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Total phenolic and flavonoid contents,antioxidant and antimicrobial activitiesof Alnus glutinosa (L.) Gaertn., Alnusincana (L.) Moench and Alnus viridis(Chaix) DC. extractsSabina Dahijaa, Jasmina Čakarb, Danijela Vidicc, Milka

Maksimovićc & Adisa Parićaa Department of Biology, Faculty of Science, University ofSarajevo, Sarajevo, Bosnia and Herzegovinab Institute for Genetic Engineering and Biotechnology, Sarajevo,Bosnia and Herzegovinac Department of Chemistry, Faculty of Science, University ofSarajevo, Sarajevo, Bosnia and HerzegovinaPublished online: 27 Jun 2014.

To cite this article: Sabina Dahija, Jasmina Čakar, Danijela Vidic, Milka Maksimović & Adisa Parić(2014): Total phenolic and flavonoid contents, antioxidant and antimicrobial activities of Alnusglutinosa (L.) Gaertn., Alnus incana (L.) Moench and Alnus viridis (Chaix) DC. extracts, NaturalProduct Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.931390

To link to this article: http://dx.doi.org/10.1080/14786419.2014.931390

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SHORT COMMUNICATION

Total phenolic and flavonoid contents, antioxidant and antimicrobialactivities of Alnus glutinosa (L.) Gaertn., Alnus incana (L.) Moench andAlnus viridis (Chaix) DC. extracts

Sabina Dahijaa*, Jasmina Cakarb, Danijela Vidicc, Milka Maksimovicc and Adisa Parica

aDepartment of Biology, Faculty of Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina;bInstitute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina; cDepartment ofChemistry, Faculty of Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina

(Received 22 April 2014; final version received 1 June 2014)

The objective of this study was to determine total phenolic and flavonoid contents,antioxidant and antimicrobial activities of methanolic extracts from the leaves and barksof three Alnus species. The phenolic and flavonoid contents of extracts were determinedspectrophotometrically using Folin–Ciocalteau and aluminium chloride methods,respectively. In addition, antioxidant activity of the extracts was determined using1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antimicrobial activitywas performed by disc diffusion assay against six reference bacterial strains includingGram-negative and Gram-positive bacteria and two fungal strains. Extract of Alnusviridis bark contained the highest amounts of total phenolics (780mgCAT/g), whileextract of A. viridis leaves had the highest amount of flavonoids (30.01mgRUT/g).All extracts showed antioxidant activity higher than thymol, which was used as apositive probe. The largest diameters of inhibition zone (25mm) were recorded withBacillus subtilis 168M and Staphylococcus aureus ATCC 6538.

Keywords: phenolics; flavonoids; antioxidant activity; antibacterial activity;antifungal activity; Alnus

1. Introduction

For centuries, plants and their products have been used extensively in traditional medicine for

human diseases. Numerous studies have been carried out to extract various natural products for

assessment of antimicrobial and antioxidant activities (Moyer et al. 2002; Mittler et al. 2004;

Kumar et al. 2006).

The Alnus genus comprises about 30 species of trees and shrubs found in the northern

Hemisphere (O’Rourke et al. 2005). This genus is represented in Bosnia and Herzegovina by

three species: Alnus glutinosa (L.) Gaertn. (black alder), Alnus incana (L.) Moench (grey alder)

and Alnus viridis (Chaix) DC. (green alder).

The infusion of A. glutinosa barks has been used to treat swelling, inflammation,

rheumatism, sore throat and pharyngitis (Middleton et al. 2005), as well as an astringent, bitter,

emetic and haemostatic (Leporatti & Ivancheva 2003). A. incana and A. viridis are used in

folk medicine in the form of infusion for the treatment of gastrointestinal and skin diseases

as well as for gargling in bacterial infections of the mouth and throat (Ritch-Krc et al. 1996;

Stevic et al. 2010).

q 2014 Taylor & Francis

*Corresponding author. Email: sabina_dudevic@yahoo.com

Natural Product Research, 2014

http://dx.doi.org/10.1080/14786419.2014.931390

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Various types of plant secondary metabolites contained in the barks, buds, leaves and pollens

of A. glutinosa have previously been reported (Daniere et al. 1991; O’Rourke et al. 2005).

Previous reports have indicated the predominance of diarylheptanoids in various plant parts of

the Alnus species (Lv & She 2010; Siddiqui et al. 2010).

Phenolic and flavonoid compounds, which are widely found as secondary metabolites in

plants, are important due to their ability to act as antioxidants (Wang et al. 2008). Many phenolic

compounds are known to have potent antioxidant, anticancer, antibacterial, antiviral or anti-

inflammatory activities (Cassidy et al. 2000; Tapiero et al. 2002). Flavonoids, which are found

normally in the leaves, flowering tissues and pollens (Larson 1998), are important antioxidants,

as they have been shown to be highly effective scavengers of most types of oxidising molecules,

including singlet oxygen and various free radicals (Bravo 1998).

This work represents the first report on the phytochemical study of three Alnus species

naturally growing in Bosnia and Herzegovina.

2. Results and discussion

2.1. Total phenolic content and total flavonoid content

Total phenolic and flavonoid content, antioxidant and antimicrobial activities of methanolic

extracts of A. glutinosa, A. incana and A. viridis leaves and barks were determined. The amount

of total phenols in extracts ranged between 333 and 780mgCAT/g (Supplementary Table S1).

One way ANOVA showed statistically significant differences in phenolic contents among all

tested extracts ( f ¼ 6.264; p , 0.05). In addition, Student–Newman–Keuls test has shown

that the extract of A. viridis bark contained statistically significant higher content of phenolic

compounds than all other tested extracts. Similarly, the flavonoid content was markedly higher

in the methanolic extract of A. viridis leaves, with a value of 30.01mgRUT/g. Differences

between flavonoid contents among all tested extracts were significant at p , 0.05 ( f ¼ 132.578).

Results obtained in this study strongly suggest that phenolics are important components of these

plants.

2.2. DPPH radical scavenging activity

Values of IC50 for all extracts ranging from 0.11 to 0.43mg/mL (Supplementary Table S1) were

lower than those obtained for thymol that was used as an antioxidant standard, indicating the

strong antioxidant activity. In this study, the highest antioxidant activity was shown by

methanolic extracts of the leaves and bark of A. incana (0.21 and 0.11mg/mL, respectively).

Extract from the leaves of A. glutinosa had higher antioxidant activity than the bark extract, in

contrast to A. viridis where the bark extract showed higher antioxidant activity than leaves

extract.

In this work, the antioxidant activity of three Alnus species might be attributed to the

presence and synergistic effects of phenolic and flavonoid compounds, besides any other active

compounds present.

The findings of this work show that methanolic extracts of three Alnus species from Bosnia

and Herzegovina possess antioxidative potential and are in agreement with data obtained by

Stevic et al. (2010), who reported the antioxidant activity of Alnus extracts.

2.3. Antimicrobial activity of Alnus extracts

In our study, antimicrobial activity of methanolic extracts obtained from the leaves and bark

of A. glutinosa, A. incana and A. viridis was screened against eight microorganisms

(Supplementary Table S2). The data indicated that the Gram-negative Escherichia coli and the

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Gram-positive Staphylococcus aureuswere the most sensitive strains tested for all extracts. Only

extract of A. viridis leaves had both antibacterial and antifungal properties.

As shown in Supplementary Table S2, all tested Alnus extracts with antimicrobial activity,

except those with capital letters in superscript, inhibited microorganism growth with efficiency

similar to used reference standards. The most active one was methanolic extract of leaves

ofA. viridis comparable to that of standard chloramphenicol (5mg/mL) and kanamycin (5mg/mL).

Gram-negative Pseudomonas aeruginosa is known to have a high level of intrinsic resistance

to practically all known antibiotics, due to a very restrictive outer membrane barrier. However,

extracts of A. glutinosa leaves and barks as well as A. viridis leaves and barks inhibit growth of

this bacterium. These results are consistent with previous reports (Middleton et al. 2005; Stevic

et al. 2010).

However, it is difficult to attribute the activity of a complex mixture to a single or particular

constituent. Major or trace compounds might give rise to the antimicrobial activity exhibited.

Possible synergistic and antagonistic effect of compounds in the extracts should also be taken

into consideration (Cavar et al. 2011).

3. Conclusions

In this work, total phenolic and flavonoid contents of A. glutinosa, A. incana and A. viridis from

Bosnia and Herzegovina and their related antioxidant and antimicrobial activities were

demonstrated for the first time. Considerable antioxidant and antimicrobial abilities of Alnus

methanolic extracts are mainly attributed to the high level of phenolic and flavonoid substances.

Further purification of the active compounds and in vivo evaluation of antioxidant and

antimicrobial activities along with toxicity studies of the extracts of Alnus glutinosa, A. incana

and A. viridis are therefore suggested.

Supplementary material

Supplementary material relating to this article is available online, alongside Tables S1 and S2.

Acknowledgements

The authors wish to acknowledge the financial support of Canton Sarajevo Ministry of Education andScience [grant number 11-14-19953.1/07] for funding the research. The authors are thankful to assistantprofessor Nedad Basic for plant determination and Mr sci. Erna Karalija for assistance in our work.

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