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ISSN (Online): 2349-056X
International Journal of Pharmacy Education and Research
Apr-Jun 2016; 3(2): 4-8.
Available online: www.ijper.net
Email: editor.ijper@gmail.com
Research Article
Antibacterial and Antifungal activities of Linum Usitatissimum
(Flax seeds)
Bongoni RAJA NARENDER*1, Sirikonda TEJASWINI1, Mekala SARIKA1, Nallagonda
KARUNA1, Racharla SHIRISHA1, Sirikonda PRIYANKA1
*1Department of Pharmaceutics, Sree Chaitanya Institute of Pharmaceutical Sciences, LMD Colony,
Thimmapur, Karimnagar - 505527, Telangana State, INDIA.
INTRODUCTION
Plants have long provided mankind with a
source of medicinal agents, with natural products once
serving as the source of all drugs. Dependence on
plants as the source of medicine is prevalent in
developing countries where traditional medicine plays
a major role in health care. The rural population of a
country is more disposed to traditional ways of
treatment because of its easy availability and cheaper
cost. Herbal therapy, although still an unwritten
Received on: 30 Apr, 2016
Revised on: 5 May, 2016
Accepted on: 6 May, 2016
*Corresponding author:
B. Raja Narender,
Department of Pharmaceutics,
Sree Chaitanya Institute of
Pharmaceutical Sciences,
LMD Colony, Thimmapur,
Karimnagar - 505527,
Telangana State, INDIA.
Mobile #: +91-97625-37250.
Email:
rajanarenderbongoni@gmail.com
ABSTRACT
Flax seeds are one of dietary sources containing considerable
amount of phenolics named lignans. The aim of this study was to
evaluate the potential activities of lignan extracts as a potential source
of antimicrobial agents like secoisolariciresinol diglucoside (SDG) in
variables levels.
In the present investigation, we present the antibacterial
activity and antifungal activity of Flax seeds extracts with unique
composition of different phenylopropanoid compounds. Due to the
elevated level of secoisolariciresinol diglucoside (SDG), ferulic acid, p
–coumaric acid, their glucosides and also their multi directional mode
of action the Flax seeds (powder extract) were effective in inhibiting
bacterial growth and fungal growth. We aimed to evaluate activity of
Flax seeds powder extract against the bacteria and fungi of clinical
relevance: Escherichia coli, Salmonella paratyphii, Lactobacillus and
Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae,
Saccharomyces cerevisiae known to be causes of antibiotic-resistant
infections. We performed the agar diffusion method and indicated the
bacteriostatic or bactericidal action and fungistatic or fungicidal action.
We suggest that extracts derived from Flax seeds might be the effective
source of antibacterial compounds and the promising alternative to
antibiotic therapy.
Keywords: Flax seeds, lignans, phenylopropanoids, antibacterial
activity and antifungal activity.
Raja Narender et al. IJPER | Apr-Jun, 2016; 3(2): 4-8.
5
science, is well established in some cultures and
traditions, and has become a way of life in almost 80%
of the people in rural areas, especially those in Asia,
Latin America and Africa. Medicinal plants, which
form the backbone of traditional medicine, have in the
last few decades been the subject of very intense
pharmacological studies. This has been brought about
by the acknowledgement of the value of medicinal
plants as potential sources of new compounds of
therapeutic value and as sources of lead compounds in
drug development. Besides, there also exists a very
large market of minimally processed medicinal plant
parts especially in Europe and America, which are
usually dispensed as over-the-counter medication [1, 2].
Studies have reported that Flax is a plant and
has been a stable part of farming for hundreds of years
and every part of it was usable. The seeds for food and
the production of linseed oil (another name for Flax
and is a good preserver of wood as well as good for
our bodies) and the stems for example can be used to
produce linen fiber for clothes and other products. The
oil industry (petroleum) removed some of the needs of
Flax and so its production took a decline especially
after the II World War commonly cultivated Flax
plants are named Linum usitatissimum L [3]. Cultivated
Flax plants grow to 1.2m (3ft 11in) tall, with slender
stems. The leaves are glaucous green,
slender lanceolate, 20-40mm long and 3mm broad.
The flowers are pure pale blue, 15–25mm diameter,
with five petals. The fruit is a round, dry capsule 5-
9mm diameter, containing several glossy
brown seeds shaped like an apple pip, 4–7mm long.
Common names: Flax seed, linseed, Alsi or
teesi (Hindi, Gujarati, and Punjabi), Ali vidai in Tamil
,Atasi and Jawas in Marathi, Tishi in Bengali, Pesi in
Oriya, Agasi in Kannada, Avise ginzalu in Telugu, and
Cheruchana vithu in Malayalam [4].
Another potent healing element found in the
fibrous shell of the Flax seed are plant ‘lignans’.
Lignans are phyto-hormone precursors that exhibit
immune stimulatory and anti-bacterial, anti-fungal and
anti-viral effects [5]. In the intestines, lignans are
structurally modified by bacteria into mammalian
lignans – enterlactone and enterodiol. These natural
estrogen-like compounds have a buffering effect on
estrogen metabolism [6].
Figure 1(a): showing the Capsules of Flax seed plant; 1(b): showing the Flowers of Flax seed plant;
1(c): showing the Flax seeds (Yellow); and 1(d): showing Flax seeds (Brown)
MATERIALS AND METHODS
SAMPLE
Flax seeds were obtained from the local
market. The seeds were washed, cleaned of
extraneous matter and shade dried completely. The
seeds were dried at room temperature under shade for
a period of one week. The dried seeds were grinded
using a laboratory grinder.
MICROBIAL CULTURES
Pure cultures were collected from the
Department of Microbiology, Kakatiya University,
Warangal, Telangana State, India.
CULTURE MEDIA
For culturing of the microorganisms, nutrient
agar medium was used. Nutrient broth was used for
the incubation and standardization of the
microorganisms. The following cultures were used for
the study: Salmonella paratyphi, Escherichia coli,
Klebsiella pneumonia and Staphylococcus aureus,
Proteus vulgaris, Lactobacillus sporogens,
Saccharomyces cereviseae.
MEDIA
Nutrient agar was procured from HiMedia
and fungal media was procured from standard YPD
media.
Raja Narender et al. IJPER | Apr-Jun, 2016; 3(2): 4-8.
6
PREPARATION OF CRUDE EXTRACT
Crude extract prepared was divided into two
portions; one portion (10g) was taken into glass vials
to be tested as crude methanol extract for antimicrobial
activity while the second portion (30g) was taken into
a glass beaker for fractionation with different solvents.
The crude extract for fractionation was suspended into
80 ml of distilled water and methanol (water: methanol
at the ratio of 8:2) and they are taken into separating
funnel then extracted with 200 ml ethyl acetate. The
separating funnel was shaken and compounds soluble
in ethyl acetate phase were collected and the aqueous
phase was extracted thrice with ethyl acetate. All
fractions of ethyl acetate were combined and poured
into round bottom flask of rotary evaporator and ethyl
acetate was isolated from the fraction leaving behind
semisolid ethyl acetate fraction. The semisolid ethyl
acetate fraction was dried in a China dish through
water bath at about 50°C and was stored in the glass
vials until used. Solvent extraction methodology was
carried out for ether, chloroform, distilled water and
butanol [8].
PREPARATION OF MEDIA
The required quantities of nutrient agar (2.8g
100ml-1) and nutrient broth (1.3g 100ml-1) were
prepared by dissolving it in distilled water in conical
flasks.
PREPARATION OF NUTRIENT BROTH
Broth was sterilized in an autoclave at 15psi
pressure and 121°C for 15 min. After sterilization,
some of the nutrient broth (approximately 20ml test
tube-1) was also poured into the test tubes. Then pure
cultures are taken and with the help of inoculating loop
the cultures are inoculated and then they are kept for
incubation for 24 hours at 37oC [9].
PREPARATION OF NUTRIENT AGAR MEDIA
Nutrient agar media of 50ml was separated in
other conical flasks and is inoculated with 0.5ml of
cultures in the same way fungal media is also prepared
but standard media that is YPD media is used and
poured aseptically into sterilized Petri plates. The
media was allowed to be solidified in petri plates and
then wells are made with sterile borer having 1cm
diameter and then placed in an incubator at 37°C for
24 h for bacteria and 30oC for 48 hr for fungi [10].
STANDARD REFERENCE ANTIBIOTIC
The reference antibiotics used were
Streptomycin and Flucanazol.
DISC DIFFUSION SUSCEPTIBILITY METHOD
In this method, nutrient agar media plates
were seeded with 18 to 24 h cultures of microbial
cultures and then different Flax seed extracts were
applied on the discs. Media plates were incubated at
37°C for 18 to 24 h in an incubator. The chemicals
diffused from the discs into the agar media thus
preventing the growth of microorganisms (if
susceptible) in the area around the disc known as zone
of inhibition. The next day, zones of inhibition were
calculated from each treatment [11].
POSITIVE CONTROLS
For Gram positive bacteria and for Gram
negative bacteria, Streptomycin was used and for
fungal activity Flucanazol was used.
ANTIMICROBIAL ACTIVITY
The antimicrobial activities were carried out
according to the conventional disk diffusion method,
using cultures of Klebsiella pneumoniae, Samonella
paratyphii, Lacto bacillus, Escherichia coli,
Staphylococcus aureus, Proteus vulgaris and
Saccharomyces cereviceae. The bacterial strains were
cultured on nutrient medium, while the fungi were
cultured on fungal medium, respectively. For bacteria,
the broth media were incubated for 24 h.
As fungi, the broth media were incubated for
approximately 48hr, the solution containing the spores
was used for inoculation. For preparation of plate
inoculation, 0.5ml of cultured were inoculated to 50ml
of agar media (50ºC) and mixed by simple inversion.
The agar was poured into 120mm petri dishes and
allowed to cool to room temperature. The microbial
growth inhibition zone for bacteria was measured after
incubation at 37°C and for fungi was measured after
incubation at 30oC, the appearance of the clear
microbial free inhibition zones, begin within 24 h for
bacteria and 48-72 h for fungi [12, 13].
Different microbial species were used to
screen the possible antimicrobial activity of different
extracts of flax seeds such as butanol, chloroform,
ether, methanol, ethyl acetate, distilled water.To
determine antimicrobial activity, Flax extracts were
tested against different organisms. This was assumed
to be sufficient for the antimicrobial screening. Very
clear differences were found between the effects of
different extracts in the study.
Maximum antimicrobial activity was
observed with aqueous extract of Flax seeds against
Gram negative bacteria, gram positive bacteria and
fungi. The different resistant patterns are likely to be
related to differences in fungi and bacteria cell wall
structures and protein synthesis. The presence of
lignans may bind both Ca+2 and Mg+2, thereby
reducing the Ca+2 and Mg+2 from lipopolysaccharide
of the outer membrane causing a release of
lipopolysaccharide, thereby destabilizing the
membrane, which may increase the activity of lignans.
STATISTICAL ANALYSIS
Values are given as mean ± standard
deviation (SD). In all analyses, P<0.05 was
considered significant.
Raja Narender et al. IJPER | Apr-Jun, 2016; 3(2): 4-8.
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RESULTS AND DISCUSSION
Among all the seed extracts aqueous extract
found to have maximum extractive yield followed by
chloroform extract followed by methanol extract
followed by ethyl acetate followed by ether extract
followed by butanol extracts. The different extracts of
Flaxseed powder were subjected for antibacterial and
antifungal activities and the results were investigated,
the crude extracts of seeds revealed the presence of
antibacterial and antifungal activities.
The results of antibacterial screening by agar
cup plate method indicates the highest antibacterial
and antifungal activities was shown by the aqueous
extract of against of all the organisms followed by
chloroform followed by methanol followed by ether
followed by ethyl acetate followed by butanol extracts
which showed activity against E.coli, S. aureus, K.
pneumonia, Lacto bacillus, Proteus vulgaris,
Samonella paratyphii, Saccharomycecs cerevisiae
only. Extract of powder showed good activity of all
organisms.
Standard antibiotic Streptomycin was
effective against all organisms and showed a zone of
inhibition 1-5cms. The results of the investigation
showed that seeds extract from Linum usitatisimum
have good antibacterial activity against E.coli,
Lactobacillus, Staphylococcus aureus, Salmonella
paratyphii, Klebsiella pneumoniae, Proteus vulgaris,
Saccharomycecs cerevisiae.
Standard antibiotic streptomycin and
flucanazole was effective against all organisms and
showed a zone of inhibition of 50-55mm the result of
the investigation showed that the seed extract have
good antibacterial activity against E.coli,
Staphylococcus aureus, Salmonella paratyphii,
Lactobacillus, Proteus vulgaris, Klebsiella
pneumonia, saccharomyces cerevisiae due to the
presence of lignans.
Table 1: Plant profile
Kingdom
Plantae
Order
Malpighiales
Family
Linaceae
Genus
Linum
Species
L. usitatissimum
Binomial name
L. usitatissimum L.
Table 2: Antifungal activity of Linum usitatisimum
Name of the
organism
Conc.
(mg/ml)
Zone of inhibition (in cm.)
Aqueous
Ether
Chloroform
Ethyl
acetate
Butanol
Methanol
Standard
(Flu.)
S.
cerevireae
10
-
-
-
-
-
-
-
20
20
1.9
2.0
2.2
1.8
1.9
5.0
30
30
1.5
1.4
2.0
1.3
1.5
5.5
Values are mean ± SD of three results. Flu. = Fluconazole
Table 3: Antibacterial activity of Linum usitatisimum
Name of the
organism
Conc.
(mg/ml)
Zone of inhibition (in cm.)
Aqueous
Ether
Chloroform
Ethyl
acetate
Butanol
Methanol
Standard
(Strep.)
S. aureus
10
-
-
-
-
-
-
-
20
1.8
1.8
1.9
2.3
2.6
1.8
5.5
30
1.2
1.4
1.3
1.6
1.9
1.2
6.1
Proteus
vulgaris
10
-
-
-
-
-
-
-
20
1.7
1.6
1.9
1.8
1.7
2.1
3.5
30
1.2
1.3
1.5
1.4
1.3
1.6
4.5
Klebsiella
pneumonia
10
-
-
-
-
-
-
-
20
1.9
1.9
1.7
1.8
1.6
2.0
3.5
30
0.7
0.5
0.7
1.0
1.1
1.6
5.5
Lactobacillus
10
-
-
-
-
-
-
-
20
1.8
2
1.9
2.0
2.0
2.1
3.0
30
1.6
1.5
1.4
1.5
1.0
1.0
4.7
E. coli
10
-
-
-
-
-
-
-
20
2.0
2.5
1.9
1.5
1.8
2.3
4.0
30
1.5
1.6
1.0
0.8
1.0
1.2
4.9
Salmonella
paratyphi
10
-
-
-
-
-
-
-
20
3.0
1.8
1.8
1.9
1.7
2.0
3.0
30
1.5
1.4
1.3
1.5
1.5
1.8
4.2
Raja Narender et al. IJPER | Apr-Jun, 2016; 3(2): 4-8.
8
Values are mean ± SD of three results. Strep. = Streptomycin
CONCLUSION
The work states that in the presence of
extracts of Linum usitatisimum were responsible for
antibacterial and antifungal activity these extracts
exhibit a maximum zone of inhibition against E.coli,
Proteus.vugaris, Staphylococcus aureus, Salmonella
paratyphii, Klebsiella pneumoniae, Lactobacillus,
Saccharomycecs cereviseae. It is interesting to
observe the results of high antibacterial effects of
aqueous and chloroform.
This study gave the way for further attention
and research to identify the active compounds
responsible for the seed biological activity. Further
studies should be undertaking to elucidate the exact
mechanism of action by which extracts exert there
antibacterial and antifungal activities.
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