Assessment of morphological and chemical changes in Himalayan thyme

Assessment of morphological and chemical changes
during plant growth and antimicrobial activity of
Thymus linearis Benth. from Uttarakhand
A DISSERTATION
SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
AWARD OF THE DEGREE OF
MASTER OF SCIENCE
IN
BIOTECHNLOGY
BY
Ms. ADITI KAMAL
Enrolment No: KU12111641
DEPARTMENT OF BIOTECHNOLOGY
M.B. GOVT. P.G. COLLEGE, HALDWANI, NAINITAL
SUPERVISOR
Ram Swaroop Verma
Senior Scientist
CSIR-CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS
RESEARCH CENTRE PANTNAGAR,
UDHAM SINGH NAGAR, UTTARAKHAND
YEAR- 2017

Date: 01.07.2017
CERTIFICATE
This is to certify that work embodied in this dissertation entitled “Assessment of
morphological and chemical changes during plant growth and antimicrobial activity
of Thymus linearis Benth. from Uttarakhand” has been carried out by Ms. Aditi Kamal
under my supervision at CSIR-Central Institute of Medicinal and Aromatic Plants,
Research Centre Pantnagar during January to June, 2017. The work embodied in this
dissertation is original and submitted to Department of Biotechnology, M.B. Govt. P.G.
College Haldwani (Kumaun University, Nainital) in the partial fulfilment of the
requirements for the award of the degree of Master of Science in Biotechnology.
.
[Ram Swaroop Verma]
Senior Scientist
Department of Phytochemistry
CSIR-CIMAP Research Centre Pantnagar
Phone: 09756021222 (M), 05944-234712 (O); E-mail: rs.verma@cimap.res.in

Department of Biotechnology and Allied Sciences
M. B. Govt. PG. College
Haldwani, 263139 Distt-Nainital
0ff. - +91-05946-281296
CERTIFICATE
It gives us great pleasure to testify the abilities of Ms. ADITI KAMAL
D/O Mr. R K SRIVASTAVA who is presently a student of M.Sc.
BIOTECHNOLOGY IV semester in the Department of Biotechnology and
Allied Sciences, M. B. Govt. PG. College, Haldwani (Nainital).
ADITI KAMAL has successfully completed the project work on
“Assessment of morphological and chemical changes during plant growth
and antimicrobial activity of Thymus linearis Benth. from Uttarakhand” to
fulfill the partial requirement of Master‟s Degree in Biotechnology. She is a
consistently hard working, honest, sincere and conscientious person.
We wish all the success in every path of her life and career.
Dr. Naveen Bhagat
Coordinator
Department of Biotechnology
& Allied Sciences
M. B. Govt. PG. College
Haldwani (Nainital)
Date: 4 /07 /2017

4
DECLARATION
I hereby declare that the present dissertation entitled “Assessment of morphological and
chemical changes during plant growth and antimicrobial activity of Thymus linearis
Benth. from Uttarakhand” is a original research work carried out by me at CSIR-CIMAP
Research Centre Pantnagar Uttarakhand in partial fulfillment of the requirement for the award
of degree of Master of Science in Biotechnology from Department of Biotechnology, M.B.
Govt. P.G. College Haldwani, Kumaun University Nainital, during the academic year 2015–
2017. It is further stated that no part of this dissertation has been submitted previously either
in part or full, for the award of any other degree or diploma by me, to this or any other
University.
Date: 4/07/2017
Place: HALDWANI [Aditi Kamal]

5
ACKNOWLEDGMENTS
Presentation, inspiration and motivation have always played a key role in the success of any
venture. I would firstly like to express my sincere gratitude to my supervisor, Mr. Ram
Swaroop Verma, Senior Scientist, CSIR-Central Institute of Medicinal and Aromatic Plants
(CSIR-CIMAP), Research Center Pantnagar whose valuable guidance, support and inspiring
suggestions have been precious for the development of this thesis content.
I take this opportunity to express my gratitude to the Management of CSIR-CIMAP,
Lucknow for providing necessary facilities for the successful completion of this research
work and for also giving an opportunity to get an exposure of this esteemed organization. I am
thankful to the staff of CSIR-CIMAP, Research Centre Pantnagar for his support during my
project training.
I am thankful to Dr. V.R Singh (Scientist-in-Charge), Dr. R.C. Padalia (Senior
Scientist), Dr Venkatesha KT (Scientist) and Dr Amit Chauhan (Technical Officer), CSIR-
CIMAP, Research Centre, Pantnagar for their help and guidance during the project work. I
also express my gratitude to Dr. Dharmendra Saikia (Principal Scientist) and Mr. Ajay Kumar
(SRF), CSIR-CIMAP, Lucknow for help during biological activity evaluation.
I am deeply and strongly obliged to Dr. Naveen Bhagat, Head, Department of
Biotechnology, M.B. Govt. P.G. College, Haldwani (Kumaun University Nainital) for
valuable suggestions and giving an opportunity to pursue my dissertation work at CSIR-
CIMAP, Research Centre Pantnagar.
I am thankful to my Biotechnology teachers for their kind support. They were always
a source of knowledge and inspiration to me.
My joy knew no bounds in expressing the heartfelt thanks to my beloved parents.
They have been selfless in giving me the best of everything and I express my deep gratitude
for their love without which this work would not have been completed. Also I would like to
thanks all my friends for their constant encouragement and support.
Last but not the least, my special thanks expends to those who could not find separate
names but had helped me directly or indirectly.
ADITI KAMAL

6
ABBREVIATIONS
% Percentage
DAP Days after planting
Mm Millimeter
˚C Degree centigrade
Cc Cubic centimeter
M Meter
µm Micrometer
µL Microliter
Ml Milliliter
Psi Per square inch
FID Fluid Ionization Detector
GC Gas chromatography
GC-MS Gas chromatography-mass spectrometry
ZOI Zone of inhibition
MIC Minimum inhibitory concentration
MBC Minimum bactericidal concentration
MFC Minimum fungicidal concentrations
BS Bacteriostatic

7
LIST OF CONTENTS
S. No. CONTENTS PAGE No.
1 ABSTRACT 10
2 INTRODUCTION 11-35
3 REVIEW OF LITERATURE 36-37
4 OBJECTIVE OF THE RESEARCH 38
5 MATERIALS AND METHODS 39-45
6 RESULTS AND DISCUSSION 46-71
7 CONCLUSIONS 72
8 REFERENCES 73-86

8
LIST OF TABLES
S.No. TABLES PAGE
No.
Table 1 Some common medicinal plants and their uses 15
Table 2 Some common essential oil producing aromatic plants 18
Table 3 Different Thymus spp. and their therapeutic property 35
Table 4 Growth and yield characters of Thymus linearis under the subtropical
conditions of north India
48-49
Table 5 Essential oil composition of the leaves of Himalayan thyme (Thymus
linearis) collected at different developmental stages in the foot-hills of
Uttarakhand, India
51-53
Table 6 Essential oil composition of the inflorescence of Himalayan thyme (Thymus
linearis) collected at different developmental stages in the foot-hills of
Uttarakhand, India
56-58
Table 7 Essential oil composition of the whole aerial-parts and stem of Himalayan
thyme (Thymus linearis) collected at full bloom stages (S4) in the foot-hills
of Uttarakhand, India
60-62
Table 8 Antibacterial activity of Thymus linearis essential oils and extracts 67
Table 9 Antifungal activity of Thymus linearis essential oils and extracts 70

9
LIST OF FIGURES
S. No. FIGURES PAGE
No.
Figure 1 Thymus linearis grown at the experimental field of CSIR-CIMAP, Research
Centre Pantnagar Uttarakhand.
40
Figure 2 Solvent extracts of Thymus linearis collected at full bloom stage 41
Figure 3 Gas chromatograph 42
Figure 4 Distribution of phenolic (phenols, phenolic ethers and phenolic esters) and
terpenoid (monoterpene hydrocarbons, oxygenated monoterpenes and
sesquiterpene hydrocarbons) constituents in the essential oils of different plant-
parts of Thymus linearis at full bloom stage.
50
Figure 5 Changes in thymol, γ-terpinene and p-cymene content in leaves of Thymus
linearis during plant ontogeny.
54
Figure 6 A representative GC-FID chromatogram of the leaf essential oil of Thymus
linearis.
54
Figure 7 Changes in thymol, γ-terpinene and p-cymene content in inflorescence of
Thymus linearis during plant ontogeny.
55
Figure 8 A representative GC-FID chromatogram of the inflorescence essential oil of
Thymus linearis.
59
Figure 9 Activity index (mean zone of inhibition of six bacterial strains) of the leaf
essential oil (EO-L), inflorescence essential oil (EO-I), whole aerial-parts
essential oil (EO-AP), hexane extract (HE-AP), acetone extract (AE-AP) and
methanol extract (ME-AP) of Thymus linearis.
66
Figure 10 Activity index (mean zone of inhibition of three fungal strains) of the leaf
essential oil (EO-L), inflorescence essential oil (EO-I), whole aerial-parts
essential oil (EO-AP), hexane extract (HE-AP), acetone extract (AE-AP) and
methanol extract (ME-AP) of Thymus linearis.
71

10
ABSTRACT
The aromatic and medicinal properties of the genus Thymus L. (Lamiaceae) has made it one
of the most popular plants all over the world. In India, the genus Thymus is represented by
two species, namely Thymus linearis Benth. (native) and Thymus serpyllum L. (exotic). T.
linearis, commonly known as „Himalaya thyme‟ is widely distributed in the Himalayan
region. The aim of the present research was to assess the morphological and chemical changes
occurring during the annual growth, and to investigate the antibacterial and antifungal
activities of T. linearis grown in the foothills of Uttarakhand. The morphological data of the
crop was recorded for ten quantitative characters (plant height, plant canopy, branch, leaf-
stem ratio, leaf yield, inflorescence yield, essential oil content in leaves, essential oil content
in inflorescence, essential oil yield of leaves and essential oil yield of inflorescence) at six
different stages, namely S1: early-vegetative-stage (120 DAP); S2: late-vegetative-stage (135
DAT); S3: flower-initiation-stage (150 DAT); S4: full-bloom-stage (175 DAT); S5: late-
bloom-stage (190 DAT); and S6: seed-shattering-stage (200 DAT). The freshly harvested
plant materials (whole aerial-parts, leaves, inflorescences and stem) were dried in shade and
subjected to hydrodistillation in a Clevenger apparatus for isolation of essential oil. The
extract of the plant was prepared in hexane, acetone and methanol. The essential oils were
analysed by GC-FID and GC-MS techniques. The antimicrobial activity of the oils was
determined against six pathogenic bacterial strains (Mycobacterium smegmatis, Enterococcus
faecalis, Escherichia coli, Streptococcus mutans, Staphylococcus epidermidis and
Staphylococcus aureus) and three fungal strains (Candida albicans ATCC, Candida kefyr and
Candida albicans clinical isolate) using disc diffusion and microdilution broth assays. The
essential oil yields of leaves and inflorescence were higher at S5 (0.826 mL plant-1
and 0.605
ml plant -1
, respectively). Major components of the oils, isolated from different plant-parts at
different stages (S1–S6) were thymol (35.5–59.0%), γ-terpinene (13.3–19.8%), p-cymene
(4.2–11.8%), borneol (0.9–8.2%), thymyl acetate (0.9–7.6%) and thymol methyl ether (2.9–
6.0%). In full bloom stage, thymol content was distributed in different samples in the
following order: whole-aerial-parts (59.0%)> leaves (54.6%)> inflorescence (51.2%)> stem
(35.5%). The essential oils and solvent extracts of T. linearis differed substantially in their
efficacy against different bacterial and fungal strains. Based on the activity index, the
antimicrobial potency of different oils and extracts can be arranged in following order: leaf
oil>inflorescence oil>whole aerial-parts oil>hexane extract>methanol extract>acetone extract.

11
1. INTRODUCTION
1.1. Status of Medicinal and Aromatic Plants
Medicinal Plants- The vast majority of people on this planet still rely on their traditional
material medica (medicinal plants and other materials) for their everyday health care needs. It
is also a fact that one quarter of all medical prescriptions are formulations based on substances
derived from plants or plant-derived synthetic analogs. According to the WHO, 80% of the
world‟s population of developing countries relies on plant-derived medicines for their primary
healthcare (Gurib-Fakim, 2006). Ethnomedicinal plants are not only used for primary health
care, but also used for treating chronic diseases such as AIDS, cancer, hepatitis disorders,
heart and old age related diseases like memory loss, osteoporosis and diabetic wound. In the
Indian coded system (Ayurveda, Unani, Siddha, Amchi), Ayurveda currently utilizes as many
as 1000 single drugs and over 8000 compound formulations of recognized merit. Similarly,
600-700 plants are utilized by other systems like Unani, Siddha and Amchi (Gurib-Fakim,
2006; Zahin et al., 2010). Therefore, medicinal plants are collected by local and folk
communities all over the world for their use, but these are generally collected in low
quantities. However, some medicinal plants are collected in huge quantities to supply them to
the market which is used as a raw material in various herbal industries (Uniyal et al., 2006).
The example of some common medicinal plants and their medicinal properties are presented
in Table 1 (Joy et al., 1998; Khare, 2007).
According to the World Health Organization “a medicinal plant” is any plant, in which
one or more of its organ contains substances that can be used for the therapeutic purposes or

12
which, are precursors for the synthesis of useful drugs (WHO, 1977). The use of medicinal
plants in the traditional system of medicine in most the developing countries for the
maintenance of good health has been widely observed (UNESCO, 1996). These plants contain
inherent active ingredients used to cure various diseases (Okigbo et al., 2008). A number of
chemical components are present in medicinal plants, which can be utilized for the treatment
of infectious as well as chronic diseases. These unique therapeutic agents are screened
repeatedly by clinical microbiologist (Lai and Roy, 2004; Periyasamy et al., 2010). Moreover,
medicinal plants are used as raw materials for the extraction of pure bioactive constituents
(e.g., quinine and quinidine from cinchona bark, emetine from Ipecacuanha root, glycosides
from digitalis leaves, sennosides from senna leaves), precursors for synthetic vitamins or
steroids, and as preparations for herbal and indigenous medicines. Products such as ginseng,
valerian and liquorice roots are part of the herbal and health food market, as well as the food
flavors, fragrance and cosmetic industries. Several formulations like herbal teas, extracts,
decoctions, infusions, tinctures, etc are prepared from medicinal plants (Kraisintu, 1997).
Moreover, the drugs are derived either from the whole plant or from a specific plant-part say
for example, root, leaves, bark, stem, flower, seed, etc. Some drugs are prepared from the
excretory parts of plant like gum, resins, and latex. The allopathic system of drugs is adopted
by a number of plant derived drugs which form an important segment of the modern
pharmacopoeia. Diosgenin, solasodine, β-ionone are some chemical intermediates required
for manufacturing the modern drugs which are also obtained from plant parts.
A huge population all over the world relies on medicinal and aromatic products for
their primary health care. Thus, medicinal plants play an important role in not only traditional
medicines, but also in trade commodities, meeting the demands of leading markets.

13
Worldwide a huge number of plant species is used to provide the raw materials needed by the
various systems of traditional medicine. The population of the world using traditional
medicine ranges between 70-80% (Shengji, 2001). In Europe and North America, the sector
has grown at 10-20% annually during 1990s (Kate and Laird, 1999). Not just in parts of Asia,
but also in America increased interest has been observed from 3% in 1991 to 37% in 1998
(Brevoort, 1998). Amongst Asian countries India, China, Japan, Indonesia, etc have higher
amounts of medicinal and aromatic plants. Out of thousands of higher plant species existing
on earth, more than 80,000 are medicinal. Angiosperms (flowering plants) are the original
source of most of the medicinal plants, but other classes like thallophytes, bryophytes,
pteridophytes and gymnosperms also have certain examples of medicinal plant as shown in
(Joy et al., 1998). China and India alone use thousands of plant species for medicinal benefits
(Farnsworth and Soejarto, 1991). With the revival of Indian traditional medicinal systems, the
domestic demand for medicinal plants has seen a rapid increase. The market of traditional
systems of medicine in India is estimated to be about Rs. 4000 crores. The ayurvedic drug
market alone accounts for about Rs. 3500 crores. Indian market for ayurvedic medicines is
estimated to be expanding at 20% annually (Subrat, 2002). India has a small share of 1.6% in
the growing global market. The world market for plant derived drugs may account for about
Rs. 2, 00,000 crores. Presently Indian contribution is less than Rs. 2000 crores. The export of
raw drugs has readily grown at 26% to Rs.165 crores in 1994-95 from Rs.130 crores in 1991-
92. The annual production of medicinal and aromatic plant‟s raw material is worth about Rs.
200 crores. This is likely to touch US $1150 by the year 2000 and US $5 trillion by 2050 (Joy
et, al., 1998). Over one and a half million practitioners of the Indian System of Medicine in
the oral and codified streams use medicinal plants in preventive and curative applications.

14
There are estimated to be over 7,800 manufacturing units in India. In addition, there is a large
and growing market for food supplements and cosmetics. The overall demand for medicinal
plants is expected to increase at about 15 to 16 percent between 2002 and 2005 (CRPA,
2000). In addition to the domestic market, there is a large and expanding international trade in
medicinal plants. It is estimated that about 2500 species of medicinal plants are traded in the
international market (Schippmann, et al., 2002). An average of 400,000 tonnes of medicinal
plants, valued at $1.2 billion was estimated to be traded annually during 1990s (Lange, 2000).
The main markets include Europe, North America and Asia. Europe, which accounts for
about 50 percent of the world market, is the largest. The three leading exporting countries are
China, India and Germany. India‟s export of medicinal and herbal plants is expected to grow
from about Rs. 446 crore in year 2000 to Rs.3000 crore annually by 2005 (Lange, 2000).
Table 1: Some common medicinal plants and their uses
Common
name
Scientific name Parts used Medicinal uses
Amla Emblica officinalis Fruit Vitamin-C, cough, cold,
diabetes, laxative and hyper
acidity
Aswagandha Withania somnifera Roots/leaves Restorative, tonic, stress,
nerves disorder,
aphrodiasiac
Bael or bilva Aegle marmelous Fruit/bark Diarrhoea, dysentery
constipation
Chiraita Swertia chiraita Whole plant Skin diseases, burning
sensation, fever
Guggal Commiphora wightii Gum/resin Rheumatism, arthritis,
paralysis, laxative

15
Calihari Gloriosa superb Seed/tuber Skin diseases, labour pain,
abortion
Long pepper/
pippali
Pepper longum Fruit/root Appetizer, enlarged spleen,
bronchitis, cold and
antidote
Makoi Solanum nigrum Fruit/whole plant Dropsy, general debility,
diuretic, anti dysenteric
Sandal wood Santalum album Heart wood /oil Skin disorder, burning
sensation, jaundice, cough
Sarpagandha Rauwolfia serpentia Root Hyper tension, insomnia
Tulsi Ocimum sanctum Leaves/seed Cold, cough, bronchitis,
expectorant
Grithkumari Aloe vera Leaves Laxative, wound healing,
skin diseases, burns, ulcer
Sada bahar Catharanthus roseus Whole plant Leukemia, hypertension,
antispasmodic, antidote
Bach Acorus calamus Rhizome Sedative, analgesic,
hypertension,
Dalchini Cinnamomum
zeylanicum
Bark/oil Bronchitis, asthma, cardiac
disorder, fever
Aromatic Plants- Herbs or shrubs or trees which accumulate odorous molecules are known as
aromatic plants. The aromatic plants are found in every vegetation covered areas of the world.
These odorous principles are volatile in nature and commonly known as essential oils. The
aroma of the plant can easily be experienced by gentle touch of aromatic part / parts of the
plant. The floral scent (aroma) even can feel by coming in the vicinity of plant. These
aromatic compounds are synthesized and stored in a special structure called gland which is
located in different parts of plant such as leaves, flowers, fruits, seeds, barks and roots. These

16
essential oils can be extracted by various physical processes such as steam-distillation,
maceration, expression and enfleurage. They are mainly used as flavors and fragrances.
However, from ancient times, these plants have been used as raw materials for cosmetics,
pharmaceuticals, botanical pesticides, etc. (Chomchalow, 2001). The major use of aromatic
plants is reported as raw materials for essential oil extraction. These are plants in which their
non leafy parts are used as a flavoring or seasoning that contribute to spices and the plants in
which their leafy or soft flowering parts are used as a flavoring or seasoning can be denoted as
herbs. These are also used in some ways other than the ones mentioned above, for example, as
medicines, cosmetics, dyes, air fresheners, disinfectants, botanical pesticides, herbal
drinks/teas, pot pourri, insect repellents, etc. (Joy et al., 1998).
Asian people since prehistoric era had made use of aromatic plants in various
traditional ways. India is regarded as the traditional home of oriental perfumes (Sharma,
1996). Presently, few countries in Asia produced essential oils on an industrial scale. These
are China, India, Indonesia, Nepal, Sri Lanka and Thailand (Chomchalow, 2001). India is
considered to be ancient home of perfumes and aromatic plants, because it is blessed with a
wide varieties of soil and climatic conditions, which support the enormous plant wealth of the
18,000 native species found in this country, some 1,300 species are known to contain
odoriferous principles, but only about 65 of these plant have large and consistent demand in
the world trade and are accordingly grown in different parts of globe. India, however,
produces limited items of commercial value both from its rich natural forest vegetation and
cultivation (Chowdhury, 2002). Throughout the long history of almost 5,000 years, the
Chinese have continued to put faith in spices for medicinal purposes and for preserving and
flavoring foods. At present, Chinese people have made use of more than 400 species of

17
aromatic plants, not only from their flavor and fragrant properties, but also as medicines.
China now produces more than 120 natural essential oils for domestic consumption as well as
for export markets (Xiao, 1996).
Recent advances in biotechnological research have been applied to genetically
improve aromatic plants for commercial exploitation. These are used for increasing genetic
variability, culturing and selecting desirable genotypes, rescuing embryo of selected
genotypes, rapid multiplication of clones of selected genotypes and transferring genes
(Chomchalow, 2002).
1.2. Essential Oils
An oil is „essential‟ in the sense that it contains the „essence of‟ the plant's fragrance-the
characteristic fragrance of the plant from which it is derived. Essential oil is a concentrated
hydrophobic liquid containing volatile aroma compounds from plants. The advantages of
essential oils are their flavor concentrations and their similarity to their corresponding
sources. The majority of them is fairly stable and contains natural antioxidants and natural
antimicrobial agent as on citrus fruits (Mehra et al., 2015). These aromatic or volatile oils are
obtained from aromatic plant materials, including flowers, roots, bark, leaves, seeds, peel,
fruits, wood, and whole plants (Hyldgaard et al., 2012). The examples of some common
aromatic plants and their uses are presented in Table 2 (Kumar and Tripathi, 2011; Mishra et
al, 2000; Joy et al., 1998).

18
Table 2: Some common essential oil producing aromatic plants
Common
name
Scientific name Parts used Medicinal uses
Almond Prunus communis Nut Flavoring
Clove Eugenia caryophyllus Bud Dentistry & flavoring
Cinnamon Cinnamomum
zeylanicum
Leaves Flavoring
Coriander Coriandrum sativum Seed Flavoring
Eucalyptus Eucalyptus globules Leaves Decongestant
Lavender Lavendula officinalis Flower Perfumery
Sandalwood Santalum album Wood Perfumery
Lemongrass Cymbopogon flexuosus leaves Flavoring & medicines
Geranium Pelargonium graveolens Leaves & shoots Perfumery & cosmetics
Cardamom Elettaria cardamomum Fruit Perfumery & flavoring
Patchouli Pogostemon patchouli leaves Perfumery & flavoring
Chamomile Matricaria chamomilla herb Flavoring & cosmetics
Mentha Mentha arvensis Leaves and stem Pharmaceutical, food flavour
& cosmetics
Vetiver Vetiveria zizanioides roots Perfumery
Citronella
(java)
Cymbopogon
winterianus
leaves Pharmaceutical, flavoring &
cosmetics
Jasmine Jasminum officinale flower Perfumery
Rose Rosa damascene flower Perfumery & cosmetics
Physio-Chemical Properties- Essential oils have several common physical properties like
characteristic fragrances and high refractive indices. They are mostly optically active and
immiscible with water, but sufficiently soluble to impart their characteristic fragrance to

19
water. Factually, the aromatic waters are dependable on this slight solubility. Essential oils are
however soluble in ether, alcohol and organic solvents (Joy et al., 1998)
Chemical Constituents- Plant essential oils are usually the complex mixture of natural
compounds, both polar and nonpolar compounds (Masango, 2005). The major constituents in
essential oils are terpenes (monoterpenes and sesquiterpenes), their oxygenated derivatives
(aldehyde, ketones, esters, oxides, lactones, ethers, etc), and phenolic compounds (Bakkali et
al., 2008; Mohamed et al., 2010).
1.3. Extraction of Essential Oils
The essential oils are obtained from plant raw material by several extraction methods.
However, on commercial scale, it is generally extracted by physical means, such as water
distillation and cold methods (Dick and Starmans, 1996; Wang and Weller, 2006). New
methods of essential oils extraction are entering the mainstream of aromatherapy, offering
new choices in oils never before available. With the new labels of CO2 and Super Critical
CO2, along with the traditional „steam‟ and „hydro‟ distillations, 'absolutes', and 'cold
pressing', a little education for the aromatherapy enthusiast can go a long way in essential oil
selection. The methods of extraction of essential oils are described below.
Conventional or Traditional Methods
i) Hydrodistillation (HD)
ii) Steam distillation (SD)
iii) Enfleurage
iv) Maceration

20
v) Cold pressing
vi) Solvent extraction
Hydrodistillation- Water or hydrodistillation is one of the oldest and easiest methods of
essential oil extraction (Meyer-Warnod et al., 1984). The essential oils are evaporated by
heating a mixture of water and plant materials followed by the liquefaction of the vapors in a
condenser. The setup comprises also a condenser and a decanter to collect the condensate and
to separate essential oils from water, respectively. The principle of extraction is based on the
isotropic distillation. It is a multilateral process that can be utilized for large or small
industries. The distillation time depends on the plant material being processed (Rassem et. al.,
2016)
Steamdistillation- Steam distillation is a type of separation or extraction process for
temperature-sensitive aromatic compounds. The plant sample is heated by steam. The heat
applied is the main cause of burst and break down of cell structure of plant material. As a
consequence, the aromatic compounds or essential oils from plant material are released
(Perineau et al., 1992; Babu and Kaul., 2005). The temperature of heating must be accurate to
break down the plant material and release aromatic compound or essential oil. A new process
design and operation for steam distillation of essential oils to increase oil yield and reduce the
loss of polar compounds in wastewater was developed by (Masango, 2005). The system
consists of a packed bed of the plant materials, which sits above the steam source. Only steam
passes through it and the boiling water is not mixed with plant material. Thus, the process
requires the minimum amount of steam in the process and the amount of water in the distillate
is reduced. Also, water soluble compounds are dissolved into the aqueous fraction of the
condensate at a lower extent (Masango, 2005).

21
Enfleurage- It is an intensive and traditional way of extracting oil from flowers. The process
involves layering fat over the flower petals. After the fat has absorbed the essential oils,
alcohol is used to separate and extract the oils from the fat. The alcohol is then evaporated and
the essential oil collected (Rao and Pandey, 2006).
Maceration- In this process, the whole or coarsely powdered crude drug is placed in a
stoppered container with the solvent and allowed to stand at room temperature for a period of
at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then
is strained, the marc (the damp solid material) is pressed, and the combined liquids are
clarified by filtration or decantation after standing (Handa, 2008).
Cold Pressing Method-The term cold pressed theoretically means that the oil is expeller-
pressed at low temperatures and pressure. The cold pressed method is also known as
scarification method. Cold pressed method is mainly used for extracting essential oils from
plants, flower, seeds, lemon, tangerine oils. In this process, the outer layer of the plants
contains the oil are removed by scrubbing. Then the whole plant is pressed to squeeze the
material from the pulp and to release the essential oil from the pouches. The essential oil rises
to the surface of the material and is separated from the material by centrifugation (Arnould et
al., 1981).
Solvent Extraction- Solvent extraction also known as liquid–liquid extraction or partitioning,
is a method to separate a compound based on the solubility of its parts. This is done using two
liquids that don't mix, for example, water and an organic solvent. It is used on delicate plants
to produce higher amounts of essential oils at a lower cost (Chrissie, 2000). The quality and
quantity of extracted mixture are determined by the type of extra heat applied because of the

22
method is limited by the compound solubility in the specific solvent used. Although the
method is relatively simple and quite efficient, it suffers from such disadvantages as long
extraction time, relatively high solvent consumption and often unsatisfactory reproducibility
(Dawidowicz et al., 2008). The plant components extracted with organic solvents to make
oleoresins, concretes and absolutes or extracted with close to or critical solvent like carbon
dioxide to supply terribly prime quality extracts. These oleoresins and extracts contain not
solely the volatile oil however additionally the targeted non-volatile flavour elements and
these have wide application within the food and pharmaceutical industries. The solvent
extraction processes are harder and sophisticated than steam distillation and can ordinarily be
on the far side the money resources of most little scale processors, however activity the raw
materials to those extraction plants will be a market choice (Calvo et al., 2011).
Disadvantages of Conventional Methods- The essential oils are thermo-labile in nature so
their components undergo chemical alterations (hydrolysis, isomerization, oxidation etc.) due
to the high applied temperatures, which effects the quality of extracted essential oils
(particularly when the extraction time is long). The extraction methods couldn‟t maintain
essential oils chemical composition and natural proportion at its original state (Rassem et. al.,
2016).
Non Traditional Methods
With technological advancement, new techniques have been developed which may not
necessarily be widely used for commercial production of essential oils, but are considered
valuable in certain situations, such as the production of costly essential oils in a natural state
without any alteration of their thermo sensitive components or the extraction of essential oils
for micro-analysis (Rassem et al., 2016).

23
These techniques are as follows:
 Solid phase micro-extraction (SPME)
 Solvent-free microwave extraction (SFME)
 Microwave hydro-diffusion and gravity (MHG)
 Ultrasound-assisted extraction (UAE)
 Microwave-Assisted Hydrodistillation (MAHD)
 Supercritical Fluid Extraction (SFE)
Purification and Storage of Essential Oils- The essential oils as obtained from the
conventional distillation techniques are in crude form. It may have suspended impurities and
appreciable moisture content. Therefore, purification of the oils is essential before storage.
The essential oil should be freed from metallic impurities and moisture, only then it should be
stored in well filled tightly closed container, at low temperature and protected from light. For
small packing amber colored glass bottles are suitable, but for large quantities they should be
stored in metal drums, like tin. A layer of carbon dioxide or nitrogen gas is blown inside
container before it is sealed in order to replace air above the oil and hence to protect it from
oxidation. In order to remove moisture, which is one of the worst factors in the spoilage of
essential oil the smaller lots can be made free from moisture by addition of anhydrous sodium
sulphate, by thoroughly shaking, keeping aside and then filtering. Calcium chloride should not
be used for removal of moisture as it forms complex salt with certain alcohols. In case of
viscous oil like Vetiver the problem of moisture could be tackled by addition of common salt
and then allowing mixture to stand until supernatant oil has become clear. The lower layer
could be filtered. Centrifuging in high-speed centrifuges (rpm greater than 15,000) is an

24
excellent mode of clarifying the oils. If, it is done along with freezing it can also remove
waxes. In case of metallic impurities for oils like Clove, Bay the tartaric acid is employed and
then filtration is done (Kumar and Tripathi, 2011)
1.4. Analysis of Essential Oils
The essential oils are complex matrices that need to be analyzed by different techniques to
ensure quality, consumer safety and fair trade. Thus, there is a wide range of instrumental
techniques available (e.g., physical, organoleptic, chemical, chromatographic, and
spectroscopic analysis) to achieve characterization. Physical and chemical properties of any
essential oil are of prime importance, and chemists are now working in an era when highly
sophisticated instruments are available for quality and quantity analysis. Still, the specific
gravity, optical rotation, solubility in dilute alcohol, and the refractive index have its own
importance for essential oils and their isolates. However, gas chromatography (GC) and gas
chromatography-mass spectroscopy (GC-MS) are used almost exclusively for the quantitative
and qualitative analysis of the essential oils (Zhao et al., 2005).
Gas Chromatography (GC)
Gas chromatography was invented by Martin and Synge. GC is a very powerful and one of
the most common instrumental analysis techniques in use. When properly utilized, it provides
both qualitative and quantitative information about individual components of sample. GC
effectively separates the different compounds of a sample from each other (Martin and Synge,
1941). Many compounds are not suitable for gas chromatographic analysis due to their

25
physical and chemical properties. For a compound to be suitable for GC analysis, it must
fulfill following criterion:
 It must possess appreciable volatility at operating temperatures of the instrument.
 The compound must be able to withstand high temperatures and be rapidly
transformed into a vapour without degradation or reacting with other compounds.
The principles of GC are similar to those of HPLC but the instrument is signiﬁcantly
different. It exploits differences in the partition coefficients between a stationary liquid phase
and a mobile gas phase of the volatilized analytes as they are carried through the column by
the mobile gas phase. Its use is therefore confined to analytes that are volatile, but thermally
stable. The partition coefficients are inversely proportional to the volatility of the analytes so
that the most volatile elute first. The temperature of the column is raised to 50-300°C to
facilitate analyte volatilization. The stationary phase consists of a high-boiling-point liquid
material such as silicone grease or wax that is either coated onto the internal wall of the
column or supported on an inert granular solid and packed into the column. There is an
optimum flow rate of the mobile gas phase for maximum column efficiency (minimum plate
height). Hence, the technique is very useful for the analysis of complex mixtures. GC is
widely used for the qualitative and quantitative analysis of a large number of low-polarity
compounds, because it has high sensitivity, reproducibility and speed of resolution.
Analytically, it is a very powerful technique when coupled to mass spectrometry (Wilson,
2010)
The experimental setup of GC is described below (Wilson, 2010).
 A supply of a carrier gas (N2 or H2) from a high pressure cylinder having a pressure
regulator and flow meters

26
 A sample injection system
 The separation column made from variety of materials including glass, copper,
stainless steel, cupro-nickel or organic polymer (Teflon)
 The detector is situated at the exit of the separation column, which senses and
measures the small amount of the separated components present in the carrier-gas
leaving the column. Commonly used detectors are flame ionization detector and
thermal conductivity detectors
 The recorder is fed by the output of the detector
 Thermo stated compartment for the column and detector.
Gas Inlets: Gas is fed from cylinders through supply piping to the instrument. It is usual to
filter gases to ensure high gas purity and the gas supply may be regulated at the bench to
ensure an appropriate supply pressure.
Pneumatic Controls: The gas supply is regulated to the correct pressure (or flow) and then
fed to the required part of the instrument. Control is usually required to regulate the gas
coming into the instrument and then to supply the various parts of the instrument. A GC fitted
with a split/split-less inlet, capillary GC column and flame ionization detector may have the
following different gas specifications: Carrier gas supply pressure, column inlet pressure
(column carrier gas flow), inlet split flow, inlet septum purge flow, detector air flow, detector
hydrogen flow, detector make-up gas flow.
Injector: Here the sample is volatilized and the resulting gas entrained into the carrier stream
entering the GC column.

27
Column: In GC, retention of analyte molecules occurs due to stronger interactions with the
stationary phase than the mobile phase. The sample is separated into its constituent
components in the column. Columns vary in length and internal diameter depending on the
application type and can be either packed or capillary. Packed columns (typical dimension
1.5m × 4mm) are packed with a solid support coated with immobilized liquid stationary phase
material (GLC). Capillary columns (typical dimension 30m × 0.32 mm × 0.1 mm film
thickness) are long hollow silica tubes with the inside wall of the column coated with
immobilized liquid stationary phase material of various film thickness.
Column Oven: Temperature in GC is controlled via a heated oven. The oven heats rapidly to
give excellent thermal control. The oven is cooled using a fan and vent arrangement usually at
the rear of the oven. A hanger or cage is usually included to support the GC column and to
prevent it touching the oven walls as this can damage the column.
Detectors: The detector responds to a physicochemical property of the analyte, amplifies this
response and generates an electronic signal for the data system to produce a chromatogram.
Many different detector types exist and the choice is based mainly on application, analyte
chemistry and required sensitivity. Detector choices include: Flame Ionization (FID) Electron
Capture (ECD) Flame Photometric (FPD) Nitrogen Phosphorous (NPD) Thermal
Conductivity (TCD) and Mass Spectrometer (MS)
Data System: The data system receives the analogue signal from the detector and digitizes it
to form the record of the chromatographic separation known as the „Chromatogram‟. The data
system can also be used to perform various quantitative and qualitative operations on the
chromatogram, assisting with sample identification and quantification (Berger, 1996)

28
Gas Chromatography-Mass Spectroscopy (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of
gas-liquid chromatography and mass spectrometry to identify different substances within a
test sample. The use of a mass spectrometer as the detector in gas chromatography was
developed during the 1950s by Roland Gohlke and Fred McLafferty (Gohlke, 1959; Gohlke
and McLafferty, 1993). As mentioned, the GC-MS is composed of two major building blocks:
the gas chromatograph and the mass spectrometer (Skoog et al., 2007). The gas
chromatograph utilizes a capillary column which depends on the column's dimensions (length,
diameter, film thickness) as well as the phase properties (e.g. 5% phenyl polysiloxane,
polyethylene glycol). The difference in the chemical properties between different molecules
in a mixture will separate the molecules as the sample travels the length of the column. The
molecules take different amounts of time (called the retention time) to come out of (elute
from) the gas chromatograph, and this allows the mass spectrometer downstream to capture,
ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer
does this by breaking each molecule into ionized fragments and detecting these fragments
using their mass to charge ratio (Prathap et al., 2013). These two components, used together,
allow a much finer degree of substance identification than either unit used separately. It is not
possible to make an accurate identification of a particular molecule by gas chromatography or
mass spectrometry alone. The mass spectrometry process normally requires a very pure
sample while gas chromatography using a traditional detector (e.g. flame ionization detector)
detects multiple molecules that happen to take the same amount of time to travel through the
column (i.e. have the same retention time) which results in two or more molecules to co-elute.

29
Sometimes two different molecules can also have a similar pattern of ionized fragments in a
mass spectrometer (mass spectrum). Combining the two processes makes it extremely
unlikely that two different molecules will behave in the same way in both a gas
chromatograph and a mass spectrometer. Therefore, when an identifying mass spectrum
appears at a characteristic retention time in a GC-MS analysis, it typically tends to increased
certainty that the analyte of interest is in the sample (Charde, 2013)
1.5. Uses of Essential Oils
Essential oils are products of the secondary metabolism of plants, and generally are fragrant
volatile materials consisting of complex mixtures of mono- and sesquiterpene hydrocarbons,
and oxygenated materials derived from them, hence the essential oils are used in flavorings,
perfumes, in aromatherapy, as insect and animal repellents, in pharmaceutical preparations, as
anti-microbial agents and in many other ways (Rios, 2016). Plant essential oils and extracts
have been used for many thousands of years, especially in food preservation, pharmaceuticals,
alternative medicine and natural therapies (Lis-Balchin and Deans, 1997). Essential oils are
potential sources of novel antimicrobial compounds especially against bacterial pathogens
(Prabuseenivasan et al., 2006). Essential Oils are very powerful components of plants - they
have the capability of being harmful if improperly used. Essential Oils can be very helpful for
some cases, supportive in others, and have little to no effect in others. There are three
traditional uses of essential oils in Aromatherapy.
Inhalation- Inhalation is often effective for mood-altering effects of essential oils. Rosemary
oil is used for mental 'stimulation', lavender for relaxation, etc. One may certainly blend
essential oils in a diffuser or burner, adding a couple drops of the oil desired. Often a nice

30
result can be had from mixing a brighter or sweeter oil (Rosemary, Basil, Orange) with one
more earthy and grounding (Patchouli, Frankincense, Cedar).
Topical Application- Perhaps even more common than inhalation, topical application is the
preferred method of use for many essential oils. However, most essential oils require
significant dilution as they can cause skin irritation. Lavender oil and chamomile oil are two
essential oils that can be applied 'neat' or without dilution. Other oils such as cinnamon oil and
oregano oil should not be applied topically in most cases. They may be applied once highly
diluted to the bottoms of the feet. The interesting thing about topical application is that
essential oils tend to pass through the skin fairly readily, as they are lipophilic (fat soluble)
and their molecular structure is fairly small. Essential oils can pass into the bloodstream and
surrounding tissues.
Ingestion- Some essential oils are ingested, usually either in water or in capsules. As this
technique is rare, and not really considered effective in most cases (Rao and Pandey, 2006).
1.6. Biological Activity
The essential oils have recently begun to receive much attention as possible sources of safe
and natural alternative medicines because they have been known to possess various medicinal
activities. Consequently, studies on essential oils to evaluate the pharmacological properties in
order to find possible alternative medicines have become active in recent years. Researchers
from all over the world are trying to characterize a range of biological properties of essential
oils which includes antimicrobial, antiviral, antimutagenic, anticancer, antioxidant,
antiinflammatory, immunomodulatory, and antiprotozoal activities (Bakkali et al., 2008).

31
Efficiencies of various essential oils are compared by analyzing the concentrations required to
inhibit the growth of target organisms. Generally, minimum growth inhibitory concentrations
(MICs), minimum lethal concentrations (MBCs or MFCs), MIC50 and LD50 values are used
for comparison of bioactivities. These values are obtained with standardized methodologies
(Raut and Karuppayil, 2014).
Disc Diffusion Method-Agar disc diffusion method used for the determination of
antimicrobial activities of the essential oil or natural extracts against bacteria and fungi
(NCCLS, 1997; NCCLS, 1999). The incubation period for bacteria is 24h (37°C) under
anaerobic conditions and for fungi 48h at (30°C) in aerobic conditions. The diameters of the
inhibition zones are measured in millimeter (mm) (Alim et al., 2009).
Microdilution Broth Method- A broth microdilution susceptibility assay is used for the
determination of minimum inhibitory concentration (MIC), minimum bactericidal
concentration (MBC) and minimum fungicidal concentration (MFC) (NCCLS, 1997; NCCLS,
1999). MIC is the lowest concentration of a chemical that prevents visible growth of a
bacterium or the concentration at bacteriostatic activity. The MBC is the concentration that
results in microbial death or the concentration at which it is bactericidal. (Tripathi, 2013). The
tests performed in Mueller Hinton Broth (MHB; OXOID-CM405) with the exception of the
yeasts (sabouraud dextrose broth-SDB; DIFCO). Bacterial strains are cultured overnight at
37°C in Mueller Hinton Agar (MHA) whereas for yeasts they are cultured overnight at 30°C
in Sabouraud Dextrose Agar (SDA) (Alim et al., 2009).
Essential oils are complex mixtures of volatile constituents, biosynthesized by plants,
which mainly include two biosynthetically related groups (Picherskey et. al., 2006). These

32
main groups include terpenes and terpenoids and aromatic and aliphatic constituents, all
characterized by low molecular weight. Most of the antimicrobial activity in EOs is found in
the oxygenated terpenoids (e.g., alcohols and phenolic terpenes), while some hydrocarbons
also exhibit antimicrobial effects (Burt, 2004; Koroch et. al., 2007). Interactions between
these components may lead to antagonistic, additive or synergistic effects. Some studies have
demonstrated that whole essential oils usually have higher antibacterial activity than the
mixtures of their major components, suggesting that the minor components are critical to the
synergistic activity, though antagonistic and additive effects have also been observed (Gill et
al., 2002; Davidson and Parish, 1989; Mourey et al., 2002).
1.7. Thymus spp.
The genus Thymus L. (Lamiaceae), commonly known as „thyme‟, consists of about 215
species of herbaceous perennial and sub-shrubs (Stahl-Biskup and Saez, 2002). The aromatic
and medicinal properties of the genus Thymus have made it one of the most popular plants all
over the world. Thymus species are commonly used as herbal tea, flavoring agents (condiment
& spice) and medicinal plants (Stahl-Biskup and Saez, 2002). Thyme is believed to be a
native of the Mediterranean region. It grows wild in almost all the countries bordering
Mediterranean area, Asia and in parts of Central Europe. It is extensively cultivated in
Germany, France, Spain, England and various other neighboring countries both for seasoning
and for its volatile oil (Grieve, 1974). Major volatile constituents obtained from the aerial-
parts of the plant are geranial, linalool, γ-terpineol, carvacrol, thymol and trans-thujan-
4ol/terpinen-4-ol (Piccaglia et al., 1993). Recent studies have shown that Thymus species have

33
strong antibacterial, antifungal, antiviral, antiparasitic, spasmolytic and antioxidant activities
(Stahl-Biskup & Saez, 2002).
Thyme is an ever green perennial herb the essential oil of which has a powerful fresh
odour masking other unpleasant smells. Its used as a pot herb in cooking, perfumery and in
liquor distillery. Thyme oil finds its major use in the perfumery industry in soap and detergent
work. Thymol has a powerful medicinal odour and finds more applications in flavours than in
perfumes. Owing to the presence of thymol the oil shows germicidal properties and is
effective against a variety of pathogenic bacteria. It is employed in dental preparations, oral
hygiene products, vermifuges and antigastro-intestinal products. In aromatherapy, garden
thyme is regarded as one of the most important elements because of its antiseptic properties.
The essence is effective in treating whooping cough as well as parasitic infestations. Dried
flowers and leaves are used to preserve linen from insects and to impart characteristic smell
(Joy et al., 1998). Thyme is a well-known medicinal plant having diverse pharmacological
properties, such as spasmolytic, antiseptic, antitussive, expectorant and antispasmodic
activities. The antiseptic, antioxidative, insecticidal, preservative and anaesthetic properties of
thyme oil are mainly due to the presence of thymol, carvacrol, geraniol and other volatile
components in the species of Thymus. The antiseptic, antioxidative, insecticidal, preservative
and anaesthetic properties of thyme oil are mainly due to the presence of thymol, carvacrol,
geraniol and other volatile components (Van-Den Broucke and Lemli, 1981). Different
Thymus spp. and their therapeutic property are summerised in Table 3. The genus Thymus has
numerous species and varieties. Some important one are mentioned below.
Thymus serpyllum- It is commonly known as “Ban-Ajwain” is a small much branched and
strongly scented shrub. It bears tiny purple colored flowers (Dymock, 1972; Nasir, 1973). It is

34
considered to be very beneficial whether used as an article of food or as a medicament. Its
volatile oil is good for invertebrate cough and also as antiseptic, anthelmintic and carminative
(Nandkarni, 1982). The essential oil of T. serpyllum possesses thymol and carvacrol along
with the other terpenes in minor amount (Ahmad, 1995).
Thymus vulgaris- It is endemic in the Mediterranean region (Dommee et al., 1978; Gigord et
al., 1999). Oblong oval dark green leaves with lengths between 6 and 12mm and winding leaf
edge are characteristic traits of T. vulgaris. The underside of the leaves is white velvety hairy.
The stems are short, round and green and woody in older plants. In June/August, the plants
begin to develop pink, white or violet ﬂowers (Mewes et al., 2008). The essential oil is well
recognized for its medicinal properties in the treatment of bronchitis, whooping cough and
tooth ache. Flavonoid may be important in spasmolytic activity of smooth muscles of guinea
pig ileum and trachea. Also it has antimicrobial activity against fungi (some aflatoxins
producers), viruses, helminthes, Gram positive and Gram negative bacteria (Farag et al.,
1989). The chemical composition of the essential oil is characterized by high amounts of
thymol, p-cymene and γ-terpinene (Porte and Godoy, 2008).
Thymus hyemalis- It commonly known as winter thyme, can be found mainly in Alicante,
Murcia and Almeria. This plant is normally present in siliceous and calcareous extensions,
from sea level to 400-700m above sea level. It is able to resist long dry periods, but its winter
flowering conditions doesn‟t allow it to grow in areas having very cold weather (Jordan et al.,
2006). It is a potential impulse of new trends in food, pharmaceuticals and cosmetics
industries (Echeverrigaray et al., 2002). The essential oil of the plant is reported to possess
antifungal, pesticide and antibacterial activities (Daferera et al., 2002; Kalemba and Kunicka,

35
2003). Moreover, thymol, carvacrol, borneol and linalool were the most abundant constituents
of T. hyemalis essential oil (Jordan et al., 2006).
Table 3: Different Thymus spp. and their therapeutic property
Thyme Common
name
Uses References
Thymus vulgaris Garden
thyme
Antispasmodic, antimicrobial,
treatment of bronchitis,
whooping cough & tooth ache
Imelouane et al.,
2009
Thymus serpyllum Creeping
thyme
antiseptic, anthelmintic &
carminative
Ahmad et al., 2006;
Verma et al., 2009
Thymus praecox Mother of
thyme
Ornamental plant Stahl-Biskup, 1986
Thymus citriodorus Lemon
thyme
Antiseptics, respiratory
problems, aromatherapy,
deodorant & disinfectant
Tatrai et al., 2016
Thymus herba
barona
Caraway
thyme
Antiseptic, perfumery,
disinfectant
Corticchiato et al.,
1998
Thymus fontanesii Antispasmodic, carminative,
stomachic, expectorant,
antitussive, antiseptic and
anthelmintic remedy in some
gastrointestinal and cold
diseases
Ghannadia et al.,
2004
Thymus linearis Himalayan
thyme
Aromatic, antiseptic,
diaphoretic, analgesic, diuretic,
carminative, stimulant, prevent
hair loss also for weak vision,
complaints of liver, suppression
of urine and menstruation.
Verma et al., 2010
Thymus mastichina Mastic
thyme
Antiseptic, deodorant, &
disinfectant
Tovar et al., 2015
Thymus cilicicus Cilician
thyme
Antiseptic, deodorant,
disinfectant & flavoring
Tumen et al., 1994
Thymus camphoratus Camphor
thyme
Antiseptic, deodorant &
disinfectant
Miguel et al., 2004

36
2. REVIEW OF LITERATURE
Himalayan Thyme (Thymus linearis Benth.)
2.1. Origin and Distribution
In India, the genus Thymus is represented by two species, namely Thymus linearis (native)
and Thymus serpyllum (exotic). T. linearis is a rather variable species, widespread in the
Himalayas and reaching its western extremities in Pakistan and Afghanistan. Jalas (1973)
recognized subspecies hedgei apart from the subspecies typical. The former subspecies was
described from Quetta, Ziarat (Pakistan) and is reported only from Pakistan and eastern
Afghanistan, while the latter was described from Kumaun region (India) with a much wider
distribution in northeast Afghanistan, Pakistan, India (Jammu and Kashmir, Himachal Pradesh
and Uttarakhand) and West Nepal (Jalas, 1973).
2.2. Morphological Characters
It is characterized by shorter petioles and a more elliptical blade. It is a creeping mat forming
herb, verticillasters in terminal head and some in axils of leaves along the branch. The leaves
elliptic-obovate with prominent lateral nerves, bracts are slightly smaller than leaves, ciliate at
the margins (Jamzad, 2009).
2.3. Traditional Uses
Dried leaves are also reported to be used in whooping cough, asthma, expelling round worm
and as an antiseptic in Gilgit region (Wazir et al., 2004). In Nepal, the young flowers are
reported to be taken in oral problems (Kunwar and Adhikari, 2005). The powder of dried herb
is used in the treatment of weak vision and menstrual regulation. Moreover, it is also used in

37
skin diseases such as eczema and psoriasis by the inhabitants of Uttarakhand Himalaya (Rana
et al., 2010).
2.4. Chemical Composition and Biological Activity
The essential oil of T. linearis is characterized by higher amounts of thymol (52.2–66.6%), p-
cymene (1.8–21.6%), and γ-terpinene (1.9–12.4%) (Verma et al., 2010) and possesses
antioxidant, antimalarial and antiproliferative activities (Hussain et al., 2013). The essential
oil of T. linearis, isolated from the herb (primary oil) and its corresponding hydrosol
(secondary oil) obtained during distillation process was investigated for chemical composition
and antimicrobial properties. The primary oil was composed of thymol (44.2%), γ-terpinene
(25.1%), p-cymene (13.1%), terpinen-4-ol (2.5%), α-terpinene (2.4%), α-pinene (2. 1%) and
β-bisabolene (1.9%); while the secondary oil of was mainly composed of thymol (92.4%) and
carvacrol (4.0%). The bioassay showed that the secondary oil exhibited stronger antibacterial
and antifungal activities than primary oil (Verma et al., 2016b). Considering the huge scope of
uses of the Himalayan thyme in pharmaceutical, food flavour, and fragrance industries, it was
introduced in the subtropical region of north India from the temperate Himalayan region. The
productivity and essential oil quality of successfully domesticated T. linearis was assessed at
during different harvests. The essential oil of Himalayan thyme produced in this region
fulfilled the criterion set by European Pharmacopeia for common thymes (Verma et al.,
2016a).

38
3. OBJECTIVE OF THE RESEARCH
The present research was undertaken with the main objective to assess the morphological and
chemical changes occurring during the annual growth cycle, and to investigate the
antibacterial and antifungal activities of Himalayan thyme (Thymus linearis) grown in the
foothills of Uttarakhand.
Specific objectives of the study:
 Assessment of morphological and yield contributing characters of Himalayan thyme
(Thymus linearis) at different growth stages
 Assessment of the changes in the essential oil profile of Himalayan thyme (Thymus
linearis) due to plant-parts and growth stage
 Evaluation of the antibacterial and antifungal activities of the essential oil and extract
of Himalayan thyme (Thymus linearis)

39
4. MATERIALS AND METHODS
4.1. Plant Materials, Growing Conditions and Rising of Crop
Well rooted stem cuttings of T. linearis were transplanted in the experimental field of CSIR-
Central Institute of Medicinal and Aromatic Plants, Research Centre Pantnagar, Uttarakhand
in the end of September, 2016. The crop was raised under normal and uniform agricultural
practices (Figure 1). The morphological data of the crop was recorded for ten quantitative
characters (plant height, plant canopy, branch/plant, leaf/stem ratio, leaf yield/plant,
inflorescence yield/plant, essential oil content in leaves, essential oil content in inflorescence,
essential oil yield of leaves/plant and essential oil yield of inflorescence/plant) in six different
stages, namely S1: early-vegetative-stage (120 DAP); S2: late-vegetative-stage (135 DAT);
S3: flower-initiation-stage (150 DAT); S4: full-bloom-stage (175 DAT); S5: late-bloom-stage
(190 DAT); and S6: seed-shattering-stage (200 DAT). The experiment was performed in a
randomized block design with six treatments as harvesting stages in three replicates. The
experimental site is located between latitude 29 N and longitude 79.38 E, and at an altitude
of 243m above mean sea level, experiencing the subtropical, humid climate. The experimental
soil was mollisol with neutral in reaction (pH 7.1).
4.2. Isolation of Essential Oil
The freshly harvested plant materials, namely whole aerial-parts, leaves, inflorescences and
stem (100g) of T. linearis were dried in shade and then subjected to hydrodistillation (3h) in a

40
Clevenger apparatus for isolation of their essential oils. The essential oils obtained were dried
over anhydrous sodium sulphate and yields (%) determined on dry weight basis. All oil
samples were kept in the refrigerator until their further analysis.
Figure 1: Thymus linearis grown at the experimental field of CSIR-CIMAP, Research Centre
Pantnagar Uttarakhand.
4.3. Preparation of Solvent Extract
The herb (whole aerial-parts) collected at full bloom stage (S4) was dried in shade and
powdered using electrical grinder. The extracts were prepared using three different solvents,
namely hexane, acetone and methanol (Figure 2). The powdered material was mixed with
solvent at a 1:20 ratio (50g herb powder in 1.0 liter solvent) and macerated for 48h. Then the

41
extract was filtrated and solvent evaporated (at 35◦
C) under reduced pressure to obtain
concentrated extracts. The crude extracts were then kept at -20 ◦
C until further bioassay.
Figure 2: Solvent extracts of Thymus linearis collected at full bloom stage
4.4. GC and GC-MS Analysis of Essential Oil
The essential oil analysis was carried out using GC-FID and GC-MS techniques. GC was
performed for quantification of the essential oil constituents, using Nucon gas chromatograph
(model 5765) equipped with DB-5 (30m  0.32 mm; 0.25 µm film thickness) fused silica
capillary column and flame ionization detector (FID) (Figure 3). Hydrogen was used as
carrier gas at 1.0mL min-1
. Temperature programming was done from 60–230°C at 3°C min-1
.
The injector and detector temperatures were 220°C and 230°C, respectively. The injection
volume was 0.03µL neat with a split ratio of 1:40. GC-MS, performed for identification of the
essential oil constituents, was done using a Clarus 680 GC interfaced with a Clarus SQ 8C

42
mass spectrometer of PerkinElmer fitted with Elite-5 MS fused-silica capillary column (5%
phenyl polysiloxane, 30m × 0.25mm internal diameter, film thickness 0.25µm). The oven
temperature program was from 60–240°C, at 3°C min-1
, and programmed to 270°C at 5°C
min-1
. Injector temperature was 250°C; transfer line and source temperatures were 250°C;
injection size 0.03µL neat; split ratio 1:50; carrier gas He at 1.0mL min-1
; ionization energy
70 eV; mass scan range 40–500amu.
Figure 3: Gas chromatograph
4.5. Identification of Essential Oil Constituents
Identification of the essential oil constituents was carried out on the basis of retention index
(RI), determined with reference to homologous series of n-alkanes (C7-C30), MS Library
search (NIST and WILEY), and by comparing RI and mass spectral data with the literature
(Adams, 2007). The relative amounts of individual components were calculated based on the
relative % peak areas (FID response), without using correction factor.

43
4.6. Antibacterial Assays
The antibacterial activity of the essential oils and extracts was determined against six
pathogenic strains, namely Mycobacterium smegmatis (UDSC-MC2
155), Enterococcus
faecalis (MTCC-439), Escherichia coli (MTCC-723), Streptococcus mutans (MTCC-890),
Staphylococcus epidermidis (MTCC-435) and Staphylococcus aureus (MTCC-96) using disc
diffusion assay as per CLSI guidelines (CLSI, 2006). Inoculums of the test bacteria were
prepared equivalent to McFarland Standard 0.5. Uniform bacterial lawns prepared using
100µL inoculums on a Mueller Hinton agar plate. 8µL of essential oil was placed over sterile
discs (6.0 mm; Himedia), then it was kept over seeded plates. The plates were incubated at 37
°C for 24 h. Activity was measured in terms of zone of growth inhibition (mm) determined by
subtracting the disc diameter (i.e. 6.0 mm) from the total zone of inhibition shown by the test
disc in terms of clear zone around the disc. The bacterial strains were procured from the
Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology (IMT)
Chandigarh, India. Antibacterial efficacy of the essential oil was also determined by Micro
dilution broth assay using 96 „U‟ bottom micro-titer plates as per CLSI guidelines (CLSI,
2012). Samples were serially diluted two folds (in the range of 1000–1.95µL/mL) in Mueller
Hinton Broth (MHB). The broth was inoculated with 10.0µL of diluted 24h grown culture of
test organisms with a titre equivalent to 0.5 McFarland standards. The inoculated plates were
incubated at 37ο
C for 16–24h and the growth was recorded visually using resazurin dye as an
indicator. The minimum inhibitory concentration (MIC) value was determined from the
turbid-metric data as the lowest concentration showing growth inhibition as compared to
control. Tetracycline and DMSO were used as a positive and negative control, respectively.

44
Bactericidal end points were obtained by spread plating known volume (100μL) from each
well on solid media, and the end point for complete inhibition was defined as the minimum
bactericidal concentration (MBC) of test samples in the original tube which failed to yield
discernible growth when sub-cultured. All the experimental observations were performed in
triplicate to rule out any error during the antibacterial assay.
4.7. Antifungal Assays
The antifungal activity of the essential oils and extract was determined against three strains of
Candida spp, namely Candida albicans (ATCC 14053), Candida kefyr (ATCC 204093) and
Candida albicans (clinical isolate). Cultures of fungi were grown on Sabouraud Dextrose
Broth (Hi Media Pvt, Ltd., India) for 24h at 37ο
C and then turbidity was adjusted to 0.5
McFarland standards (approximately 1.2×106
CFU/mL). 100µL of inoculum (0.5 McFarland)
of the fungal culture was withdrawn with caution and spread uniformly over the surface of
Sabouraud Dextrose agar plate to get even lawn. 8µL of oil was impregnated on the sterile
paper disc (6mm diameter, Himedia) and placed on the fungal lawns. The plates were then
incubated for 24h (37ο
C) following which the diameter of the inhibition zone was measured.
The net zone of growth inhibition was determined by subtracting the disc diameter (i.e. 6mm)
from the total zone of growth inhibition shown by the test disc in terms of clear halo fungal
lawn around the disc. MIC was estimated using macro dilution broth assays. For this purpose
two-fold serial dilution series was employed to assess the MIC of given oils. In each assay,
10µL of fungal culture (0.5 McFarland) prepared as before was added to the 1.0mL medium
and incubated at 37±1ο
C and the killing or inhibition was examined by visible turbidity.

45
Minimal fungicidal concentration (MFC) was determined by plating 100µL from each tube
used for determining MIC and observed for any growth after 2 days of incubation.
Ketoconazole was used as standard in antifungal activity evaluation (Saikia et al., 2001). All
the experimental observations were performed in triplicate.
4.8. Statistical Analysis
The morphological data (yield and yield contributing characters) are reported as the mean of
three replicates. The numerical data of all the components were subjected to analysis of
variance (ANOVA) using randomized block design (RBD). Statistical analysis of data was
done following standard procedures (Snedecor and Cochran, 1967).

46
5. RESULTS AND DISCUSSION
5.1. Morphological (Yield and Yield Contributing) Characters
The morphological data of T. linearis observed at different developmental stages (S1–S6) is
summarized in Table 4. Plant height was found to vary from 7.1–18.6cm during growth cycle
with the significantly highest (18.6cm) at S6 (seed-shattering-stage), which was at par with S5
(late-bloom-stage). Plant canopy was found to vary from 32.3–55.4cm during different stages
of plant growth. Significantly highest canopy was recorded at S4 (full bloom stage) and it was
at par with S5 and S6. Number of branches per plant was found to vary from 25.0–183.3 with
the significantly highest at S6 (183.3) and S5 (177.3). The leaf/stem ratio was found to vary
from 2.5–5.7 during different growth stages considered. However, significantly highest
leaf/stem ratio was recorded at S1 (early vegetative stage). The leaf yield was found to vary
from 4.7-24.4g plant-1
with the significantly highest at S5. However, the inflorescence yield
varied from 2.4-23.2g plant-1
during blooming process with the significantly highest at S5.
The essential oil content of dried leaves varied from 2.65–3.48%, with the significantly
highest at S4 (3.48%), followed S5 (3.39%) and S3 (3.25%). The essential oil content of dried
inflorescence varied from 1.77–3.54%, with the significantly highest at S4 (3.54%).
Consequently the essential oil yields of leaves and inflorescence were found to vary from
0.124–0.826mL plant-1
and 0.066-0.605mL plant-1
, respectively. However, the leaves and
inflorescence gave maximal yields at S5 (0.826 and 0.605mL plant-1
, respectively). There are
several examples of the existence of seasonal and ontogenic variations in the essential oil
yield of Thymus and other plant species (Miguel et al., 2005; Figueiredo et al., 2008; Verma et
al., 2011; Verma et al., 2016a). Moldao-Martins et al (1999) observed that the essential oil

47
yield of Thymus zygis L. subsp. sylvestris was maximal at the flowering stage. However, in
present study maximum essential oil yields of leaves and inflorescences were recorded at late-
flowering stage (S5). Similar results have also been observed earlier in case of whole aerial-
parts of T. linearis during first harvest (Verma et al., 2016a).
5.2. Chemical Composition
The essential oils obtained from different plant-parts, collected at different growth/ harvesting
stages (S1–S6) of T. linearis in the foot-hills of Uttarakhand were analysed by GC-FID and
GC-MS. The results pertaining to chemical constituents and their relative amounts in different
essential oils are summarized in Table 5-7. Altogether, forty-two constituents, forming 93.4–
99.9% of the total oil composition were identified. The essential oils were dominated by
phenolic compounds (42.9–66.3%; represented by free phenols, phenolic ethers and phenolic
esters) and terpenoids (33.6–50.5%; mainly represented by monoterpene hydrocarbons,
oxygenated monoterpenes and sesquiterpene hydrocarbons). The distribution of different
classes of components in different plant-parts of T. linearis is presented in Figure 4. Major
components in the essential oils of different samples, collected at different stages (S1–S6)
were thymol (35.5–59.0%), γ-terpinene (13.3–19.8%), p-cymene (4.2–11.8%), borneol (0.9–
8.2%), thymyl acetate (0.9–7.6%), thymol methyl ether (2.9–6.0%), β-bisabolene (1.6–5.3%),
(E)-caryophyllene (1.5–3.5%), α-terpinene (2.0–3.2%), carvacrol (<0.05–2.8%), myrcene
(1.1–2.1%), and α-thujene (1.0–2.1%).

48
Table 4: Growth and yield characters of Thymus linearis under the subtropical conditions of north India
Plant
stage
Plant
height
(cm)
Canopy
(cm)
Branch
/plant
Leaf/stema
Leaf
yield
(g/plant)b
Essential
oil
content
of leaves
(%)b
Essential
oil yield of
leaves
(mL/plant)b
Inflorescence
yield
(g/plant)b
Essential oil
content of
Inflorescence
(%)b
Essential oil
yield of
Inflorescence
(mL/plant)b
S1 7.1 32.3 25.0 5.7 4.7 2.65 0.124 - - -
S2 10.6 39.3 51.0 3.8 11.8 3.15 0.371 - - -
S3 11.1 43.1 92.0 4.2 13.8 3.25 0.449 2.4 2.73 0.066
S4 16.7 55.4 152.7 4.3 21.8 3.48 0.760 13.6 3.54 0.483
S5 18.2 55.1 177.3 4.0 24.4 3.39 0.826 23.2 2.61 0.605
S6 18.6 54.0 183.3 2.5 5.7 3.07 0.175 18.9 1.77 0.332
SEM 0.33 1.54 4.82 0.21 0.84 0.10 0.03 1.71 0.05 0.04
CD
(5%) 0.73 3.43 10.73 0.47
1.87 0.23 0.07
3.81
0.11 0.10
CV (%) 10.81 27.61 55.35 12.77 27.81 7.12 5.94 67.26 4.53 10.73

49
S1: early-vegetative-stage (120 DAP); S2: late-vegetative-stage (135 DAT); S3: flower-initiation-stage (150 DAT); S4: Full-bloom-
stage (175 DAT); S5: late-bloom-stage (190 DAT); S6: seed-shattering-stage (200 DAT); a
S3-S6: Inflorescence treated as leaves for
leaf/stem ratio calculation; b
determined on dry weight basis; SEM: standard error of mean; CD: critical difference; CV: coefficient of
variance.

50
Figure 4: Distribution of phenolic (phenols, phenolic ethers and phenolic esters) and
terpenoid (monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpene
hydrocarbons) constituents in the essential oils of different plant-parts of Thymus linearis at
full bloom stage.
Essential Oil Composition of Leaves
The leaf oil of T. linearis collected at different stages of plant growth (S1–S6) showed
substantial variation in their quantitative chemical composition (Table 5). Major component
of the oil were thymol (47.7–54.6%), γ-terpinene (13.3–18.4%) and p-cymene (5.3–11.8%),
borneol (0.9–1.9%), thymyl acetate (1.3–4.2%), thymol methyl ether (3.1–4.4%), β-bisabolene
(1.6–5.3%), (E)-caryophyllene (1.5–3.0%), α-terpinene (2.0–2.9%), carvacrol (1.2–2.7%),
myrcene (1.4–2.1%) and α-thujene (1.1–2.1%). Thymol content was found to be highest at S4
(54.6%) followed by S1 (54.5%). However, γ-terpinene and p-cymene were recorded higher at
S3 (18.4%) and S6 (11.8%), respectively (Figure 5). A representative GC-FID chromatogram
of the leaf essential oil is presented in Figure 6.
0
10
20
30
40
50
60
70
EO-L EO-I EO-S EO-AP
Phenolic compounds (%) Terpenoids (%)

51
Table 5: Essential oil composition of the leaves of Himalayan thyme (Thymus linearis)
collected at different developmental stages in the foot-hills of Uttarakhand, India
S. No. COMPOUNDa
RIb
RIc
CONTENT (%)/
LEAF OIL
S1 S2 S3 S4 S5 S6
1. Tricyclene 920 921 - - t - - -
2. α-Thujene 925 924 1.8 1.8 2.1 1.5 1.7 1.1
3. α-Pinene 935 932 0.8 1.0 0.8 0.6 0.8 0.5
4. Camphene 945 946 0.4 0.4 0.5 0.3 0.4 0.4
5. Sabinene 972 969 0.1 0.1 0.2 0.1 0.1 0.1
6. β-Pinene 978 974 0.2 0.2 0.2 0.2 0.2 0.2
7. 3-Octanone 981 979 t t t T t t
8. Myrcene 989 988 1.9 1.9 2.1 1.8 1.8 1.4
9. α-Phellandrene 1002 1002 0.3 0.3 0.3 0.3 0.3 0.2
10. δ-3-Carene 1010 1008 0.1 0.1 0.1 0.1 0.1 0.1
11. α-Terpinene 1016 1014 2.2 2.6 2.9 2.4 2.6 2.0
12. p-Cymene 1021 1020 6.5 5.3 5.9 6.3 8.6 11.8
13. Limonene 1025 1024 0.4 0.4 0.4 0.4 t t
14. (E)-β-Ocimene 1045 1044 0.1 0.1 0.1 0.1 0.1 0.1
15. γ-Terpinene 1053 1054 13.3 16.0 18.4 16.0 17.3 14.2
16. cis-Sabinene hydrate 1068 1065 0.9 0.9 1.0 1.0 0.9 1.2
17. Terpinolene 1088 1086 0.1 0.1 0.1 0.1 0.1 0.1
18. Linalool 1098 1095 0.2 0.2 0.2 0.2 0.2 0.3

52
19. trans-Sabinene hydrate 1100 1098 t t t T 0.1 0.1
20. cis-Thujone 1103 1101 t t t T - t
21. Borneol 1167 1165 0.9 0.9 1.1 0.9 1.2 1.9
22. Terpinen-4-o1 1174 1174 0.3 0.2 0.3 0.3 0.3 0.4
23. p-Cymen-8-ol 1182 1179 t t t T t t
24. α-Terpineol 1188 1186 0.1 0.1 0.1 0.1 0.1 0.1
25. Octanol acetate 1210 1211 t t t T 0.1 0.1
26. Thymol methyl ether 1235 1232 3.2 3.3 3.6 3.2 3.1 4.4
27. Carvacrol methyl ether 1245 1241 t t t T t 0.1
28. Bornyl acetate 1287 1284 t t t 0.1 t t
29. Thymol 1292 1289 54.5 52.9 50.8 54.6 51.2 47.7
30. Carvacrol 1302 1298 1.5 2.0 2.7 2.2 1.6 1.2
31. Thymyl acetate 1351 1349 4.2 2.8 2.6 1.7 1.3 1.3
32. Carvacryl acetate 1373 1370 0.1 t t T t t
33. (E)-Caryophyllene 1419 1417 1.8 2.1 1.5 2.1 1.8 3.0
34. Aromadendrene 1442 1439 t t - - t t
35. α-Humulene 1454 1452 0.1 0.1 t 0.1 0.1 0.1
36. allo-Aromadendrene 1460 1458 t t - T t -
37. Germacrene D 1484 1480 0.2 0.4 0.2 0.2 0.1 0.1
38. Viridiflorene 1495 1496 0.1 0.1 t T t 0.1
39. β-Bisabolene 1510 1505 3.4 3.4 1.6 2.9 2.8 5.3
40. γ-Cadinene 1518 1513 0.1 0.1 0.1 0.1 0.1 0.2
41. Spathulenol 1580 1577 - - - - - -

53
42. Caryophyllene oxide 1584 1582 - - t - - -
CLASS COMPOSITION
Phenolic compounds
Phenols 56.0 54.9 53.5 56.8 52.8 48.9
Phenolic ethers 3.2 3.3 3.6 3.2 3.1 4.5
Phenolic ester 4.3 2.8 2.6 1.7 1.3 1.3
Terpenoids
Monoterpene hydrocarbons 28.2 30.3 34.1 30.2 34.1 32.2
Oxygenated monoterpenes 2.4 2.3 2.7 2.6 2.8 4.0
Sesquiterpene hydrocarbons 5.7 6.2 3.4 5.4 4.9 8.8
Oxygenated sesquiterpenes - - t - - -
Others t t t T 0.1 0.1
Total identified (%) 99.8 99.8 99.9 99.9 99.1 99.8
Note- a
Identification based on the retention index (RI), mass fragmentation pattern (NIST &
WILEY library) and literature data (see experimental section); b
retention index, determined on
DB-5 gas chromatography column; c
retention index value from literature; S1: early-
vegetative-stage (120 DAP); S2: late-vegetative-stage (135 DAT); S3: flower-initiation-stage
(150 DAT); S4: Full-bloom-stage (175 DAT); S5: late-bloom-stage (190 DAT); S6: seed-
shattering-stage (200 DAT); t: trace (<0.05%).

54
Figure 5: Changes in thymol, γ-terpinene and p-cymene content in leaves of Thymus linearis
during plant ontogeny.
Figure 6: A representative GC-FID chromatogram of the leaf essential oil of Thymus linearis.
0
10
20
30
40
50
60
S1 S2 S3 S4 S5 S6
Thymol (%) γ-Terpinene (%) p-Cymene (%)

55
Essential Oil Composition of Inflorescence
The inflorescence oil of T. linearis collected at different stages of plant growth (S3–S6)
showed substantial variation in their quantitative chemical composition (Table 6). Major
component of the oil were thymol (40.6–53.5%), γ-terpinene (14.4–19.8%) and p-cymene
(4.2–10.6%), borneol (1.2–2.4%), thymyl acetate (0.9–7.6%), thymol methyl ether (2.9–
4.5%), β-bisabolene (2.0–3.7%), (E)-caryophyllene (1.9–3.5%), α-terpinene (2.0–3.2%),
carvacrol (0.8–2.8%), myrcene (1.5–2.0%) and α-thujene (1.1–1.9%). Thymol content was
found to be highest at S6 (53.5%), followed by S4 (51.2%) and S5 (49.4%). However, γ-
terpinene and p-cymene were recorded higher at S5 (18.4%) and S6 (11.8%), respectively
(Figure 7). A representative GC-FID chromatogram of the inflorescence essential oil is
presented in Figure 8.
Figure 7: Changes in thymol, γ-terpinene and p-cymene content in inflorescence of Thymus
linearis during plant ontogeny.
0
10
20
30
40
50
60
S3 S4 S5 S6
Thymol (%) γ-Terpinene (%) p-Cymene (%)

56
Table 6: Essential oil composition of the inflorescence of Himalayan thyme (Thymus linearis)
collected at different developmental stages in the foot-hills of Uttarakhand, India
S. No. COMPOUNDa
RIb
RIc
CONTENT (%)/
INFLORESCENCE OIL
S3 S4 S5 S6
1. Tricyclene 920 921 - - - t
2. α-Thujene 925 924 1.5 1.9 1.5 1.1
3. α-Pinene 935 932 0.8 0.8 0.7 0.6
4. Camphene 945 946 0.5 0.4 0.6 0.4
5. Sabinene 972 969 0.2 0.2 0.1 0.1
6. β-Pinene 978 974 0.2 0.2 0.2 0.2
7. 3-Octanone 981 979 t t t t
8. Myrcene 989 988 1.8 2.0 1.7 1.5
9. α-Phellandrene 1002 1002 0.4 0.3 0.3 0.2
10. δ-3-Carene 1010 1008 0.1 0.1 0.1 0.1
11. α-Terpinene 1016 1014 3.0 3.2 2.8 2.0
12. p-Cymene 1021 1020 4.2 4.5 5.1 10.6
13. Limonene 1025 1024 0.5 0.5 0.5 t
14. (E)-β-Ocimene 1045 1044 0.1 0.1 0.1 0.1
15. γ-Terpinene 1053 1054 19.6 18.8 19.8 14.1
16. cis-Sabinene hydrate 1068 1065 1.3 1.1 1.0 1.4
17. Terpinolene 1088 1086 0.1 0.1 0.1 0.1
18. Linalool 1098 1095 0.2 0.2 0.2 0.3

57
19. trans-Sabinene hydrate 1100 1098 t t t t
20. cis-Thujone 1103 1101 t t - -
21. Borneol 1167 1165 1.5 1.2 1.8 2.4
22. Terpinen-4-o1 1174 1174 0.3 0.3 0.3 0.3
23. p-Cymen-8-ol 1182 1179 t t - t
24. α-Terpineol 1188 1186 0.1 t - t
25. Octanol acetate 1210 1211 t t t t
26. Thymol methyl ether 1235 1232 4.5 3.5 2.9 4.0
27. Carvacrol methyl ether 1245 1241 t t t t
28. Bornyl acetate 1287 1284 t t t 0.1
29. Thymol 1292 1289 40.6 51.2 49.4 53.5
30. Carvacrol 1302 1298 2.3 0.8 2.8 1.4
31. Thymyl acetate 1351 1349 7.6 3.9 1.4 0.9
32. Carvacryl acetate 1373 1370 0.1 t t t
33. (E)-Caryophyllene 1419 1417 3.5 2.2 2.4 1.9
34. Aromadendrene 1442 1439 t t - -
35. α-Humulene 1454 1452 0.1 0.1 0.1 0.1
36. allo-Aromadendrene 1460 1458 0.1 t 0.1 0.1
37. Germacrene D 1484 1480 0.5 0.2 0.1 0.1
38. Viridiflorene 1495 1496 0.1 t t t
39. β-Bisabolene 1510 1505 3.7 2.0 3.1 2.2
40. γ-Cadinene 1518 1513 0.2 0.1 0.1 0.1
41. Spathulenol 1580 1577 t - - -

58
42. Caryophyllene oxide 1584 1582 0.1 - - -
CLASS COMPOSITION
Phenolic compounds
Phenols 42.9 52.0 52.2 54.9
Phenolic ethers 4.5 3.5 2.9 4.0
Phenolic ester 7.7 3.9 1.4 0.9
Terpenoids
Monoterpene hydrocarbons 33.0 33.1 33.6 31.1
Oxygenated monoterpenes 3.4 2.8 3.3 4.5
Sesquiterpene hydrocarbons 8.2 4.6 5.9 4.5
Oxygenated sesquiterpenes 0.1 - - -
Others t t t t
Total identified (%) 99.8 99.9 99.3 99.9
Note- a
retention index value from literature; S3: flower-
initiation-stage (150 DAT); S4: Full-bloom-stage (175 DAT); S5: late-bloom-stage (190
DAT); S6: seed-shattering-stage (200 DAT); t: trace (<0.05%).

59
Figure 8: A representative GC-FID chromatogram of the inflorescence essential oil of
Thymus linearis.
Essential Oil Composition of Whole Aerial-Parts
The essential oil composition of whole aerial-parts of T. linearis collected at full bloom stage
(S4) is presented in Table 7. The oil was characterised by the presence of higher amount of
thymol (59.0%), γ-terpinene (15.1%) and p-cymene (4.9%), borneol (1.2%), thymyl acetate
(2.8%), thymol methyl ether (3.0%), β-bisabolene (1.9%), (E)-caryophyllene (1.6%), α-
terpinene (2.3%), carvacrol (1.5%), myrcene (1.5%) and α-thujene (1.4%). Comparison of the
chemical compositions of different oils showed that thymol content was relatively higher in
aerial-parts compared to other studied oils.

60
Table 7: Essential oil composition of the whole aerial-parts and stem of Himalayan thyme
(Thymus linearis) collected at full bloom stages (S4) in the foot-hills of Uttarakhand, India
S. No. COMPOUNDa
RIb
RIc
CONTENT (%)
Whole aerial-parts Stem
1. Tricyclene 920 921 t -
2. α-Thujene 925 924 1.4 1.0
3. α-Pinene 935 932 0.6 1.2
4. Camphene 945 946 0.4 1.6
5. Sabinene 972 969 0.1 t
6. β-Pinene 978 974 0.1 0.2
7. 3-Octanone 981 979 t -
8. Myrcene 989 988 1.5 1.1
9. α-Phellandrene 1002 1002 0.2 0.3
10. δ-3-Carene 1010 1008 0.1 -
11. α-Terpinene 1016 1014 2.3 2.3
12. p-Cymene 1021 1020 4.9 9.7
13. Limonene 1025 1024 0.4 0.5
14. (E)-β-Ocimene 1045 1044 0.1 -
15. γ-Terpinene 1053 1054 15.1 15.3
16. cis-Sabinene hydrate 1068 1065 0.7 1.3
17. Terpinolene 1088 1086 0.1 -
18. Linalool 1098 1095 0.1 0.5

61
19. trans-Sabinene hydrate 1100 1098 t -
20. cis-Thujone 1103 1101 t -
21. Borneol 1167 1165 1.2 8.2
22. Terpinen-4-o1 1174 1174 0.4 -
23. p-Cymen-8-ol 1182 1179 t -
24. α-Terpineol 1188 1186 0.1 -
25. Octanol acetate 1210 1211 t -
26. Thymol methyl ether 1235 1232 3.0 6.0
27. Carvacrol methyl ether 1245 1241 t -
28. Bornyl acetate 1287 1284 t -
29. Thymol 1292 1289 59.0 35.5
30. Carvacrol 1302 1298 1.5 t
31. Thymyl acetate 1351 1349 2.8 1.4
32. Carvacryl acetate 1373 1370 t -
33. (E)-Caryophyllene 1419 1417 1.6 2.8
34. Aromadendrene 1442 1439 t -
35. α-Humulene 1454 1452 0.1 -
36. allo-Aromadendrene 1460 1458 t -
37. Germacrene D 1484 1480 0.1 -
38. Viridiflorene 1495 1496 t -
39. β-Bisabolene 1510 1505 1.9 4.5
40. γ-Cadinene 1518 1513 0.1 -
41. Spathulenol 1580 1577 t -

62
42. Caryophyllene oxide 1584 1582 t -
CLASS COMPOSITION
Phenolic compounds
Phenols 60.5 35.5
Phenolic ethers 3.0 6.0
Phenolic ester 2.8 1.4
Terpenoids
Monoterpene hydrocarbons 27.3 33.2
Oxygenated monoterpenes 2.5 10.0
Sesquiterpene hydrocarbons 3.8 7.3
Oxygenated sesquiterpenes - -
Others t -
Total identified (%) 99.9 93.4
Note- a
retention index value from literature; t: trace (<0.05%).

63
Essential Oil Composition of Stem
The stem oil composition of T. linearis analysed at full bloom stage (S4; essential oil yield
≤0.05%) is presented in Table 7. The oil showed a quite different chemical composition as
compared to leaf, inflorescence and whole aerial-parts oils. Major components of the stem oil
were thymol (35.5%), γ-terpinene (15.3%), p-cymene (9.7%), borneol (8.2%), thymol methyl
ether (6.0%), β-bisabolene (4.5%), and (E)-caryophyllene (2.8%). Thymol content was quite
low in stem oil as compared to leaf and inflorescences. However, borneol and thymol methyl
ether were higher in stem oil than leaf and inflorescence oils.
Thus, it was clear from the study that the essential oil yields and chemical composition
of T. linearis substantially influenced by the plant-parts and harvesting time in the foot-hills of
Uttarakhand. Previously, the essential oil composition of T. linearis has been studied from
natural habitat, i.e. temperate Himalayan region. Major component of the oil was thymol
(52.28–66.65%) (Verma et al., 2010). Considering contents of the major constituent, oil
examined from the foot-hill region (thymol, leave: up to 54.6%; inflorescence: up to 53.5%;
and whole aerial-parts: 59.0%) was comparable with that produced from natural habitat.
Further, T. linearis grown in Pakistan possessed 36.5% thymol (Hussain et al., 2013). These
variations in the essential oil composition of T. linearis across countries might be due to the
varied agro-climatic conditions of the regions. However, various studies have also indicated
that the essential oil yield and chemical composition of thyme is influenced by environmental
and agricultural management factors (Abu-Darwish et al., 2012).

64
5.3. Antibacterial Activity
The antibacterial activity of the essential oils and extracts of Thymus Linearis was determined
against six pathogenic bacterial strains. The results in terms of net zone of inhibition (mm);
minimumm inhibitory concentration (µL/mL) and minimum bactericidal concentration
(µL/mL) are summarized in Table 8. The leaf and inflorescence essential oils exhibited
activity against all six bacterial strains; however, oil of the aerial-parts exhibited activity
against five bacterial strains. The leaf oil showed very good activity against all tested bacterial
strains (ZOI: 20–32mm; MIC: 0.26-1.04 µL/mL; MBC: bacteriostatic-4.16 µL/mL).
According to ZOI of leaf oil, different strains can be arranged as follows: M. smegmatis
(32mm) > E. faecalis (28mm) = E. coli (28mm) > S. aureus (26mm) > S. epidermidis (25mm)
> S mutans (20mm). The MIC and MBC value of the leaf oil were lowest for E. coli and S.
aureus (0.26µL/mL and 0.52µL/mL); however this oil was bacteriostatic for M. smegmatis.
Likewise, the inflorescence oil also exhibited very good activity against all tested bacterial
strains (ZOI: 20–26mm; MIC: 0.26–1.04 µL/mL; MBC: bacteriostatic–2.08µL/mL).
According to ZOI of inflorescence oil, different strains can be arranged as follows: E. faecalis
(26mm) = E. coli (26mm) = S. epidermidis (26mm) > S. aureus (25mm) > S mutans (21mm) >
M. smegmatis (20mm). However, MIC and MBC value of the inflorescence oil were lowest
for E. coli and S. aureus (0.26µL/mL and 1.04µL/mL). Nevertheless, MBC of the oil was also
low for E. faecalis (1.04µL/mL). Moreover, this oil was bacteriostatic for M. smegmatis. As
far as the antibacterial activity of aerial-parts oil was concern, it was found to be active against
five bacterial stains. This oil showed very good activity against S. aureus (ZOI: 28mm; MIC:

65
0.52µL/mL; MBC: 1.04µL/mL), S. epidermidis (ZOI: 25mm; MIC: 0.52µL/mL; MBC:
1.04µL/mL), M. smegmatis (ZOI: 22mm; MIC: 1.04µL/mL; MBC: bacteriostatic) and S.
mutans (ZOI: 22mm; MIC: 2.08µL/mL; MBC: 4.16 µL/mL); however it was moderately
active against E. faecalis (ZOI: 10mm; MIC: 1.04µL/mL; MBC: 1.04µL/mL). Further, this oil
was not active against E. coli. Further, the solvent extracts (hexane, acetone and methanol) of
T. linearis, in general, showed low to moderate antibacterial activity, except the acetone
extract which has not shown activity against E. coli and S. epidermidis. In general, hexane
extract (ZOI: 2–9mm; MIC: 1.04–16.67 µL/mL; MBC: bacteriostatic–33.34µL/mL) was
found to be more active than methanol (ZOI: 2–7mm; MIC: 4.16–16.67µL/mL; MBC:
bacteriostatic–66.67µL/mL) and acetone (ZOI: 0–7mm; MIC: 8.32–16.67µL/mL; MBC:
bacteriostatic–33.34µL/mL) extracts. Thus, based on the activity index (mean zone of
inhibition against tested bacterial strains) data (Figure 9), the antibacterial potency of
different essential oils/extracts can be explained as: leaf oil (26.5mm)> inflorescence oil
(24.0mm)> whole aerial-parts oil (17.8mm)> hexane extract (6.3mm)> methanol extract
(4.3mm)> acetone extract (2.5mm).

66
Figure 9: Activity index (mean zone of inhibition of six bacterial strains) of the leaf essential
oil (EO-L), inflorescence essential oil (EO-I), whole aerial-parts essential oil (EO-AP), hexane
extract (HE-AP), acetone extract (AE-AP) and methanol extract (ME-AP) of Thymus linearis.
0
5
10
15
20
25
30
EO-L EO-I EO-AP HE-AP AE-AP ME-AP
Activity index (mm)

67
Table 8: Antibacterial activity of Thymus linearis essential oils and extracts
Sample*
Bacterial Strains
MS EF EC SM SE SA
ZOI MIC MBC ZOI MIC MBC ZOI MIC MBC ZOI MIC MBC ZOI MIC MBC ZOI MIC MBC
EO-L 32 00.52 BS 28 00.52 04.16 28 00.26 00.52 20 01.04 02.08 25 00.52 02.08 26 00.26 00.52
EO-I 20 00.52 BS 26 00.52 01.04 26 00.26 01.04 21 01.04 02.08 26 00.52 02.08 25 00.26 01.04
EO-AP 22 01.04 BS 10 01.04 01.04 -- -- -- 22 02.08 04.16 25 00.52 01.04 28 00.52 01.04
HE-AP 07 04.16 BS 02 01.04 02.08 04 08.33 33.34 07 04.16 04.16 09 08.33 16.67 09 16.67 33.34
AE-AP 02 08.32 BS 02 08.33 16.67 -- -- -- 04 08.33 16.67 -- -- -- 07 16.67 33.34
ME-AP 05 08.32 BS 04 04.16 08.33 02 16.67 66.67 04 04.16 08.34 04 08.33 16.67 07 08.33 16.67
*
Full bloom stage (S4); EO-L: essential oil of leaves; EO-I: essential oil of inflorescence; EO-AP: essential oil of whole aerial-parts;
HE-AP: hexane extract of whole aerial-parts; AE-AP: acetone extract of whole aerial-parts; ME-AP: methanol extract of whole aerial-
parts; MS: Mycobacterium smegmatis, EF: Enterococcus faecalis, EC: Escherichia coli, SM: Streptococcus mutans, SE:
Staphylococcus epidermidis, SA: Staphylococcus aureus; ZOI: net zone of inhibition (mm); MIC: minimumm inhibitory concentration
(µL/mL); MBC: minimum bactericidal concentration (µL/mL).

68
5.4. Antifungal Activity
The antifungal activity of the essential oils and extracts of T. linearis was determined against
three fungal strains. The results in terms of net zone of inhibition (mm); minimumm
inhibitory concentration (µL/mL) and minimum fungicidal concentration (µL/mL) are
summarized in Table 9. The leaf oil was found to be active against all three fungal strains,
while inflorescence and the whole aerial-parts oils showed activity against only two strains.
The leaf oil exhibited very good activity against C. kefyr (ZOI: 27mm; MIC: 0.52µL/mL;
MFC: 0.52µL/mL) and C. albicans ATCC (ZOI: 26mm; MIC: 0.52µL/mL; MFC:
1.04µL/mL), while it was moderately active against C. albicans AI (ZOI: 10mm; MIC:
8.33µL/mL; MFC: 16.67µL/mL). Further, the inflorescence oil showed very good activity
against C. albicans ATCC (ZOI: 27mm; MIC: 0.52µL/mL; MFC: 1.04µL/mL) and C. kefyr
(ZOI: 25mm; MIC: 0.52µL/mL; MFC: 1.04µL/mL). However, the oil of whole aerial-parts
showed very good activity against C. kefyr (ZOI: 25mm; MIC: 0.52µL/mL; MFC:
1.04µL/mL) and C. albicans AI (ZOI: 25mm; MIC: 0.52µL/mL; MFC: 0.52µL/mL). On the
other hand, solvent extracts, in general, showed low to moderate activity against different
fungal strains (ZOI: 2–6mm), except the hexane extract which showed very good activity
against C. albicans ATCC (ZOI: 25mm; MIC: 0.52µL/mL; MFC: 0.52µL/mL). Thus, based
on the activity index (mean zone of inhibition of three tested fungal strains) data (Figure 10),
the antifungal potency of different essential oils/ extracts can be summerised as follows: leaf
oil (21.0mm)> inflorescence oil (17.3mm)> whole aerial-parts oil (16.7mm)> hexane extract
(11.3mm)> methanol extract (5.3mm)> acetone extract (4.0mm).

69
Biological activity of the essential oils/extract depends on their chemical composition,
which is determined by the genotype/chemotype, influenced by environmental factors,
extraction methods, and agronomical conditions. In case of genus Thymus much of the
antimicrobial activity in essential oils appears to be associated with phenolic compounds, viz.
thymol and carvacrol (Davidson and Naidu, 2000; Skocibusic et al., 2006; Rota et al., 2007).
It seems possible that phenol components may interfere with cell wall enzymes like chitin
synthase/chitinase as well as with the α- and β-glucanases of the fungus (Adams et al., 1996).
Therefore, the high content of phenol components may account for the high antifungal
activity (Sokovic et al., 2009). However, it was also considered that minor components, as
well as a possible interaction between the substances could also affect the antimicrobial
activities. In fact, other constituents, such as γ-terpinene, have been considered to display
relatively good activity due to their possible synergistic or antagonistic effects (Didry et al.,
1993; Vardar-Unlu et al., 2003; Ebrahimi et al., 2008).

70
Table 9: Antifungal activity of Thymus linearis essential oils and extracts
Sample*
Fungal Strains
ATCC DS-02 AI
ZOI MIC MFC ZOI MIC MFC ZOI MIC MFC
EO-L 26 00.52 1.04 27 00.52 00.52 10 08.33 16.67
EO-I 27 00.52 1.04 25 00.52 1.04 -- -- --
EO-AP -- -- -- 25 00.52 1.04 25 00.52 00.52
HE-AP 25 00.52 00.52 04 33.34 33.34 05 33.34 66.67
AE-AP 05 16.67 16.67 02 16.67 33.34 05 16.67 33.34
ME-AP 06 16.67 16.67 06 16.67 33.34 04 16.67 33.34
*
Full bloom stage (S4); EO-L: essential oil of leaves; EO-I: essential oil of inflorescence; EO-
AP: essential oil of whole aerial-parts; HE-AP: hexane extract of whole aerial-parts; AE-AP:
acetone extract of whole aerial-parts; ME-AP: methanol extract of whole aerial-parts; ATCC:
Candida albicans (ATCC), DS-02: Candida kefyr, AI: Candida albicans (clinical isolate);
ZOI: net zone of inhibition (mm); MIC: minimumm inhibitory concentration (µL/mL); MFC:
minimum fungicidal concentration (µL/mL).

71
Figure 10: Activity index (mean zone of inhibition of three fungal strains) of the leaf essential
oil (EO-L), inflorescence essential oil (EO-I), whole aerial-parts essential oil (EO-AP),
hexane extract (HE-AP), acetone extract (AE-AP) and methanol extract (ME-AP) of Thymus
linearis.
0
5
10
15
20
25
EO-L EO-I EO-AP HE-AP AE-AP ME-AP
Activity index (mm)

72
6. CONCLUSIONS
It was clear from the study that the yield, yield contributing traits and chemical composition
of T. linearis substantially influenced by the plant-parts and harvesting stages considered. The
essential oil content of dried leaves (3.48%) and inflorescence (3.54%) were significantly
highest at full bloom stage (S4). However, dry leaf yield (24.4 g plant-1
) and inflorescence
yield (23.2g plant-1
) recorded significantly highest at late-bloom stage (S5). Consequently, the
essential oil yields of leaves and inflorescence were higher at S5 (0.826 mL plant-1
and 0.605
ml plant -1
, respectively). Major components of the oils, isolated from different plant-parts at
different stages (S1–S6) were thymol (35.5–59.0%), γ-terpinene (13.3–19.8%), p-cymene
(4.2–11.8%), borneol (0.9–8.2%), thymyl acetate (0.9–7.6%) and thymol methyl ether (2.9–
6.0%). In leaves, thymol content varied from 47.7–54.6% with the highest at full bloom stage
(S4; 54.6%). In inflorescence, thymol content varied from 40.6–53.5% with the highest at
seed shattering stage (S6; 53.5%). In full bloom stage, thymol content was distributed in
different samples in following order: whole-aerial-parts (59.0%)> leaves (54.6%)>
inflorescence (51.2%)> stem (35.5%). Moreover, thymol content was higher in leaves than
inflorescence during different stage of blooming (S3–S5), except S6 when thymol reached
higher in inflorescence. The essential oils (leaves, inflorescence and whole aerial-parts) and
solvent extracts (hexane, acetone and methanol) of T. linearis differed in their efficacy against
different bacterial and fungal strains. Based on the activity index, the antimicrobial potency of
different oils and extracts can be arranged in following order: leaf oil> inflorescence oil>
whole aerial-parts oil> hexane extract> methanol extract> acetone extract. However, the
results pertaining to the antibacterial and antifungal activities, reported here can be considered
as a preliminary step towards the possible practical application.

73
REFERENCES
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Adams RP. 2007. Identification of essential oil components by gas chromatography/mass
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Adams S, Kunz B, Weidenborner M. 1996. Mycelial deformations of Cladosporium
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