See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/337926395

Phytochemical and antimicrobial activity of Medicago sativa (alfalfa) as source

of animal food against some animal pathogens

Article · January 2015

CITATIONS READS

7 1,054

1 author:

Rehab Eldesoukey
Shaqra University
36 PUBLICATIONS 152 CITATIONS

SEE PROFILE

All content following this page was uploaded by Rehab Eldesoukey on 13 December 2019.

The user has requested enhancement of the downloaded file.

Global Veterinaria 14 (1): 136-141, 2015
ISSN 1992-6197
© IDOSI Publications, 2015
DOI: 10.5829/idosi.gv.2015.14.01.92109

Phytochemical and Antimicrobial Activity of Medicago sativa (Alfalfa)

as Source of Animal Food Against Some Animal Pathogens

Rehab Mohamed Atta El-Desoukey

Department of Microbiology and Immunology, National Research Center, Giza, Egypt

Abstract: Many plants have so far been used for the treatment and management of various ailments since the
beginning of human civilization. One of the common problems in the medical world, spreading of bacterial
resistance against antibiotics, so one of the most important steps in microbiological researches is to find a new
antimicrobial compound with minimal side effects. Due to the presence of biological active compounds in plant
Medicago sativa and its use in traditional objective medicine and animal nutrition, it seems that this plant
contain considerable antimicrobial capacity. So the aim of this study is to investigate the antimicrobial activity
of Medicago sativa aqueous and solvent extracts on some medically important animal pathogens and to
determine some phytochemical compounds. Hot and cold aqueous extracts in addition to successive solvent
extracts.( acetone, ether, ethanol and chloroform)of Medicago sativa were evaluated for their antimicrobial
activity against some medically important pathogens isolated from animals and poultry farms (Staphylococcus
aureus, Escherichia coli, Streptococcus pyogens, Pseudomonas aerogenes, Salmonella typhimurium,
Enterococcus, Bacillus cereus, Klebsiella pneumonia and Candida albicans)by agar well diffusion method.
The hot aqueous extract is the most effective against Staphylococcus aureus followed by cold aqueous extract
showed significant antibacterial activity against B.cereus, S.typhimurium, K.pneumoniae, P.aerogenes and
E.coli followed by ethanol extract followed by acetone extract followed by chloroform while the ether extract
did not show any significant antibacterial or antifungal activity against all examined microbes except
Enterococcus. However ethanol extract is the only extract showed high antifungal effect against Candida
albicans. Also, phytochemical compound of ethanol extract was determined, results of the chemical tests
explain the M. sativa extracts contain flavonoids, tannins, alkaloids, saponin and glycosides
compounds. So it could be concluded that the M. sativa extract possess remarkable antimicrobial activity
against microbial pathogens and to be introduced as an alternative to chemical antimicrobial drugs, is required
wider investigation.

Key words: Natural Antimicrobial Father Of Food New Antibiotic Alfalfa

INTRODUCTION B. cereus was identified as an in frequent abortigenic

agent in cattle. Necrotizing placentitis with no or sporadic
Microbes are the most common cause of infectious lesions in fetal tissues [3].
diseases which participate in about half percent of the K. pneumoniae is a common cause of clinical mastitis
deaths cases in animals. As well as morbidity and in dairy cattle. Wood products are considered to be the
mortality due to diarrhea in many developing countries main source of Klebsiella on dairy farms [4].
which act as a major problem, The infections due to Antibiotic resistance has become a global
variety of bacterial etiologic agents such as pathogenic concern. as multi-drug resistant pathogen mostly affects
Escherichia coli(E. coli),Salmonella spp. and the clinical efficacy of many existing antibiotics [5].
Staphylococcus aureus (S. aureus) are most common [1]. Throughout the history of mankind many infectious
Also systemic fungal infections due to Candida albicans diseases have been known to be treated with herbal
(C. albicans) have emerged as important causes of remedies where medicinal plants still act as the most
morbidity and mortality [2]. common source of antimicrobial agents. This traditional

Corresponding Author: Rehab Mohamed Atta El-Desoukey, Department of Microbiology and Immunology,
National Research Center, Giza, Egypt. E-mail: [email protected].
136
 Global Veterinaria, 14 (1): 136-141, 2015

health remedies use is the most popular for 80% of world MATERIALS AND METHODS
population all over the world and was reported to have
minimal side effects pharmacological or biological tests Collection of Plant Materials: Fresh plant materials were
have been submitted to approximately 20% of the plants collected from farm in Giza. The collected different parts of
found in the world. A continuous effort is done by the plant were washed with distilled water, air dried, grinded
microbiologists using the systemic screening of to a fine powder using pestle and mortar and stored in
antimicrobial plant extracts to find new compounds have air-tight bottles at room temperature.
the potential to act against multidrug resistant pathogenic
bacteria and fungi [6]. Aqueous Extraction: About 10 ml of each hot and cold
M. sativa can survive more than 20 years, depending distilled water was added to 5g of the selected plant in
on climate and variety so it was described as a perennial sterile test tubes. The tubes were kept for 1 week at room
forage legume with height up to 1 m (3 ft) and deeply root. temperature until use.
These make it very resistant, especially to droughts. Also
it is called as “Father of all food” [7]. Solvent Extraction: About 5 g of dried plant material was
M. sativa (Leguminosae), one of the most medicinal extracted with 10 ml of each solvent (Ether, Acetone,
plant traditionally used to cure kidney pain, improve the Chloroform and Ethanol) kept for 24 h. Then, it was
memory, cough, sore muscles, as anti-inflammatory, filtered using Watman No.1 filter paper. The solvent was
antidiabetic, rejuvenator, antioxidant,, antimicrobial and in evaporated to make the final volume as 1/2 of the original
CNS disorders. In addition to its long tradition use as volume [11].
ayurvedic and homoeopathic medicine in CNS disorders
[8]. Preparation of Inoculums: The inoculums (bacterial
M. sativa was reported phytochemicaly as it contain strains and fungi) were isolated from large animals and
tannin, flavonoids, digestive enzymes, alkaloids, poultry farms on the outskirts of Cairo. The strains of
coumarins, phytosterols, phytoestrogens, triterpenes and bacteria (S. aureus, E. coli, S. pyogens, P.aerogenes,
saponins. The reduction of cholesterol absorption and Salmonella typhimurium, Enterococcus, B. cereus,
atherosclerotic plaque formation in the arteries due to the K. pneumoniae)& fungi (C. albicans) were inoculated on
ingestion of M. sativa was reported by several clinical and Sabaroud dextrose agar (SAB) (Purchased from Watin
animal studies. More over its benefits in convalescence, – Biolife Company produced by Jalil Medicals
cardiovascular complaints, diabetes and debility and also Company)and in nutrient broth (Purchased from Watin –
used as a tonic during anemia and after blood loss [8]. Biolife Company produced by Jalil Medicals Company).for
Tannins have been reported to be bacteriostatic or overnight at 37°C for bacteria and 25°C for fungi.
bactericidal against S. aureus [9]. The antimicrobial
mechanisms of tannins can be summarized as follows. (i) Antimicrobial Screening: The preliminary study of
The astringent property of the tannin may induce antimicrobial activity of different extracts of M. sativa
complexation with enzymes or substrates. Many microbial was performed by using agar well diffusion method
enzymes in raw culture filtrates or in purified forms are [12]. The sensitivity of all extracts was tested against
inhibited when mixed with tannins. (ii) A tannin's toxicity (S. aureus, E. coli, S. pyogens, P.aerogenes, Salmonella
may be related to its action on the membranes of the typhimurium, Enterococcus, B. cereus, K. pneumonia )&
microorganisms. (iii) Complexation of metal ions by fungi (C. albicans). The anti-microbial activity was
tannins may account for tannin toxicity [10]. measured by the inhibition zones produced in milliliter.
Medicago sativa was reported to have selective All experiments were duplicated. Ciprofloxacin (10 µ g)
toxicity in dog cancer cells grown in vitro and anti-tumor and penicillin (10 µ g) used as positive control while
activity against certain types of leukemia cells in mice [8]. distilled water (100 µg) used as negative control for
So the aim of this study is to investigate the antibacterial screening aqueous extract while (0.01%)
antimicrobial activity of Medicago sativa aqueous and DMSO used as negative control in solvent extracts.
solvent extracts on some medically important animal Nystatin (10 µg) was used as positive control while
pathogens(S. aureus, E coli, S. pyogens, P.aerogenes, distilled water (100 µg) used as negative control for
Salmonella typhimurium, Enterococcus, B.cereus, K. antifungal screening. All chemicals used (Purchased from
pneumoniae and C. albicans)by agar well diffusion Witan – Biolife Company produced by Jalil Medicals
method. Company).

137
 Global Veterinaria, 14 (1): 136-141, 2015

Phytochemical Tests in (Table 1). The screening step in the preliminary study
Tannins Test:A modified methods stated in [13] was used for antimicrobial activity was done using the agar well
to be presented of tannins on the extracts, Afew drops of diffusion method. The inhibition activity was determined
ferric chloride reagent were added for 3 ml of extract. A by measuring the diameter of clear zone. The diameter of
blue black color refereed to the present of tannins. the clear zone indicated the inhibition activity. The hot
aqueous extract is the most effective against S. aureus
Alkaloids Test: A few drops of Marqus reagent (prepared followed by cold aqueous extract showed significant
from mixing 0.5 ml of formaldehyde with 5ml of antibacterial activity against B. cereus, Salmonella
concentration H2SO4), added to the 5 ml of extract. typhimurium, K. pneumoniae, Ps.aerogenes and E. coli.
Turbidity refereed to the present of alkaloids [14]. Followed by ethanol extract followed by acetone extract
followed by chloroform and ether extract while the ether
Saponins Test: 3 ml of extractwas added to the 2 ml of extract did not show any significant antibacterial or
Ferric chloride, awhite residue to be formed as evidence to antifungal activity against all examined microbes except
the present of Saponins [15]. Enterococcus as shown in Table (2). However Ethanol
extract is the only extract showed high antifungal effect
Flavonoids Test: Flavonoids test were implement in against C. albicans. Phytochemical screening indicated
conformity with [15]. 2 ml of extract mix with Alcoholic that the ethanolic extract most abundantly contained
KOH (0.5 mol.), a yellow color as proofed to the present of tannins, saponins and flavonoids, alkaloids and
Flavonoids. glycoside. as shown in Table (3).

Glygosides Test: 0.5g of dried extract was dissolved in DISCUSSION

2ml of glacial acetic acid containing one drop of Ferric
chloride solution and then under laid with 1 ml of The use of herbs in medicine is common all over the
concentration H2SO4A brown ring indicated the present world. About one third of adults in the world use
of Glycosides [16]. All chemicals used (Purchased from alternative therapies, including plants which may be used
Watin – Biolife Company produced by Jalil Medicals singly or combined in mixtures. In the contrary to chemical
Company). drugs, plant sainted to be non-toxic, due to its natural
origin. However, problems may originate due to
RESULTS adulteration, contamination, toxicity, substitution;
misidentification and lack of standardization [17].
This investigation of antimicrobial activity was This undesirable fact encourage the study of medicinal
performed on six different extracts of M. sativaas shown plants and plant compounds used in medicine.

Table 1: List of Medicago sativa different extracts used to evaluate antimicrobial activity.
No Name Colour of used extract
1 Ether Light green
2 Ethanol Dark green
3 Chloroform Green yellow
4 Acetone Green yellow
5 Hot aqueous extract Green
6 Cold aqueous extract Green

Table 2: Antimicrobial activity of Medicago sativa different extracts against some animal pathogen in (mm).
Type of extract
-------------------------------------------------------------------------------------------------------------------------------------------------------
Typeofmicroorganism ETH A C E CAE HAE P Ci N D.W DMSO
G-Ve E. coli 0 14 11 0 20 0 0 20 0 0 0
Salmonella 0 7 11 0 25 21 15 30 0 0 0
Enterococcus 14 11 0 13 0 32 20 35 0 0 0
Ps.aerogens 17 14 0 0 12 19 0 30 0 0 0
G+Ve Bacillus 20 11 0 0 28 30 0 34 0 0 0
S aures 0 19 0 0 0 22 11 25 0 0 0
S.pyogens 15 12 0 0 10 0 0 0 0 0 0
K. pneumoniae 25 11 0 0 20 19 0 38 0 0 0
fungi C. albicans 30 0 0 0 0 0 0 0 16 0 0
A= acetone/ C= chloroform/E= ether/ETH= ethanol/CAE= Cold aqueous extract/HAE= Hot aqueous extract/P= penicillin/Ci= Ciprofloxacin/N=
Nystatin/DW= distelled water/ DMSO=dimethyle sulfoxide.

138
 Global Veterinaria, 14 (1): 136-141, 2015

Fig. 1: Antimicrobial activity of Medicago sativa different extracts against some animal pathogen in (mm).

Table 3: The phytochemical compounds in ethanolic extract of Medicago According to Venkataswamy et al. [22] the Gram-
sativa
positive bacterial strains were more susceptible to the
Plant extracts Phytochemical testsMedicagosativa
activity of the aqueous M. sativa leaf extracts when
Flavonoids Test +
compared to gram negative bacteria. This may be
Alkaloids Test +
Glygosides Test + explained due to the fact that these two groups have
Saponins Test + different cell wall structure. The ability of tannin
Tannins Test + compounds which reported to be present in M. sativa
+ (contain this phytochemical compound) disintegrates the bacterial colonies, by its interference
with the bacterial cell wall.
However the revolution of synthetic antibiotics, the Considering the above results, our result was
infections due to bacteria and fungi are still of major found the maximum antibacterial activity in
concern in medicine, due to the presence of antibiotic Medicago sativa against Gram positive S.aureus as
resistant strains which increasing the interest to use comparing to Gram negativeE.coli.as shown in Table (2)
natural products due to their better biodegradability and and Figure (1)
availability [18]. The antimicrobial activity of saponins isolated from
In this regard, the study was performed to M. Sativa against selected medically important yeasts,
investigate the antimicrobial activity of Medicago Gram-positive and -negative bacteria has been
sativa aqueous and solvent extracts on some investigated. Increasing antibiotic activity was observed
medically important animal pathogen (S. aureus, E. going from the saponin extracts. Activity was found
coli, S. pyogens, Pseudomonas aerogens, especially high against Gram-positive bacteria (B.cereus)
Salmonella typhimurium, Enterococcus, B. cereus, K. [8]
pneumoniae and C. albicans) by agar well diffusion (Xiao-kang and Pinarosa ) mentioned that the better
method. antibacterial activity of Alfalfa extract to P. aeruginosa
All plant parts in order to perform their physiological and S. dysgalactiae where the antibacterial effects on
activities form some chemicals by themselves, in the E.coli and S. aureus were ordinary [23, 24].
present study, M. sativa produce different kinds of However this investigation of antimicrobial activity
secondary metabolites which play an important role was performed on six different extracts of M. sativa as
medicinally. shown in (Table 1). The screening step in the preliminary
A lot of studies involve extraction of the active study for antimicrobial activity was done using the Agar
component in the plants using organic solvents. But well Diffusion Method. The diameter of the clear zone
plants as used in traditionally, using organic solvent indicated the inhibition activity. The hot aqueous extract
extraction for the antibacterial properties should not be is the most effective against S. aureus followed by
done often; therefore, in the present study the commonly cold aqueous extract showed significant antibacterial
used plants extracts are made with hot and cold distilled activity against B. cereus, Salmonella typhimurium,
water in addition to organic solvent extracts and tested for K. pneumoniae, Ps. aerogens and E. coli followed by
its antimicrobial effect against S. aureus, E. coli and C. ethanol extract followed by acetone extract followed by
albicans. chloroform and ether extract while the ether extract did not
All plants used in the traditional medicine mostly show any significant antibacterial or antifungal activity
have moderate antimicrobial activities as reported earlier against all examined microbes except Enterococcus as
[19-21]. shown in Table (2) and Figure (1).

139
 Global Veterinaria, 14 (1): 136-141, 2015

The results of our study showed that ethanol extract 5. Parekh Jigna and Sumitra V. Chanda, 2007. In-vitro
of Medicago sativa possess very potential antifungal antimicrobial activity and phytochemical
effect on C. albicans as shown in Table (2) and Figure analysis of some Indian Medicinal Plants. Turk J.
(1).Also All phytochemical tests results indicated that Biol., 13: 53-58.
Medicago sativa contain flavonoids, tannins, alkaloids, 6. Safary, A., H. Motamedi, S. Maleki and S.M.
saponin and glycosides compounds as shown in Table (3) Seyyendnejad, 2009. A preliminary study on the
Antimycobacterial properties of the plant could be antibacterialactivity of Quercus brantii against
due to the abundant flavonoids, saponins and tannins bacterial pathogens, particularly enteric pathogens.
that were found in it Table (3). Phytochemical Intl. J. Botany, 5(2): 176-180.
constituents such as tannins, saponins, flavonoids, 7. Alfalfa (plant) – Britannica Online Encyclopedia.
alkaloids and several other aromatic compounds are Britannica.com. Archived from the original on 7 June
secondary metabolites of plants that serve as defense 2011. Retrieved 29 June 2011.
mechanisms against predation by many microorganisms, 8. Kundan Singh Bora and Anupam Sharma, 2011.
insects and other herbivores [25]. Evaluation of antioxidant and cerebroprotective
Therefore, in order to obtain effective antimicrobial effect of Medicago sativa Linn against ischemia
activity at low concentrations without any side effects on and reperfusion insult. Evidence-Based
the health researches should focus on the optimization of Complementary &Alternative Medicine. doi: 10.
purification and applications. 1093/ecam/neq019.
9. Chung, K.T., S.E. Stevens, Jr, W.F. Lin and C.I. Wei,
CONCLUSIONS 1993. Growth inhibition of selected food-borne
bacteria by tannic acid, propyl gallate and
So it could be concluded that the Medicago sativa
related compounds. Letters in Applied Microbiology,
extract exhibited remarkable antimicrobial activity against
17: 29-32.
microbial pathogens and can be introduced as an
10. Chung, K.T., T.Y. Wong, C.I. Wei, Y.W. Huang and
alternative to chemical antimicrobial drugs, but it requires
Y. Lin, 1998. Tannins and human health: a review.
wider investigation. Also the data suggest that the
Critical Reviews in Food Science and Nutrition,
ethanolic extract of M. sativa could be a rich source of
38: 421-64.
antimicrobial agents, especially antifungals. Those results
11. Sasikumar, J.M., A. Pichi Anthoni Doss and A. Doss,
have been encourage to conduct additional tests to
2005. Antibacterial activity of Eupatorium
confirm the specific use of the studied extracts to treat the
glandulosum leaves. Fitoterapia, 76(2): 240-243.
bacterial and fungal infections caused by bacteria and
12. Mahon, C.R. and G. Manuselis, 1995. Textbook of
fungi found to be sensitive to our extracts.
diagnostic microbiology. W.B.Saunders,
Pennsylvania.
REFERENCES
13. Trease, G.E. and W.C. Evans, 1996. Atext book of
1. Parastoo Karimi Alavijeh, Parisa Karimi Alavijeh and pharmacognosy.14th ed.Bailliere Tindall Ltd.London.
Devindra Sharma, 2012. A study of antimicrobial 14. Harborne, J.B., 1984. Pytochemical methods :Aguide
activity of few medicinal herbs. Asian Journal of to modern techniques ofplant analysis. 2nd
Plant Science and Research, 2: 496-502. ed.Chapman and Hall, London. UK, pp: 288.
2. Beuchat, L.R. and D.A. Golden, 1989. 15. AlKhazaragi, S.M., 1991. Biopharmacological study
Antimicrobials occurring naturally in foods. J. Food of Artemisia herba alba. MSC. Thesis. Univ.
Technol, 43: 134-142. Baghdad.
3. Schuh, J. and D. Weinstock, 2009. Bovine abortion 16. Oloyede, O.O., 2005. Chemical profile of unripe pulp
caused by Bacillus cereus. J Am Vet Med Assoc. of Carica papaya. Pak. J. Nutri., 4(6): 379-381.
1985 Nov 15;187(10): 1047-8. 17. Atta, R.M., 2014. Microbiological Studies on Some
4. Munoz, M.A., C. Ahlström, B.J. Rauch and Herbs Used in Folk Medicine, World Applied
R.N. Zadoks, 2006. Fecal shedding of Sciences Journal, 32(9): 1795-1799.
Klebsiella pneumoniae by dairy cows. J. Dairy Sci., 18. Parrotta, J.A., 2001. Healing plants of peninsular
89(9): 3425-30. India. A.B. International Wallingford, UK. pp: 944.

140
 Global Veterinaria, 14 (1): 136-141, 2015

19. Soliman, O.E., 2004. Evaluation of myrrh (Mirazid) 23. Xiao-kang, L.U., 2008. (Gansu Agricultural
therapy in fascioliasis and intestinal schistosomiasis University, Lanzhou, Gansu 730000), Study on the
in children: immunological and parasitological study. Antibacterial Activity of Alfalfa Extract, Journal of
J Egypt Soc Parasitol. Dec., 34(3): 941-966. Anhui Agricultural Science, 23: 31-47.
PubMed.gov 24. Pinarosa Avato, Rossella Bucci, Aldo Tava, Cesare
20. Takazawa, H., F. Tajima and C. Miyashifa, 1982. An Vitali, Antonio Rosato, Zbigniew Bialy and Marian
antifungal compound from shitake (Lentinus edodes) Jurzysta 18 APR, 2006. Antimicrobial activity of
Yaku Zass(Japanese), 102: 489-491. saponins from sp.: structure activity Medicago
21. Wondill Froman, 2005. Biblical Facts About Wine: Is relationship, African journal of Biotechnology,
It a Sin to Drink Wine?. AuthorHouse.pp. 307-. ISBN 10(55): 11433-11441.
978-1-4184-0964-7http:// books.google.com/ 25. Salama, H.M. and N. Marraiki, 2008. Antimicrobial
books?id=7DsPWFnTEFgC&pg=PA307.Retrieved15 activity and phytochemical analysis of Polygonum
November 2012. aviculare L. (Polygonaceae), naturally growing in
22. Venkataswamy, R., A. Doss, M. Sukumar and Egypt. Aust J. Basic. Appl. Sci., 3: 2008-2015.
H.M. Mubarack, 2010. Preliminary phytochemical
screening and antimicrobial studies of Lantana indica
roxb. Indian J. Pharmaceutical Sci., 72(2): 229-231.

141

View publication stats