Iraqi J Pharm Sci, Vol.21(1) 2012               Antibacterial and phytochemical study of salvia officinali 

93 

 

Antibacterial and Phytochemical Study of Iraqi Salvia officinalis 

Leave Extracts 
Lana Y. Muttalib*      and       Alaadin M. Naqishbandi*

,1 

 

*Department of Pharmacognosy, College of Pharmacy, Hawler Medical University, Erbil, Iraq. 

 

Abstract 
Sage (Salvia officinalis), belong to Labiatae family is indigenous to Iraq and other 

Mediterranean areas but now cultivated world- wide, principally for its use as culinary herb. In the 

present study preliminary screening for the important phytochemical natural product groups indicated 

the presence of flavonoid, saponin, hyrolysable and condensed tannin groups. The antibacterial  

activity of two concentrations 10 mg/ml and 100 mg/ml of chloroform and hydroalcoholic extracts 

from  Salvia officinalis leaves was evaluated against four strains of gram negative bacteria (Escherichia 

coli,  Pseudomonas arigenossa,  Klebsiella pneumonia, and  Proteus spp) and two strains of gram 

positive bacteria (Staphylococcus aureus,  Bacillus cerus) using agar well diffusion method. 

Chloroform extract 100mg/ml was found active against two types of bacteria, Staphylococcus aureus 

with MIC of 90 mg/ml and Proteus spp with MIC of 80 mg/ml. Bioassay guided separation using TLC 

led to the separation of 6 constituents from the active extract, one of them was identified as thujone. 

Key words: Salvia officinalis, Antibacterial, TLC, Thujone. 
 

دراسة الفعالية التثبيطية للبكتيريا والمكونات الكيميائية لمستخلصات أوراق الميرمية التي تنمو 

 في العراق
عالء الذين محمذ النقشبنذي*

،1
 و     النا يوسف مطلب*     

 *فشع انعمالُش وانُباتاث انطبُت  ، كهُت انصُذنت ، جايعت هىنُش انطبُت ، اسبُم ، انعشاق  

 الخالصة
وانتٍ تًُىا بصىسة طبُعُت فٍ انعشاق و يُاطك  Labiataeانً انعائهت انشفىَت   (Salvia officinalis)انًُشيُت  تُتًٍ

اخشي فٍ انششق األوسظ و تزسع فٍ يُاطك يختهفت يٍ انعانى بانذسجت انشئُسُت كعشبت نألكم.  انبحث تًهُذٌ نُىاتج انكًُُائُت 

( .  و لذ أختبشث  flavonoid, saponin, hyrolysable and condensed tanninتج )  انطبُعُت فٍ انُباث ادث انً كشف انُىا

 C1 10mg/ml , C2 ( بتشكُزٍَ )ethanol 75% ( و يزَب انكحىنٍ )chloroformيستخهصاث انًزَب انعضىٌ ) 

100mg/mlَ ٍىع انصبغت انسانبت ) ( نغشض يعشفت انفعانُت انتثبُطُت نهبكتُشَا يفابم أسبعت سالالث يٍ انبكتُشَا ي Escherichia 

coli,  Pseudomonas arigenossa,  Klebsiella pneumonia  ، Proteus spp وَىعٍُ يٍ انصبغت انًىجبت  )
(Staphylococcus aureus, Bacillus cerus( باستخذاو تمُُت )agar well diffusion ٌاظهشث انُتائج باٌ انًزَب انعضى .)

100mg/ml تثبُطُت  يمابم سالنتٍُ يٍ انبكتُشَا،  رو فعانُتStaphylococcus aureus  انحذ األدًَ نهتشكُز انًثبظ )و ب 

MIC)90 mg/ml   و Proteus spp  ب( انحذ األدًَ نهتشكُز انًثبظ MIC) 80 mg/ml  الحما تى فصم و يطابمت انًكىَاث.
( و لذ تى فصم ستت TLC)  تمُُت كشويىتىغشافُا انصفائح انشلُمتان  باستخذاوانشئُسُت نًستخهص انفعال ألوساق َباث انًُشيُت 

 . thujone بأَها إحذاهًايكىَاث سئُسُت وتى تشخُص 

 ، الميرمية ، الفعالية التثبيطية للبكتيريا   TLCالكلمات المفتاحية :  
 

Introduction 
Sage (in Kurdish and Arabic, 

Meramea) derived from (Salvia officinalis) 

belong to Labiatae family, indigenous to Iraq 

and other Mediterranean areas but now 

cultivated world- wide, principally for its use 

as culinary herb. Salvia is a perennial 

herbaceous to shrubby herb growing up to 

50cm in height (
1, 2)

. Sage is used as 

therapeutic agent and in food, beverages, 

spirits, and cosmetics and in the production of 

relaxing and curative herbal tea 
(3)

. Sage 

reportedly has antibacterial, fungistatic, 

virustatic, astringent, secretion-stimulating, 

and perspiration-inhibiting effects.  Phenolic 

acids (e.g., salvin and salvin monomethyl 

ether) isolated from sage have antimicrobial 

activities, especially against Staphylococcus 

aureus 
(4)

.The volatile oil of sage consists of 

about 5% of α- and β- thujone together with 

cineole, borneol and other constituents 
(2, 3)

. 

Non volatile components of the leaf include 

diterpenes, phenolic glycosides based on 

caffeic acid, p- hydroxybenzoic acid and 

tannins (3-8%) both hydrolysable and 

condensed tannins 
(2)

. Thus, the present study 

is aimed to carry out preliminary screening for 

the important phytochemical natural product 

groups and to investigate and establish the 

antibacterial activity of Iraqi Salvia officinalis 

leave extracts and finally bioassay guided 

separation and identification of the main active 

constituents in the active extract (s).

 

1Corresponding author E- mail : alaadinmn@hotmail.com 

Received : 12/5/2011 

Accepted : 3/4/2012

mailto:alaadinmn@hotmail.com


Iraqi J Pharm Sci, Vol.21(1) 2012               Antibacterial and phytochemical study of salvia officinali 

94 

 

Materials and Methods 
Plant material 

Salvia officinalis leaves were collected 

during September 2009 in Erbil and 

authenticated by the Department of 

Pharmacognosy, College of Pharmacy-Hawler 

Medical University. 

 

Phytochemical screening 

Fifty g of dried powdered Salvia 

officinalis leaves were extracted using 75% 

ethanol for the phytochemical investigation 

using ultra sonic bath for 1hr at 40 
0
C 

(5)
 . 

Preliminary qualitative tests were carried out 

on the plant extracts to detect the most 

important natural product groups 
(6)

. 

Alkaloid test: Hydroalcoholic extract of sage 

leaves (75% ethanol) was treated with dilute 

sodium hydroxide (5% NaOH) solution; 

extraction is then carried out with chloroform. 

The concentrated organic liquid is then shaken 

with 5% HCl and allowed to separate. The 

aqueous extract was used for detection of 

alkaloidal compounds 
(2)

. Few drops of 

Dragendorff reagent were added to 1ml of 

extract. The appearance of a reddish-brown to 

orange precipitate indicated the presence of 

alkaloid 
(2, 7, and 8)

. 

 Flavonoid test: 2 ml of hydroalcoholic extract 

of sage leaves were tested for the presence of 

flavonoid glycoside by the addition of few 

drops of NaOH solution. The formation of 

intense yellow color which turn to colorless on 

addition of few drops of dilute acid solution 

indicated the presence of flavonoids 
(9)

. 

Anthraquinone test: The tested sage extract 

was boiled with 1ml of dilute Hcl in a test tube 

over a pre-heated water bath for 5 minute. The 

contents were cooled and extracted with 

chloroform, the chloroform layer was 

separated and ammonia solution was added. 

The appearance of a rose-pink color in 

ammonia layer is indicated the presence of 

anthraquinone glycoside 
(9)

. 

Cardioactive glycoside test: The dried sage 

extract was dissolved in chloroform, and then 

evaporated to dryness. Dissolve the residue in 

(0.4ml) glacial acetic acid with few drops of 

FeCl3, concentrated sulphuric acid (H2SO4) 

was added along the side of the test tube to 

settle at the bottom. The reddish brown color 

changing to bluish green color appears at the 

junction of the two reagents within 2-5 min. 

spreading slowly into the acetic acid layer 

indicated the presence of cardioactive 

glycoside 
(9)

.  

 Saponin test: A volume of 2ml of 

hydroalcoholic sage extract were shaken with 

1ml of water. The formation of semi-

permanent foam (15 min) indicated the 

presence of saponin natural products 
(6)

.  

Tannin test: A few drops of ferric chloride 

reagent 1% were added to 1ml of sage extract. 

The appearance of blue color indicated the 

presence of hydrolysable tannins and 

appearance of green color indicated the 

presence of condensed tannins 
(2, 6)

. 

 

Antibacterial evaluation 

Extraction: Plant material was collected, dried 

in air for seven days, and all plants were 

powdered with mechanical grinder. 50gm of 

dried powdered plant material was extracted 

separately with 1000 ml of chloroform using 

ultra sonic bath for one hour at 40 
0
C 

(5)
. The 

extracts were obtained by filtration through 

Buckner funnel, and evaporated to dryness by 

rotary evaporator yielding chloroform extract 

(5.4 g). The residual plant materials were dried 

then re-extracted using 75% ethanol using ultra 

sonic bath for one hour at 40 
0
C 

(5)
. The 

obtained extracted from filtration by Buckner 

funnel evaporated to dryness by rotary 

evaporator yielding ethanol extract (9.2 g). 

Plant extract preparation: The plant extracts 

were evaluated separately at two different 

concentrations 10mg and 100mg. the 

chloroform extracts and ethanol extracts were 

dissolved separately in 1ml of 20% tween 80 

and 10% dimethyl sulfoxide (DMSO) 

respectively.  

Tested microorganism: Bacteria which were 

used in the process of investigation are 

obtained from biology department, Science 

College, Salahaddin University. Bacterial 

strains include two gram positive bacteria 

(Staphylococcus aureus, Bacillus cerus) and 

four gram negative bacteria (Escherichia coli, 

Psedomonus arigenossa, Klepssila spp and 

Proteus spp). The bacterial samples are frozen 

at -4 
0
C in cooled incubator, later reactivated 

before it’s used. 

Method of antibacterial evaluation: The 

antibacterial activity of the two types of plant 

extracts were tested against six strains of 

bacteria using agar- well diffusion method 
(10, 

11)
. From the frozen bacteria inoculation was 

done into nutrient agar media, and incubated at 

37
o
C for 24 hr.The grown bacteria were 

suspended in a normal saline solution (0.85% 

sodium chloride w/v) to a turbidity of 0.5 Mc 

Farland standards (108 cfu/ml).The prepared 

bacterial suspension was used to inoculate into 

Muller-Hinton agar plate with a sterile non-

toxic cotton swab on a wooden applicator. 

Four wells were done by a sterile cork borer of 

5mm in diameter in each plate.100 µl of 

dilution of plant extracts in 10% DMSO for 

ethanol extract and 20% tween 80 for 



Iraqi J Pharm Sci, Vol.21(1) 2012               Antibacterial and phytochemical study of salvia officinali 

95 

 

chloroform extract to give final concentration 

of 1mg and 10mg of each plant extract was 

added in each well, 10% DMSO and 20% 

tween 80 were used as negative control and 

streptomycin antibiotic used as positive control 

in concentration of (100mg/ml) added in a well 

in each plate. Plates are incubated at 37ºC for 

24 hr.   

Determination of minimum inhibitory 

concentration (MIC): MIC values for biolog- 

ically active extracts against Staphylococcus 

aureus and Proteus spp. were determined by 

agar well diffusion method 
(10, 11)

. 

 

Separation and Identification of the main 

active constituents  

Thin layer chromatography (TLC) 

technique was applied for the separation and 

identification of active constituents using silica 

gel GF 254 nm, layer thickness 0.2 mm; 20x20 

cm   aluminium cards (fluka, Switzerland)
(7, 12)

. 

Different solvent systems were tried as a 

mobile phase [Toluen: Ethyl acetate (93:7), 

Toluen: Ethyl acetate: Formic acid (5:4:1), 

Ethyl acetate: Formic acid: glacial acetic acid: 

water (10:1.1:1.1:2.6), Benzene: Pyridine: 

Formic acid (7.2:1.8:1)] 
(13, 14)

.Fifty mg of 

dried chloroform extract was dissolved in 1 ml 

chloroform and 1µLof thujone reference 

substance (Chromadex, USA) was dissolved in 

1ml toluene, 10 µL of both solutions were 

applied to TLC plate and developed in 

different mobile phase systems. The plate was 

dried at room temperature. The TLC plate was 

sprayed with anisealdehyde - sulphuric acid 

reagent after the development process. The 

plate was heated to 100–110°C for 5–10 min. 

and visualized under visible light 
(13)

. A 

number of constituents were separated, and 

their Rf values were calculated, the procedure 

was triplicated and mean values were taken, 

table (1). 

 

Table 1: Rf value and spot color of separated constituent (S3) 
 

Separated 

constituent 

Rf value 

Spot color 
Mobile 

phase (1) 

Mobile 

phase (2) 

Mobile 

phase (3) 

Mobile 

phase(4) 

S3 0.5 0.31 0.13 0.43 Violet 

Standard Thujone 0.52 0.34 0.15 0.44 Violet 
 

Mobile phase (1): Toluen: Ethyl acetate (93:7)  

Mobile phase (2): Toluen: Ethyl acetate: Formic acid (5:4:1)  

Mobile phase (3): Ethyl acetate: Formic acid: glacial acetic acid: water (10:1.1:1.1:2.6)  

Mobile phase (4): Benzene: Pyridine: Formic acid (7.2:1.8:1)  

 
Results 

Sage leaves chloroform extract 

shows activity against two strain of bacteria 

Staphylococcus aureus and Protues spp. at 

100mg\ml concentration with inhibition zone 

diameter (20±0.265 and 10±0.2) respectively 

with corresponding MIC value 90 mg\ml 

against Staphylococcus aures and 80 mg\ml 

against Proteus spp, table (2). the 

phytochemical screening tests showed positive 

results for flavonoid, saponin hydrolysable and 

condensed tannin, while the other natural 

product groups including alkaloid, 

anthraquinone, and cadioactive  were found 

absent. The results of separation by TLC 

revealed the presence of a number of 

constituents in chloroform extract, one of the 

constituent (S3) was found to be with identical 

color and Rf value with that of thujone 

reference substance,figure (1). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: TLC chromatogram of Sage 

leaves, mobile phase [Toluen: Ethyl acetate 

(93:7)]; chloroform extract (CE), thujone 

reference substance (R). 



Iraqi J Pharm Sci, Vol.21(1) 2012               Antibacterial and phytochemical study of salvia officinali 

96 

 

Table 2: Antibacterial activity for Sage leaves chloroform extract, C1 (10mg/ml) and C2   

               (100mg/ml). 
 

 

 

No. Microorganism 

Inhibition zone diameter  (mm),                                  

MIC value ( mg/ml) 

Chloroform extracts Ethanol extracts 

C1 C2 C1 C2 

1- Staphylococcus aureus --- 20±0.265,      (90)* --- --- 

2- Pseudomonas aeroginosa --- --- --- --- 

3-  Escherichia coli --- --- --- --- 

4- Klebsila pneumonae --- --- --- --- 

5- Bacillus cerus --- --- --- --- 

6- Proteus spp. --- 10±0.2,        (80)* --- --- 

*All readings were repeated three times, the mean of triplicates ± SD values were taken. 

 
Discussion 

Sample preparation is the crucial 

first step in the analysis of herbs, because it is 

necessary to extract the desired chemical 

components from the herbal materials for 

further separation and characterization. Thus, 

the development of modern sample preparation 

techniques with significant advantages over 

conventional methods analysis of medicinal 

plants is likely to play an important role in the 

overall effort of ensuring and providing high 

quality herbal products to consumers 

worldwide 
(15)

. The plant expresses 

antibacterial activity against two strains of 

bacteria. From the results, the chloroform 

extract was active while hydroalcoholic extract 

was inactive. The activity of the plant as 

showed in the literature review may be due to 

phenolic acids (example, salvin and salvin 

monomethyl ether) previously isolated from 

sage, especially against Staphylococcus aureus 
(4)

. The obtained results were in agreement 

with the finding of Zhi-He and Hiroyuki, 

(1996) 
(16)

 who were found that sage leaves 

extract at 0.2% concentration was gave strong 

activity against Staphylococcus aureus, in the 

same time Rogério et al, (2004) 
(17)

 found that 

the essential oils of Salvia officinalis was 

inhibited 83.3% of Proteus spp. while Gislene 

et al, (2000) 
(18)

 were found that the collected 

sage in Brazil was inactive against 

Staphylococcus aureus and Proteus spp. 

Literature reviews on Salvia officinalis 

revealed that there was few phytochemical 

studies conducted on the Iraqi species of sage, 

and due to the antibacterial activity of its 

chloroform extract and from phytochemical 

screening which was found to contain four 

groups of phytochemical natural product 

groups, it was tried to separate and identify the 

main active constituents in the active extract 

by using TLC. The proposed TLC method was 

regarded as suitable for quality control of 

herbal products containing Salvia officinalis 

and for its extracts. The method is simple, 

sensitive, and specific, and both the standards 

and the samples can be analyzed 

simultaneously, without the requirement for 

sophisticated equipment 
(13)

. Separation of the 

main active constituents in chloroform extract 

by the TLC revealed the presence of six 

constituents figure (1). One of the constituents 

(S3) had the same Rf value and color as thujone  

the reference compound. Chemotaxonomic 

markers are sometimes used as indicators of 

botanical identity; thujone is the marker for 

Salvia officinalis 
(19)

. Miladinović and 

Miladinović (2000) 
(20)

 found that the main 

component of the Salvia officinalis essential 

oils was thujone (24.88%) and camphor 

(16.3%). The carriers of antimicrobial activity 

of the sage oil were found to be thujone and 

camphor 
(19, 21)

. Further study using advanced 

spectroscopical methods and gas-liquid 

chromatography is recommended for more 

confirmation of the identity of (S3) constituents 

and to identify the other separated constituents 

in the chloroform extract. 

 

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