In vitro Antimicrobial Activity of Angelica sylvestris Roots

Abstract

Angelica sylvestris, which is commonly known as wild angelica, has been used as a medicinal herb in different traditions. Especially its roots are known to be a traditional medicine in different cultures. Mesir paste was prepared about 500 years ago during Ottoman period as a medicinal paste and A. sylvestris was one of its ingredients. In this study the in vitro antimicrobial activity of ethanol extract of A. sylvestris roots was investigated against 17 bacterial and 1 fungal strain, ATCC 25923 and Staphylococcus epidermidis DSMZ 20044 by using the disk diffusion method. It is observed that ethanol extracts of A. sylvestris roots has antimicrobial activity against several Gram positive and Gram negative microorganisms tested.

Full text

International Journal of Biological Sciences 1(1) :1-7, 2016
ISSN: 2415-2560
© Noble Science Publishers, 2016
1
In vitro Antimicrobial Activity of Angelica sylvestris Roots
Kerem CANLI
1*
, Ali YETGIN
2
,
Ilgaz AKATA
3
, Ergin Murat ALTUNER
4
1
Dokuz Eylül University, Faculty of Science, Department of Biology, Buca, İzmir, TURKEY
2
Izmir Institute of Technology, Faculty of Science, Department of Molecular Biology, Izmir, TURKEY
3
Ankara University, Faculty of Science, Department of Biology, TR 06100, Ankara, TURKEY
4
Kastamonu University, Faculty of Science and Arts, Department of Biology, TR 37150, Kastamonu,
TURKEY*Corresponding author: biyoloji@gmail.com
Abstract.
Angelica sylvestris, which is commonly known as wild angelica, has been used as a medicinal herb in
different traditions. Especially its roots are known to be a traditional medicine in different cultures.
Mesir paste was prepared about 500 years ago during Ottoman period as a medicinal paste and A.
sylvestris was one of its ingredients. In this study the in vitro antimicrobial activity of ethanol extract of
A. sylvestris roots was investigated against 17 bacterial and 1 fungal strain, namely, Bacillus subtilis
DSMZ 1971, Candida albicans DSMZ 1386, Enterobacter aerogenes ATCC 13048, Enterococcus
durans, Enterococcus faecalis ATCC 29212, Enterococcus faecium, Escherichia coli ATCC 25922,
Klebsiella pneumoniae, Listeria innocula, Listeria monocytogenes ATCC 7644, Pseudomonas
aeruginosa DSMZ 50071, Pseudomonas fluorescence P1, Salmonella enteritidis ATCC 13075,
Salmonella infantis, Salmonella kentucky, Salmonella typhimurium SL 1344, Staphylococcus aureus
ATCC 25923 and Staphylococcus epidermidis DSMZ 20044 by using the disk diffusion method. It is
observed that ethanol extracts of A. sylvestris roots has antimicrobial activity against several Gram
positive and Gram negative microorganisms tested.
KEYWORDS: Angelica sylvestris, Mesir paste, antimicrobial activity, antimicrobial screening,
ethanol extract.
INTRODUCTION
Using herbal remedies against diseases is
assumed to be as old as human history. Today
there is an increasing interest by the scientists to
define the secrets of these traditional herbal
medicines [1]. Attempts to discover new
antimicrobial agents have increased mainly
because of the excess need to new drugs against
resistant microorganisms [2]. It is a very well-
known issue that most of the antimicrobial
agents are developed from natural products,
which have an antimicrobial potential [3, 4, 5].
The antimicrobial potential of these plants were
discovered by human beings without scientific
knowledge mostly through trial and error
method in the history [6]. Mesir paste is a
traditional special mixture of several herbs and
spices used as a medicine, which was founded
about 500 years ago during Ottoman period,
including Zingiber officinale (Ginger),
Terminalia citrina (black chuglam or citrine
myrobalan), Cuminum cyminum (Cumin) and
Angelica sylvestris (wild angelica). All
ingredients of Mesir paste separately have been
used to heal several diseases in Turkish folk

International Journal of Biological Sciences 1(1) :1-7 , 2016
2
medicine for long centuries [7]. But the healing
effect of Mesir paste mostly depends on the
synergistic antimicrobial effect of the
ingredients the paste.
World Health Organization (WHO) has
predicted the bacterial evolution and increasing
antimicrobial resistance as a major threat for the
public health for the 21
st
century [8]. To prevent
spreading of antibiotic resistant microorganisms
scientists has been conducting researches to find
new antimicrobial agents [9, 10].
Huge numbers of studies clearly showed that
natural products have been used for hundreds of
years to treat several diseases caused by
bacterial and fungal infections [11]. Current
researches especially conducted in the last
decades showed that natural herbal remedies
have an important potential of providing
opportunities for new antibiotic drug
development. As far as the current literature is
concerned, it is obvious that only a very small
amount of the available diversity among plants
have yet been explored for such purposes [12,
13].
In this study the antimicrobial activity
screening of A. sylvestris roots, one of the
ingredients of Mesir paste, is investigated
against 17 bacterial and 1 fungal strains by using
the disk diffusion method. Although Sarker et
al. [14] studied the antibacterial activity of the
dichloromethane and methanol extracts seeds of
A. sylvestris by broth dilution method against 8
strains previously, our results are the first report
of the antimicrobial activity of the ethanol
extracts of A. sylvestris roots.
MATERIALS AND METHODS
Extraction procedure
All A. sylvestris samples were ground by a
pestle and a mortar. Ethanol (Sigma-Aldrich)
was chosen as an extraction solvent in order to
extract active substances. Ground samples were
shaken in the extraction solvent at 100 rpm for 3
days at room temperature. The extract was
filtered through filter paper (Whatman No. 1)
into evaporation flasks. The filtrate was
evaporated by a rotary evaporator (Buchi R3) at
35°C. After evaporation the residues were
collected and used to prepare 90 mg.mL
-1
of
ethanolic extracts.
Microorganisms
A wide range of Gram negative and Gram
positive bacteria and yeast were selected to test
the antimicrobial effect of A. sylvestris. These
strains are Bacillus subtilis DSMZ 1971,
Candida albicans DSMZ 1386, Enterobacter
aerogenes ATCC 13048, Enterococcus durans,
Enterococcus faecalis ATCC 29212,
Enterococcus faecium, Escherichia coli ATCC
25922, Klebsiella pneumoniae, Listeria
innocula, Listeria monocytogenes ATCC 7644,
Pseudomonas aeruginosa DSMZ 50071,
Pseudomonas fluorescence P1, Salmonella
enteritidis ATCC 13075, Salmonella infantis,
Salmonella kentucky, Salmonella typhimurium
SL 1344, Staphylococcus aureus ATCC 25923
and Staphylococcus epidermidis DSMZ 20044.
The strains were chosen from standard strains
as much as possible. Other strains which are not
standard were all isolated from food and
identified in Ankara University, Faculty of
Science, Department of Biology.
Preparation of inocula
All bacterial strains were incubated at 37 ˚C
for 24 hours [15]. But since the requirements for
C. albicans is different, C. albicans was
inoculated at 27 ˚C for 48 hours. Inocula were
prepared by transferring morphologically similar
colonies of each organism into 0.9% sterile
saline solution until the visible turbidity was
equal to 0.5 McFarland standard having

International Journal of Biological Sciences 1(1) :1-7 , 2016
3
approximately 10
8
cfu.mL
-1
for bacteria and
10
7
cfu.mL
-1
for C. albicans [16-19].
Disk diffusion method
Disk diffusion test was performed as
described previously by Andrews [20]. The
culture medium was poured into 90 mm sterile
Petri dish to give a mean depth of 4.0 mm ± 0.5
mm [21, 22]. 70 µL and 20 µL aliquots of
extract was applied on sterile disks of 6 mm
diameter end up with 1800 µg and 6300 µg
sample on each disk [23, 24]. To get rid of any
residual solvent which might interfere with the
results, disks were left to dry overnight in sterile
conditions [24, 25]. The surfaces of the plates
were inoculated using previously prepared
inocula containing saline suspension of
microorganisms. Inoculated plates were then left
to dry for 5-6 minutes at room temperature in
aseptic conditions before applying the disks
[26]. Disks were firmly applied to the surface of
the plate which had an even contact with the
agar. Plates were incubated and inhibition zone
diameters were expressed in millimetres [27-
28].
Controls
Empty sterile disks and extraction solvent
(ethanol) loaded on sterile disks which were
dried at sterile conditions to remove solvent as
done in the study were used as negative controls.
Ciprofloxacin 5 µg used as positive control.
Statistics
All extracts were tested in triplicate and
MACANOVA (version 5.05) was used for
statistical analysis of the data. P values of <0.05
were considered statistically significant.
RESULTS and DISCUSSION
The main aim of this study was to identify
the antimicrobial activity of ethanol extracts of
A. sylvestris roots. To do this, disk diffusion test
was performed in the study. In this test, extracts
were loaded on empty sterile disks and these
disks were then applied on a culture medium
inoculated with microorganisms. If the extracts
were shown activity against these
microorganisms, they have caused an inhibition
zone. The diameters of the inhibition zones
recorded in millimetres are given in Table 1. No
activity was observed for the negative controls;
extraction solvent and empty sterile disks.
Results given in Table 1 clearly show that 20
µL (1800 µg.µL
-1
) of A. sylvestris root samples
caused an inhibition zone of 14 mm against E.
faecium, L. monocytogenes ATCC 7644 and S.

International Journal of Biological Sciences 1(1) :1-7 , 2016
4
aureus ATCC 25923, 13 mm against S.
epidermidis DSMZ 20044, 10 mm against B.
subtilis DSMZ 1971, 8 mm against C. albicans
DSMZ 1386, K. pneumonia, L. innocula and P.
aeruginosa DSMZ 50071, where 70 µL (6300
µg.µL
-1
) of A. sylvestris root samples caused an
inhibition zone of 15 mm against L.
monocytogenes ATCC 7644, 14 mm against E.
faecium, S. aureus ATCC 25923 and S.
epidermidis DSMZ 20044, 10 mm against B.
subtilis DSMZ 1971, 9 mm against K.
pneumonia and L. innocula, 8 mm against C.
albicans DSMZ 1386 and P. aeruginosa DSMZ
50071, 7 mm against E. faecalis ATCC 29212
and P. fluorescens P1.
On the other hand, no inhibition zone was
observed against E. aerogenes ATCC 13048, E.
durans, S. enteritidis ATCC 13075, S. infantis,
S. kentucky and S. typhimurium SL 1344.
Although Sarker et al. [14] studied the
antibacterial activity of the dichloromethane and
methanol extracts seeds of A. sylvestris by broth
dilution method against Citrobacter freundii
NCTC 9750, E. faecalis NCIMB 775, E. coli
NCIMB 8110, E. coli NCIMB 4174,
Lactobacillus plantarum NCIMB 6376,
Salmonella goldcoast NCTC 13175, S. aureus
NCTC 10788 and S. aureus NCTC 11940
previously, our results are the very first report of
the antimicrobial activity of the ethanol extracts
of A. sylvestris roots. Sarker et al. [14] identified
that dichloromethane extracts of seeds has an
antimicrobial activity only against Citrobacter
freundii NCTC 9750 and S. aureus NCTC
11940 however methanol extracts of seeds
observed no activity, our study clearly show that
ethanol extracts of A. sylvestris roots are active
against most of the strains tested in terms of its
antimicrobial activity.
S. aureus is known one of the common
nosocomial infections in medical intensive care
units [29]. Several researchers study
antimicrobial activity of some plant extracts on
S. aureus strains. For example, Nair and Chanda
[30] compared 10 medicinal plants antimicrobial
effects on S. aureus strains, namely Anethum
gravelons, Commiphora wightii, Emblica
officinalis, Ficus benghalensis, Ficus racemosa,
Ficus religiosa, Ficus tisela, Hibiscus
cannabinus, Mentha arvensis and Mimusops
elengi. In this study maximum activity of
ethanol extract was shown by E. officinalis with
9 mm of inhibition zone. In our study we
observed 14 mm zone for 1800 µg.µL
-1
of A.
sylvestris roots. Comparing these results clearly
puts forward how A. sylvestris roots are active
against S. aureus when compared to some other
higher plants.
It is a fact that the Gram-negative bacteria are
more resistant because of the nature of their
outer membrane, containing proteins and
lipopolysaccharids that is impermeable to most
of the antimicrobial substances [31, 32]. It was
also indicated previously that Gram-negative
bacteria are the dominant killers among
infections in the Intensive Care Units (ICU)
[33]. Klebsiella is one of the Gram-negative
bacteria that cause death in ICUs [33], so
showing antibacterial activity against K.
pneumoniae may be very important.
Although the pathogenic potential of B.
subtilis is described as low or absent [34], rarely
B. subtilis known to cause serious infections in
immunocompromised patients [35]. Several
researchers study antibacterial activity of some
plant extracts on B. subtilis strains. For example,
Parekh et al. [36] compared six different ethanol
plant extracts, namely Acyranthus aspera,
Calotropis gigantea, Carissa congesta, Fagonia
cretica, Mangifera indica and Rauwolfia
serpentina. In this study inhibition zones were
found to be 14 mm for M. indica, 11 mm for C.
congesta and 10 mm for A. aspera, C. gigantea,
F. cretica and R. serpentina. In our study we

International Journal of Biological Sciences 1(1) :1-7 , 2016
5
observed 10 mm zone for both 1800 µg.µL
-1
and
6300 µg.µL
-1
of A. sylvestris roots extract.
Conter et al. [37] reported that L.
monocytogenes strains are susceptible to the
antibiotics commonly used in human listeriosis
treatment, but L. monocytogenes is slowly
becoming antibiotic resistant and a perpetual
surveillance of emerging antimicrobial
resistance of this pathogen is critical to ensure
effective treatment of human listeriosis. From
this point of view, having antibacterial activity
against L. monocytogenes may very important.
Ates and Erdogrul [38] identified that ethanol
extract of Juniperus oxycedrus caused 7 mm of
inhibition zone against L. monocytogenes
whereas Cinnamomum cassia, Glycyrrhiza
glabra, Coriandrum sativum and Pimpinella
anisum observed no activity. In our study we
observed 14 mm zone for 1800 µg.µL
-1
of A.
sylvestris roots and 15 mm zone for 6300 µg.µL
-
1
mg of A. sylvestris roots. Comparing these
results clearly presents that A. sylvestris roots
are highly active against L. monocytogenes.
CONCLUSION
As a result, it can be concluded that there is
clear antimicrobial activity of A. sylvestris roots
against most of the strains tested. The results of
our study clearly presents that A. sylvestris roots
could have a possible medicinal uses especially
against E. faecium, L. monocytogenes ATCC
7644, B. subtilis DSMZ 1971, S. epidermidis
DSMZ 20044 and S. aureus ATCC 25923.
But further researches are needed to be
conducted in order to analyse the active
substances and their activity mechanisms in
details.
CONFLICT OF INTERESTS
The authors declare that there is no conflict of
interests regarding the publication of this paper.
REFERENCES
[1] Abbasi AM, Khan MA, Ahmad M, Jahan S
& Sultana S. Ethnopharmacological application
of medicinal plants to cure skin diseases and in
folk cosmetics among the tribal communities of
North-West Frontier Province. Pakistan. J
Ethnopharmocol 2010; 128: 322–335.
[2] Okeke IN, Laxmaninarayan R, Bhutta ZA,
Duse AG, Jenkins P, O’Brien, TF, Pablos-
Mendez A & Klugman KP. Antimicrobial
resistance in developing countries. Part 1: recent
trends and current status. Lancet Infectious
Diseases 2005; 5: 481-493.
[3] Clardy J & Walsh C. Lessons from natural
molecules. Nature 2004; 432:829-837.
[4] Altuner EM, Çetin B & Çökmüş C.
Antimicrobial activity of Tortella tortulosa
(Hedw.) Limpr. Extracts. Kastamonu University
Journal of Forestry Faculty 2010; 10: 111-116.
[5] Altuner EM, Çetin B & Çökmüş C.
Antimicrobial Screening of Some Mosses
Collected From Anatolia. Pharmacogn Mag
2010; 6: 56.
[6] Karasu A. Bulgaristan Tırgovişte’den
Osmanlı Dönemine Ait Bir Tıp Metni. Turkish
Studies 2015; 10(8): 1597-1612.
[7] Oskay M, Karayıldırım T, Ay E & Ay K.
Determination of some chemical parameters and
antimicrobial activity of traditional food: Mesir
paste. Journal of Medicinal Food 2010; 13 (5):
1195–1202.
[8] Syed GW, Syed AS & Oh LA. Risk
Evaluation Under Various Speculations of
Antibiotic Usage; A Cohort Survey Among
Outpatients of Pinang, Malaysia. Eur J Gen Med
2010; 7: 303-309.
[9] Paudel B, Bhattarai HD, Lee JS, Hong SG,
Shin HW & Yim JH. Antibacterial Potential of
Antarctic Lichens against Human Pathogenic
Gram-positive Bacteria. Phytotheraphy
Research 2008; 22; 1269-1271.

International Journal of Biological Sciences 1(1) :1-7 , 2016
6
[10] Ozkan OE, Zengin G, Akca M, Baloglu
MC, Olgun C, Altuner EM, Ates S, Aktumsek A
& Vurdu H. DNA protection,
antioxidant, antibacterial and enzyme inhibition
activities of heartwood and sapwood extracts
from juniper and olive woods. RSC Advances
2015; 5: 72950-72958.
[11] Jones FA. Herbs - useful plants. Their role
in history and today. European Journal of
Gastroenterology and Hepatology 1996; 8:
1227-1231.
[12] Cos P, Vlietinck AJ, Vanden Berghe D &
Maes L. Anti-infective potential of natural
products: How to develop a stronger in vitro
‘proof-of-concept’. Journal of
Ethnopharmacology 2006; 106: 290-302.
[13] Altuner EM. Investigation of antimicrobial
activity of Punica granatum L. fruit peel ash
used for protective against skin infections as
folk remedies especially after male
circumcision. Afr J Microbiol Res 2011; 5(20):
3339-3342.
[14] Sarker SD, Eynon E, Fok K, Kumarasamy
Y, Murphy EM, Nahar L & Siakalima M.
Screening the extracts of the seeds of Achillea
millefolium, Angelica sylvestris and Phleum
pratense for antibacterial, antioxidant activities
and general toxicity. Oriental Pharmacy and
Experimental Medicine 2003; 3(3): 157-162.
[15] Canli K, Altuner EM & Akata I.
Antimicrobial screening of Mnium stellare.
Bangladesh J Pharmacol 2015; 10: 321-325.
[16] Hammer KA, Carson CF & Riley TV.
Antimicrobial activity of essential oils and other
plant extracts. Journal of Applied Microbiology
1999; 86: 985-990.
[17] Altuner EM, Akata I & Canli K. In vitro
antimicrobial screening of Bovista nigrescens
(Pers.). Kastamonu U J For Fac 2012; 12: 90-96.
[18] Altuner EM, Akata I & Canli K. In vitro
antimicrobial screening of Cerena unicolor
(Bull.) Murrill (Polyporaceae Fr. Ex Corda).
Fresen Environ Bullet 2012; 21(1B): 3704-3710.
[19] Canli K, Çetin B, Altuner EM, Türkmen Y,
Üzek U & Dursun H. In vitro antimicrobial
screening of Hedwigia ciliata var.
leucophaea and determination of the ethanol
extract composition by Gas
Chromatography/Mass Spectrometry (GC/MS).
J Pure Appl Microbiol 2014; 8(4):2987-2998.
[20] Andrews JM. BSAC standardized disk
susceptibility testing method (version 6). J
Antimicrob Chemother 2003; 60: 20-41.
[21] Altuner EM & Çetin B. Antimicrobial
activity of Thuidium delicatulum (Bryopsida)
extracts, Kafkas U Fen Bil Enst Der 2009; 2: 85-
92.
[22] Altuner EM & Akata I. Antimicrobial
activity of some macrofungi extracts. SAÜ Fen
Bil Der 2010; 14: 45-49.
[23] Mahasneh AM & El-Oqlah AA.
Antimicrobial activity of extracts of herbal
plants used in the traditional medicine of Jordan.
Journal of Ethnopharmacology 1999; 64: 271-
276.
[24] Silici S & Koc AN. Comparative study of
in vitro methods to analyse the antifungal
activity of propolis against yeasts isolated from
patients with superficial mycoses. Letters in
Applied Microbiology 2006; 43: 318-324.
[25] Altuner EM & Canli K. In vitro
antimicrobial screening of Hypnum andoi A.J.E.
Sm.. Kastamonu U J For Fac 2012; 12: 97-101.
[26] Altuner EM & Canli K, Akata I.
Antimicrobial Screening of Calliergonella
cuspidata, Dicranum polysetum and Hypnum
cupressiforme. J Pure Appl Microbiol 2014;
8(1): 539-545.
[27] Altuner EM, Çeter T, Bayar E, Aydın S,
Arıcı F, Süleymanoğlu G & Edis A.
Investigation on antimicrobial effects of some
moss species collected From Kastamonu

International Journal of Biological Sciences 1(1) :1-7 , 2016
7
Region. Commun Fac Sci Univ Ank Series C
2011; 23: 33-43.
[28] Altuner EM, Çeter T, Demirkapı D, Özkay
K, Hayal U & Eser G. Investigation on
Antimicrobial Effects of Some Lichen Species
Collected From Kastamonu Region. Commun
Fac Sci Univ Ank Series C 2011; 23(1-2): 21-
31.
[29] Richards MJ, Edwards JR, Culver DH &
Gaynes RP. Nosocomial infections in medical
intensive care units in the United States:
National Nosocomial Infections Surveillance
System. Crit Care Med 1999; 27: 887–892
[30] Nair R & Chanda SV. Antibacterial
activities of some medicinal plants of the
Western Region of India. Turkish Journal of
Biology 2007; 31: 231–236.
[31] Faucher JL & Avril JL. Bactériologie
générale et médicale. Tome 1, (Ellipses (Ed.),
Paris), 2002; 214pp.
[32] Nikaido H. Antibiotic resistance caused by
Gram-negative multidrug efflux pumps. Clin
Infect Dis 1998; 27: 32-41.
[33] Villegas MV & Quinn JP. An update on
antibiotic-resistant gram-negative bacteria.
Infections in Medicine 2004; 21: 595-599.
[34] De Boer AS & Diderichsen B. On the
safety of Bacillus subtilis and B.
amyloliquefaciens: A review. Appl Microbiol
Biotechnol 1991; 36: 1-4.
[35] Galieni P & Bigazzi C. Recurrent
septicemia in an immuno-compromised patient
due to probiotic strains of Bacillus subtilis. J
Clin Microbiol 1998; 36: 325-326.
[36] Parekh J, Nair R & Chanda S. Preliminary
screening of some folkloric plants from Western
India for potential antimicrobial activity. Indian
J Pharmacol 2005; 37: 408-409.
[37] Conter M, Paludi D, Zanardi E, Ghidini S,
Vergara A, & Ianieri A. Characterization of
antimicrobial resistance of foodborne Listeria
monocytogenes. International journal of food
microbiology 2009; 128(3): 497-500.
[38] Ates DA & Erdogrul OT. Antimicrobial
activities of various medicinal and commercial
plant extracts. Turk J Biol 2003; 27: 157-162.