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Analysis of Wintergreen Oil 

Analysis of Wintergreen Oil Extracted from Leaves of Wintergreen in Distillation Units of Dolakha

A Report Submitted To Ecology Agriculture and Rural Development Society (ECARDS)-Dolakha Charikot, Dolakha

Submitted By
Khilendra Gurung June 2007

Analysis of Wintergreen Oil 
Table of contents

1. Introduction 2. Objectives 3. Materials and methods 3.1 Collection and extraction of essential oils 3.2 Determination of organoleptic properties 3.3 Determination of physico-chemical properties 3.3.1 Specific gravity 3.3.2 Optical rotation 3.3.3 Refractive index 3.3.4 Solubility 3.3.5 Determination of acids 3.3.6 Determination of esters 3.3.7 Determination of ester number after acetylation 4. Results 4.1 Characteristics 4.2 Active constituents of Wintergreen oil 5. Conclusion 5.1 Actions 5.2 Uses 5.2.1 Aromatherapeutic uses 5.2.2 Other uses References Lists of tables Table 1: Specification of Wintergreen oil Table 2: Monoterpene composition of Wintergreen Oil Table 3: Sesquiterpene composition of Wintergreen oil

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Analysis of Wintergreen Oil 
Abbreviations and acronyms
C: Centigrade CFUGs: Community Forest User Groups DMC: Deudhunga Multipurpose Cooperative GC-MS: Gas Chromatography-Mass Spectra GEF: Global Environment Facility HPPCL: Herbs Production and Processing Company Limited M/S: Mild steel m: Meter ml: Millilitre NTFP: Non Timber Forest Products S/S: Stainless steel SGP: Small Grant Program UNDP: United Nations Development Program VDC: Village Development Committee

Analysis of Wintergreen Oil 
1. Introduction Dolakha district is rich in plant diversity due to the presence of wide range of geographical and climatic conditions. Wintergreen plant (Gaultheria fragrantissima Wall.; family: Ericaceae) locally known as Dhasingre/machhino/patpate is widely distributed in open places, shrubberies, rocky slopes between 1100-2800m of Dolakha district (Press et al. 2000). Leaves of wintergreen are aromatic, stimulant and carminative. Juice of the leaves, mixed with water, is taken for coughs. This juice, about 2 teaspoons, is given as an anthelmintic. It acts as a vermicide on hookworms. Young leaves are also taken as an anthelmintic. Oil extracted from the leaf is applied to treat rheumatism and scabies. Immature fruits are chewed or their paste is given for stomach troubles. Juice of unripe fruit is taken to treat stomachaches. Ripe fruits are eaten fresh. The fruits are also distilled locally for alcohol (Manandhar, 2002). The leaves with twigs bear essential oil, popularly known as Wintergreen oil. In spite of such privileges, the locals were not able to reap rich rewards from wintergreen plants. Several problems associated with wintergreen plant were lack of data on the quantity of herbs available and scientific research on the weather, growing conditions and life cycle of these herbs. There were no systematic and sustainable practices regarding the management, conservation and marketing of these herbs. Against the backdrop of these problems, Deudhunga Multipurpose Cooperative (DMC) came into existence in 1995 (2051 BS) and by coordinating the Community Forest User Groups (CFUGs) who depend on Non Timber Forest Products (NTFP) for their livelihoods. The cooperative started its activities by obtaining raw materials from the CFUGs and by distilling Wintergreen oil. The cooperative now runs 4 locally made Mild steel (M/S) distillation units and 3 newly installed Stainless steel (S/S) distillation units along with S/S testing units and distills various essential oils like Abies, Anthopogon, Artemisia, Juniper and Wintergreen oils. M/S distillation units were established with individual cash contribution and raw material provided by CFUGs. While S/S distillation units were installed with the assistance from UNDP, GEF/SGP with the increments of share holders as Community Forest User Groups (CFUGs) and identified pro-poor. The cooperative has established itself as an ideal NTFP based industry, adopting social inclusive model. In this respect, it is essential to analyze the chemical constituents of wintergreen oil for its quality control and quality assurance for its marketing to national and international markets.

Analysis of Wintergreen Oil 
2. Objectives The objectives of the research are as follows: 1. To analyze the organo-leptic and physico-chemical test of wintergreen oil in the authorized lab of Nepal 2. To incorporate the results of active constituents analysis of wintergreen oil performed by other researchers 3. To recommend the quality control mechanism of wintergreen oil 3. Materials and methods 3.1 Collection and extraction of essential oils The leaves of wintergreen were collected from different CFUGs of Dolakha district. The collected leaves were steam distilled to produce wintergreen oils in three distillation units located at Lakuri Danda VDC, Suspa VDC and Hawa VDC of Dolakha district. About 10 ml sample from three distillation units were collected and mixed in 30 ml bottle. The sample was taken to the quality control laboratory of Herbs Production and Processing Company Limited (HPPCL) for the analysis work. 3.2 Determination of organoleptic properties Organoleptic properties of wintergreen oil were detected by expert nose and eyes. Appearance of oil was determined by eyes and aroma by nose. 3.3 Determination of physico-chemical properties The physico-chemical properties of wintergreen oil were determined as according to Guenther (1972). 3.3.1 Specific gravity Specific gravity bottle (5ml, 10ml or 25ml) was used to determine specific gravity of wintergreen oil. Dry and clean bottle was filled with oil to be determined and the weight was taken upto three digits in an electronic balance. The same bottle was cleaned and the weight of bottle with freshly boiled and cooled distilled water was taken. The weight of oil contained in the specific gravity bottle divided by the water equivalent gives the specific gravity of oil at specific temperature. 3.3.2 Optical rotation Polarimeter was used to determine the optical rotation of wintergreen oil. The extent of optical activity of oil was measured in degrees of rotation. The angle of rotation was dependent upon the nature of the liquid. In recording rotations the direction by the use of (+) sign to indicate dextrorotary (rotation to the right i.e. clockwise) or a (-) sign to indicate levorotatory (rotation to the left i.e. anti clock wise)

Analysis of Wintergreen Oil 
3.3.3 Refractive index Refractometer was used for this determination. Few drops of sample was put into funnel shaped aperture between the prisms of refractometer, closed the prism firmly by tightening the screw and reading was noted by rotating alidade backward or to forward until yield of vision was divided into a light and dark portion. Reading was taken as the refractive index of the sample directly on the scale of the sectors. 3.3.4 Solubility The number of volumes of dilute alcohol required for the complete solubility of one volume of oil was calculated as the solubility of the given oil. 3.3.5 Determination of acids In determining the acid number dilute alkali was used. The acid number of oil was calculated as the number of milligrams of Potassium hydroxide required neutralizing the free acids in 1 gm of oil using Phenolphthalein as an indicator. Acid number: 28.05 x a/S Where, a= No. of CC of 0.5N NaOH used to neutralize S= Weight of oil taken in grams 3.3.6 Determination of esters Ester number of oil was calculated as the number of milligrams of Potassium hydroxide required to saponify the esters present in 1 gram of oil. Ester number: 28.05 x a/S Where, a= Number of CC of 0.5 NHCL used in saponification. S= Weight of oil taken in grams. 3.3.7 Determination of ester number after acetylation The ester number after acetylation was determined as the number of milligrams of Potassium hydroxide required to saponify the ester present in 1 gram of acetylated sample. Ester number after acetylation: 28.05 x a/20 S Where, a= Number of CC of 0.5N HCL used in saponification S=Weight of acetylated oil taken in grams.

Analysis of Wintergreen Oil 
4. Results The organo-leptic and physico-chemical analysis of wintergreen oil from 3 distillation units of Dolakha revealed the following results as follows: 4.1 Characteristics Table 1: Specification of Wintergreen oil Tests Results Organo-leptic properties
Appearance Color Aroma A fluid liquid Pale yellow or pinkish Strongly aromatic with a sweet woody odor, displaying a peculiar creamy-fruity top note and a sweet-woody dryout

Physico-chemical properties
Specific gravity Optical rotation Refractive index Acid number Ester number Ester number after acetylation Solubility 1.1735 to 1. 1 855 at 25o C. [-] 0.3 o to [-] 10.5 o at 25o C 1.537 to 1.5405 at 25o C 10 to 25 335 to 365 88.8 to 98% (calculated as Methyl salicylate) Soluble in 2.0 to 3.5 volumes of 80% alcohol

4.2 Active constituents of Wintergreen oil Juliani et al. (2004) conducted Gas Chromatography-Mass Spectra (GC-MS) of wintergreen oil distilled in Lakuri danda VDC of Dolakha. The results of the analysis revealed that the wintergreen oil was dominated by methyl salicylate (97%) with minor and traces amounts of monoterpenes (alpha-pinene, myrcene, delta-3-carene, and limonene) and sesquiterpenes (delta-cadinene, 3, 7 Guaiadiene). Similarly, Baruah and Bhagat (1976) conducted active constituent analysis of Indian wintergreen oil and found the major constituent as methyl salicylate (99.6%). Whereas, Clark (1999) reported that wintergreen oil from other related species (Gaultheria procumbens) were also dominated by methyl salicylate and is the commercial source of this component. The table below shows the details of the composition of wintergreen oil distilled in Lakuri danda VDC, Dolakha.

Analysis of Wintergreen Oil 
Table 2: Monoterpene composition of Wintergreen Oil SN Components % 1 alpha-pinene 0.4 2 Myrcene 0.2 3 delta-3-Carene 0.9 4 Limonene 0.5 5 Methyl salicylate 97.4 Total monoterpene 99.4 Table 3: Sesquiterpene composition of Wintergreen oil SN Composition % 1 3, 7 Guaiadiene 0.1 2 delta-Cadinene 0.3 Total sesquiterpene 0.4 Source: Juliani et al. (2004) 5. Conclusion The composition of oil of Gaultheria fragrantissima was similar to the oil of G. procumbens, but the Nepalese sample can be an alternative source of the flavoring agent methyl salicylate. The study showed that Nepali wintergreen oil showed a great chemical diversity thus providing new aroma profiles. The chemical diversity found in Nepali wintergreen oil can be used to open new market opportunities, due to their novel and unique properties. However, the development of grades and standards as well as quality assurance and quality control system are key strategies for further development of the oil quality. 5.1 Actions Analgesic, anti-inflammatory, anti-rheumatic, antitussive, astringent, carminative, diuretic, emmenagogue, galactogogue, stimulant. 5.2 Uses 5.2.1 Aromatherapeutic uses Beneficial for muscle and joint discomfort, arthritis, cellulite, obesity, edema, poor circulation, headache , stimulates liver, heart disease, coronarities crisis and preventative, hypertension, rheumatism, tendentious, cramps, anti-inflammatory, high in cortisone-like functions, eczema, hair care, psoriasis, gout, ulcers, broken or bruised bones. 5.2.2 Other uses 1. Used in some perfumery applications especially in forest type fragrances. 2. Used as a flavoring agent for toothpaste, chewing gum and soft drinks.

Analysis of Wintergreen Oil 
References Baruah, A.K.S. and Bhagat, S.D. 1976. Oil of Indian Wintergreen. Indian Journal of Pharmacy. 38(2): 56-57. Clark, G. 1999. An Aroma-chemical Profile. Methyl salicylate, or Oil of Wintergreen. Perfumer and Flavorist. 24(1): 5-11. Guenther E. 1972. The Essential Oils: History-Origin in Plants ProductionAnalysis. Volume 1. Robert E. Krieger Publishing Company, Huntington, New York. Juliani R, Moharram H, Wang M and Simon J. 2004. Chemical diversity of Nepalese Essential oils. In: New Use Agriculture and Natural Plant Products Program, Cook College, Rutgers University, USA. Lawless J. 1995.Complete Essential Oils: A Guide to the Use of Oils in Aromatherapy and Herbalism. Element Books Limited. Manandhar N P. 2002. Plants and People of Nepal. Timber Press, Portland, Oregon USA. Press, J R, K K Shrestha and D A Sutton. 2000. Annotated Checklist of the Flowering Plants of Nepal. The Natural History Museum, London.

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