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 1
2. Objectives 2
3. Materials and methods 2
3.1 Collection and extraction of essential oils 2
3.2 Determination of organoleptic properties 2
3.3 Determination of physico-chemical properties 2
3.3.1 Specific gravity 2
3.3.2 Optical rotation 2
3.3.3 Refractive index 3
3.3.4 Solubility 3
3.3.5 Determination of acids 3
3.3.6 Determination of esters 3
3.3.7 Determination of ester number after acetylation 3
4. Results 4
4.1 Characteristics 4
4.2 Active constituents of Wintergreen oil 4
5. Conclusion 5
5.1 Actions 5
5.2 Uses 5
5.2.1 Aromatherapeutic uses 5
5.2.2 Other uses 5
References 6

Lists of tables
Table 1: Specification of Wintergreen oil 4
Table 2: Monoterpene composition of Wintergreen Oil 5
Table 3: Sesquiterpene composition of Wintergreen oil 5
 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 A fluid liquid
Color Pale yellow or pinkish
Aroma Strongly aromatic with a sweet woody odor, displaying a peculiar
creamy-fruity top note and a sweet-woody dryout
Physico-chemical properties
Specific gravity 1.1735 to 1. 1 855 at 25o C.
Optical rotation [-] 0.3 o to [-] 10.5 o at 25o C
Refractive index 1.537 to 1.5405 at 25o C
Acid number 10 to 25
Ester number 335 to 365
Ester number after 88.8 to 98% (calculated as Methyl salicylate)
acetylation
Solubility 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
¾ Baruah, A.K.S. and Bhagat, S.D. 1976. Oil of Indian Wintergreen. Indian
Journal of Pharmacy. 38(2): 56-57.
¾ Clark, G. 1999. An Aroma-chemical Profile. Methyl salicylate, or Oil of
Wintergreen. Perfumer and Flavorist. 24(1): 5-11.
¾ Guenther E. 1972. The Essential Oils: History-Origin in Plants Production-
Analysis. Volume 1. Robert E. Krieger Publishing Company, Huntington, New
York.
¾ 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.
¾ Lawless J. 1995.Complete Essential Oils: A Guide to the Use of Oils in
Aromatherapy and Herbalism. Element Books Limited.
¾ Manandhar N P. 2002. Plants and People of Nepal. Timber Press, Portland,
Oregon USA.
¾ Press, J R, K K Shrestha and D A Sutton. 2000. Annotated Checklist of the
Flowering Plants of Nepal. The Natural History Museum, London.