ANTICANCER COMPOUNDS FROM MEDICINAL PLANTS


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Original Article 

 
Preliminary investigation of free radical scavenging activity and 
total phenolic content of three cultivated Thymus chemotypes 
from Bulgaria  
 

Milena Nikolova
1*

 Genka Zhekova
2
 Anatoli Dzhurmanski

2 

 

1 
Institute of Biodiversity and Ecosystem Research, 23, Acad.G. Bonchev St., 1113 Sofia, 

Bulgaria 
2
 Institute of Roses, Essential and Medicinal Cultures, bul. Osvobozhdenie 49, 6100 

Kazanlak, Bulgaria 

 

 

* E-mail: milena_n@bio.bas.bg 

 
Abstract:  

Nikolova, M., Jekova, G., Dzhurmanski, A.: Preliminary investigation of free radical scavenging activity 

and total phenolic content of three cultivated Thymus chemotypes from Bulgaria , Biologica Nyssana, 3 

(1), September 2012: 11-15. 

 

Methanol extracts of three varieties of Thymus “Slava”, “German winter” and “Pagane”, representatives 

respectively of citral, thymol and geraniol chemotype were evaluated for their antioxidant potential and 

phenolic content. Total content of phenols was determined by spectrophotometric method using Folin-

Ciocalteu reagent. The antioxidant potential of methanol extracts was estimated by 1,1-diphenyl-2-

picrylhydrazyl radical (DPPH) free radical assay. All of the examined extracts exhibited significant free 

radical scavenging activity and their IC50 values were below 50 μg/mL. The highest antioxidant activity was 

determined for the extract of thymol chemotype “German winter” with IC50 value – 19.34 µg/mL. The extract 

of this variety had the highest amount of phenols 75.61 mg/g extract. The other two varieties “Slava” and 

“Pagane” have a lower content of phenols and antiradical activity. Influence of three stages of flower 

development on antiradical activity and total phenolic content was examined for variety “Pagane”. The 

highest antioxidant activity was determined on the extract prepared from plant material collected at the stage 

“end of flowering”. For the first time, provides data on the phenolic content and antiradical activity of 

varieties “Slava” and “Pagane”. These results are the basis for selection of varieties with high antioxidant 

potential for the preparation of cosmetic and herbal products.  

 

Key words: Thymus vulgaris, Thymus marschalianus, DPPH, citral, thymol, geraniol 

 

Introduction 
 
The genus Thymus L. (Lamiaceae) comprises 

about 300 species of perennial aromatic, herbaceous 

plants with many subspecies, varieties, subvarieties 

and forms. The plants are extensively used in 

phytotherapy, cosmetic and food industries 

(S t a n e v , 1974; N i k o l o v , 2006). Essential oils, 

flavonoids (highly methylated flavonoids, luteolin 

derivatives), biphenils and phenolic compounds 

have been determined as main constituents of the 

thyme with antioxidant, antiseptic, antiinflamatory 

and antimicrobial properties (J o v a n o v i ć  et al., 

1995; H a r a g u c h i  et al., 1996; F e c k a  & 

T u r e k , 2008; B o r o s a  et al., 2010). 

The thyme shows a great species diversity 

which reflects to the composition of the essential oil 

content. Based on the dominant monoterpene in the 

essential oils of different Thymus chemotypes, 

compounds such as geraniol, thymol, α-terpineol, 

linalool, carvacrol can be distinguished 

(T h o m p s o n  et al., 2003; C h i z z o l a  et al., 

3 (1) • September 2012: 11-15 



BIOLOGICA NYSSANA  3 (1)  September 2012: 11-15 Nikolova, M. et al.  Preliminary investigation of free radical… 

 

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2008). In recent years, several reports have been 

published concerning the antioxidant activity on the 

essential oils on different thyme chemotype (J u k i  

et al., 2005; B o u n a t i r o u  et al., 2007; M i g u e l  

et al., 2007; S t o i l o v a  et al., 2008; G r i g o r e  et 

al., 2010; T e p e  et al., 2011) while the studies on 

antioxidant activity of methanol extracts from 

different chemotypes and varieties are insufficient 

(J o v a n o v i ć  et al., 1995; K u l i š i ć  et al., 2006). 

A positive correlation between antioxidant activity 

and phenolic content in plant extracts has been well 

documented (M i l i a u s k a s a  et al., 2004; 

K i s e l o v a  et al., 2006). Widely used methods for 

assessing the total content of phenols and 

antioxidant properties of plant extracts are based on 

Folin-Ciocalteu reagent and 1-diphenyl-2-

picrylhydrazyl (DPPH) radicals (K i s e l o v a  et al., 

2006, G i o r g i  et al., 2009; N i ć i f o r o v i ć  et al., 

2010; M a r i n o v a  & B a t c h v a r o v , 2011).  

The purpose of the present work is to examine 

antioxidant activity and total phenolic content 

varieties created and cultivated in Bulgaria: thyme 

“Slava” (Thymus vulgaris, citral chemotype) and 

“Pagane” (Thymus marschalianus Wild., geraniol 

chemotype) as well as of the introduced variety 

“German winter” (Thymus vulgaris, thymol 

chemotype). This study contributes to the 

knowledge of the antioxidant properties of the 

Thymus chemotypes. 

 

Materials and methods 
 
Plant material 

The plants used for the present study were 

cultivated in the experimental field of Institute of 

Roses, Essential and Medicinal Cultures, Kazanlak. 

Aerial parts of two varieties “Slava” and “German 

winter” were collected at full flowering stage. Aerial 

parts of the variety “Pagane”, collected in three 

stages of flowering – beginning of flowering, full 

flowering and end of flowering were analyzed.  

 

Preparations of extracts 

Air-dried, powdered plant material (1 g) was 

extracted with 80% methanol in an ultrasonic bath. 

After evaporation of the solvent the crude extract 

was subjected to subsequent analyses. 

 

Determination of total phenolic content 

Total phenolic content of the methanol 

extracts was determined by employing the methods 

given in the literature including Folin–Ciocalteu 

reagent and gallic acid as standard (G i o r g i  et al., 

2009; N i ć i f o r o v i ć  et al., 2010). Plant extracts 

were diluted to the concentration of 1 mg/mL, and 

aliquots of 0.25 mL were mixed with 2.5 mL of 

Folin–Ciocalteu reagent (previously diluted 10-fold 

with distilled water) and 2 mL of Na2CO3 (6%). 

After 1 h of staying at the room temperature, the 

absorbances of the samples were measured at 765 

nm on spectrophotometer versus blank sample. 

Total phenols were determined as gallic acid 

equivalents (mg GA/g extract) by the following 

formula: 

 

C = c x V / m  

 

where C - total content of phenolic compounds, 

mg/g plant extract, in GAE; c - the concentration of 

gallic acid established from the calibration curve, 

mg/mL; V - the volume of extract, mL; m - the 

weight of pure plant methanolic extract, g. 

 

Free radical scavenging activity 

Free radical scavenging activity of plant 

extracts was evaluated using a 1,1-diphenyl-2-

picrylhydrazyl (DPPH) assay (C h o i  et al., 2002; 

S t a n o j e v i ć  et al., 2009). Different 

concentrations of plant extract (10, 20, 50, 100 and 

200 μg/mL, in methanol) were added at an equal 

volume (2.5 mL) to methanol solution of DPPH (0.3 

mM, 1 mL). After 30 min at room temperature, the 

Ab (absorbance) values were measured at 517 nm 

on a spectrophotometer (Jenway 6320D) and 

converted into the percentage antioxidant activity 

using the following equation:  

 

DPPH antiradical scavenging capacity (%) = 

[ 1- ( Absample – Abblank ) / Abcontrol ] x 100 
 

Plant extract solution (2.5 mL) in methanol 

(1.0 mL) was used as a blank, while DPPH solution 

in methanol was used as a control. The IC50 values 

were calculated by sigmoid non-linear regression 

model using plots, where the abscissa represented 

the concentration of tested plant extracts and the 

ordinate the average percent of scavenging capacity 

(Software Prizm 3.00). IC50 values denote the 

concentration of sample required to scavenge 50% 

of DPPH radical.  
 

Statistical analysis  

Statistical analysis was carried out using 

excel. All experiments were performed in triplicate. 

Results are presented as a value ± standard deviation 

(SD). Significant levels were defined at p<0.05.  

 

 
 



BIOLOGICA NYSSANA  3 (1)  September 2012: 11-15 Nikolova, M. et al.  Preliminary investigation of free radical… 

 

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Results and discussion 
 

Methanol extracts of three chemotypes of 

Thymus (citral, thymol and geraniol) were 

examined for their antiradical potential using a 

DPPH assay and expressed as IC50 value- extract 

concentration providing 50% inhibition of the 

DPPH solution (Table 1).  

The all examined extracts exhibited 

considerable free radical scavenging activity and 

their IC50 values were below 50 μg/mL. The extract 

of variety “German winter” (thymol chemotype) 

showed the strongest antioxidant activity with IC50 

value 19.34 µg/mL, followed by citral chemotype 

“Slava” (33.18µg/mL)  and geraniol chemotype 

“Pagane” (47.72 µg/mL). The established 

differences in the antioxidant potential of studied 

varieties can be explained by different chemotype – 

different composition of essential oils. The thyme 

with the highest content of thymol (“German 

winter”) has the strongest antioxidant activity. This 

is in accordance with results reported by Chizzola et 

al. (2008) which prove that the essential oil with 

high content of thymol and/or carvacrol have the 

highest antioxidant activity. These data allow us to 

assume antioxidant potential of thyme based on the 

essential oil composition and individual components 

(geraniol, linalool, thymol, etc.).  

A total phenolic content of studied varieties 

of Thymus “Slava”, “German winter” and “Pagane” 

was determined (Table 1). The extracts of variety 

“German winter” contain the highest amount of 

phenols (75.61 mg/g),  followed by “Slava” (56.6 

mg/g) and “Pagane” (45.42 mg/g). The greater 

amount of phenolic compounds positively correlated 

to more potent radical scavenging effect. 

Carried out correlation analyses shows strong 

positive mutual dependence between total phenolic 

content and antioxidant (DPPH scavenging) activity 

of investigated extracts (R=0, 9812). This is in 

accordance of several reports for such positive 

correlation between phenols and antioxidant activity 

of plant extracts (M i l i a u s k a s a  et al., 2004; C a i  

et al., 2004; S h a n  et al., 2005; K i s e l o v a  et al., 

2006; V i l l a ñ o  et al., 2007).  

The antioxidant potential and total phenolic 

content of extracts obtained from material collected 

at differing stages of flower development of the 

variety “Pagane” was examined. It was found that 

extracts of plant material gathered at stage “end of 

flowering” had the highest antioxidant activity with 

IC50 value of 34.28 µg/mL (Table 2). The extracts 

prepared from plant material collected at the stages 

“beginning of flowering” and “full flowering” 

showed IC50 values 47.72 and 67.00 µg/mL, 

respectively. Thyme is recommended to be picked 

up at the “end of flowering” stage, when their 

antioxidant activity is 40% higher than in a “full 

flowering” stage and 95% higher than in stage 

“beginning of flowering”. Opposite to the results on 

antioxidant activity, it was found that there are no 

statistical significant differences (p>0,05) in the 

phenolic content of the examined extracts during 

flowering period. Total phenols ranges from 44.08 

to 46.82 mg/g. This fact suggests that radical 

scavenging activity of the studied extracts is not 

only due to phenol compounds.  

 

 

Table 1. Total phenolic content and free radical scavenging activity of methanol extracts of three Thymus 

varieties  

Thymus varieties Total phenols 

mg GAE/g extract* 

Free radical scavenging activity 

IC50 (µg/mL)* 

Pagane 46.82±1.22 47.72±5.41 

Slava 56.6±1.25 33.18±1.89 

German winter 75.61±2.71 19.34±1.76 
* 
values represent mean ±SD, n=3 

 

Table 2. Total phenolic and antiradical activity of extracts of variety “Pagane” collected at three stage of 

flowering 

Stage of flowering Total phenols 

mg GAE/g extract* 

Free radical scavenging activity 

IC50 (µg/mL)* 

beginning of flowering  44.08±1,80 67.00±7.63 

full flowering 46.82±1.22 47.72±5.41 

end of flowering 45.42±2.62 34.28±1.98 
* 
values represent mean ±SD, n=3 



BIOLOGICA NYSSANA  3 (1)  September 2012: 11-15 Nikolova, M. et al.  Preliminary investigation of free radical… 

 

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Conclusion 
 

In the present study, the total phenolic content 

and antioxidant capacity of three varieties of 

Thymus “Slava”, “German winter” and “Pagane” 

were determined. High positive correlation was 

found between investigated indexes. Bulgarian 

varieties thyme – “Slava” and “Pagane” was studied 

for the first time for their total phenolic content and 

the antioxidant activity. The methanol extracts of all 

examined samples showed high radical scavenging 

activity (IC50 <50 µg/ml). Extracts of thymol 

chemotype “German winter” exhibited the strongest 

antiradical activity and highest content of phenols 

among studied varieties. Therefore, thymol 

chemotype can be priority for utilization in 

phytoterapy and cosmetics. The recommended 

concentration of thyme solution for lipid stabilizing 

in cosmetics and food products is 50 µg/ml, when it 

provides 95% free radical inhibition whilst for the 

other types of thyme, it is twice higher. Thyme is 

recommended to be picked up at the “end of 

flowering” stage, because then the content of 

antioxidants is the highest. Obtained data will be 

employed as a basis for selection of varieties with 

high antioxidant potential for the preparation of 

cosmetic and herbal products, as well as for the 

optimization of harvesting time.  

 

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