A systematic review on ethnopharmacology, phytochemistry and pharmacological aspects of Thymus vulgaris Linn

Abstract

Thymus vulgaris Linn. is a medicinal and culinary herb from the Southern European region known for its anti-infective, cardioprotective, gastroprotective, anti-inflammatory, and immunomodulatory activities since the Egyptian era. The reported pharmacological activities of T. vulgaris L. include antibacterial, antioxidant, anti-inflammatory, antiviral, and anti-cancerous activities. In this review, a comprehensive approach is put forth to scrutinize and report the available data on phytochemistry, ethnopharmacology, pharmacology, and toxicology of the plant. The different extracts and essential oil obtained from the plant have been assessed and reported to treat ailments like microbial infections, inflammation, non-communicable diseases like cancer, and sexually transmitted diseases like HIV-1 and Herpes. The literature review has also indicated the use of volatile oils, phenolic acids, terpenoids, flavonoids, saponins, steroids, tannins, alkaloids, and polysaccharides in pharmacotherapy. Applications of these compounds including antidiabetic, anti-Alzheimer's, cardio, neuro and hepatoprotective, anti-osteoporosis, sedative, immunomodulatory, antioxidant, anti-tyrosinase, antispasmodic, antinociceptive, gastroprotective, anticonvulsant, antihypertensive, antidepressant, anti-amnesia, and anti-helminthic activities have been mentioned. Further, based on research gaps, recommendations have been provided to evaluate T. vulgaris L. systematically to develop plant-based drugs, nutraceuticals, and to evaluate their clinical efficiency and safety.

Full text

Research article
A systematic review on ethnopharmacology, phytochemistry and
pharmacological aspects of Thymus vulgaris Linn.
Shashank M. Patil
a
, Ramith Ramu
a
,
*
, Prithvi S. Shirahatti
b
, Chandan Shivamallu
a
,
Raghavendra G. Amachawadi
c
,
**
a
Department of Biotechnology and Bioinformatics, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, 570 015, Karnataka, India
b
Department of Biotechnology, Teresian College, Siddhartha Nagara, Mysuru, 570 011, Karnataka, India
c
Departments of Clinical Sciences, Kansas State University, Manhattan, KS, 66506-5606, USA
ARTICLE INFO
Keywords:
Thymus vulgaris L.
Ethnopharmacology
Pharmacology
Phytochemistry
Toxicology
ABSTRACT
Thymus vulgaris Linn. is a medicinal and culinary herb from the Southern European region known for its anti-
infective, cardioprotective, gastroprotective, anti-inflammatory, and immunomodulatory activities since the
Egyptian era. The reported pharmacological activities of T. vulgaris L. include antibacterial, antioxidant, anti-
inflammatory, antiviral, and anti-cancerous activities. In this review, a comprehensive approach is put forth to
scrutinize and report the available data on phytochemistry, ethnopharmacology, pharmacology, and toxicology of
the plant. The different extracts and essential oil obtained from the plant have been assessed and reported to treat
ailments like microbial infections, inflammation, non-communicable diseases like cancer, and sexually trans-
mitted diseases like HIV-1 and Herpes. The literature review has also indicated the use of volatile oils, phenolic
acids, terpenoids, flavonoids, saponins, steroids, tannins, alkaloids, and polysaccharides in pharmacotherapy.
Applications of these compounds including antidiabetic, anti-Alzheimer's, cardio, neuro and hepatoprotective,
anti-osteoporosis, sedative, immunomodulatory, antioxidant, anti-tyrosinase, antispasmodic, antinociceptive,
gastroprotective, anticonvulsant, antihypertensive, antidepressant, anti-amnesia, and anti-helminthic activities
have been mentioned. Further, based on research gaps, recommendations have been provided to evaluate
T. vulgaris L. systematically to develop plant-based drugs, nutraceuticals, and to evaluate their clinical efficiency
and safety.
1. Introduction
T. vulgaris L. or thyme, known as “garden thyme”is an aromatic and
perennial flowering plant belonging to the Lamiaceae family [1,2]. The
Greek form of the word ‘thyme’depicts ‘to fumigate’, owing to its use as
incense or for its balsamic odour or it belonged to the class of
sweet-smelling herbs [1]. Being native to Southern Europe, T. vulgaris L.
is reported to have a worldwide distribution [2]. The plant grows well in
an arid climate and unshaded areas in coarse, rough, and well-drained
soil that is generally unsuitable for many plants. It appears as a short
and bushy plant with several small flowers [3]. It is usually grown for
commercial purposes in several countries for its dried leaves, plant ex-
tracts, plant oil, and oleoresins [4,5]. T. vulgaris L. is commercially used
as aflavouring agent in food industries due to its extensive aromaticity
[1]. It is also used for preserving meat, chicken, and fish [6], along with
its use for flowering and ornamental purposes [7]. In addition, the per-
fumery and cosmetic industries also use T. vulgaris L. for its characteristic
aroma [5].
T. vulgaris L. is reported with an array of ethnobotanical applications
due to its extensive pharmacological properties. The plant was chiefly
used for the treatment of wounds, as it possesses healing and antiseptic
properties [4,5]. Usage of its aerial parts for fumigation, treating skin
and respiratory diseases in ancient Europe deliberates on the
anti-infective property of the plant [5]. Besides, T. vulgaris L. was used in
monasteries in food preparations. These properties depict its efficacy as a
culinary and medicinal plant as well. It is believed that Romans and
Greek fumigated their surroundings by burning the entire plant [8].
Treatment of skin diseases like Black Death during the 1340s and several
foodborne illnesses was also done using the T. vulgaris L. aqueous extract
[5,9,10]. This is supported by modern studies, which have proved the
* Corresponding author.
** Corresponding author.
E-mail addresses: ramithramu@jssuni.edu.in (R. Ramu), agraghav@vet.k-state.edu (R.G. Amachawadi).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2021.e07054
Received 22 December 2020; Received in revised form 23 February 2021; Accepted 7 May 2021
2405-8440/©2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Heliyon 7 (2021) e07054

antibacterial efficacy using both normal and MDR strains of virulent
bacteria and fungi [11]. In the 1980s, T. vulgaris L. was recommended for
treating respiratory disorders raised due to the inflammation of upper
respiratory tract mucous membranes, including whooping cough, bron-
chitis, and catarrh [9]. However, application of thyme (T. vulgaris L.) and
primrose (Primula vulgaris) extract has demonstrated beneficiary in
clinical trials against bronchitis and other respiratory disease related
symptoms [12]. Experts have also recommended the use of T. vulgaris L.
in cases of bacterial and fungal infections. The plant is expected to render
a possible inhibition of bacterial adsorption and biofilm matrix formation
[13]. In addition, T. vulgaris L. was believed to possess antiseptic,
astringent, carminative, tonic, and anthelminthic properties. The plant
has gained much popularity due to the profound activity shown towards
intestinal infections and infestations caused by ascarids, hookworms,
fungi, yeast, and bacteria. It has also found its importance in dermato-
logical issues like acne, oily skin, dermatitis, bug bites as well as sciatica
and rheumatic aches. In support of this, modern pharmacological in-
vestigations have come up with several findings that satisfactorily deci-
pher the antifungal nature of different extracts and compounds of the
plant [14,15,16]. It is reported that, T. vulgaris L. relieves bites and
stings, and the neurological issues associated with it. In aromatherapy,
red T. vulgaris L. oil and white T. vulgaris L. oil are used to treat skin
disorders as well as body pains [17,18]. Modern anti-inflammatory in-
vestigations have revealed the efficacy of T. vulgaris L. in the amelioration
of oxidative stress and cell mediated immune response [19,20,21].
These initial findings prevail the significant therapeutic potential of
the plant with respect to pharmacological properties, which may be the
results of its active components. In recent years, T. vulgaris L. has been the
centre of research interest and colossal amount literature is already
available. Although a few small reviews have been carried out on limited
aspects of T. vulgaris L. [3,22], no comprehensive review has been
published till date including details of the potential therapeutic aspects.
Therefore, we deliberate on summing up the present and up-to-date in-
formation with respect to chemical composition, ethnopharmacology,
pharmacology, and toxicology of T. vulgaris L. We also aim to document a
few uncommon pharmacological activities that can completely change
the present perspectives on the plant.
2. Materials and methods
2.1. Databases, softwares, e-sources, and keywords search
Literature survey was conducted to gather all the essential informa-
tion surrounding T. vulgaris L. using electronic databases including
Google Scholar, PubMed, SpringerLink, Wiley-Blackwell, and Web of
Science. An extensive number of studies published in peer reviewed
journals like Food Chemistry, International Journal of Molecular Sci-
ences, Fitoterapia, Journal of Science in Food and Agriculture, Parasi-
tology, Toxicology research and many others were collected. E-books
“Thyme: the genus Thymus”and “Medicinal spices and vegetables from
Africa”were utilized as well. Authors searched for the data using key-
words including “Thymus vulgaris,”“T. vulgaris,”“Ethnopharmacology,”
“Traditional uses,”“Ethnobotany,”“Chemical profiling,”“Pharmaco-
logical properties,”“Medicinal properties”, which resulted in the gath-
ering of much literature. A special search was conducted to get the
ethnomedicinal details of the plant using words “root”,“stem”, and
“leaves”.Plant.org was used for the correct names of the plants used in
the article. Also, chemical structures and IUPAC names were added using
PubChem. The systematic arrangement of studies and their management
till the end of the drafting was completed using the software Mendeley.
2.2. Literature management
Authors searched different databases using keywords to end up with a
mammoth literature that included research articles, review articles, book
chapters, and books. The literature was divided into different sections
based on the title and abstract available along with the removal of non-
relevant information and duplicates. Later, a second screening was car-
ried out, where studies with unique methodology covering maximum
research aspects were retained. Further, a few of the review articles with
limited amount of information were removed. During the drafting of the
review, a few research articles were removed to retain the articles with
unique methodology and significant results. However, it was inevitable
to retain a few of the articles despite their insignificant results and older
methodology, due to lack of relevant and advanced information. From
2791 identified studies, a total of 118 studies were retained on comple-
tion of the different levels of screening. The outline was planned to meet
up the PRISMA regulations (Figure 1). The article was primarily divided
into different sections based on the previous work of authors on phyto-
chemistry and pharmacological reviews. Further, a few additional
changes were brought according to the amount and type of literature
available.
3. Botanical background
3.1. Taxonomy
Being a member of the Lamiaceae family, the genus Thymus comprises
a total of 928 species [23]. The most related genera are Origanum,Sat-
ureja,Micromeria and Thymbra [24]. The classification of Thymus species
attributes to the chromosomal information, which is an important key for
taxonomy. It is difficult to identify the chromosomes of Thymus species
due to their small size and similar morphology [25].
3.2. Botanical description
T. vulgaris L. is an aromatic, perennial, straight growing plant which
measures up 10–30 cm in height with woody base [24,26]. Leaves are
small, opposite, greyish green coloured, oblong–lanceolate to linear, and
are gland-dotted. They are measured up to 5–10 mm long and 0.8–2.5
mm wide with recurved margins. Flowers are light violet in colour,
two-lipped, and possess a hairy glandular calyx. They measure up to 5
mm long with leaf-like bracts in loose whorls arranged in axillary clusters
on the branchlets or in terminal oval or rounded heads [26]. Figure 2
details the different parts of T. vulgaris L. However, the morphological
characters may vary according to environmental conditions. T. vulgaris L.
grows well in arid, temperate, and unshaded areas. T. vulgaris L. grows
well in hot, arid conditions with well-drained soil, and is usually planted
in the spring. The plant can be propagated using seed, cuttings, or by
dividing rooted sections. The plant also takes up deep freezes and can be
found on mountain highlands [4].
3.3. Variations
T. vulgaris L. shows aneuploidy where the number of chromosomes
varies in different plants of the same species. Among these, 2n ¼28, 30,
56 and 60 are said to be the most frequent numbers for other plants
belonging to the genus Thymus. Aneuploidy has played an important role
during evolution and is responsible for varying numbers. This is true for
T. vulgaris L. with 2n ¼28, 58 [24]. Thus, it becomes evident that the
plant has a diverse genetic constitution. This may also contribute to its
morphological variation. However, there are not available data on the
restriction of these genetically diverse species to a specific region or
country. Several sub-species are present that further add to the com-
plexities of genetic research. In addition, several chemotypes or chemo-
vars of T. vulgaris L. exist. As the phytochemical profile of T. vulgaris L.
depicts the presence of extensive amount of essential oil components,
chemotypes vary based on the composition of the same [27]. There are as
many as 13 different chemotypes have been identified based on the
predominance of monoterpenes in the essential oil. Recently, a study
identified 6 different chemotypes namely linalool, borneol, geraniol,
sabinene hydrate, thymol, and carvacrol [10]. Pharmacological effects of
S.M. Patil et al. Heliyon 7 (2021) e07054
2

different chemotypes vary due to their diverse essential oil composition
[28,29]. More research needs to be conducted in terms of chemotypes to
identify the potent chemicals that can be used to target diseases at a
molecular level. Based on the chemical profile and mode of employment
of these chemotypes against specific diseases, significant results can be
expected in phytotherapy.
3.4. Distribution
Native to Southern Europe, T. vulgaris L. has a worldwide distribution.
It is indigenous to the Mediterranean region and other neighbouring
countries. It is also found in Northern Africa including Egypt and Saharan
countries like Morocco, Algeria, Tunisia, and Libya [9]. Also, the plant is
cultivated in Nigeria, Cameroon, and South Africa. It is also cultivated in
European countries, such as Spain, France, Bulgaria, Italy. Along the
Mediterranean coastal region, it can be found growing up to 800 m from
sea level. The plant can be cultivated by vegetative propagation, using
seeds, cuttings, or divided root sections [2].
4. Ethnopharmacology
Thymus species have been used since ancient times for the treatment
of different health aberrations. The plant possesses a variety of the me-
dicinal properties that positively affect human health (Table 1). Authors
were not able to find most of the specific parts of the plant used in
traditional medicinal practices. Even though search was conducted using
the words “root,”“stem,”or “leaves”, there was no available records for
specific parts of the plant used for traditional medicine. Few available
studies were able to depict the details limited to “aerial parts”of the
plant, which includes stem, leaves, flowers, and buds. Also, these studies
were not able to reveal the accurate use of the plant. Mechanisms of
ethnopharmacological uses were not available. The information was
limited to “whole plant”or “thyme water”. Investigations need to be done
in this regard to decipher the unknown medicinal properties, which may
benefit the pharmacological studies.
4.1. Treatment for poisoning
The aqueous extract of the plant is reported to possess the properties
of an antidote. It is believed that Romans used to consume the plant
extract before or during meal to protect themselves from getting
poisoned. Bathing in warm water dosed with T. vulgaris L. could stop the
effects of poison, making it a favourite herb for the emperors [8]. These
examples indicate the effectiveness of T. vulgaris L. as a natural antidote.
In support of this, the present-day studies show presence of compounds
like thymol and carvacrol, which can serve as a profound antidote [31,
32].
4.2. Disinfection and wound healing
T. vulgaris L.is reported to be used as a disinfectant, where dried plant
bundles were burned to purify the surroundings. Romans and Greeks
evoked a spirit of courage by burning these bundles to purify their homes
and temples [8]. This reminds us of the modern-day fumigation, where
the disinfection is done with various antimicrobial substances. However,
studies have proven the presence of γ-terpinene and p-cymene, which are
the biochemical precursors of thymol and carvacrol, to be responsible for
the observed antimicrobial properties [16,33]. Nurses in the 19th cen-
tury used to apply bandages soaked in thyme water, as this plant was
believed to be a natural healer and an antiseptic. Also, the examination of
the aqueous extract of T. vulgaris L. has been proved to improve immu-
nomodulatory functions [34].
4.3. Treatment of skin diseases
T. vulgaris L. was also extensively used against plague in the late
1340s, an era that was known as the age of Black Death. T. vulgaris L. was
directly applied on plague-blistered skin [5]. The beneficiary effects of
such an application was later identified to be owing to the presence of a
chemical compound known as thymol, which is widely used in hand
sanitizers, mouthwashes, and acne medication in the present day. Also,
several studies have proved the use of T. vulgaris L. for the treatment of
skin problems like dermatitis [9]. These effects are observed due to the
presence of volatile oils, which principally comprises linalool,
α
-pinene
along with thymol. Other compounds like mono and sesquiterpenes
(β-caryophyllene, germacrene-D and nerolidol) also reported to play an
important role [33].
4.4. Treatment of foodborne illnesses
Apart from medicinal purpose, T. vulgaris L. has found its importance
as a cooking herb alongside rosemary and sage in Europe. Monastries that
served food, also used this plant as a food additive to wade away the
microbial contamination as well as a part of the medicinal formulations.
Food products like soups, roasts, and breads contained the plant formu-
lation [10]. However, most of the studies have reported the antimicrobial
activity of T. vulgaris L. extracts (ethanol and water) as well as its use in
the form of essential oil against foodborne pathogens. In addition to
thymol and carvacrol, phenolic compounds in the extracts are attributed
for this property [35,36].
4.5. Treatment of respiratory diseases
The whole plant extract was reported to be used against several res-
piratory disorders. It was believed to be an effective remedy for
Figure 1. PRISMA outline followed for literature search.
S.M. Patil et al. Heliyon 7 (2021) e07054
3

bronchitis, asthma, whooping cough, and pharyngitis. T. vulgaris L. tea
was extensively used to treat cough and cold. They were used to treat sore
throat [37]. In addition, flowering shoots were used to treat cold, chest
infections and sore throat [38]. Recently, the phytochemical analysis of
the plant extracts revealed the presence of carvacrol and γ-terpinene that
possess antiviral and anti-inflammatory activities, which could be the
reason for the observed effects during its traditional use [32,39].
4.6. Other traditional uses
T. vulgaris L. has been an important medication in several other health
maladies. The plant possesses the ability to cure gastrointestinal aber-
rations, as the extract was given to treat worms in children [37]. The
modern-day studies have reported the anti-parasitic/anti-helminthic role
of T. vulgaris L., which is attributed to the presence of monoterpenes and
phenolic compounds [40]. Also, the topical use of T. vulgaris L. oil
reduced rheumatic aches and sciatica, where the aromatherapy method
used the oil to treat such body pains [17]. This property of reducing pain
is mainly attributed to thymol, which possesses the greatest
anti-inflammatory potential among all the reported constituents. Flower
shoots were used to treat mouth and skin infections, high blood pressure,
heart problems, fluid retention, cystitis, digestive system disorders,
rheumatism, and arthritis [38]. However, no specific phytochemicals
have been attributed to these medicinal properties. In addition, reports
have revealed the anthelminthic, antifungal, antispasmodic, diuretic
[41], intestinal anti-inflammatory, buccal antiseptic, ocular antiseptic,
Figure 2. A) Plant B) flowers C) leaves D) seeds of Thymus vulgaris L.
Table 1. Summary of the ethnomedicinal uses of Thymus vulgaris L.
Ethnomedicinal Use Part of the Plant Palnt Material Mode of Application References
Treatment of poisoning Aerial parts/stem/leaves Dried whole plant/water extract Oral, Topical [8]
Disinfection and wound healing Aerial parts/stem/leaves Dried whole plant/vapours, water extract Topical [8]
Plague-blistered skin, Acne, oily skin, dermatitis Aerial parts/stem/leaves Water extract, volatile oil Topical [9,18]
Treatment of foodborne illnesses Aerial parts/stem/leaves Dried whole plant/Water extract Oral [10]
Asthma, bronchitis, whooping cough, pharyngitis Aerial parts/stem/Leaves Water extract Oral [37]
Cough, cold and sore throat Aerial parts/stem/Leaves/Flower shoots Water extract Oral [37]
Intestinal worms Aerial parts/stem/Leaves Dried whole plant/water extract Oral [37]
Rheumatic aches, body pain and sciatica Aerial parts/stem/Leaves Volatile oil Topical [17]
Emphysema, sedative, anthelmintic, antifungal,
antispasmodic, diuretic
Not defined Not defined Not defined [41]
Skin infections, high blood pressure, heart problems,
fluid retention, cystitis, digestive system disorders,
rheumatism and arthritis
Flower shoots Not defined Not defined [38]
S.M. Patil et al. Heliyon 7 (2021) e07054
4

internal antiseptic, laxative, antiodontalgic, anticatarrhal, and urinary
antiseptic [38] properties of the plant, yet without attributing the specific
protogonistic phytochemicals.
5. Phytochemistry
T. vulgaris L. primarily consists of a myriad of chemical compounds
categorised as phenolic compounds, terpenoids, flavonoids, steroids, al-
kaloids, tannins, and saponins. Most of them are volatile compounds
extracted from plant oil. The studies have assessed the phytochemical
composition mostly using gas chromatography-mass spectroscopy (GC-
MS) and high-performance liquid chromatography/thin layer
chromatography (HPLC/HPTLC) techniques [42,43,44]. The revelations
depict the prevalence of essential oil components over the other
metabolites.
Further, only a few studies could decipher the presence of steroids,
alkaloids, tannins, and saponins, yet no information is available on in-
dividual compounds belonging into these categories [45,46]. Apart from
the plant oil characterisation, ethanolic and aqueous extracts need to be
studied in detail to reveal information on the presence of various other
important components. Several phytochemicals identified from this plant
are of substantial pharmacological significance (Table 2). Though
chemicals are present in T. vulgaris L., their isolation and identification
has not been performed. Researchers have reported the activities of these
phytochemicals from independent origin.
Table 2. Summary of the individually reported phytochemicals and their pharmacological significance present in Thymus vulgaris Linn.
Class Compound Name IUPAC Name Pharmacological Activity Reference
Phenolic
compounds
Quinic acid 6-methoxyquinoline-4-carboxylic acid Anti-cancer, immunomodulatory,
anti-fungal, antioxidant, neuroprotective
[47,83,84]
Rosmaric acid (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-
3-(3,4-dihydroxyphenyl) prop-2-enoyl]
oxypropanoic acid
Anti-alzheimer's, anti-cancer, antidiabetic,
antimicrobial, cardioprotective, nephroprotective,
anti-ageing, hepatoprotective, anti-inflammatory,
anti-allergic, anti-depressant
[85,86]
Caffeic acid (E)-3-(3,4-dihydroxyphenyl) prop-2-enoic acid Antioxidant, antimicrobial, anticancer, adipogenetic,
lipolytic, anti-alzheimer's, antiviral, antidiabetic,
cardioprotective, hepatoprotective, anti-atherosclerotic
[87,88,89]
p-coumaric acid (E)-3-(4-hydroxyphenyl) prop-2-enoic acid Immunomodulatory, anti-inflammatory, antioxidant,
gastroprotective, antidiabetic, anti-hyperlipidemia,
anti-tyrosinase, anticancer, hepatoprotective
[90,91,92]
p-hydroxybenzoic acid 4-hydroxybenzoic acid Anticancer, antimicrobial, antiviral [93,94,95]
Gentisic acid 2,5-dihydroxybenzoic acid Anticancer, antioxidant, antimicrobial, cardioprotective,
antimicrobial, anti-inflammatory, analgesic, nephroprotective,
hepatoprotective, neuroprotective, muscle relaxant
[91,96,97]
Syringic acid 4-hydroxy-3,5-dimethoxybenzoic acid Anticancer, antioxidant, antidiabetic, anti-inflammatory,
neuroprotective, antimicrobial, antiendotoxic, hepatoprotective,
anti-osteoporotic
[98,99]
Ferulic acid (E)-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoic acid Anticancer, antidiabetic, antioxidant, cardioprotective,
neuroprotective, anti-alzheimer's,
[100,101]
Terpenoids Thymol 5-methyl-2-propan-2-ylphenol Antibacterial, antifungal, antispasmodic, antitussive, anxiolytic,
neuroprotective, antihypertensive, antioxidant, antihyperlipidemic,
anti-inflammatory, immunomodulatory, anti-cancerous, analgesic,
growth promoting
[41]
Carvacrol 2-methyl-5-propan-2-ylphenol Antimicrobial, antimutagenic, antitumor, analgesic, anti-inflammatory,
antihepatotoxic, antiparasitic, antispasmodic, and hepatoprotective
[32]
Geraniol (2E)-3,7-dimethylocta-2,6-dien-1-ol Anti-cancerous, anti-inflammatory, antioxidant, hepatoprotective,
antimicrobial, cardioprotective, antidiabetic, neuroprotective
[102]
Linalool 3,7-dimethylocta-1,6-dien-3-ol Sedative, antiviral, anti-inflammatory, antioxidant, anti-nociceptive,
analgesic, anesthetic, antimicrobial, anxiolytic, anti-hyperlipidemic,
antinoceptive, antidepressive, neuroprotective
[41,103]
ρ
-Cymene 1-methyl-4-propan-2-ylbenzene Antimicrobial, antinociceptive and anti-inflammatory, antioxidant,
anxiolytic, anticancer, vasorelaxant, immunomodulatory, antinoceptive
[104,105,106]
γ-terpinene 1-methyl-4-propan-2-ylcyclohexa-1,4-d iene Antibacterial, antioxidant, anti-inflammatory, antinociceptive. [107,108,109]
Limonene 1-methyl-4-(1-methylethenyl)-cyclohexene Antibacterial, antifungal, anti-inflammatory, antinociceptive, antioxidant, [110,111]
β-Caryophyllene (1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo
[7.2.0] undec-4-ene
Antimicrobial, cardioprotective, hepatoprotective, gastroprotective,
neuroprotective, nephroprotective, antioxidant, anti-inflammatory,
immunomodulatory
[48]
β-Pinene 6,6-dimethyl-2-methylidenebicyclo [3.1.1] heptane Anti-cancerous, antimicrobial, antioxidant, anti-inflammatory, analgesic,
cytogenetic, gastroprotective, anxiolytic, cytoprotective, anticonvulsant,
neuroprotective
[49]
α
-Terpineol 2-(4-methylcyclohex-3-en-1-yl) propan-2-ol Anti-cancerous, antioxidant, antinociceptive, anticonvoluscent,
gastroprotective, cardioprotective, antihypertensive, sedative
[112]
Flavonoids Apigenin 5,7-dihydroxy-2-(4-hydroxyphenyl) chromen-4-one Antidiabetic, anti-cancerous, antidepressive, anti-insomnia, anti-amnesia,
anti-alzheimer's, antiviral
[113]
Luteolin 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one Anti-alzheimer's, anti-cancerous [114,115]
Cirsimaritin 5-hydroxy-2-(4-hydroxyphenyl)-6,
7-dimethoxychromen-4-one
Antioxidant, anti-inflammatory, antimicrobial, antidiabetic, anticancer,
neuroprotective, cardiovascular, hepatoprotective
[116]
Xanthomicrol 5-hydroxy-2-(4-hydroxyphenyl)-6,7,
8-trimethoxychromen-4-one
Anti-inflammatory, anti-spasmodic, anti-platelet, anti-cancerous effects [117,118]
S.M. Patil et al. Heliyon 7 (2021) e07054
5

5.1. Phenolic compounds
T. vulgaris L. is one of the principal sources of phenolic compounds.
Heidari et al. (2018) identified new phenolic compounds using GC-MS
technique, in which a few of the phenolic compounds were already re-
ported. They include creosol 2-Methoxy-4-methylphenol), thiophenol
(benzenethiol), quininic acid (6-methoxyquinoline-4-carboxylic acid),
loliolide [(6S,7aR)-6-hydroxy-4,4,7a-trimethyl-6,7-dihydro-5H-1-
benzofuran-2-one], phenol 4-(3- hydroxy-1- propenyl-2- methoxy), and
3-Methoxy-5- methylphenol [47]. Also, previous studies have identified
the presence of rosmaric acid, caffeic acid, p-coumaric acid, geranic acid,
p-hydroxybenzoic acid, gentisic acid, syringic acid, and ferulic acid [4].
Among the various others reported, the highest number of compounds
with pharmacological significance were the phenolic compounds
(Figure 3).
5.2. Terpenoids
Characterization of the essential oil of T. vulgaris L. reveals a list of
hydrocarbons, oxides, alcohols/esters, and aldehydes/ketones. Many of
the studies have reported the uses of a variable number of compounds
with different concentrations. Among all the reported volatile com-
pounds, thymol, carvacrol, geraniol, linalool,
α
- and β-pinene, p-cymene,
and γ-terpinene have been reported with major pharmacological activ-
ities [48,49,50]. In addition, hydrocarbons like 2,6-Octadienal,
cis-Sabinene hydrate, germacrene D, limonene, β-ocimene, myrcene,
β-caryophyllene,
α
-thujene,
α
-phellandrene and
α
-humulene are present.
Oxides like 1,8-cineol, caryophyllene oxide; alcohol/esters including
α
-terpineol, borneol, 1-octen-3-ol, 3-octanol, p-cymen-8-ol,
terpinen-4-ol, thymol methyl ether, carvacrol methyl ether; aldehy-
des/ketones like 3-octanone, camphor, thymoquinone, and geranial are
present. The GC-MS analysis also showed the presence of esters including
butanoic acid, 2-methyl-, methyl ester, bornyl acetate, and geranyl
propanoate. The majority of these compounds need to be assessed indi-
vidually to decipher their pharmacological potential [51]. However,
some of the terpenoids have been reported with profound pharmaco-
logical significance (Figure 4).
5.3. Flavonoids
Flavones like 6-hydroxyluteolin, apigenin, luteolin, methyl-flavones
including cirsimaritin or genkwanin, cirsilineol, 5-desmethylnobiletin,
8-methoxycirsilineol, 7-methoxyluteolin, gardenin B, salvigenin, thy-
monin, sideritoflavone, xanthomicrol and thymusin [4]. There have been
no reports on pharmacological activities of these compounds except
apigenin, luteolin, cirsimaritin, genkwanin, and xanthomicrol. Studies
need to focus on the extraction and biosynthesis of individual compounds
which can further become a basis for phytomedicines. Although only a
few of the flavonoids are reported (Figure 5), they are found to exhibit
profound pharmacological activities.
5.4. Steroids, tannins, alkaloids, saponins
A study that used HPTLC to assess the methanolic extract of T. vulgaris
L. documented the presence of 9 alkaloids, 14 saponins, 12 steroids, and
8 tannins. The study revealed a good resolution with 10 bands of
essential oils, along with bands corresponding to tannins (9.2%) and
saponins (23.1%), where a remarkable antimicrobial activity is possessed
Figure 3. Phenolic compounds with pharmacological activity in Thymus vulgaris L.
Figure 4. Terpenoids with pharmacological activity in Thymus vulgaris L.
S.M. Patil et al. Heliyon 7 (2021) e07054
6

by saponins [46]. However, it could only reveal the presence of a class of
compounds but not decipher the presence and role of individual com-
pounds. As most of the chemical components belong to essential oils and
phenolic compounds, metabolites like steroids, tannins, alkaloids, and
saponins have not been evaluated in detail. Therefore, it now becomes
essential to focus on these compounds to reveal their biological
significance.
5.5. Other phytochemical compounds
Banerjee et al. (2019) assessed polysaccharide content using aqueous
extraction of the alcohol insoluble residue of T. vulgaris L. leaf and re-
ported the presence of four types of polysaccharides. They include
homogalacturonan, starch, cellulose and a unique type of polysaccharide
known as rhamnogalacturonan I (RG-I). They also reported the associa-
tion of RG-I with ester linked phenolic acids shows profound antioxidant
activity [52]. However, much remains unknown about other bio-
molecules present in T. vulgaris L.. Identification of the other bio-
molecules like proteins and vitamins using the whole plant extracts could
lead to the elucidation of nutritive profiling of the plant.
6. Pharmacology
6.1. Antibacterial activity
Bacteria obtained from the feces of the red deer (Cervus elaphus)
including Escherichia coli, Klebsiella pneumoniae, Yersinia enterocolitica,
Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis were
found sensitive towards the ethanol extract of T. vulgaris L., where min-
imum inhibitory concentration (MIC) was observed as >6.25,
3.12,>6.25, 0.2, 0.39, 0.78 mm respectively at 100
μ
l concentration (p <
0.05). These bacteria showed mixed response towards antibiotics [53]. In
another study, methanolic extract of T. vulgaris L. was found to be
effective against methicillin-resistant S. aureus (MRSA), where bacteria
isolated from the infected mice body parts were tested under in vivo
conditions. It was found that for the bacteria isolated from throat and
lungs, MIC was found to be 2.93 and 3.83 colony forming unit (CFU)
(log
10
)/ml, respectively (p <0.05) [54]. In case of Salmonella typhirium,
thyme oil from T. vulgaris L. showed MIC of 25.5 mm at 100
μ
l concen-
tration, in accordance with amoxicillin (23.0 mm) and cefotaxime (15.0
mm) [55]. Biofilm of Salmonella enteritidis (48 h) was inhibited by thyme
oil at an MIC/MBC of 0.156/0.315
μ
l/ml, in accordance with that of the
control. The oil also reduced the metabolic activity by 9.6–70.5%, where
both biofilm and metabolic activity reductions were found to be signif-
icant (p <0.05). The study also included the individual assessment of
thymol and carvacrol, whose results were found to be strikingly similar in
case of antibacterial assay [13].
Further, the essential oil from T. vulgaris L. was found to inhibit the
growth of multi-drug resistant (MDR) variants of S. aureus. A significant
inhibition was observed with a zone of 35–40 mm at 2.8–11.5
μ
g/ml for
MDR variants, whereas cefotaxime showed MIC at 32
μ
g/ml concentra-
tion (p <0.05) [11]. In addition, Pseudomonas aeruginosa swabs were
taken from surgical wounds at the end of the hip implant surgery, where
minimum bactericidal concentration (MBC) was found to be 8% at 100
μ
l
concentration [56]. T. vulgaris L. essential oil or thyme oil was also found
to be effective against porcine respiratory bacteria. Actinobacillu spleur-
opneumoniae, Streptococcus suis, Actinobacillus suis, Haemophilus parasuis,
Pasteurella multocida, and Bordetella bronchiseptica were tested with
thyme oil and considerable MIC values were obtained, ranging from
0.039% to 0.078% at dilutions 0.01–1.25% v/v (p <0.05) [57].
These studies indicate the antibacterial potential of thyme oil and
other extracts on bacteria isolated from various sources. A greater
inhibitory activity was observed by the use of thyme oil against bacteria
in comparison with that of the aqueous, ethanolic extracts, and even a
few antibiotics [55]. Thyme oil is also capable of inhibiting biofilm for-
mations, which are regarded to be highly infective [13]. Although both
bacteriostatic and bactericidal values were discovered, dose-dependent
activities need to be conducted using animal models and human cell
lines. It is noteworthy that thyme oil has exhibited a significant bacte-
riostatic activity against MDR and MRSA strains [54,56]. Though the oil
serves as a better antibacterial agent owing to its facile extraction method
and significant activity, studies need to focus on individual compounds to
assess their effects either in single or in combination. Many of the studies
have reported the synthesis as well as extraction of individual com-
pounds present in thyme oil that are proved to have profound antibac-
terial effects [11,13,56,57]. Further, mechanisms of the bacteriostatic
and bactericidal functions of the fractions need to be elucidated with
respect to physicochemical and physiological changes in bacterial cell.
6.2. Antioxidant activity
Antioxidant enzymes like catalase, glutathione, glutathione-S-
transferase, and superoxide dismutase are primarily responsible for the
reduction of free radicals in the body. Abdel-Gabbar et al. (2019)
assessed the in vivo activity of these enzymes by the administration of
T. vulgaris L. aqueous extract on the experimental rabbits, where the
levels were found to be increased by 14.12%, 27.69%, 98.75% and
78.29%, respectively (p <0.05) compared with that of the control
(water). Total antioxidant capacity was increased at 100 mg/kg and 50
mg/kg concentration in rabbits, with no adverse effects on kidney and
liver function parameters [58]. Similarly, the levels of alanine amino-
transferase, aspartate aminotransferase, and alkaline phosphatase were
also significantly increased (2 units/ml) upon the administration of 500
mg/kg of T. vulgaris L. aqueous extract for 14 days. In combination with
paracetamol, (200 mg/kg), the enzyme levels increased by 15–20
units/ml, compared with that of the control (p <0.01) [59]. In addition,
the antioxidant potential of the aqueous extract was also assessed using
conventional methods. The aqueous extract of T. vulgaris L. leaf and stem
was analysed with 2,2-diphenyl-1-picrylhydrazyl (DPPH) resulting in the
free radical scavenging activity (92.0%) at a concentration of 1.5 mg/ml,
in accordance with butylated hydroxyanisole (BHA-95.7%) and butyl-
ated hydroxytoluene (BHT-96.6%). The potential benefits observed in
the study were attributed to the presence of polysaccharides like starch,
homogalacturonan, rhamnogalacturonan I (RG-I) and cellulose in
T. vulgaris L. leaves. The binding of RG-I with bovine serum albumin
(BSA) indicated the formation of water-soluble complexes that further
induces antioxidant activity [52].
In case of thyme oil, DPPH, Ferric reducing antioxidant power
(FRAP), Ferrous ion-chelating ability assay (FIC), and 2,20-azinobis-(3-
ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging
activities were reported with the IC
50
values 12.69, 13.29, and 6.46 mg/
ml, respectively (p <0.05) [60]. The unused plant material obtained
after extracting the oil using steam distillation process was also found to
be the reservoir of phenolic compounds. El-Guendouz et al. (2019)
Figure 5. Flavonoids with pharmacological activity in Thymus vulgaris L.
S.M. Patil et al. Heliyon 7 (2021) e07054
7

demonstrated the ability of the ethanolic extract obtained using the oil
extraction by-product of T. vulgaris L. in preventing the formation of
primary and secondary lipid oxidation products from oil in water (O/W)
emulsions. Assessment of the antioxidant activity through DPPH showed
an IC
50
of 93
μ
g/ml, similar to that of BHT (89
μ
g/ml). But these values
were found to be of lower activity compared with that of the pure
aqueous and ethanol extracts [61].
The antioxidant potential of the oil is proved to be higher than those
of the aqueous and ethanolic extracts [60,61]. However, novel appli-
cations like silver and zinc nanoparticles could be used in place of pure
extracts, as they possess lower amount of cytotoxicity. Usage of cell lines
and animal models could provide a better insight to the studies, revealing
the biochemical changes that could occur before and after the adminis-
tration of the T. vulgaris L. fractions. It is noteworthy that the antioxidant
enzymes have been focused rather than the conventional assays like
DPPH, ABTS, and FRAP [58,59]. However, it is advisable that these
enzymatic analyses could be carried out using human cell lines with
correlation in terms of its toxicity based on dose-dependent studies. In
addition, studies have proved that different T. vulgaris L. extracts could
alone be able to reduce the oxidative stress levels using the conventional
antioxidant assays. But there are no available controls to compare its
efficacy [58,59]. In addition, the treatment with aqueous T. vulgaris L.
extract alone has proved its potential to prevent or reverse the
drug-induced toxic biochemical changes to normal via its antioxidant
activity [58]. As a key finding, studies have revealed that an array of
bioactive compounds showing antioxidant activities are also present in
leaves and other parts of the plant, other than the oil alone [52].
6.3. Antifungal activity
Thyme essential oil was found to be effective against fungal species
including Sclerotinia sclerotiorum,Botrytis cinerea,Phytophthora parasitica,
Pythium aphanidermatum,Fusarium oxysporum,Alternaria brassicae,Tri-
choderma aggressivum f.sp. europaeum, and Cladobotryum mycophilum. The
sensitivity of these fungi were assessed with the radial growth inhibition
assay, which revealed an inhibitory value ranging from 13.9 to 41.4 mm
at 5% concentration and ED
50
values ranging from 9.3 to 18.0% for all
the species tested (p <0.05) [14]. Further, a group of 183 clinical isolates
of Candida albicans and 76 isolates of C. glabrata species were tested for
sensitivity towards T. vulgaris L. extract. Both MIC and minimum fungi-
cidal concentration (MFC) were found to be in the range of 0.04–22.9
mg/ml for all the isolates. In the time kill assay, thyme oil reduced the
fungal growth in the initial hours (4–8). Interestingly, thyme oil inhibited
the fungal growth supplemented with sorbitol, an osmoprotectant with a
much lower MIC (0.08 mg/ml) (p <0.05) [15]. In addition, an in vivo
assessment using thymol on C. albicans in a Caenorhabditis elegans nem-
atode model resulted in the complete inhibition of the fungus at 64 mg/l
and 128 mg/ml, better than that of the standard antibiotics used (p <
0.05). Thymol is also reported to enhance the expressions of pmk-1and
sec-1 genes, thereby resulting in the enhancement of p38 MAPK signal-
ling pathway, which exhibits signalling events to combat C. albicans [62].
Scalas et al. (2018) assessed the antifungal activity against Crypto-
coccus neoformans where MIC and MFC were found to be in the range of
56–1.12 mg/ml, in accordance with the standards fluconazole (FLC),
itraconazole (ITC), and voriconazole (VRC). But individual antifungal
assay showed profound results with thymol (0.02–0.08 mg/ml) (p <
0.05) [63]. Thyme oil in vapor and liquid phase was also found to be
effective against fungi, where a total reduction in mycelial growth was
detected at the concentrations of 20 and 400
μ
g/ml, respectively for
Aspergillus flavus. Also, treatment with 10
μ
g/ml of thyme oil reduced
production of aflatoxin by 97.0 and 56.4% through vapour and liquid
phases, respectively (p <0.05). In addition to this, the study also
reported the downregulation of a few genes related to fungal develop-
ment including brlA, abaA, and wetA and genes related to aflatoxin
biosynthesis -aflR, aflD, and aflK[16].
It is noteworthy that a majority of the studies have performed both
fungistatic and fungicidal assessments which determine the complete
inhibition of fungi. A few of the studies have performed the time kill
assay and in vivo inhibition assays to effectively determine the antifungal
activity [15,62]. The literature search was not able to find the antifungal
studies completed using water and alcohol extracts. Also, there is a lack
in antifungal studies performed, using the extracts and compounds on
MDR species. The fungal resistance needs to be dealt with using variable
number of doses of the extracts along with concerns regarding its cyto-
toxicity on the living cells. Furthermore, there is a lack of information on
the fungicidal and fungistatic mechanism of the plant extracts and
essential oil [15,16,62]. Owing to a lack of studies on host-pathogen
interaction, advanced studies to decipher this mechanism should be
carried out in order to identify the optimal dose. These studies must
evaluate the cytotoxic effects as well as cell survivability so that clinical
trials using plant fractions could be performed.
6.4. Anti-inflammatory activity
Though nitric oxide (NO) radicals act as intracellular messengers
under normal conditions, their elevated numbers can bring up the
cytotoxicity and inflammation issues. A study has evaluated the anti-
inflammatory potential of T. vulgaris L. aqueous extract. Significant
scavenging of NO radicals with 80.3% of the activity at 16
μ
g/ml con-
centration was observed in accordance with the values obtained using
dexamethasone (p <0.05) in murine macrophage cell line J774A.1 [19].
In case of inflammation, immune cells generally express the genes
encoding for 5-lipoxygenease (5-LOX) production, which in turn acti-
vates the synthesis of leukotrienes. Tsai et al. (2011) targeted the inhi-
bition of 5-LOX, showing an inhibition at 0.005
μ
g/ml (IC
50
) of thyme oil
concentration. This was more effective than
α
-bisabolol (0.049
μ
g/ml).
They also assessed the effect of essential oil on lipopolysaccharide
(LPS)-induced TNF-
α
, interleukins IL-1β, and IL-8 secretions using THP-1
cells at 0.01
μ
g/ml [20].
In an in vivo study, Abdelli et al. (2017) analysed thyme oil in a dose
dependent manner (100, 200 and 400 mg/kg) against mice with
carrageenan-induced paw edema to determine the anti-inflammatory
activity. Paw thickness was found reducing at a dose of 400 mg/kg.
Results were in accordance with both the controls Tween 80 and diclo-
fenac (p <0.001). The study also determined the toxic level of thyme oil
(4500 mg/kg), where sedation was observed at 5000 mg/kg [43].
Similarly, in the carrageenan-induced pleurisy model thyme oil at 250,
500 and 750 mg/kg reduced inflammatory exudates as well as migrated
leucocytes in ear edema. Individual assessments revealed that thymol
(34.2%) and carvacrol (47.3%) are attributable for the anti-inflammatory
activity (p <0.05). This study also reported the toxicity level of thyme oil
as 4000 mg/kg, which stands in accordance with the findings of Abdelli
et al. (2017) [64].
It was reported that anti-inflammatory response can be effectively
generated by both extracts and essential oil from T. vulgaris L[19,20,43,
62]. It is a laudable approach by the researchers to assess the levels of
inflammatory cytokines and tumour necrosis factor (TNF) [20]. A study
was even able to determine the toxicity level of the thyme oil as 4500
mg/kg in mice models [43]. However, a few notable parameters like
cyclooxygenase-2 (COX-2), cholestasis, vascular lesions, c-reactive pro-
tein (CRP) in chronic hepatitis, hepatic fibrosis, drug-induced autoim-
munity could be assessed, which can support the antioxidant potential. In
addition, autoimmune models like rheumatoid arthritis could be focused
to assess the effect of T. vulgaris L. fractions.
S.M. Patil et al. Heliyon 7 (2021) e07054
8

Table 3. Summary of the pharmacological properties of Thymus vulgaris L.
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
Antibacterial activity in vitro Escherichia coli, Klebsiella
pneumoniae,
Yersinia enterocolitica,
Staphylococcus aureus,
Listeria monocytogenes,
Enterococcus faecalis
Aerial parts Ethanol 100
μ
l of 6.25–0.025%
serial dilutions
Solvent ethanol Not defined MIC was observed as
>6.25, 3.12,>6.25, 0.2,
0.39, 0.78 mm
respectively at 100
μ
l
concentration (p <0.05).
These bacteria showed
mixed response towards
antibiotics.
[53]
in vivo Methicillin-resistant
Staphylococcus
aureus (MRSA) in mice
Not defined Methanol 200 mg/ml per kg of
body weight
Positive control-infected,
negative
control-normal mice,
Antibiotics
Not defined For the bacteria isolated
from throat and lungs,
MIC was found to be 2.93
and 3.83 CFU (log
10
)/ml,
respectively (p <0.05).
[54]
in vitro Salmonella typhirium Aerial parts Essential oil 100
μ
l of 1/20–1/200 v/v
serial dilutions
Amoxicillin, cefotaxime Not defined MIC of 25.5 mm at 100
μ
l
concentration, in
accordance with
amoxicillin (23.0 mm)
and cefotaxime (15.0
mm) (p <0.05).
[55]
in vitro Salmonella Enteritidis
Biofilm
Dried plant Essential oil 5–0.0024
μ
l/ml. Growth control (broth þ
microbe),
negative control (broth þ
propylene glycol
þmicrobe), sterility
control (broth þtest oil),
positive control (broth þ
streptomycin þmicrobe)
Possible inhibition of
bacterial adsorption and
biofilm matrix formation
Biofilm inhibition at
MIC/MBC 0.156/0.315
μ
l/ml by oil, thymol, and
carvacrol. Oil reduced the
metabolic activity by
9.6–70.5%, (p <0.05).
[13]
in vitro Staphylococcus aureus
(MDR)
Not defined Essential oil 10
μ
l of 2.87–11.5
μ
g/ml Cefotaxime Not defined A significant inhibition
with 35–40 mm
inhibition zone at
2.8–11.5 at
μ
g/ml was
observed for MDR
variants, whereas
cefotaxime showed MIC
at 32
μ
g/mL
concentration (p <0.05)
[11]
in vitro Pseudomonas aeruginosa Leaves and branches Essential oil 100
μ
l oil of different
concentrations
Not defined Not defined Minimum bactericidal
concentration (MBC) was
found to be 8% at 100
μ
l
concentration
[56]
in vitro Actinobacillus
pleuropneumoniae,
Streptococcus suis,
Actinobacillussuis,
Haemophilus parasuis,
Pasteurella multocida,
and Bordetella
bronchiseptica
Not defined Essential oil 100
μ
l of 1.25 to 0.01%
(v/v)
Media þmicrobes þPBS Not defined MIC values ranging from
0.039% to 0.078% at
dilutions 0.01–1.25% v/v
(p <0.05)
[57]
Antioxidant activity in vivo Antioxidant enzyme
levels in rabbits
Not defined Aqueous extract 50 mg/kg of body weight Water Not defined Levels of antioxidant
enzymes catalase,
glutathione, glutathione-
S-transferase, and
[58]
(continued on next page)
S.M. Patil et al. Heliyon 7 (2021) e07054
9

Table 3 (continued )
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
superoxide dismutase
increased by 14.12%,
27.69%, 98.75% and
78.29%, respectively (p <
0.05)
in vivo Antioxidant enzyme
levels in rats
Dried leaves Aqueous extract 500 mg/kg body weight Paracetamol (200 mg/kg) Not defined Alanine
aminotransferase,
aspartate
aminotransferase, and
alkaline phosphatase
content increased by 2
units/mL. In combination
with paracetamol, the
enzyme levels increased
by 15–20 units/ml (p <
0.01)
[59]
in vitro Radical scavenging
activity using DPPH
Leaf and stem Aqueous extract 0.0125–3.0 mg/ml Not defined Polysaccharide biding
with BSA brings out the
radical scavenging
Radical free scavenging
activity of 92.0% at the
concentration of 1.5 mg/
mL, in accordance with
butylated hydroxyanisole
(BHA-95.7%) and
butylated hydroxytoluene
(BHT-96.6%).
[52]
in vitro FRAP, ABTS, and FIC Not defined Essential oil 0.23–30 mg/ml Not defined Not defined FRAP, FIC, ABTS assays
showed IC
50
values
12.69, 13.29, and 6.46
mg/ml respectively (p <
0.05)
[60]
in vitro Primary and secondary
lipid oxidation
products in oil in water
(O/W)
emulsions through DPPH
Dry waste plant Ethanolic extract 100
μ
l of different
concentrations
Not defined Not defined IC
50
of 93
μ
g/ml
compared to BHT (89
μ
g/
mL) (p >0.05)
[61]
Antifungal activity in vitro Sclerotinia sclerotiorum,
Botrytis cinerea,
Phytophthora parasitica,
Pythium aphanidermatum,
Fusarium oxysporum,
Alternaria brassicae,
Trichoderma
aggressivumf.sp.
europaeum,
Cladobotryum mycophilum
Not defined Essential oil 5,10,15,20,30% (v/v) Media þTween 20 Not defined Mycelial growth
inhibition values were
found to be ranging
between 13.9 to 41.4 mm
at 5% concentration with
ED
50
values ranging from
9.3-18.0% for all the
species (p <0.05)
[14]
in vitro Clinical isolates of
Candida albicans and
C. glabrata species
Not defined Essential oil 0.005–2.5% (v/v) Amphotericin B Possible ergosterol
binding
MIC and MFC were in the
range of 0.04–22.9 mg/
ml for all the isolates.
Thyme oil reduced the
fungal growth in the
initial hours (4–8). It
inhibited the growth with
sorbitol at lower MIC
(0.08 mg/ml) (p <0.05)
[15]
(continued on next page)
S.M. Patil et al. Heliyon 7 (2021) e07054
10

Table 3 (continued )
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
in vivo C. albicans in a
Caenorhabditis elegans
nematode model
Not defined Thymol 32, 64, and 128 mg/l Kanamycin (45
μ
g/ml),
ampicillin (100
μ
g/mL),
and streptomycin (100
μ
g/ml)
Enhancing pmk-1and sec-
1 gene expressions, which
in turn enhance p38
MAPK signalling pathway
Complete inhibition of
fungi and biofilm at 64
mg/l and 128 mg/ml,
compared to control used
(p <0.05). Growth
reduction at 12 h,
compared to control (36
h). Thymol enhances the
expressions of pmk-1and
sec-1 genes, in turn p38
MAPK signalling pathway
[62]
in vitro Cryptococcus neoformans Not defined Essential oil 0.07–10 mg/ml FLC (0.06–128
μ
g/mL),
ITC (0.0078–2
μ
g/ml),
VRC (0.0078–32
μ
g/mL)
Possible membrane
deterioration by thymol,
Possible ergosterol
binding
MIC and MFC were found
to be in 0.56–1.12 mg/ml
in accordance with
controls. Thymol showed
better activity 0.02–0.08
mg/ml (p <0.05)
[63]
in vitro Aspergillus flavus Not defined Essential oil vapour
and liquid phases
0, 1, 5, 10, and 20
μ
g/ml Aflatoxin B Downregulating of fungal
development genes brlA,
abaA, wetA and aflatoxin
biosynthesis genes aflR,
aflD, and aflK
Vapor and liquid phases
reduced growth at 20 and
400
μ
g/ml, respectively.
Thyme oil 10
μ
g/mL of
reduced production of
afltoxin by 97.0 and
56.4% through vapour
and liquid phases,
respectively. (p <0.05).
[16]
Anti-inflammatory
activity
in vitro NO radical scavenging in
murine
macrophage cell line
J774A.1
Flowering tops Aqueous extract 8.5, 16, 50.4, 84
μ
g/ml Dexamethasone Possible cellular
mechanisms of
suppression of iNOS
induction by flavonoids
Significant scavenging of
NO radicals with 80.3%
of the activity at 16
μ
g/ml
concentration was
observed in accordance
with control (p <0.05)
[19]
in vitro 5-lipoxygenease (5-LOX)
production,
lipopolysaccharide (LPS)
induced TNF-
α
,
IL-1β, and IL-8 secretions
using THP-1 cells
Dried plant Essential oil 30
μ
l of different
concentrations
α
-bisabolol Not defined 5-LOX got inhibited at
0.005
μ
g/ml (IC
50
)of
thyme oil, compared to
α
-bisabolol (0.049
μ
g/
mL). TNF-
α
, IL-1β, and IL-
8 got inhibited at 0.01
μ
g/ml.
[20]
in vivo Mice with carrageenan-
induced paw edema
Aerial parts and
dried leaves
Essential oil 100, 200 and 400 mg/kg Tween 80 and diclofenac Not defined Paw thickness was found
reducing at a dose of 400
mg/kg. Results were in
accordance with both the
controls Tween 80 and
diclofenac (p <0.001).
Toxic level of thyme oil
was found (4500 mg/kg),
where sedation was
observed at 5000 mg/kg.
[43]
in vivo Mice with carrageenan-
induced pleurisy
Leaves Essential oil 250, 500 and 750 mg/kg Croton oil Carvacrol may act by
inhibiting cytokines and
leukotrienes, and these
mediators are likely not
All the concentrations
reduced inflammatory
exudates as well as
migrated leucocytes in
[64]
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S.M. Patil et al. Heliyon 7 (2021) e07054
11

Table 3 (continued )
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
involved in the
mechanism of action of
thymol
ear edema. Individual
assessment showed
thymol (34.2%) and
carvacrol (47.3%) are
attributable for the anti-
inflammatory activity (p
<0.05)
Anti-cancerous activity in vitro MCF7 (breast
adenocarcinoma), HCT15
(colon carcinoma), HeLa
(cervical carcinoma),
HepG2 (hepatocellular
carcinoma), and NCI-
H460
(non-small cell lung
cancer) cell lines
Dried aerial parts Essential oil 10–100
μ
g/ml Ellipticine (0.24–65.2
μ
g/
ml)
Possible involvement of
thymol in the stimulation
of active proliferation of
pulp fibroblasts
T. vulgaris L. oil showed
inhibition of growth at
76.02–180.40
μ
g/ml
concentration (GI
50
). It
did not show any effect
on non-tumour liver PLP
cells, even at a high
concentration of 400
μ
g/
ml (p <0.05)
[65]
in vitro THP-1 leukemia cell line Not defined Essential oil 10–500
μ
g/ml DMSO Not defined At a concentration of 100
μ
g/ml and >200
μ
g/ml,
thyme oil prevented the
proliferation of THP-1
leukemia cells
[66]
in vitro H460 lung cancer cell line Not defined Hydroalcoholic extract 0.04–0.6% Glyceraldehyde 3-phos-
phate dehydrogenase
Possible interference in
pro-inflammatory
cytokines
H460 lung cancer cell line
was found to be sensitive
at 0.11% of
hydroalcoholic extract (p
<0.05) and
downregulated NF-κB
p65 and NF-κB p52
proteins along with the
reduction of IL-1βand IL-
8 gene expression in LPS
model
[21]
in vivo Mammary carcinoma rat
and 4T1 mouse models
Dried plant Thyme powder 50 mg/kg body weight Untreated models Possible interference with
pro-inflammatory
cytokines, Possible
upregulation of caspase
genes at epigenetic level
Thyme powder reduced
the volume of 4T1
tumours by 85% at 1%
concentration. In rat
model, the same
concentration decreased
the tumour frequency by
53% (p <0.05).
Upregulation of caspase-2
and caspase-3 enzymes,
along with bcl-2 and Bax
proteins
[67]
in vitro HL-60 acute promyelotic
leukemia
cell line, human
peripheral
blood mononuclear cell
(PBMC)
Not defined Thymol 5, 25, 50, 75 and 100
μ
M
for 24 h
Camptothecin (5
μ
M) Apoptosis induced by
thymol in HL-60 cells was
associated with ROS
production, increase in
mitochondrial H
2
O
2
production, decrease in
Bcl-2 protein, increase in
Bax protein levels,
enhancing apoptosis
inducing factor (AIF) in
Thymol showed no
cytotoxic effect on human
peripheral blood
mononuclear cell (PBMC)
at 5 and 25
μ
M
concentrations. However,
extensive cytotoxicity
was observed at >50
μ
M,
after 24 h
[68]
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S.M. Patil et al. Heliyon 7 (2021) e07054
12

Table 3 (continued )
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
mitochondria and caspase
activation
in vitro Synthesized silver
nanoparticles against
T47D human breast
cancer cells
Dried leaves Silver nanoparticles and
ethanol extract
12.5–200
μ
g/ml Untreated cells Nanoparticles could trig-
ger translocation of
phosphatidylserine (PS)
from the inner membrane
indicating apoptosis
pathway rather than
necrosis
T47D cells showed high
sensitivity towards
nanoparticles (90%)
compared to the extract
(75%). T47D cells treated
with nanoparticles
showed 18.40% early and
0.69% late apoptosis with
varying IC
50
concentrations (12.5–100
μ
g/mL). Same was
observed in case of plant
extract, where 15.67%
early and 1.70% late
apoptosis was found (p <
0.05)
[47]
Antiviral activity in vitro Influenza virus Not defined Essential oil vapour and
liquid phases
3.12–100
μ
l/ml Canova oil Possible interaction with
hemagglutinin (HA)
Liquid phase at 3.1
μ
l/ml
concentration completely
inhibited the viral
growth, which was better
than that of control used
(canola oil). Significant
inhibition of HA was
observed. Also, 50% of
the culture was reduced
depicted as TC
50
14.34
μ
l/ml (p <0.05)
[33]
in vitro Herpes simplex virus
(HSV) on RC-37
(African green monkey
kidney cells)
Not defined Essential oil 10–750
μ
g/ml Untreated cells Not defined Cytotoxicity ranged
between 20
μ
g/ml for
citral and 1250
μ
g/ml for
1,8-cineole. IC
50
values
for1,8-cineole was 1200
μ
g/ml. Thyme oil proved
to reduce the viral load by
>96%, whereas all
monoterpenes by >80%
(p <0.05)
[69]
in vitro HIV-1 in HeLa HL3T1 cell
line
Not defined Essential oil 7.5–240
μ
g/ml
Neomycin, cisplatin Possible alteration in the
structure of Tat/TAR-
RNA complex
EMSA showed a notable
inhibitory potential of oil
(3–6
μ
g/ml), compared to
the control in case of Tat/
TAR-RNA complex
inhibition. Reduction
activity test against Tat-
induced HIV-1 LTR
transcription resulted in
RT
50
¼0,83
μ
g/ml, a
notable inhibitory
potential which reduced
viral transcription to 52%
(p <0.05)
[70]
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S.M. Patil et al. Heliyon 7 (2021) e07054
13

Table 3 (continued )
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
in vitro HIV-1 subtype A in PBMC
cell line
Dried plant Methanol extract 10, 100, 200, 800 and
1600
μ
g/ml
DMSO, Zidovudine Not defined The cytotoxicity value
(CC
50
) on PBMC was
found to be 200
μ
g/ml.
Antiviral assay revealed
EC
50
value of >500
μ
g/
ml. Mean fluorescent
intensity (MFI) of the
CD4þexpressions were
found to be 22.72 in
PBMC (p <0.05)
[71]
Antidiabetic activity in vitro Inhibition of
α
-glucosidase and
α
-amylase enzymes
Not defined Aqueous, methanol and
ethanol extracts
4, 8, 15, and 20
μ
g/ml Acarbose Not defined Methanol extract resulted
in maximum inhibition of
α
-glucosidase (IC
50
4.35,
22.04, 30.77, 43.13),
though less compared to
Acarbose (IC
50
16.11,
44.6, 53.03, 63.70).
Similarly,
α
-amylase got
reduced maximally by the
same extract (IC
50
6.39,
11.47, 17.01, 22.93), less
compared to Acarbose
(IC
50
12.37, 25.16, 36.08,
44.97)
[72]
Anxiolytic activity in vivo Elevated plus-maze
(EPM) rat model
Dried plant Aqueous extract 50 mg/kg, 100 mg/kg,
and 200 mg/kg
Saline fed groups Possible relation with
antioxidant activity of
phytochemicals
The aqueous extract
exhibited a significant
increase in rat movement
into the open arms at 100
mg/kg (p <0.05) and
200 mg/kg (p <0.01).
[74]
UV-protective activity in vitro Human skin cells Not defined Aqueous extract, thymol 1.82
μ
g/ml extract and 1
μ
g/ml thymol
Normal cells without UV
treatment, but with
extract treatment
Reduction of ROS
induced DNA damage,
Possible involvement of
polyphenols in
protectivity
Aqueous extract of thyme
leaf (1.82
μ
g/ml) and
thymol (1
μ
g/ml) reduced
the release lactic acid
dehydrogenase (LDH), in
cultured skin cells treated
with UV rays. Cell
proliferation was
observed in thyme pre-
treated skin cells in
accordance with control,
along with the reduction
in DNA damage (p <
0.01)
[75]
Anthelminthic activity in vitro Eimeria spp. oocysts from
Turkey fowls
Not defined Essential oil 0, 1, 2, 4, 8, 10, 20, 40,
80, and 800 mg/ml
Ammonia and diclazuril Not defined Thyme oil showed
significant anti-
helminthic activity
against 4 species of
Eimeria spp. at IC
50
53.42
mg/ml for 510
4
oocysts
(p <0.05)
[40]
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S.M. Patil et al. Heliyon 7 (2021) e07054
14

Table 3 (continued )
Pharmacological
Activity
Type of
Study
Models used Plant part/material Type of extract/
compound
Doses used Controls Possible/reported
mechanisms
Results References
Anti-anti-alzheimer's
activity
in vivo Acetylcholine esterase
and nicotinic
acetylcholine receptor in
C. elegans
nematode model
Aerial parts and leaves Essential oil 10, 20, 40, 60, 80, and
100 ppm
10% DMSO Upregulation of unc-17,
unc-50, and cho-1 genes
by
ρ
-Cymene
Enhancement of the
nicotinic acetylcholine
receptor activity,
upregulation of unc-17,
unc-50, and cho-1 genes
at 40 and 60 ppm
ρ
-Cymene was attributed
for gene upregulation
activity along with
downregulating ace-1
and ace-2 at 20 and 100
ppm (p <0.05). Thymol
and γ-terpinene enhanced
synaptic acetylcholine
levels in combination (40
ppm)
[76]
Anti-osteoporotic activity in vivo Rat model with low
calcium intake
Dried leaves Leaf powder 5% w/w Standard diet þnormal
calcium (Ca 0.5% w/w),
standard diet þlow
calcium (Ca 0.1% w/w),
Thyme powder (5% w/w)
þlow calcium (Ca 0.1%
w/w)
Possible promotion of
calcium resorption in the
gut
Significant increase in the
bone mass (2.93 g/kg),
length 32.8 mm), and
density (0.13 g/cm
2
),
compared to low calcium
diet control (2.46 g/kg,
32.2 mm, and 0.09 g/
cm
2
, respectively) (p <
0.05)
[77]
Anti-pulpotomy activity in vivo Formocresolpulpotomy in
humans
Not defined Ethanolic extract Suitable consistency Formocresol Not defined Thyme ethanolic extract
along with zinc oxide
reduced pain and
tenderness, Enhanced
bone and root resorption.
Clinical and radiographic
evaluations showed
94.4% and 88.2%
success, respectively with
no statistical significance
compared to the control,
formocresol 88.2% (p >
0.05)
[78]
S.M. Patil et al. Heliyon 7 (2021) e07054
15

Table 4. Summary of clinical trials of T. vulgaris preparations.
Pharmacological activity Type of study Models Plant part/material Type of Extract/
compound
Doses Controls Mechanisms Results References
Adipogenetic and anti-
aging
in vitro Humans Leaves and flowers ThymLec gel 2% prepared
from leaf and flower
extract (1.0%–3.0%) þ
water, propanediol,
glycerine þ5.0%–13.4%)
lecithin (additive) þ
benzyl alcohol, potassium
sorbate, tocopherol
(preservatives)
2 mg/cm
2
of the facial
skin applied with 2%
concentration (20 mg/g
w/w), gently spreading,
twice a day (morning and
evening).
Placebo (formulation
containing the same
vehicle of ThymLec gel)
and Benchmark 2%
ThymLec topical
application leads into the
adipogenesis and lipid
production, further
augmenting cell volume
and better remodelling of
face oval features.
ThymLec modulates the
PPAR-γsignalling
pathway, increasing
adiponectin production
and adipocyte lipid
accumulation.
Reduction of area (7.0%),
depth, and length
(10.2%) of the perioral
wrinkles on day 60,
whereas benchmark
produced a reduction of
5.4% and 7.5%,
respectively in case of
humans. Length of the
nasolabial lines (8.9%),
Crow's feet wrinkles' area
(8.9%), breadth (3.9s%),
and length (11.1%) were
also decreased. Face oval
remodelling evaluation
resulted in the reduction
of total face volume (4.9
fold) in comparison with
benchmark on day 60. In
vitro adiponectin
synthesis increased in
3T3-L1 embryonic
fibroblasts treated with
ThymLec 2% (122% at
0.0195% concentration)
and (137% at 0.039%
concentration).
[79]
Anti-dysmenorrhea in vitro Humans Not defined Essential oil 25 drops of essential oil Ibuprofen, Placebo
(Ibuprofen 200mg þoil
25 drops)
Anti-prostaglandin and
antispasmodic activity of
T. vulgaris L.
Pain intensity mean
values reached lowest
point (6.57–1.14 VAS) in
case of oil consumers'
group in comparison with
placebo (6.13–3.45) and
ibuprofen (5.3–1.48) (p <
0.05). No significant
results were obtained in
case of bleeding control
by both oil and placebo.
Oil was able to reduce
clinical symptoms like
lower abdominal pain,
nausea, mood swing, and
fainting after 48 h of
consumption (p <0.05).
[80]
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S.M. Patil et al. Heliyon 7 (2021) e07054
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6.5. Anti-cancerous activity
Cytotoxic activity of thyme oil was analysed against MCF7 (breast
adenocarcinoma), HCT15 (colon carcinoma), HeLa (cervical carcinoma),
HepG2 (hepatocellular carcinoma), and NCI-H460 (non-small cell lung
cancer) cell lines. T. vulgaris L. oil showedgrowth inhibition for all the cell
lines tested at 76.02–180.40
μ
g/ml concentration (GI
50
). However,
thyme oil did not show any effect on non-tumour liver PLP cells, even at a
high concentration of 400
μ
g/ml (p <0.05) [65]. In case of THP-1 leu-
kemia cell line, at a concentration of 100
μ
g/ml and >200
μ
g/ml, thyme
oil prevented the proliferation [66]. Apart from the oil, thymus extracts
also proved to be effective against lung cancer cells. In a study, H460 lung
cancer cell line was found to be sensitive at 0.11% of hydroalcoholic
extract (p <0.05) and downregulated NF-κB p65 and NF-κB p52 proteins
along with the reduction of IL-1βand IL-8 gene expression in LPS model.
However, there was no cytotoxicity reported within a concentration
range of 0.04–0.60% [21].
In an in vivo study, administration of dried T. vulgaris L. powder to
mammary carcinoma rat and 4T1 mouse models led to a remarkable
reduction in the volume of 4T1 tumours by 85% at 1% concentration. In
the rat model, the same concentration decreased the tumour frequency
by 53% compared with that of the control, besides suppressing the genes
associated with tumour inducing properties. This property is attributed to
the resultant upregulation of caspase-2 and caspase-3 enzymes, along
with bcl-2 and Bax proteins that result in cell apoptosis. Results were in
accordance with the controls used, and these results were significant (p <
0.05 [67].
It was noteworthy to find that thyme oil and extracts show a notable
cytotoxic effect on tumour cells, but not on normal human cells. A study
conducted by Deb et al. (2011) showed the effect of thymol on HL-60
acute promyelotic leukemia cells. Thymol showed no cytotoxic effect
on human peripheral blood mononuclear cell (PBMC) at 5 and 25
μ
M
concentrations. However, extensive cytotoxicity was observed at >50
μ
M, after 24 h [68]. Heidari et al. (2018) employed a different approach,
where both the plant extract and synthesized silver nanoparticles from
the plant extract were evaluated against T47D human breast cancer cells.
T47D cells showed a high sensitivity towards nanoparticles (90%) when
compared with that of the extract (75%). T47D cells treated with nano-
particles showed 18.40% early and 0.69% late apoptosis with a varying
IC
50
concentrations (12.5–100
μ
g/ml) (p <0.05), whereas in case of
plant extract, 15.67% early and 1.70% late apoptosis was observed [47].
The selectiveness of thyme oil and other extracts towards tumour cells
needs to be studied, for the possible presence of molecular pathways that
mediate the anti-cancerous effect. The mechanisms of T. vulgaris L.
fractions on different cell lines need to be elucidated with respect to
biochemical and physiological changes occurring within the proliferating
cell. These findings may serve as key sources during drug development
process. Most of the studies have attributed the anti-proliferative activity
to the monoterpenes, carvacrol and thymol that are predominantly pre-
sent in thyme oil [66,68]. According to Aazza et al. (2014) carvacrol and
thymol possess higher anti-cancerous potential than p-cymene and
borneol [66]. Thus, isolation and identification of individual components
for anti-cancerous potential needs to be done. Although Deb et al. (2011)
have deduced the mechanism of thymol in causing damage to the cancer
cell via inducing the activity of apoptosis inducing factor (AIF) [68],
much remains unknown about the other compounds. In a new approach,
nanoparticles have induced better cell apoptosis compared with that of
the plant extract, but with more cytotoxic effect [47]. Therefore, studies
need to decipher the appropriate doses for consumption, prior to the
complete decoding of the mechanism of action. Accurate determination
of these doses using human cell lines and animal models could be useful
to conduct clinical trials for drug development.
Also, effect of the plant fractions needs to be correlated with the
expression of oncogenes, which may deduce druggable targets ensuring a
check on its toxicity. For example, thymol showed higher cytotoxic ef-
fects in animal models at >50
μ
M, compared with the other extracts [68].
Table 4 (continued )
Pharmacological activity Type of study Models Plant part/material Type of Extract/
compound
Doses Controls Mechanisms Results References
Anti-bronchitis in vitro Humans Whole plant extract Dry powder extract 160 mg oral dose along
with 60 mg primrose
(P. vulgaris) root extract
for 11 days
Placebo tablet without
ingredients
Not defined The extract reduced the
cough symptoms on day 9
with an efficacy of 73.7%
in comparison with
placebo (day 11; 57.8%)
(p <0.0001). Reduction
of bronchitis severity
score was observed only
after 4 days in case of
extract (-6.2) in
comparison with placebo
(-4.1) (p <0.05). The
extract also reduced
minor symptoms like
disturbance in sleeping,
rising body temperature,
chest pain, and difficulty
in breathing.
[12]
S.M. Patil et al. Heliyon 7 (2021) e07054
17

It becomes essential to focus on the other compounds present in the
essential oil with lower toxicity level. Minimum toxicity levels have been
reported with plant extracts and dried powder, which can be utilized for
further analysis. Furthermore, much remains unknown about cancers like
Hodgkin and non-Hodgkin's lymphomas, Kaposi sarcoma and leukemia.
These cancer models along with the other rare cancer types also need to
be evaluated.
6.6. Antiviral activity
Thyme oil in the form of vapour and liquid was found to be effective
against the influenza virus. However, partial activity was observed in
vapour phase, whereas the liquid phase at 3.1
μ
l/ml concentration
completely inhibited the viral growth, which was better than that of
control used (canola oil). In addition, the researchers also evaluated the
effects on the principle external proteins of the virus, namely the hem-
agglutinin (HA) and neuraminidase (NA), where significant inhibition of
HA was observed. In addition, the TC50 value, which is 50% reduction of
the culture, was found to be 14.34
μ
l/ml (p <0.05) [33].
Apart from influenza, thyme oil was found to be effective on causative
viruses of sexually transmitted diseases (STDs) like herpes simplex virus
(HSV) and human immunodeficiency virus 1 (HIV-1). HSV possesses two
antigenic types, type 1 (HSV-1) and type 2 (HSV-2), resulting in flu like
symptoms in humans. Thyme oil along with the major monoterpene
compounds
α
-terpinene, terpinen-4-ol,
α
-terpineol,
α
-pinene, p-cymene,
thymol, citral and 1,8-cineole were analysed for their antiviral activity.
RC-37 kidney cells were used for non-cytotoxic dose determination,
which ranged between 20
μ
g/ml for citral and 1250
μ
g/ml for 1,8-
cineole. IC
50
values for 1,8-cineole was 1200
μ
g/ml. Thyme oil proved
to reduce the viral load by >96%, whereas all monoterpenes by >80% (p
<0.05) [69].
The human immuno deficiency virus is another STD which has no
available vaccine yet. A study conducted by Feriotto et al. (2018) tar-
geted the Tat protein that aids in the transcription of the viral genome.
This study evaluated the interaction of the essential oil from T. vulgaris L.
with the transcription of the Tat/TAR-RNA complex as well as the Tat-
induced HIV-1 long terminal repeat (LTR) [70]. An electrophoretic
mobility shift assay (EMSA) for this complex showed a notable inhibitory
potential (3–6
μ
g/ml), compared with that of the control. Similarly, a
reduction activity test against the Tat-induced HIV-1 LTR transcription
resulted in RT
50
¼0,83
μ
g/ml, a notable inhibitory potential which
reduced viral transcription to 52% (p <0.05) [70]. Similarly, the
methanol extract of the plant was evaluated on the infected PBMC cells
with HIV-1 subtype A. The cytotoxicity value (CC
50
) on PBMC was found
to be 200
μ
g/ml. Further, the antiviral assay revealed EC
50
value of >500
μ
g/ml. The study also focused on CD4þexpressions, where mean fluo-
rescent intensity (MFI) of the cells was found to be 22.72 in PBMC (p <
0.05) [71].
The studies have primarily focused on determining cytotoxicity
values using animal models, which may not give an accurate account in
case of human trials [69,71]. Thus, it becomes essential to conduct ex-
periments using human cell lines. Human cells can be infected in vitro,
subsequently treated with different extracts, oil, and individual com-
pounds to provide conclusive evidence of its effects on human system.
Effect of plant fractions on viral genome and protein synthesis needs to be
studied along with the gene expression levels. Using bioinformatic tools
like molecular docking to study the binding efficiency of plant-based
compounds to viral proteins could be a feasible approach. For example,
Feriotto et al. (2018) have deduced the role of TAT/TAR-RNA complex in
the virulence of HIV-1. The same complex could be assessed in terms of
its interaction with the plant phytochemicals [70]. Including HIV-1,
diseases like influenza and Herpes are often difficult to identify
because they are asymptomatic and have an extended incubation period.
Herpes has a substantial effect on skin, which could be evaluated by the
application of thyme oil. As the viruses affect targeted organs, an
organ-targeted delivery of the modified plant fraction could be studied in
a dose-dependent manner.
6.7. Other pharmacological activities
Apart from major pharmacological properties, T. vulgaris L. is re-
ported to comprise a few other properties which are yet be focused on.
Studies are yet to be conducted to assess the antidiabetic and anti-
hyperglycemic activities regarded as the indispensable properties of a
medicinal plant. A study by Aljarah and Hameed (2018) depicts the in
vitro inhibition of
α
-glucosidase and
α
-amylase enzymes known to be the
potential targets of antidiabetic drugs. Out of aqueous, methanol and
ethanol extracts at different concentrations (4, 8, 15, and 20
μ
g/ml),
methanol extract resulted in maximum inhibition of
α
-glucosidase (IC
50
4.35, 22.04, 30.77, 43.13) and
α
-amylase (IC
50
6.39, 11.47, 17.01,
22.93), yet lesser than the standard drug acarbose [72]. As stress and
anxiety levels reported to play a crucial role in diabetes [73], T. vulgaris L.
extract was also evaluated for its impact on anxiety levels in T. vulgaris L.
on elevated plus-maze (EPM) rat model. The aqueous extract exhibited a
significant increase in rat movement into the open arms at 100 mg/kg (p
<0.05) and 200 mg/kg (p <0.01) [74].
Further, thyme oil and extracts were evaluated for their protectant
ability against UV-A and UV-B rays, by inhibiting the proliferation of skin
cells leading to cancer. Aqueous extract of T. vulgaris L. leaf (1.82
μ
g/ml)
and thymol (1
μ
g/ml) reduced the release of lactic acid dehydrogenase
(LDH), in cultured skin cells treated with UV rays. Significant cell pro-
liferation was observed in T. vulgaris L. pre-treated skin cells in accor-
dance with that of the control used, along with the reduction in DNA
damage (p <0.01) [75]. In addition, an in vitro evaluation of thyme oil
showed a significant anti-helminthic activity against 4 species of Eimeria
spp. at IC
50
53.42 mg/ml for 510
4
oocysts (p <0.05). These parasites
affect poultry and cattle, reducing the yield [40].
Cholinergic affliction is found in Alzheimer's disease, a progressive
neurodegenerative disorder involving the death of cholinergic neurons.
Administration of thyme oil to C. elegans led to an enhancement of the
neurotransmission by regulating synaptic acetylcholine levels [76].
Enhancement of the nicotinic acetylcholine receptor activity is also
observed because of the upregulation of unc-17, unc-50, and cho-1 and
genes at 40 and 60 parts per million (ppm). p-cymene, a monoterpene
present in thyme extract, was attributed for the gene upregulation ac-
tivity along with a corresponding downregulation of ace-1 and ace-2 at
20 and 100 ppm (p <0.05). Interestingly, thymol and γ-terpinene
enhanced synaptic acetylcholine levels in combination (40 ppm) but
failed to do the so when administered individually [76].
Aging, a condition characterized by a reduced bone density, can be
treated by increasing calcium uptake in the body. Elbahnasawy et al.
(2019) conducted a study depicting the effect of T. vulgaris L. powder on
rats with low calcium intake. The study resulted in a significant increase
in the bone mass (2.93 g/kg), length 32.8 mm), and density (0.13 g/cm
2
),
compared with that of the low calcium diet control (2.46 g/kg, 32.2 mm,
and 0.09 g/cm
2
, respectively) (p <0.05). This study proved the potential
of T. vulgaris L.in the treatment of osteoporosis and other bone related
diseases [77]. T. vulgaris L. is also effective on dental caries and proved its
efficacy over the formed ocresolpulpotomy, due to profound antibacte-
rial properties. T. vulgaris L. ethanolic extract along with zinc oxide was
used in patients with dental caries. Clinical and radiographic evaluations
showed 94.4% and 88.2% improvement, respectively with no statistical
significance compared to that of the standard drug formocresol 88.2% (p
>0.05) [78].
With the studies depicting different pharmacological potentials of
T. vulgaris L., it becomes evident that more studies need to be conducted
using different approaches. For example, antidiabetic study conducted by
Aljarah and Hammed (2019) using in vitro enzyme inhibition studies lack
the usage of animal models and human cell lines [72]. In addition, a
dose-dependent evaluation of the plant extracts is essential in order to
understand the optimum dosage and toxicity. Analysis of binding of
S.M. Patil et al. Heliyon 7 (2021) e07054
18

individual compounds could be done using molecular docking, which can
become a pavement for drug development process. Also, studies need to
focus on feasible experimental designs, which can give better yields, less
toxicity and more pharmacological effect. If more studies with novel
pharmacological models are available, one can compare these studies
and suitable methods can be assayed to develop a specific dosage and
formulation. The anxiolytic EPM model by Komaki et al. (2016) has
highlighted the need for further evaluation of the mechanism behind the
observed effect [73]. More elucidations need to be done with respect to
the brain activity, secretion of hormones, and blood pressure upon the
administration of different doses of T. vulgaris L. extracts. Further, a study
conducted by Elbahnasawy et al. (2019) has discussed the anthelminthic
activity of T. vulgaris L. in vitro that could be better represented using
turkey as an animal model, instead of isolating the parasite from its feces
[74]. Furthermore, the evaluation of T. vulgaris L. activity on dental caries
need to be conducted using anti-infective or antibacterial assay primarily
because the caries is principally caused by bacteria. In total, these studies
depict T. vulgaris L.as a storehouse of different pharmacological proper-
ties attributed to the presence of various phytochemical compounds.
Thus, isolation, identification and determination of the pharmacological
properties of individual compounds needs to be focused. A summary of
the pharmacological properties of different extracts and isolated com-
pounds of T. vulgaris L. is given (Table 3). With this, one can understand
the pharmacological evaluations done so far, and can further refine the
research methodologies.
7. Clinical trials of T. vulgaris L. preparations
Despite many explorations in the aspects of pharmacological effi-
ciency, very little data are available on the clinical trials of T. vulgaris L.-
based products. Our literature survey could find only few studies eval-
uating the pharmacological potential of T. vulgaris L.-based products
using humans as study models [79,80]. These clinical trials used
different forms of T. vulgaris extracts and have resulted in the ameliora-
tion of the patients'/volunteers' health conditions (Table 4). Chemical
characterization of these herbal products is yet to be done, which could
decipher the specific action of phytochemicals involved in the pharma-
cological activity. Recently, a study by Caverzan et al. (2020) prepared a
gel named ThymLec gel 2%, which was used as a phytocosmetic to
improve facial health conditions [79]. A preparation from leaf and flower
extract (1.0%–3.0%) þwater, propanediol, glycerine þ5.0%–13.4%)
lecithin (additive) þbenzyl alcohol, potassium sorbate, tocopherol
(preservatives), application of this gel2 mg/cm
2
on female facial skin
with 2% concentration (20 mg/g w/w) twice a day (morning and eve-
ning) resulted in the reduction of area (7.0%), depth, and length (10.2%)
of the perioral wrinkles on day 60, whereas the benchmark produced a
reduction of 5.4% and 7.5% of the same parameters, respectively. The
length of the nasolabial lines was also decreased (8.9%). Similarly, area
(8.9%), breadth (3.9%), and length (11.1%) of the crow's feet wrinkles
were decreased with a corresponding reduction in the smile lines by
6.9%, 4.1%, and 10.1%, respectively. Face oval remodelling evaluation
using ThymLec 2% resulted in the reduction of total face volume (4.9
fold) in comparison with benchmark on day 60. In vitro adiponectin
synthesis was significantly increased in cultured 3T3-L1 embryonic fi-
broblasts treated with ThymLec 2% (122% at 0.0195% concentration)
and (137% at 0.039% concentration). This was 87% higher than the
benchmark used. A dose-dependent treatment of ThymLec 2% resulted in
the expression of PPAR-γmRNA levels, in comparison with that of the
control groups. ThymLec topical application led to adipogenesis and lipid
production, further augmenting cell volume and better remodelling of
face oval features. ThymLec modulates the PPAR-γsignalling pathway,
increasing adiponectin production and adipocyte lipid accumulation [79.
In another trial, which was focused on the amelioration effects of
T. vulgaris L. on dysmenorrhea (painful menstruation in females),
essential oil (25 drops) was investigated for its pain relieving prop-
erties [80]. Pain intensity during menstruation was measured in visual
analogue scale (VAS) on a 10 cm line before and after administration
of T. vulgaris L. essential oil. Pain intensity mean values reached the
lowest point (6.57–1.14) in case of oil consumers’group in compari-
son with that of the placebo (6.13–3.45) and ibuprofen (5.3–1.48) (p <
0.05). However, no significant results were obtained in case of
bleeding control by both oil and placebo (Ibuprofen 200mg þoil 25
drops). In addition, the oil was able to reduce clinical symptoms like
lower abdominal pain, nausea, mood swing, and fatigue after 48 h of
consumption (p <0.05). Around 71.4% of the volunteers marked
T. vulgaris L. oil as excellent whereas ibuprofen (28.0%) and placebo
(14.3) were found to be underscored. Here, anti-prostaglandin and
antispasmodic activity of T. vulgaris L. was attributed [80].
The use of T. vulgaris L. in the treatment of respiratory ailments
[37], was supported by a clinical trial investigating a combination of
T. vulgaris and Primula vulgaris (primrose, common thyme) dry powder
extract on bronchitis patients. The combination (oral dose of 160 mg
thyme and 60 mg primrose root extract for 11 days) reduced the cough
symptoms on day 9 with an efficacy of 73.7% in comparison with that
of the placebo (day 11; 57.8%) (p <0.0001). Reduction of bronchitis
severity score was observed only after 4 days in case of thyme and
primrose treatment group (-6.2) in comparison with the placebo (-4.1)
(p <0.05). Extract was well tolerated in most of the patients (183)
with only 1.7% adverse effects reported with 97.8 % patients rating
this combination as preferable. The extract also reduced minor
symptoms like disturbance in sleep, rising body temperature, chest
pain, and difficulty in breathing [12]. However, no underlying
mechanisms were decoded during this study. Therefore, under-
standing the mode of action along with product formulation with
lesser adverse effect and affordability are to be focused. Research also
needs to be focused on revealing the chemical components of such
herbal products made.
8. Toxicology
T. vulgaris L. is considered as a culinary herb and has been extensively
used since time immemorial in Europe and Mediterranean. In all the
records of ethnomedicinal properties, there is no mention of toxic effects.
Moreover, it has been described as the most used herb by the physician
Dioscorides in the 2nd century [8]. In support of this, present-day studies
report no cytotoxic activities of either the extracts or oil. A 28-day oral
dose toxicity assessment of thyme oil in rats was conducted in single (2,
000 mg/kg) and repeated dose (100, 250, and 500 mg/kg/day) methods,
where no signs of toxicity were detected. Even with the administration of
500 mg/kg/day, the body weight was found to be altered, yet with no
toxicity observed even on 28th day [81]. In support to this, a study
conducted by Benourad et al. (2014) assessed the activity of thyme
essential oil and found no histopathological changes in the rat liver and
kidney, except a few metabolic changes including increase in urea ni-
trogen, creatinine and uric acid levels. Besides this, alterations in
glycolysis, β-oxidative pathways, Kreb's cycle were also observed. In
total, this study revealed that injection of repeated doses of thyme oil
results in a few intermediary metabolic disturbances with no cytotoxic
activities [82]. Several studies conducted on cancer cell lines reported
the cytotoxic effect on tumour cells. However, thyme oil did not show
any effect on non-tumour liver PLP cells, even at the high concentration
of 400
μ
g/ml [66]. Even thymol is reported with no cytotoxic effect on
normal human cells except >50
μ
M[68]. From these studies, it becomes
evident that the T. vulgaris L. plant extracts and oil possess no cytotoxic
activity though there are reported cases of mild metabolic changes.
9. Perspectives and projections
Based on the available literature and gaps present with respect to
ethnopharmacology, pharmacology, and phytochemistry of T. vulgaris L.,
a few perspectives and future projections could be drawn out. Though a
tremendous amount of ethnobotanical literature is present, it lacks
S.M. Patil et al. Heliyon 7 (2021) e07054
19

specifications in terms of plant material, plant parts, and extracts used in
ancient times. With these foundational evidences, concrete modern-day
pharmacological approaches could be used, thereby supporting the
vital claims by the previous generation that in turn could be used in drug
discovery. Pharmacological findings need to be supported by the mech-
anisms. The action of plant extracts and individual compounds need to be
defined at the molecular level to find more druggable targets, which can
further be used to design a specific drug. Moreover, these studies have
used multicomponent assays with varying concentrations, which
complicate the determination of specific dose. Thus, qualitative and
quantitative assays using individual compounds in a dose-dependent
manner and a combination approach could reduce the burden of multi-
factorial results. Isolated compounds from the plant extracts could be
further modified according the requirement based on cytotoxic evalua-
tions done in primary screening. Such compounds could be analysed in
silico for effective binding and inhibitory effect on specific component or
any part of the identified target. Further, the bioavailability of the
compounds could be dealt to modify the compound to match the physi-
ological conditions of the target organ. Usage of in silico methods like
molecular modelling and docking can save a lot of animals, which are
used in laboratories.
Many of the studies quoted in this review need to be modified in
terms of their methodology. Antimicrobial evaluations need a reforma-
tion, where the zone of inhibition could be excluded. Metabolic ap-
proaches like enzyme inhibition, protein inactivation, and interference in
DNA and protein biosynthesis could be carried out. These mechanisms
support the specific drug discovery process. In case of antioxidant ac-
tivity, conventional assays like DPPH, ABTS, and FRAP need to be
excluded, as advancements including evaluation of cytokine factors and
antioxidant enzymes have been a major concern in recent years. Herein,
the use of in vivo approach in support to the in vitro findings needs to be
focused. Evaluations require animal model and cell lines with drug-
induced autoimmunity. As the inflammation is concerned with several
signalling pathways and immune cells, the fate of these cells and path-
ways need to be studied upon the introduction of plant fractions. Anti-
cancerous studies need to include animal model and more in vivo ap-
proaches than cell lines, along with a dose-dependent approach using
individual compounds.
These suggestions are utmost necessary to enhance the pharmaco-
logical studies associated with the plant products. Following up these
protocols will help in the generation of data from phytochemicals, which
can be transferred into clinical trials to get a completely processed plant
product. Furthermore, high quality, advanced phase, randomized,
placebo-controlled, multi-centred, randomized, and double-blind clinical
trials could be done.
10. Conclusion
The present-day proximate analysis of T. vulgaris L. supports the use of
the plant as an important ingredient in food since the Egyptian era.
Alongside biomolecules, the rich diversity of phytochemicals present in
the T. vulgaris L. plant may be accountable for major health benefits. The
ethnomedicinal uses of the plant have revealed the importance in treat-
ing various diseases. Several studies conducted in vitro and in vivo using
cell lines and animal models with induced pathological conditions have
proved the efficiency of the plant as a therapeutic agent. T. vulgaris L. is
found to be effective against both infectious and foodborne pathogens.
Both the oil and extract of the plant were evaluated for their effects on
inflammation and cancer. Along with the proven pharmacological as-
pects, there are several activities that are yet to be reported. For example,
the literature survey could find only 2 studies with significant results on
anti-diabetic potential of the plant. Furthermore, inhibition of acetyl-
choline esterase could be assessed for more physiological aspects, espe-
cially with diabetes. The extensive inhibitory effect on foodborne and
oral pathogens has proved its possible use as a preservative agent and an
important drug for dental caries, respectively. Meanwhile, T. vulgaris L.
also possesses anti-helminthic property, which can make it a prominent
therapeutic agent against parasitic infections. Also, a great amount of
work needs to be done with respect to its skin protection activity. The
promising evidence on the cell proliferation post-exposure to UV rays
suggests its clinical trials for establishing a potent product. Pre-clinical
studies at a large scale including in vivo cytotoxicity assays are sug-
gested for the evaluation of accurate dosage levels of different plant
fractions. Pharmacokinetics and pharmacodynamics of the compounds
need to be focused. In addition, interactions with dietary molecules and
drugs need to be assessed for the development of nutraceuticals. The
pharmacological analysis at the genetic and proteomic level, along with
commercially available drugs would determine the beneficial and
harmful effects of the plant extracts or individual compounds. In total,
T. vulgaris L.can open the doors to the development of many prominent
therapeutic agents with diverse nature of applications to resolve several
health maladies.
Declarations
Author contribution statement
Ramith Ramu; Shashank M Patil; Prithvi S Shirahatti: Conceived and
designed the experiments; Performed the experiments; Analyzed and
interpreted the data; Contributed reagents, materials, analysis tools or
data; Wrote the paper.
Raghavendra G. Amachawadi; Chandan Shivamallu: Analyzed and
interpreted the data; Contributed reagents, materials, analysis tools or
data; Wrote the paper.
Funding statement
This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.
Data availability statement
No data was used for the research described in the article.
Declaration of interests statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
All the authors thank JSS Academy of Higher Education and
Research, Mysore for constant support and encouragement to carry out
this work.
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