International Journal of Chemical Science

International Journal of Chemical Science

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Volume 1; Issue 2; November 2017; Page No. 110-116

Different extraction methods and antioxidant properties of thyme (Thymus vulgaris L.) herb
*1
Eqbal MA Dauqan, 2 Faten H Thamer, 3 Khalid M Naji, 4 Yahia Alshaibi
1
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, University Kebangsaan Malaysia,
Bangi Selangor, Malaysia
2, 3, 4
Chemistry Department, Faculty of Science, Sana'a University, Sana'a, Yemen

Abstract
Medicinal plants have played an essential role in the development of human culture. Medicinal plants are a source of traditional
medicine. Among different species of Thymus, (Thymus vulgaris L.) is used more than other species in therapeutic dosage forms.
In Traditional medicine, Thymus vulgaris L. is cultivated in many countries by most people especially in rural areas depend on
herbal medicines to treat many diseases including inflammation-related ailments such as rheumatism, muscle swelling, insect bites
and pains. Also the modern medicine in essential oil of thyme has demonstrated the compounds have shown antioxidant,
antibacterial and antifungal properties. Thus the highlight of the paper was to review the different extraction methods and
antioxidant properties of Thyme (Thymus Vulgaris) herb.

Keywords: medicinal plants, traditional medicine, Thymus vulgaris L., antioxidants

1. Introduction They are among our oldest medicines and their increasing use
Human body constantly creates free radicals culminating in an in recent years is evidence of public interest in alternatives to
“oxidative stress” when their elimination by antioxidant conventional medicine [11]. Since prehistoric times, herbs have
defense mechanisms is not sufficient [1]. Oxidative stress also been the basis for nearly all medicinal therapy until
contributes to the pathogenesis of many human diseases; synthetic drugs were developed in the 19th century [16].
therefore the intake of antioxidative agents is important for the Herbs are still found in 40% of prescription drugs [16]. In
prevention of chronic diseases [2]. Antioxidants play an addition, herbs are used for many other purposes including
important role in preserving of food too. In food industry beverages such as tea, dyeing, repellents, fragrances Many
widely used synthetic antioxidants as butylated hydroxitoluen recent studies found out that herbs contain various
(BHT) and butylated hydroxianisole (BHA) are very effective phytochemicals including antioxidants [17, 18, 19, 20, 21]. At the
because of their low cost, high thermal stability and efficiency same time it has been shown that Reactive Oxygen Species
but they are instable and they can play role as promoters of (ROS), such as hydroxyl radical, hydrogen peroxide, and
carcinogenesis [3, 4, 5, 6]. Due to these reasons, there is a superoxide anions, are produced as by-products in aerobic
growing interest in the study of natural additives as potential organisms and have been implicated in the pathology of a vast
antioxidants [7]. The presence of antioxidants in many spices variety of human diseases including cancer, atherosclerosis,
gives them food-preserving properties too, especially in diabetic mellitus, hypertension, Aids, aging, cardiovascular
preventing oxidation of lipids [6]. diseases, cataracts, immune system decline and brain
Nevertheless, the use of synthetic antioxidants in the food dysfunction [9]. It was also found out that free radical
industry has been questioned regarding its innocuousness. formation is controlled naturally by various beneficial
Studies about spices and aromatic herbs have been widely compounds, namely antioxidants. There has been lots of
emphasized, can act as an alternative to prevent the oxidative evidence that consuming foods of plant origin (fruits,
deterioration of food and reduce the use of synthetic vegetables, tea, coffee and others) is associated with reduced
antioxidants [8]. The use of natural antioxidants from food incidence of these diseases [19].
plants has the following advantages: They are accepted by the Moreover, knowledge and application of such potential
consumers; they are considered safe; they do not need safety antioxidant activities in reducing oxidative stresses in vivo has
tests; they have functional and acceptable sensory properties prompted many investigators to search for potent and cost-
[9]
. Studies found in literature have demonstrated that the effective antioxidants from various plant sources [19]. These
spices belonging to the Lamiaceae family, as well as their research activities have contributed to new or renewed public
extracts and essential oils, are efficient antioxidants [8, 10, 11, 12, interests worldwide in herbal medicines, health foods, and
13, 14, 15]
. nutritional supplements. It is therefore of big interest to
Herbs are usually considered as plants with aromatic systematically check for the presence of antioxidants and their
properties and are mainly used in spicy foods and for antioxidative capacity in medicinal herbs that are widely used
preparation of herbal teas in folk medicine [13]. Medicinal in local folk medicine [ 22].
plants have always been considered as a source of health [16]. Studying medicinal plants helps to understand plant toxicity

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and protect human and animals from natural poisons.

Therefore the objective of this paper was to review the
different extraction methods with different conditions used to
extract thymus oil and antioxidant properties of Thyme
(Thymus Vulgaris) herb.

2. Lamiaceae Family
Lamiaceae family is a group of about 210 genera and some (A) (B)
3500 species [23]. Many of them are commonly used as
culinary herbs. They are often cultivated because of their
aromatic qualities and also of their easy cultivation. Many
species of the family are reported with high phenolic contents
and antioxidant capacities [24].

2.1 Thyme (Thymus Vulgaris L)

Thyme is one herb of Lamiaceae family. It dates back to 3500
BC by Sumerians and Egyptians [25]. Its spread to Europe was (C)
due to Romans, as they used it to give aromatic flavors to Fig 1: (A)Thyme in Bani-Matar, Sana'a, Yemen, (B) thyme in Al-
liqueurs & cheese. Thyme is the general name for the many shahel, Hajah, Yemen and (C) thyme in Maswar, Amran, Yemen
herb varieties of the Thymus species, all of which are native to
Europe and Asia. Common or garden thyme is considered the Thymus vulgaris L. is one of the most popular hybrid plant
principal type, and is utilized commercially for flowering and used worldwide [31]. Locally Thymus vulgaris L species native
ornamental purposes [26]. to the Sultanate of Oman and Yemen known as “zaater” and
The name of Thyme in its Greek form was first given to the their dried whole parts are used in herbal tea, condiments, and
plant by the Greeks as a derivative of a word which meant to folk medicine. Since ancient times, this aromatic plant has
fumigate either because they used it as incense for its balsamic been used for the preparation of different aliments to cure
odor or because it was taken as a type of all sweet smelling various curable and chronic diseases [32].
herbs. Others derive the name from the Greek words Thyo, In food Thymus vulgaris L. can be used in fresh or dried form
meaning perfume or Thumus, signifying courage the plant especially for tea. From the medical point of view, thyme has
being held in ancient and medieval days to be a great source antiseptic effect due to the presence after penoidic compounds
of invigoration its pleasant qualities inspiring courage [27]. and also antifungal effects [32].
Thyme is a tiny perennial shrub, with a semi ever green As a tea it is useful for colds, asthma and bronchitis [25] or for
groundcover that seldom grows quite 40 cm tall it’s each the treatment of acne. The antiseptic effects are mainly due to
horizontal and upright habit. The stems become woody with the presence of volatile oil, which mainly contains thymol,
age [28]. Thyme leaves are terribly little, usually 2.5 to 5 mm carvacrol, p-cimen, linalool, α-pinen [27] and other mono- and
long and vary significantly in form and hair covering, sesquiterpenes (β-cariophilen, germacren D or nerolidol) [33].
depending on the variety, with every species having a rather The antioxidant activity of wild thyme is given especially by
completely different scent. T. vulgaris leaves are oval to phenolic acids (rosmarinic acid) [34] and by flavonoids
rectangular in form and somewhat fleshy aerial components (quercitin, eriocitrin, luteolin, apigenin, serpyllin) [35].
are used for volatile oil production, principally by steam Many factors such as harvest time, seasonal variations, drying
distillation [25, 28]. conditions, etc. may affect the composition of thyme. Thyme
India and Europe thyme grows well during a temperate to contains high concentrations of phenols [34]. Carvacrol and
heat, dry, sunny climate, and wherever the plants don’t seem thymol are the main phenolic components which are primarily
to be shaded. It desires full sun to grow to its best potential. responsible for its antioxidative activity [36].
Thyme doesn’t like excessive wet as a result of its condition it
will gets rot diseases [29]. Thyme prefers lightweight, well- 3. Methods of Extraction of Thyme Oils
drained soils with a pH of 5.0 to 8.0. Thyme species do best in Michalak [37] reported that the thyme essential oil quality and
coarse, rough soils that may be unsuitable for several yield depend on many factors and choosing a suitable
alternative plants [28, 30] . It is slightly spicier than oregano and extraction method is very important.
sweeter than sage. Numerous cultivars and hybrids have been Essential oil of thyme herb has usually been obtained by some
developed for ornamental purposes. Nomenclature can be very methods such as:
confusing. French, German and English varieties vary by leaf
shape and color and essential oils. The many cultivars include 3.1 Steam Distillation
'Argenteus' (silver thyme).The cultivar 'Silver Queen' possess Steam distillation procedure is widely used for essential oil
white-margined leaves [30]. In Yemen thyme is a seasonal separation; beyond its efficiency, this method gives a greater
shrub, with a green leaves that seldom grows quite 5 cm tall to or lesser compounds instability under the influence of high
extend horizontal about 50cm or more. Figure 1 shows the temperature. There are two methods use steam distillation:
varieties of thyme in different cities in Yemen. simple steam distillation and Clevenger system [38].

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3.2 Solvent Extraction compounds in reference to their ability to scavenge the radical
Solvent extraction was the main method adopted by most anion 2, 2-diphenyl-1-picrylhydrazyl (DPPH) [20].
researchers to extract phenolics from thyme leaves. This is a The DPPH is a very stable and commercially available free
process designed to separate soluble compounds by diffusion radical, able to accept an electron or a hydrogen atom creating
from a solid matrix using a liquid matrix. The aim of the diamagnetic molecule DPPH (non radical). The radical
extraction is concentrate antioxidant components of raw absorbs visible light at 515 nm appearing red-violet in color,
material [37]. but when it is mixed with a protic solution the reduced form
(non radical) is generated with the loss of the violet color and
3.3 Supercritical Fluid Extraction the appearance of a pale yellow color [41]. During early work
Can offer a good yield and preserve the properties of on DPPH, researchers concluded that the reaction takes place
antioxidants. This method can be used for the extraction of through the transfer of a hydrogen atom between the radical
polyphenol from plant tissue and greatly facilitates the and the solution. In fact, the reaction starts with an electron
extraction process and reduces extraction time due to low transfer, while hydrogen atom abstraction is a slow secondary
viscosity and relatively high density of supercritical fluid; it reaction only occurring in strong hydrogen bond-accepting
can also minimize possible degradation because it can operate solvents such as methanol and ethanol. As in other electron
in absence of light and air. Supercritical carbon dioxide is the transfer-based assays, the scavenging activity is strongly
most widely used extraction solvent [39]. influenced by the pH and the solvent properties. A good
choice for analyzing both lipophilic and hydrophilic
3.4 Pressurized Liquid Extraction (PLE) antioxidants is a 50% (v/v) water/ethanol mixture [42].
A relatively recent solvent extraction technique could in The DPPH assay is a valid and easy method for evaluating the
principle eliminate some of the drawbacks of the classical scavenging activity of antioxidant compounds because the
solvent extraction methods. Pressurized liquid extraction is radical is a stable molecule and does not need to be generated.
based on the use of solvents at temperatures above their The results are highly reproducible and comparable to other
normal boiling points and pressures enough to keep the antioxidant detection methods [41]. A drawback is that the
extracting fluid in the liquid state during the whole extraction solvent effect has to be quantified carefully. In the case of a
process. By applying these conditions, faster extraction protic solvent, the competition of hydrogen abstraction
processes result in which typically higher extraction yields are between the antioxidant and the solvent can lead to false
obtained with lower volumes of organic solvents [14]. positive results, which obviously invalidate the determination
[41].

4. Antioxidant Properties
An antioxidant is a molecule that inhibits the oxidation of 4.2 Ferric Reducing Antioxidant Power Assay (FRAP)
different molecules. Oxidation is a chemical process that The FRAP assay measures antioxidant capacity by studying
transfers electrons or hydrogen from a substance to an the reduction of the complex ferric tripyridyl-triazine (FeIII-
oxidizing agent. Oxidation reactions will produce free TPTZ) at low pH. The reduction of ferric ions to ferrous ions
radicals. In turn, these radicals will begin chain reactions. leads to an intense blue-colored ferrous tripyridyl-triazine
Once the chain reaction happens in a cell, it will cause damage complex, the formation of which can be followed
or death to the cell. Anti-oxidants stop these chain reactions spectrophotometrically [43, 44, 45]. The difference in absorbance
by removing free radical intermediates and inhibit different with respect to a reaction mixture containing ferrous ions of
oxidation reactions. The leafy parts of thyme and its oil are known concentration is directly related to the total ferric
utilized in foods for the flavor, aroma and preservation and reducing power of the antioxidant in the sample. The FRAP
additionally in folk medicines [25, 40]. Table 1 shows the assay provides fast and reliable results for plasma, single
different methods used for thyme extraction, antioxidant antioxidants in pure solution, and mixtures of antioxidants in
values and essential oil analysis. All the previous studies in aqueous solutions. Moreover, the FRAP assay is simple and
Table 1 used DPPH and FRAP methods to determine the inexpensive. The only drawback of this method is that it
antioxidants activity. cannot be used to determine antioxidants containing
oxidizable groups [46, 47, 48].
4.1 2, 2-Diphenyl-1-Picrylhydrazyl Radical Assay (DPPH)
The DPPH assay measures the antioxidant properties of

Table 1: The different methods used for thyme extraction, antioxidant values and essential oil analysis
Essential
Main Methods of Value of Total
Country Ratio of compounds Value of DPPH Oil References
compounds extraction FRAP phenols
Analysis
Thymol 61% %92.4±0.4 IC50
, Carvacrol, 20.6% 1.8-Cineole 14.2%, thymol and Solvent =1.5 GAE G.C
Ankara, Turkey - - [30]
q-Cymene, 22.2% linalool 4.8% borneol, carvacrol extraction (mmol/L HPLC
7.5% Apinene and 6.6% Camphor methanol)
Thymol (41.33%), p-Cymene (18.08%),
γ-Terpinene (13.12%), thymol and Hydrodist- IC50= 4.21 ±
south of France - - G.C - MS [5]
.1,8 Cineole (40.2%) and carvacrol illation 0.08 µg/mL
α-Pinene (13.2%).

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1-Octen-3-ol 0.48%
Supercriticl
p-Cymene 1.77% 1,8 Cineole 7.23% thymol and
Murcia, Spain fluid - - - GC–MS [39]
Sabinene 1.39% Linalool 2.05% Camphor carvacrol
extraction
9.55% Thymol 57.46% Carvacrol 3.44 %
482.92
(mmol 5.60mg
[IC50 trolox/gT equivale
Caffeic acid, ,Rosmarinic acid Soxhlet
Morocco - (mg/mL TAE)] AE) nt [11]
andQuercetin extractor
0.44 ± 0.02 65.00 ± affeicaci
9.40 d/g
TAE)
Carvacrol (21-37%) Cravacrol Solvent
Armenia - - - GC.MS [24]
Thymol (10–17%) and thymol extraction
- Cravacrol Steam
Palestine - - - GC-MS [49]
and thymol Distillation
Phellandrene2.91%
α-Pinene2.79%
β-Myrecene7.56% Cymene6.06%
γ-Terpinene26.0%
Bucharest, Cevacrol0.95% Thymol (30.86%) Cravacrol steam GC.MS-
89%. - - [50]
Romania p-Cymene (30.53%) and thymol distillation HPLC
Myrcene 1.63% Sabinene 4.24%
linalool 2.73%
Borneol 3.16%
Caryophyllene 2.48%
Soxhlet
Sultanate of method- 2000
- - 76-98% [51]
Oman Solvent mg/L
extraction
phydroxybenzoic acid 0.05 ±0.01 69.05%
Put in boiling HPLC-
Croatia Rosmarinic acid 17.45±0.21 Apigenin-7-O- - IC50= - - [52]
water PDA
glucoside 2.37±0.01 0.30g/l
Seva Flora
1.13 g 4.16 g
Valtice Solvent [9]
- - - GAE/100 GAE.10 -
extraction
g−1dw 0 g−1dw
9.07 ±
Constantine, Solvent 0.002
- - 70% - - [53]
Algeria extraction mg/g
TAE
Thymol 47%
p-cymene 34%
Thymol and Steam
Paris (France) Linalool1 .26% - - - GC-MS [54]
p-Cymene distillation
Carvacrol4 .20%g-Terpinene 2.03%a-Pinene
1.50%
Thymol 12–61% Carvacrol 0.4–20.6%,
1,8-Cineole %92.4±0.4 IC50
(0.2–14.2%), =1.5 GAE
Thymol and Solvent G.C
(Mugla, Turkey q-Cymene (mmol/L - - [55]
Carvacrol extraction HPLC
(9.1–22.2%), methanol)
Linalool (2.2–4.8%), Borneol (0.6–7.5%),a-
Pinene (0.9–6.6%), and Camphor (0–7.3%
Thymol (41.33%), p-Cymene (18.08%),
Thymol and Hydro- IC50= 4.21 ±
Iran γ-Terpinene (13.12%) - - G.C /MS [56]
Carvacrol distillation 0.08 µg/mL
.1,8 Cineole (40.2%) and α-Pinene (13.2%).
1-Octen-3-ol 0.48% p-Cymene 1.77% 1,8
Supercritical
Cineole 7.23% Sabinene 1.39% Linalool Thymol and
Greece fluid - - - GC–MS [44]
2.05% Camphor 9.55% Thymol 57.46% Carvacrol
extraction
Carvacrol 3.44 %
482.92
(mmol
5.60mg
[IC50(mg/mL trolox/gT
Catalonia Soxhlet equivale
Caffeic acid ,Rosmarinic acid andQuercetin - TAE)] 0.44 ± AE) - [8]
(Spain) extractor nt caffeic
0.02* 65.00 ±
acid/g
9.40
TAE)
Romania Cravacrol Solvent
Carvacrol (21–37%) Thymol(10–17%) - - - GC.MS [24]
(Fares) and Thymol extraction
Cravacrol Steam HS-
Ravenna, Italy - - - - [57]
and Thymol Distillation GCMS

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International Journal of Chemical Science

Phellandrene2.91%α-Pinene2.79%β-
Myrecene7.56%
m-Cymene6.06%γ-Terpinene26.0%
northern Cravacrol steam GC.MS-
Cevacrol0.95% thymol (30.86%) 89%. - - [10]
California USA and thymol distillation HPLC
p-Cymene (30.53%) Myrcene 1.63%
Sabinene 4.24% Linalool 2.73%
Borneol 3.16% Caryophyllene 2.48%
Soxhlet and
2000
Saveh,- Iran - - Solvent 76-98% [51]
mg/L
extraction
Phydroxybenzoic acid 0.05 ±0.01
Shambolia Put in boiling 69.05%IC50=0.3 HPLC-
Rosmarinic acid 17.45±0.21 - - - [52]
farm-France water 0g/l PDA
Apigenin-7-O-glucoside 2.37±0.01
1.13 g 4.16 g
Estahban Solvent
- - - GAE.100 GAE.10 - [9]
Branch, Iran extraction
g−1dw 0 g−1dw
9.07 ±
Solvent 0.002
Croatia - - 70% - - [53]
extraction mg/g
TAE
517 ±
Grabowo
Solvent 0.54 mg
Wielkie- - - - - HPLC [58]
extraction GAE/10
Poland
0 g dw
Tthymol (50.48%)
Belgrade, Thymol and Solvent
p-Cymene (24.79%), Linalool (4.69%), - - - GC-MS [59]
Serbia p-cymene. extraction
γ Terpinene (4.14%) 1,8-Cineole (4.35%)
Pinene 2.0 (0.3),
p-Cymen) 15.5 (0.8)
Terpinene 2.2 (0.3), Linalool 40.2 (3.3),
Borneol 1.2 (0.2), 1.3
Dalmatia- Thymol and
1,4-Terpineol 85.35% Fe2+/mm - GC-MS [60]
(Croatia) linalool
6.8 (0.6), hydro- ol L-1
á-Terpineol4.1 (0.4) Linayl acetate distillation
3.2 (0.2),
Thymol 16.3 (0.6) , Carvacrol 6.8 (1.7)
34.50
southern Solvent
92.17% - (µgGAE/ [11]
Morocco - extraction -
- mg DM

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