL was carried out. MIC was defined as the lowest concentration that inhibited the visible bacterial growth [8b]. A negative control was also included in the test using a filter paper disc saturated with DMSO to check the possible activity of this solvent against the bacteria assayed. The experiment was repeated twice. Acknowledgments – The first author is thankful to University Grants Commission, New Delhi for the award of FIP fellowship. References [1] Ramachandran VS, Nayar VJ. (1988) Flora of Cannanore, Botanical Survey of India, Culcutta, 370-371. [2] Sasidharan, N. (2004), KFRI – Hand book No 17, Kerala forest research institute, Peechi, Kerala, 165-166 [3] Min BS, Lee SY, Kim JH, Kwon OK Park BY, Lee KU, Moon HI, Hyeong KL. (2003) Lactones from the Leaves of Litsea japonica and Their Anti-complementActivity. Journal of Natural Products, 66, , 1388-1390. [4] Cheng HI., Lin WY, Duh CY, Lee KH, Tsai IL, Chen IS. (2001) New Cytotoxic Butanolides from Litsea acutivena. Journal of Natural Products, 64, 1502-1505. [5] Zhang H.J, Tan GT, Bernard DS, Andrew DM., Hung NV, Fong HS. (2003) New Sesquiterpenes from Litsea verticillata. Journal of Natural Products, 66, 609-615. [6] (a)Arctander S. (1969) Perfume & Flavour chemicals, Published by the editor, Mountclair; (b) Bauer K, Garle D, Surlourg H, (1990) Common fragrance & Flavour material, VCH, Weinheim. [7] Kalemba D, Kunicka K, (2005) Processing Analysis and applications of essential oil.Edited by Jirovetz L . and Buchbauer G. Har. Krishnan Bhalla and Sons, Dehradun, India. [8] (a) Rondón M, Velasco J, Morales A, Rojas J, Carmona J, Gualtieri M, Hernández V. (2005) Composition and antibacterial activity of the essential oil of Salvia leucantha Cav. cultivated in Venezuela Andes. Revista Latinoamericana de Química, 33, 40-44; (b) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Sixteenth informational supplement. CLSI document M100-S17 [ISBN 1-56238- 625-5]. Clinical and Laboratory Standards Institute, 940 (2007) West Valley Road, Suite 1400, Wayne, Pennsylvania, USA, 1887-1898.