Antioxidant, Antibacterial Activities and General Toxicity of Alnus glutinosa, Fraxinus excelsior and Papaver rhoeas

Authors

Abstract

Alnus glutinosa, Fraxinus excelsior and Papaver rhoeas have long been used in folkloric medicine for the treatment of various ailments. As part of our continuing screening of plant extracts for activities, the extracts of A. glutinosa, F. excelsior and P. rhoeas have been screened for their antioxidant and antibacterial activities, as well as their general toxicity towards brine shrimps. Among the extracts, the methanol (MeOH) extract of F. excelsior displayed the highest level of antioxidant activity (RC50=1.35x10-2 mg/mL) and the dichloromethane (DCM) extract of P. rhoeas was the most toxic extract towards brine shrimps (LD50=2.4x10-2 mg/mL). The n-hexane and DCM extracts of F. excelsior and the MeOH extract of A. glutinosa were active (MIC values were within 1.25x10-1 and 1.00 mg/mL) against all 8 bacterial species tested, including methicillin-resistant Staphylococcus aureus (MRSA).

Keywords


Antioxidant, Antibacterial Activities and General Toxicity of Alnus glutinosa, Fraxinus excelsior and Papaver rhoeas
Iranian Journal of Pharmaceutical Research (2005) 2: 101-103
Received: June 2004
Accepted: February 2005

Copyright ? 2005 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Antioxidant, Antibacterial Activities and General Toxicity of Alnus glutinosa, Fraxinus excelsior and Papaver rhoeas

Patricia Middletona, Fiona Stewarta, Salem Al-Qahtania, Paula Egana, Ciara O?Rourkea, Aysha Abdulrahmana, Maureen Byresa, Moira Middletona, Yashodharan Kumarasamya, Mohammad Shoeba, Lutfun Naharb, Abbas Delazarc and Satyajit Dey Sarkerd*

 

aSchool of Pharmacy, The Robert Gordon University, Aberdeen, Scotland, UK. bSchool of Life Sciences, The Robert Gordon University, Aberdeen, Scotland, UK. cSchool of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. dSchool of Biomedical Sciences, University of Ulster, Coleraine, Londonderry, Northern Ireland, UK

* Corresponding author: s.sarker@ulster.ac.uk

 

Abstract

 

Alnus glutinosa, Fraxinus excelsior and Papaver rhoeas have long been used in folkloric medicine for the treatment of various ailments. As part of our continuing screening of plant extracts for activities, the extracts of A. glutinosa, F. excelsior and P. rhoeas have been screened for their antioxidant and antibacterial activities, as well as their general toxicity towards brine shrimps. Among the extracts, the methanol (MeOH) extract of F. excelsior displayed the highest level of antioxidant activity (RC50=1.35x10-2 mg/mL) and the dichloromethane (DCM) extract of P. rhoeas was the most toxic extract towards brine shrimps (LD50=2.4x10-2 mg/mL).  The n-hexane and DCM extracts of F. excelsior and the MeOH extract of A. glutinosa were active (MIC values were within 1.25x10-1 and 1.00 mg/mL) against all 8 bacterial species tested, including methicillin-resistant Staphylococcus aureus (MRSA).

 

Keywords: Alnus glutinosa; Fraxinus excelsior; Papavar rhoeas; 2, 2-Diphenyl-1-picrylhydrazyl (DPPH); Antibacterial; Brine shrimp lethality assay.

Introduction

 

Alnus glutinosa (L.) Gaertn. (Family: Betulaceae), commonly known as ?black alder? or ?european alder?, native to a number of countries in northern Africa, temperate Asia and Europe, is one of the ca. 30 species of trees and shrubs of the genus Alnus (1, 2).   Various types of plant secondary metabolites including anthraquinones, phenolic glycosides, flavonol glycoside, terpenoids, xanthones, etc. have previously been reported from the barks, buds, leaves and pollens of A. glutinosa (3, 4).  The decoction of A. glutinosa barks has been used to treat swelling, inflammation and rheumatism (5).  It has also been used as an astringent, bitter, emetic and hemostatic, and for the treatment of sore throat and pharyngitis (6). Fraxinus excelsior L. (Family: Oleaceae), commonly known as ?ash? or ?European ash?, is an anemophilous tree native to the countries of temperate Asia and Europe, including Scotland (2, 7, 8). To date, various classes of compounds including benzoquinones, coumarins, flavonoids, phenylethanoids, secoiridoid glucosides, indole derivatives and simple phenolics have been reported from F. excelsior (3, 9, 10).  The barks of F. excelsior have long been used as antipyretic (11).  From the beginning of the 20th century, the leaves of this plant have been recommended in prescriptions for the treatment of fever or rheumatism (12).  The alcoholic extract of F. excelsior barks possesses an anti-inflammatory property similar to diclofenac (12, 13).  The leaf tea is popular in Europe as a mild purgative and is often used for rheumatism, while the bark is effective against intestinal worms (11).  Other medicinal uses of this plant include its use in the treatment of arteriosclerosis, hypercholesterolemia, jaundice and kidney problems (14).  Papaver rhoeas L. (family: Papaveraceae) is commonly known as ?corn poppy? and found wild in various parts of Europe, northern Africa, western Asia and Indian subcontinent (2, 15-17).  The medicinal uses of P. rhoeas are somewhat unclear.  However, as early as the 11th century, Arab physicians used this plant as a cough remedy (18).  This plant is claimed to be useful in the treatment of respiratory problems, asthma, cough, loss of voice, hay fever, insomnia, and intestinal and urinary irritations (18, 19).   Previous phytochemical investigations on this plant have revealed the presence of mainly various alkaloids (3, 9, 20-22).  As part of our on-going screening of plant extracts for activities (23-30), we now report on the antioxidant, antibacterial activities, and general toxicity of A. glutinosa and P. rhoeas extracts obtained from their seeds, and that of of F. excelsior leaves.

 

Experimental

 

General

All solvents were purchased from Fischer Scientific Ltd., Loughborough, England. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), molecular formula C18H12N5O6, was obtained from Fluka Chemie AG, u. k. Quercetin was obtained from Avocado Research Chemicals Ltd, Heysham, u.k. Resazurin tablets were purchased from BDH Laboratory Supplies, Poole, England.  Waterlife? brand brine shrimp (Artemia salina) eggs were purchased from The Waterlife Research Industries, middlesex, UK.  Podophyllotoxin was obtained from Sigma-Aldrich, Dorset, UK.

Plant materials

The seeds of Alnus glutinosa (Cat no. 225), Papaver rhoeas (Cat no. 13928) and the leaves of Fraxinus excelsior (Cat no. 402314) were purchased from B & T World Seeds, Sarl, France and voucher specimens, respectively, PH00171103-2-SDS, PH00171103-3-SDS and PH00171103-1-SDS were deposited in the herbarium of the Department of Plant and Soil Science, University of Aberdeen, Aberdeen.

Extraction

Ground seeds (~100 g) of A.glutinosa and P. rhoeas, and leaves (~100 g) of F. excelsior were Soxhlet-extracted sequentially, using solvents (1.1 L each) of increasing polarity, n-hexane, dichloromethane (DCM) and methanol (MeOH).  Solvent was evaporated from the extracts, using a rotary evaporator, at a temperature not exceeding 50?C.

Preparation of the extract solutions for bioassays

The n-hexane, DCM and MeOH extracts (0.025g) were dissolved in 5 mL DMSO (or MeOH) to obtain stock solutions of 5 mg/mL concentration.

DPPH assay

The DPPH assay was used to determine the free radical scavenging (antioxidant) activity.   The method used by Takao et al. (31) was adopted with suitable modifications (23, 24, 27).  DPPH (4 mg) was dissolved in MeOH (50 mL) to obtain a concentration of 80 ?g/ml.

Qualitative assay

Test samples were applied on a TLC plate and sprayed with DPPH solution, using an atomiser. It was allowed to develop for 30 min. The colour changes (purple to white) were noted.

Quantitative assay

Stock solutions (5 mg/mL) of the plant extracts were prepared in MeOH.  Serial dilutions were carried out to obtain concentrations of 5x10-1, 5x10-2, 5x10-3, 5x10-4, 5x10-5, 5x10-6 5x10-7, 5x10-8, 5x10-9 and 5x10-10 mg/mL.  Diluted solutions (1 mL each) were mixed with DPPH (1 mL) and allowed to stand for 30 min for any reaction to occur. The UV absorbance was recorded at 517 nm. The experiment was performed in triplicate and the average absorption was noted for each concentration. The same procedure was followed for the positive control (quercetin).

Antibacterial assay

Antibacterial activity of the extracts was tested against 8 species of Gram-positive and Gram-negative pathogenic bacteria (Table 1). The bacterial cultures used were from the properly identified and appropriately maintained stock cultures from the Micro Research Laboratory, School of Pharmacy, the Robert Gordon University.  The antibacterial test was performed using the 96 well microplate-based broth dilution methods and the resazurin solution as an indicator of bacterial growth (24, 32). All tests were performed in triplicate. 

 

Table 1. Antibacterial activity of the extracts obtained from the seeds of A. glutinosa (AG), P. rhoeas (PR) and the leaves of F. excelsior (FE)

Bacterial species

MIC (mg/mL)

n-Hexane

DCM

MeOH

Positive control (Norfloxacin)

AG

PR

FE

AG

PR

FE

AG

PR

FE

3.91 x 10-4

Citrobacter freundii  NCTC 9750

2.00

-

0.250

2.00

-

0.250

0.500

-

1.00

9.77 x 10-5

Escherichia coli NCIMB 8110

2.00

-

0.250

2.00

-

0.250

0.125

-

2.00

1.56 x 10-3

Escherichia coli NCIMB 4174

2.00

-

0.250

2.00

 

0.250

0.500

-

-

1.56 x 10-3

Klebsiella aerogenes NCTC 9528

-

-

0.250

-

-

0.250

0.250

-

2.00

1.56 x 10-3

Lactobacillus plantarum NCIMB 6376   

-

-

0.250

-

-

0.250

0.500

-

2.00

1.56 x 10-3

Pseudomonas aeruginosa NCTC 6750

2.00

-

0.250

2.00

-

0.250

0.500

-

2.00

1.56 x 10-3

Staphylococcus aureus NCTC 10788

2.00

-

0.125

2.00

-

0.125

0.250

-

2.00

3.90 x 10-4

Staphylococcus aureus NCTC 11940 ( MRSA)

-

-

0.500

-

-

0.500

1.00

-

-

3.13 x 10-3

= No inhibition of growth at the highest concentration (5 mg/mL) tested There was no significant inhibition of growth observed with the negative control DMSO

 

Preparation of bacterial species

The bacterial cultures were prepared by incubating a single colony overnight in nutrient agar at 37?C, following the procedure described by Sarker et al. (24).  The bacterial solution was diluted in order to obtain a concentration of 5 x 105 cfu/mL.

Preparation of resazurin solution

One resazurin tablet was dissolved in 40 mL sterile distilled water to obtain the standard resazurin solution.

Preparation of 96 well plates and assay

The top 96 well plates were prepared and the assays were performed according to the method described by Sarker et al. (24).  Norfloxacin, a well-known antibiotic, was used as  the positive control.  Normal saline, resazurin solution and dimethyl sulphoxide (DMSO) were used as negative controls. The presence of bacterial growth was indicated by colour changes from purple to pink.

Brine shrimp lethality assay

The method of Meyer et al. (33) was adopted to study the general toxicity of the extracts (24).  Briefly, the brine shrimp eggs were hatched in a conical flask containing brine shrimp medium (300 mL), the flasks were well aerated with the aid of an air pump, and kept in a water bath at 29-30?C, a bright light was left on, and the nauplii hatched within 48 h.  The stock solution of each extract (5 mg/mL) was serially diluted ten-times, solution of each concentration
(1 mL) was transferred into clean sterile universal vials with a pipette, and aerated seawater (20 mL) was added. About 10-15 nauplii were transferred into each vial with a pipette. A check count was performed. The number alive after 24 h was noted. The mortality endpoint of this bioassay was defined as the absence of controlled forward motion during 30 sec of observation. The experiment was carried out in triplicate and the average values were noted. The controls used were DMSO, normal saline, and podophyllotoxin (3 ?g/mL). Abbots formula was used to correct the values, i.e., P=Pi- C/1-C, where P denotes the observed non-zero mortality rate and C represents the mortality rate of the DMSO control.

 

Results and Discussion

 

Antioxidant activity

The DPPH assay (23, 24, 31) was used to determine the antioxidant potential of the extracts of A. glutinosa, P. rhoeas and F. excelsior.  The DPPH contains an odd electron which becomes paired off in the presence of antioxidant compounds.  In the stable free radical form DPPH is purple and when in contact with antioxidant compounds, it becomes yellow (34, 35).  This resulting decolourisation is stoichiometric with respect to the concentration of antioxidant.  In the qualitative DPPH assay, while the n-hexane and DCM extracts of A. glutinosa showed extremely low levels of antioxidant activity evident from faint white spots against a pink background on the TLC plate, the MeOH extract displayed a quite significant antioxidant property.  In the quantitative assay (Table 2), the RC50 value of the n-hexane and DCM extract could not be determined within the test concentrations (5 mg/mL being the concentration of stock solutions).  The RC50 value of the MeOH extract was found to be 1.27 x 10-1 mg/mL.  The antioxidant property (RC50 = 1.35 x 10-2) of the MeOH extract of F. excelsior was the most potent of all extracts among the three plants.  While all three F. excelsior extracts showed a significant activity in the qualitative DPPH assay, the RC50 value for the n-hexane and DCM extracts could not be determined due to the interference from the high amounts of chlorophyll present in these extracts: this is because chlorophyll also absorbs light significantly at the wavelength of 517 nm, which was used to determine the RC50 values spectrophotometrically.  None of the P. rhoeas extracts exhibited any antioxidant activity, either in the qualitative or quantitative DPPH assay.

 

Table 2. Antioxidant activity and brine shrimp toxicity of the seeds of A. glutinosa (AG), P. rhoeas (PR) and the leaves of F. excelsior (FE)

Assay

mg/mL

Quercetin/

podophyllotoxin*

(mg/mL)

n-Hexane

DCM

MeOH

AG

PR

FE

AG

PR

FE

AG

PR

FE

Antioxidant activity (RC50)

-

-

NO

-

-

NO

1.27 x 10-1

-

1.35 x 10-2

2.88 x 10-5

Brine shrimp toxicity    (LC50)

5.29 x 10-1

ND

2.6 x 10-2

8.3 x 10-1

2.4 x 10-2

7.0 x 10-2

1.29 x 10-1

2.6 x 10-2

8.71 x 10-1

2.4 x 10-3

*Quercetin and podophyllotoxin were used as positive controls, respectively, for antioxidant and brine shrimp toxicity assays

- = no activity at test concentrations

NO =  The RC50 value could not be obtained due to the interference from high amounts of chlorophyll present in the extracts

ND = Could not be done due to extremely oily nature of the extract

 

Antibacterial activity

The micro-plate based serial dilution checkerboard method is one of the most convenient assays for determining antibacterial activity quantitatively (MIC determination).  The convenience of this method can even be enhanced significantly by incorporating resazurin as an indicator of cell growth.  The extracts of P.rhoeas did not show any antibacterial activity, at test concentrations, against any of the 8 bacterial species (Table 1).   Among the three extracts of A. glutinosa, the MeOH extract was found to be active against all bacterial species including MRSA; the most potent activity was against E. coli (8110) with an MIC value of 1.25x10-1 mg/mL.  Despite the high MIC value against MRSA (1.00 mg/mL), this finding could be considered significant, at least qualitatively, because this activity was not due to a purified compound, but to a crude extract.  The n-hexane and DCM extracts of F. excelsior were also active against all bacterial species tested (MIC values within the range of 1.25x10-1 to 1.00 mg/mL).  Like the MeOH extract of A. glutonosa, the n-hexane and DCM extracts of F. excelsior were active against methicillin-resistant Staphylococcus aureus (MRSA), but with a lower MIC value (5.0x10-1 mg/mL).

General toxicity

Brine shrimp lethality assay is a convenient method for general screening for toxicity of the extracts or compounds towards brine shrimp, and it can give an indication regarding possible cytotoxicity of the test samples.  All cytotoxic compounds show positive results in this assay, but not necessarily all extracts or compounds that show a positive result in this assay are cytotoxic.  An LD50 value of <1 mg/mL is considered to be significant, and the lower the value the higher is the toxicity of the test sample (13).   Apart from the n-hexane extract of P. rhoease, which was too oily to disperse in the brine shrimp medium, all extracts were tested for general toxicity using the brine shrimp lethality assay.  High levels of toxicity were observed with the n-hexane and DCM extracts of F. excelsior (LD50 = 2.6x10-2 and 7.0x10-2 mg/mL, respectively), and the DCM and MeOH extracts of P. rhoeas (LD50 = 2.4x10-2 and 2.6 x10-2 mg/mL, respectively).   All three extracts of A. glutinosa showed low levels of toxicity towards brine shrimps (LD50 values were in the range of 1.29x10-1 to 8.30x10-1 mg/mL).

The antibacterial, antioxidant activities and general toxicities of various extracts of A. glutinosa, P. rhoeas and F. excelsior, found in this study, may explain some of the traditional medicinal uses of these plants.  The anti-MRSA activity of the MeOH extract of A. glutinosa and the n-hexane and DCM extracts of F. excelsior could be of particular interest in relation to the isolation and identification of new ?lead? compounds for the development of anti-MRSA drugs.

 

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