Efficacy evaluation of ethanolic extract of Brassica nigra as potential antimicrobial agent against selected microorganisms

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International Journal of Pharmaceutical Science and Health Care Issue 4, Vol. 3.May-June 2014
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Abstract:
Spices are used as additives to flavour, colour or preserve food from ancient time in India. These
are pungent or aromatic substances obtained from by dried seeds, fruits, roots, bark or leaves.
Spices are the potential source of natural products and naturally derived compounds. These
compounds show the antioxidant, antimicrobial properties. The aim of this study is to evaluate
the antimicrobial properties of Brassica nigra. In this study, antimicrobial activities of 70%
ethanolic seed extracts of Brassica nigra were evaluated against different bacterial strains
(E.coli, Pseudomonas aeruginosa, Candida albicans) by agar well diffusion method & MIC
determination by broth dilution method. The extract showed a broad spectrum of antimicrobial
activities by inhibiting the growth of respective microorganism in Agar well diffusion assay. The
present study supports the immense medicinal properties of Brassica nigra. It may be helpful for
future researches in area of new drug development of herbal origins.
Keywords: Brassica nigra, Antibacterial; MIC; broad spectrum; medicinal properties
Introduction:
The World Health Organization (WHO) reported that about 80% of the world’s population
depends primarily on traditional medicine that mainly involves the use of plant extracts [1].
There are 2600 plant species of which more than 700 are noted for their uses as medicinal herbs
[2]. In folk medicine, medicinal herbs and plant products were used in treating a wide spectrum
of infections and other diseases. [3,4] Today, a great number of different medicinal tea and other
plant products are available in market (including cosmetics and pharmaceuticals), which contains
biologically active substances. In recent years, there has been a gradual revival of interest in the
use of medicinal and aromatic plants in developed as well as in developing countries, because
plant-derived drugs have reported to be safe and without side-effects. A survey of literature
reveals that there are many essential oils which possesses antifungal activity [5]. Satia-Abouta et
al [6] in 2002 reported that the cuisines of Asia, Southeast Asia, and the Mediterranean are
perceived by many to be healthier than the typical Western diet.
The traditional use of plants as medicines provide the basis for indicating which essential oils
and plant oils may be useful for specific medical conditions. Historically, many plant oils and
extracts, such as tea tree, myrrh, clove, Ajwain and Asafoetida (Heing) have been used as topical
antiseptics and have antimicrobial properties [7,8,9]. It is important to investigate scientifically

International Journal of Pharmaceutical Science and Health Care Issue 4, Vol. 3.May-June 2014
Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738
R S. Publication, rspublicationhouse@gmail.com Page 118
those plants which have been used in traditional medicines as potential sources of novel
antimicrobial compounds [10]. Also, the resurgence of interest in natural therapies and
increasing consumer demand for effective, safe, natural products means that quantitative data on
plant oils and extracts are required.
Brassica nigra (L) (Black mustard) is an annual, erect, 2-7 degree high, high freely and widely
branching, pubscent or glabrate. Lower leaves slender petioled, deeply pinnatifid, with 1 terminal
large lobe and 2-4 smaller lateral ones, dentate all around; upper leaves shorter-petioled or
sessile, pinnatifid or dentate, the uppermost reduced to lanceolateor oblong entire blades; flowers
bright yellow, 3”-5” broad; pedicels slender , appressed,2” long in fruit; pods narrowly linear, 4
sided, 5”-7”long, ½ wide, a pressed against the stem and forming very narrow racemes; beak
slender,1”-2”long;seeds dark brown [11].
It is cultivated in the states of Assam, Bihar, Gujarat, Haryana, Himachal Pradesh, Jammu &
Kashmir, Madhya Pradesh, Orrissa, Punjab, Rajasthan, Uttar Pradesh etc.
The antimicrobial activity of Brassica nigra against different micro-organisms has been
observed in few studies so it was thought worthwhile to assess the anti-microbial activity of
Brassica nigra spice (Ethanolic Extract of seed) against different pathogens.
Materials & Methods:
Collection of sample:
The seeds of spice Brassica nigra (black mustard) were collected from local Gwalior market.
Micro-organisms Used:
1. Staphylococcus aureus
2. Escherichia coli
3. Candida albicans
These micro-organisms were isolated from clinical samples by using standard microbiological
procedures. Identification was done morphologically and biochemically following the standard
of Bergey’s Manual of Determinative Bacteriology.
Preparation of extracts:
The seed of black mustard were dried and broken into small pieces (powder) under sterile
conditions, and 10 g of black mustard seed powder were dissolve with 100 ml of 70% ethanol,
(Merck) and shaken at 160 rpm for 24 hours at ambient temperature. The mixtures were then
filtered. The filtrates were evaporated and stored at 4°C for further use. Stock solutions of crude
extracts were prepared by diluting the dried extracts with DMSO solution, to obtain a final
concentration of 200 mg/ml.
Collection of microorganism:
The bacterial strains such as E. coli , Staphylococcus aureus and Fungal strain Candida albicans
were isolated from clinical samples and used for antimicrobial assay. Bacterial strain were grown
in LB medium contains in water at pH 7.2 and incubated at 37o C for overnight. Whereas fungal

International Journal of Pharmaceutical Science and Health Care Issue 4, Vol. 3.May-June 2014
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strain Candida albicans were grown in PDB medium containing potato infusion and dextrose at
pH 5.6 incubated at 25o C for five day. The entire microorganism subcultured in 30 days.
Screening for antimicrobial activities:
The antibacterial and antifungal activity studies were carried out by agar well diffusion method
as given by Irobi et al [12]. The sterile nutrient agar plates and potato dextrose agar plates were
prepared. The bacterial test organisms like Staphylococcus aureus, and Escherichia coli were
spread over the nutrient agar plates by using separate sterile spreader. Then the fungal test
organism like Candida albicans were spread over the potato dextrose agar plates. Wells were
then bored into the plates of seeded organisms using sterile cork borer of 6 mm in diameter then
50μl extracts of concentration 200mg/ml were placed on the wells in different plates with control
plates with solvent . All bacterial plates were incubated at 37°C for 24 hrs and fungal plates at
25°C for five days. The diameter of the minimum zone was measured in mm.
Minimum inhibitory concentration (MIC):
The extracts which showed antimicrobial activity in agar well assay will be subjected to MIC
assay. The antimicrobial MIC studies were carried out by broth dilution method.
6.Results:
In the present investigation the Brassica nigra (black mustard) spice were collected from local
Gwalior market. The 70% ethanol solvent was used for the extraction of active agents from black
mustard spices. Extracts were used for investigation of the antimicrobial potential. The results of
in vitro antimicrobial activity of black mustard spices against pathogens are presented below:
Figure 1 shows the antimicrobial activity of 70% ethanol extract of Black mustard (Brassica
nigra). The 70% ethanol extracts showed antibacterial & antifungal activity against E.coli, S.
aureus & C.albicans respectively. The maximum zone of inhibition showed against C.albicans
(30 mm) Whereas S.aureus (25mm) & E.coli (20.5mm) showed moderate activity.
Figure 1. Antimicrobial activity of 70% ethanolic extract of B.nigra against microorganisms
0
5
10
15
20
25
30
E.coli
S.aureus
C. albicans
Zone of Inhibition (mm)
Microorganisms
Antimicrobial activity of B.nigra (ethanolic seed extract)
against some microorganisms
E.coli
S.aureus
C. albicans

International Journal of Pharmaceutical Science and Health Care Issue 4, Vol. 3.May-June 2014
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The extract which showed antibacterial activity in agar well assay will be subjected to MIC
assay. The antimicrobial MIC studies were carried out by broth dilution method.
Figure 2 shows the results of MIC assay, it indicates that the MIC value of crude extract against
E.Coli was10 mg and for S aureus & C. albicans it was 8 mg.
Figure 1. Minimum Inhibitory concentration of extract of B.nigra against microorganisms
Discussion:
In recent years, multiple drug resistance in human pathogenic microorganisms has been
developed due to indiscriminate use of commercial antimicrobial drugs commonly used in the
treatment of such diseases [13,14]. Plants are the richest source of natural antimicrobial agents.
Traditional healers claim that some medicinal plants are more efficient to treat infectious
diseases than synthetic antibiotics [15]. Biomolecules of plant origin appear to be one of the
alternatives for the control of these antibiotic resistant human pathogens [16].
There is widespread use of plants as ethnomedicines. After the pathogen attack, some plants may
produce certain compounds such as secondary metabolites being known as phytoalexins. These
compounds have shown remarkable in vitro antibacterial activity against potential pathogens
[17]. There is immense need of antimicrobial drugs developed from natural sources as plant
derived products are safe in comparison to chemically synthesized products [18].
Brassica family is a commonly consumed vegetable and a valuable source of glucosinolates,
polyphenols and flavonoids. On hydrolysis by enzyme myrosinase, glucosinolates can produce
d-glucose, sulfate, isothiocyanates, thiocyanates and nitriles [19]. Isothiocyanates have the
largest bactericidal, bacteriostatic and antifungal potential among these bioactive products [20].
The medicinal properties of cruciferous vegetables can be related to the activity of
isothiocyanates against several human pathogens.
0
2
4
6
8
10
E.coli
S.aureus
C. albicans
Concentration (mg/ml)
Micoorganisms
MIC of B.nigra extracts agains some microorganisms
E.coli
S.aureus
C. albicans

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In addition phytochemicals investigation of Brassica family indicate oleic acid, phenolics,
carotenoids, selenium, glucosinolates and vitamin C present in brassicaceae mustard seed
[21,22].Mustard leaves have been reported to possess many bioactive substances and antioxidant
properties [23]. Earlier reviews indicated the biocidal, bio-herbicidal, antioxidant, anticancer
activities of glucosinolates and their products from Brassicaceae [24,25].
Mamta Bhatia et al [26] in 2012, reported that the B. nigra, at different concentration levels,
displayed growth inhibitory effect towards three bacterial strains namely B. cereus, P.
aeruginosa and S. sonnei. Moreover Senanayake et al [27] reported the activity against
Aspergillus niger & Rhizopus sp. whereas Alyaa Sabti jasim [28] in 2012 showed the activity of
oils extracts of Brassica nigra seeds against pathogenic oral micro flora.
The previous studies on ethanolic extract from Brassica nigra have shown that it has potent
antibacterial activities. The extract was found to be active against Streptococcus pyogenes which
is the cause of many important human diseases, ranging from mild superficial skin infections to
life-threatening systemic diseases. The extracts were also active against Salmonella typhimurium
which is the cause of gastroenteritis in humans and other mammals [29].
The results of our study indicate the effect of 70% ethanol extract of Brassica nigra against
pathogens. In vitro antimicrobial activity of black mustard spices against pathogens showed
significant results in our study. The ethanolic extract showed broad spectrum activity since it is
effective against both Gram positive (S. aureus) and Gram negative (E. coli) bacteria & C.
albicans. Our results also extend and strengthen the previous research [29].
Conclusion:
The results of the present study highlight the fact that 70% ethanol extract possessed good
antimicrobial activity. This may be due to the presence of polar and non-polar antimicrobial
principles in the extract. Brassica nigra contains phenol, falvonoids, alkaloids, sterols, terpenes
etc., which may be responsible for the activity against microorganisms. Further study in this area
may be helpful for finding of new principle compound which may be helpful in new drug
development research.
Acknowledgment:
We wish to express our sincere acknowledgement to Dr. Ashok Kumar Chauhan, President,
RBEF parent organization of Amity University Madhya Pradesh (AUMP), Mr. Aseem Chauhan,
Additional President, RBEF and chairman of AUMP, Gwalior, Lt. Gen. V.K. Sharma, AVSM
(Retd.), Vice Chancellor of AUMP, Gwalior for providing necessary facilities, their valuable
support and encouragement throughout the work.
References:
[1]. C.A.M. Low, T.J.C. Regnier, L. Korsten, Medicinal bulbous plants of South Africa and
their traditional relevance in the control of infectious diseases. J Ethnopharmacol Vol.
82, pp. 147–154, 2002.
[2]. M.S. Ali-Shtayeh and S.I. Abu Ghdeib, Antimycotic activity of twenty-two plants
usedin folkloric medicine in the Palestinian area for the treatment of skin diseases
suggestive of dermatophyte infection. Mycoses, Vol. 42, pp. 665-672, 1999.

International Journal of Pharmaceutical Science and Health Care Issue 4, Vol. 3.May-June 2014
Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738
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[3]. V. Shrivastava, R. S. Tomar, R. K. Mishra, A. Jyoti and S. Kaushik, Medicinal potential
of some mythologically important plants of India: A Review, Int. J. of Multidisciplinary
and Current research, Vol 2 (1), pp. 99-103, 2014
[4]. R. S. Tomar, V. Shrivastava and S. Kaushik, In vitro efficacy of methanolic extract of
Mimosa pudica against selected micro-organisms for its broad spectrum antimicrobial
activity, Int.J.Curr.Microbiol.App.Sci, Vol 3(4), pp. 780-784, 2014
[5]. B.K. Dutta, S. Karmakar, A. Naglot, J.C. Aich and M. Begam, Anticandidial activity of
some essential oils of a mega biodiversity hotspot in India. Mycoses, Vol. 50(2): pp.121,
2007.
[6] J. S. Abouta, R.E. Patterson, M.L. Neuhouser, J. Elder, Dietary acculturation:
applications to nutrition research and dietetics, J Am Diet Assoc, Vol. 102(8), pp.1105–
18, 2002.
[7]. J.Lawless, The Illustrated Encyclopedia of Essential Oils. Shaftesbury, UK: Element
Books Ltd. 22, 1995.
[8]. V. Shrivastava, G. Shrivastava, R. Sharma, N. Mahajan, V. Sharma, U. Bhardwaj, Anti
microbial potential of Ajwain (Trachyspermum copticum): An Immense Medical Spice,
Journal of Pharmacy Research, Vol.5(7), pp.3837-3840, 2012.
[9]. V. Shrivastava, U. Bhardwaj, V. Sharma, N. Mahajan, V. Sharma and G. Shrivastava,
Antimicrobial activities of Asafoetida resin extracts (A Potential Indian Spice) Journal of
Pharmacy Research, Vol. 5(10), pp. 5022-5024, 2012
[10]. L.A. Mitscher, S. Drake, S.R. Gollapudi and S.K. Okwute, A modern look at folkloric
use of anti-infective agents. J. Natural Products, Vol. 50, pp.1025-1040, 1987.
[11]. Brassica nigra Koch.in Roehl, Deutsche fl. Ed.3 , 4,713-1833
[12]. O.N. Irobi, M. Moo-Young, W.A. Anderson, S.O. Daramola, Antimicrobial activity of
the bark of Bridelia ferruginea (Euphorbiaceae). Intern. J. Pharmacog. Vol. 34, pp. 87-
90, 1994.
[13]. L. Gutmann, D. Billot-Klein, R. Williamson, F.W. Goldstein, J. Mounier, J.F. Acar and
E. Collatz, Mutation of Salmonella paratyphi A conferring cross-resistance to several
groups of antibiotics by decreased permeability and loss of invasiveness, Antimicrob.
Agents Chemothe, Vol. 32, pp. 195-201, 1988.
[14]. C. Mohanasundari, D. Natarajan, K. Srinivasan, S.A. Umamaheswari and A.
Ramachandran, Antibacterial properties of Passiflora foetida L. – a common exotic
medicinal plant. African Journal of Botechnoogy, Vol. 6(23), pp. 2650-2653, 2007.
[15]. A. Mathur, R. Singh, S. Yousuf, A. Bhardwaj, S.K. Verma, P. Babu, V. Gupta, G.B.K.S.
Prasad and V.K. Dua, Antifungal activity of some plant extracts against Clinical
Pathogens. Advanced Applied Science Research, Vol. 2, pp. 260-264, 2011.
[16]. M.P. Raghavendra, S. Satish and K.A. Raveesha, Phytochemical analysis and antibacterial
activity of Oxalis corniculata; a known medicinal plant, My science, Vol. 1(1), pp.72-78,
2006.
[17] K.P. Chong, J.W. Mansfield, M.H. Bennet and J.F. Rossiter . Detection of antifungal
compounds in Arabidopsis thaliana by Brassica oleracae by thin layer chromatography,
Asean Journal on Science and Technology for Development , Vol. 23, no. 1, pp.123-13,
2007.
[18] D. Singh and S.K. Agrawal, Himachalol and β- himachalene insecticidal principles of
Himalayan cedar wood oil J Chem Ecol. Vol. 14 (4), pp.1145-51, 1988.

International Journal of Pharmaceutical Science and Health Care Issue 4, Vol. 3.May-June 2014
Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738
R S. Publication, rspublicationhouse@gmail.com Page 123
[19] J.W. Fahey, A.T. Zalcamann, P. Talalay, The chemical diversity and distribution of
glucosinolates and isothiocyanates among plants, phyto-chemistry, Vol. 56 (1), pp.5-51,
2001.
[20]. S. Tamanna, G.P. Savage, D.L. Mc Neil, N.G. Porter, The yield of isothiocyantes in
wasabi rhizomes stored at different temperatures, Science and Technology , Food
agriculture and environment, Vol. 1, pp.39-45, 2003.
[21]. C.C. Conaway, S.M. Getachun, L.L. Liebes, D.J. Pusateri, D.K.W. Tophan, M. Botero-
Omary and F.L. Chung, Disposition of glucosinolates and sulphoraphanes in human
after ingestion of steam and fresh broccoli, Nutrition and Cancer Vol. 38, pp.168–178,
2001.
[22]. C. Fimognari, M. Nusse, R. Cesari, R.Iori, G. Cantelli- Forti and P. Hrelia, Growth
inhibition, cell cycle arrest and apoptosis in human T-cell leukaemia by the
isothiocyanates sulphoraphane. Carcinogenesis, Vol. 23, pp.581–586, 2002.
[23]. H.Y. Kim, T. Yokozawa, E.J. Cho, H.S. Cheigh, J.S. Choi and H.Y. Chungh, In vitro
and in vivo antioxidant effects of mustard leaf (Brassica juncea). Phytotherapy
Research, Vol. 17, pp. 465–471, 2003.
[24]. H. Zukalova and J. Vasak, The role and effects of glucosinolates of Brassica species- a
review. Rostlinna Vyroba, Vol. 48 (4), pp. 175–180, 2002.
[25]. B.A. Halkier and J. Gershenzon, Biology and biochemistry of glucosinolates. Annual
Review of Plant Biology Vol. 57, pp. 303–333, 2006.
[26]. M. Bhatia and A. Sharma, Brassica nigra and cuminum cyminum: inhibitors of food
borne pathogens, International Journal of applied biology and pharmaceutical
biotechnology, Vol. 3 (3), 2012,
[27]. I.C. Senanayake and P.N. Yapa, Antifungal activity of black mustard (Brassica Nigra)
root exudates, Proceeding of RUSL-Research Symposium, 2010.
[28]. A. S. Jasim, Antibacterial activity of oils extracts of Brassica nigra seeds on some
bacteria isolated from plaque and healthy teeth in children (1-5) years, Basrah Journal of
Scienec (B) Vol.30(1), pp. 105-119, 2012
[29]. A.A. Sayed and M.K. Emadeldin, Chemical Constituents, Cytotoxic And Antibacterial
Activities Of The Aerial Parts Of Brassica Nigra, International Journal of Bioassays,
Vol. 02 (8), pp. 1134-1138, 2013.