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).
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
Antioxidant, Antibacterial Activities and
General Toxicity of Alnus glutinosa, Fraxinus excelsior and
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
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
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 Staphylococcusaureus
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).
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
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,
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.
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.
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.
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.
Stock solutions (5 mg/mL) of the plant extractswere
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 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)
Positive control (Norfloxacin)
3.91 x 10-4
9.77 x 10-5
Escherichia coli NCIMB 8110
1.56 x 10-3
1.56 x 10-3
Klebsiella aerogenes NCTC 9528
1.56 x 10-3
Lactobacillus plantarum NCIMB 6376
1.56 x 10-3
Pseudomonas aeruginosa NCTC 6750
1.56 x 10-3
Staphylococcus aureus NCTC 10788
3.90 x 10-4
Staphylococcus aureus NCTC 11940 ( MRSA)
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
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
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
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).
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.
Antioxidant activity and brine shrimp toxicity of the seeds of A. glutinosa
P. rhoeas (PR)and the leaves of F. excelsior (FE)
Antioxidant activity (RC50)
1.27 x 10-1
1.35 x 10-2
2.88 x 10-5
Brine shrimp toxicity (LC50)
5.29 x 10-1
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
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
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.
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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