Original paper

Yield Determination and Specification of Essential Oil Obtained from Lindera neesiana (Wall. ex Nees) Kurz Harvested from Eastern Nepal
Khilendra Gurung*, Minoba Yonzon**and Usha Rijal** *Resource Research and Development Center; Naya Baneswor, Kathmandu, Nepal, E-mail: khilendragurung@yahoo.com, Contact: 9841501159 **Herbs Production and Processing Company Limited; Koteswor, Kathmandu, Nepal, E-mail: yminoba@hotmail.com/usharijal@gmail.com; Tel: 6-630452 Abstract
The yield and specification of Lindera neesiana oil, obtained from fruits of Lindera neesiana, harvested from eastern Nepal were studied. Hydro distillation of dried fruits of Lindera neesiana with moisture content of 3% yielded 1% of oil. While dried fruits with moisture contents of 4.3% and 5.6% contain oils in 0.87% and 0.52% respectively. It was observed that the yield of essential oil in fruits of Lindera neesiana were high with fruits of low moisture content than that of high moisture content. Similarly, organo-leptic and physico-chemical properties of Lindera neesiana oil were analyzed as its specification. Key word index: Lindera neesiana, essential oil, hydro-distillation, organo-leptic, physico-chemical

1. Introduction Lindera neesiana (Wall. ex Nees) Kurz (Lauraceae), locally known as "Siltimur" is a medium size deciduous tree growing upto 4m in height. It is distributed in temperate Himalayan region from 1800-2700m; mainly in the eastern and central regions of Nepal (HMG, 1970; Stainton, 1997; Press et al., 2000). The fruits of this plant are aromatic and local people has been consuming for years as pickle and chewed for treating stomachache due to indigestion, diarrhea, toothache, nausea, as anthelmintic and flatulence (Pohle, 1990; Manandhar, 2002 and Gurung, 2003). Fruits are also given to cattle if they eat poisonous plants (Manandhar, 2002). Leaf is a good source of fodder for cattle and goats. Leaves and branches are aromatic when crushed. Fruits, leaves and barks contain an essential oil. Singh et al. (1995) extracted essential oils from fresh leaves and branches of Lindera neesiana and analyzed the active constituents via Gas Chromatography-Mass Spectra (GC-MS). In this work, the yield and specification of Lindera neesiana oil obtained from fruits harvested from eastern Nepal were studied. The results of this study could contribute to a better utilization of this raw material scrap. 2. Materials and Methods 2.1 Sample Collection Ripe fruits of Lindera neesiana were collected from Ambung Village Development Committee, Ward No.:1, Tehrathum district, Nepal during August-September, 2004 from the altitude ranges from 1800-2000m. The collected fruits were dried in aerated shade and stored at room temperature. 2.2 Extraction of Essential Oil The Lindera neesiana oil was extracted in the laboratory of Herbs Production and Processing Company Limited (HPPCL) at Koteswor, Kathmandu in May-June, 2005.

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The dried fruits were crushed and Clevenger type laboratory scale hydro-distillation system was used for the distillation process (HMG, 1986), for 15-17 hours. 2.3 Determination of Organo-leptic Properties Organo-leptic properties of Lindera neesiana oil was detected by expert nose and eyes. 2.4 Determination of Physico-chemical Properties Physico-chemical properties of Lindera neesiana oil were determined according to Guenther (1972). 2.4.1 Specific Gravity Specific gravity was determined as the weight of oil contained in the specific gravity bottle divided by the water equivalent gives the specific quantity of oil at specific temperature. 2.4.2 Optical Rotation Polarimeter was used to determine the optical rotation. The extent of optical activity of oil was measured in degrees of rotation. 2.4.3 Refractive Index Refractometer was used for the determination of refractive index. Reading was taken as the refractive index of the sample directly on the scale of the sectors. 2.4.4 Solubility The number of volumes of dilute alcohol (Ethyl Alcohol) required for the complete solubility of one volume of Lindera neesiana oil was calculated as the solubility of the given oil. 2.4.5 Determination of Acids The acid number was calculated as the number of milligrams of Potassium hydroxide required to neutralize the free acids present in 1gm of oil using Phenolphthalein as an indicator. 2.4.6 Determination of Esters Ester number was calculated as the number of milligrams of Potassium hydroxide required to neutralize the acids liberated by the hydrolysis of esters present in 1gm of oil. 2.4.7 Determination of Ester Number after Acetylation The ester number after acetylation was determined as the number of milligrams of Potassium hydroxide required to neutralize the acid liberated by the hydrolysis of 1gm of acetylated Lindera neesiana oil. 2.5 Determination of Moisture Content Five gm of crushed Lindera neesiana seeds were weighed in a tarred silica crucible and they were dried in an air oven at 60-80°C till the constant weight was obtained. The differences in the weighing give total moisture present in the seeds (HMG, 1986).

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3. Results and Discussion Hydro-distillation of dried fruits of Lindera neesiana with moisture content of 3% yielded 1% of oils. While dried fruits with moisture contents of 4.3% and 5.6% furnished oils in 0.87% and 0.52% respectively. It was observed that the yield of essential oil obtained from fruits of Lindera neesiana were high with fruits of low moisture content than that of high moisture content. Singh et al. (1995) found that hydro-distillation of fresh leaves and branches of Lindera neesiana yielded essential oils in 1.3% and 0.5% (w/w) respectively. Organo-leptic and Physico-chemical analysis of Lindera neesiana oil revealed the following properties as shown in the table below: Table 1: Specification of Lindera neesiana oil SN Organo-leptic Properties Tests Results 1 Appearance Fluid liquid 2 Color Pale yellow 3 Aroma Refreshing, pleasant, lemon type 4 5 6 7 -

Physico-chemical Properties Tests Results Specific gravity 0.893 at 27°C Optical rotation (-) 0.9° at 27°C Refractive index 1.386 at 26.5°C Solubility Soluble in 1.4 volumes of 80% ethyl alcohol 18.25 18.97 214.65

Acid number Ester number Ester number after acetylation Specific gravity is an important criterion of the quality and purity of an essential oil. Specific gravity values for essential oils vary between 0.696 and 1.188 at the specific temperature. The specific gravity of Lindera neesiana oil (0.893) lies within the given values for essential oils. Generally the gravity is less than 1.000. Most essential oils when placed in a beam of polarized light possess the property of rotating the plane of polarization as dextrorotatory or laevorotatory. Both the degree of rotation and its direction are important criteria of purity of oils. In this work, the direction of rotation is laevorotatory as indicated by the minus (-) sign. The refractive index of Lindera neesiana oil (1.386) is more than the refractive index of pure water (1.333). This indicates that Lindera neesiana oil is denser, as it is a fluid liquid, forming denser medium than pure water. Refractive index value plays an important role in the elucidation of structure in the case of many individual constituents of essential oils after separation and purification. Most essential oils are only slightly soluble in water and are miscible with absolute alcohol. The determination of solubility is a convenient and rapid aid in the evaluation of quality of oil. In this work, Lindera neesiana oil was clearly soluble in 1.4 to 2 volumes of 80% ethyl alcohol and more upto 10 volumes. Most essential oils contain only small amounts of free acids. Therefore, the acid content is usually reported as an acid number rather than as a percentage calculated as a specific acid. The acid number (18.25) of Lindera neesiana oil indicates that the oil is fresh and properly stored. Normally, the acid number of oil increases with the age of the oil, particularly if it is improperly stored. Oxidation of aldehydes and hydrolysis of esters increase the acid number.

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The determination of the ester content is of great importance in the evaluation of many essential oils. The use of ester number is especially convenient when the ester present in the oil is unknown. Ester numbers are frequently used for oils which contain very small amounts of ester. A high ester number is usually indicative of adulteration in essential oils. The low ester number (18.97) of Lindera neesiana oil implies minimal adulteration in the oil. The alcoholic constituents of an essential oil are determined by acetylation. However, Lindera neesiana oil has not been thoroughly investigated and its alcoholic constituents are not well known. So, it is convenient to report the results as an ester number after acetylation which is 214.65. GC-MS and antimicrobial test of Lindera neesiana oil should be conducted for determining its active constituents, actions and aromatherapeutic uses. Acknowledgements We would like to express our sincere gratitude to Dr. Narendra N. Tiwari, former General Manager of HPPCL and Ms. Ganga Shrestha for her painstaking assistance to carry out this study. We are thankful to the local residents of Ambung VDC-1 for providing us with the Siltimur fruits for the study. References Guenther, E. 1972. The Essential Oils: History-Origin in Plants Production-Analysis, Volume 1. Robert E. Krieger Publishing Company, Huntington, New York, pp.229-348. Gurung, K. 2003. Indigenous Knowledge on the Plant Resources Used by the People of Tinjure Area, Tehrathum District. In: Botanica Orientalis. Central Department of Botany, Tribhuvan University, Kathmandu, Nepal 3(1): 118-125. HMG. 1970. Medicinal Plants of Nepal. Department of Medicinal Plants, Kathmandu, Nepal, pp. 142-143. HMG. 1986. Standards of Ayurvedic Crude Drugs: Volume 1. Royal Drug Research Laboratory, Kathmandu, Nepal, pp.86. Manandhar, N. P. 2002. Plants and People of Nepal. Timber Press, Portland, Oregon, pp. 294. Pohle, P. 1990. Useful Plants of Manang District: A Contribution to the Ethnobotany of Nepal Himalaya. Franz Steiner Verlag Wiesbaden GmbH Stuttgart pp. 15. Press, J. R., K. K. Shrestha and D. A. Sutton. 2000. Annotated Checklist of the Flowering Plants of Nepal. The Natural History Museum, London, pp. 161. Singh, R. S., P. B. Kanjilal, M. G. Pathak and A. C. Ghosh. 1995. Volatiles of Lindera neesiana Benth., Leaf and Branch. In: Journal of Essential Oil Research 7(6): 695-696. Stainton, A. 1997. Flowers of the Himalaya: A Supplement. Oxford University Press, Delhi, India, pp. 53.

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