Ujmr 1 - 1 2016 - 009 PDF
Ujmr 1 - 1 2016 - 009 PDF
Ujmr 1 - 1 2016 - 009 PDF
61
UMYU Journal of Microbiology Research
UJMR, Volume 1 Number 1 December, 2016 ISSN: 2616 - 0668
It has been in use since ancient times, to MATERIALS AND METHODS
treat a number of human ailments and also Collection of plant materials
as household pesticide (Chattopadhyayet al., The flowers of Azadirachta indica was
1993; Chattopadhyay 1996; Chattopadhyay obtained from Audu Bako Zoological and
and Bandyopadhyay, 2005). Extracts from Botanical Garden, Kano. The collected
the bark, leaves, fruits and roots have been flowers were identified and authenticated in
used to control leprosy, intestinal Herbarium, department of Plant Biology,
helminthosis and respiratory disorders Bayero University Kano. Voucher No 118
(Ketkar and Ketkar, 1995). Every part of the was given to the plant.
neem tree has been used as traditional Preparation of the plant materials
medicine for house-hold remedy against The collected sample was washed
various human ailments from antiquity. thoroughly with running tap water and
Chemical investigation on the products of finally with sterile distilled water. The
the neem tree extensively undertaken in the material was chopped into small pieces and
middle of the isolation of nimbin, the first then air dried on a sterile blotter under shade
bitter compound isolated from neem oil, for 20-30 days.
more than 135 compounds have been Extraction procedures
isolated from different parts of neem Plant extract was prepared by the maceration
(Ganguli, 2002). Neem elaborates a vast method as demonstrated by Alade and Irobi
array of biologically active compounds that (1993). 200 g of the air dried powdered form
are chemically diverse and structurally of the flower of Azadirachtaindica was
complex (Subapriya and Nagini, 2005). soaked in 250 ml of petroleum ether for
Infectious diseases are the world’s leading three days. The mixture was stirred every 24
cause of premature deaths, killing almost 50 h using a sterile glass rod. The plant
000 people every day. In recent years, drug sediment was allowed to dry and soaked in
resistance to human pathogenic bacteria has 250ml of chloroform and finally in methanol
been commonly reported from all over the solvent for three days respectively. At the
world (Piddock and Wise, 1989; Singh et al., end of extraction each extract was passed
1992; Mulligen et al., 1993; Davis, 1994; through Whatman filter paper No. 1
Robin et al., 1998). However, the situation is (Whatman, UK). The procedure was
alarming in developing as well as developed repeated three timeswith fresh volumes of
countries due to indiscriminate use of the solvents used.The alcoholic filtrates
antibiotics. The drug-resistant bacteria and obtained were concentrated in water bath at
fungal pathogens have further complicated 30°C and stored at 4°C until further use. The
the treatment of infectious diseases in dried plant extracts/fractions obtained were
immune compromised, AIDS and cancer weighed and labeled as PFE(Petroleum ether
patients (Rinaldi, 1991; Diamond, 1993). In flower extract), CFE(Chloroform flower
the present scenario of emergence of extract), and MFE(Methanol flower extract).
multiple drug resistance to human Phytochemical screening
pathogenic organisms, this has necessitated a • Test for saponins
search for new antimicrobial substances Exactly 2ml of petroleum ether extract was
from other sources including plants.Thus, vigorously shaken with distilled water and
the choice of azadirachta indicaas a plant of allowed to stand for a while. A persistent
interest for this research work was done frothing indicates the present of saponins.
based of its diverse and immense therapeutic Same procedure was repeated using
ethno medicinal values. In view of the chloroform and methanolic extracts
forgoing, this research is aimed at studying (Sofowora, 1984).
the antibacterial activity and phytochemical • Test for alkaloids
properties of extracts of flower of Half gram of petroleum ether extract was
Azadirachta indica (neem) on some selected stirred with 5ml 1% HCL on steam bath.
clinical isolates.
UMYU Journal of Microbiology Research 62
UJMR, Volume 1 Number 1 December, 2016 ISSN: 2616 - 0668
The solution was cooled and filtered.1ml of colour from rose-pink to red indicates
the filtrate was treated separately with drops anthraquinone. Same procedure was
of Mayer’s dragendoff’s and wagner’s repeated using chloroform extracts and
reagents; and formation of dirty/dark brown, methanolic extracts (Ciulie, 1984)
yellow-brown or reddish brown precipitated • Test for steroids(Salkowaski’s test)
respectively indicates the presence of Two mills of concentrated sulphuric acid
alkaloid (El-olemy et al., 1994). was added to 2ml of each extract
• Test for phlobactannins .Appearance of effervescence after which a
A 2ml of each extract was added to 5ml clear reddish brown color appeared at the
HCL.Formation of turbidity/precipitation interface confirms the presence of steroidal
indicates the presence of phlobactannins. ring (Harbone,1973).
(Sofowora, 1984). Test organisms
• Test for tannins Clinical isolates of bacteria were used for
Exactly 2ml of each extract was treated with the bioassay studies. The isolates include
3 drops of 5% ferric chloride. A dark black Proteus mirabilis, Escherichia coli,
colored precipitate in a very dark solution, Staphylococcus aureus, Pseudomonas
which gives a green-black to blue black aeruginosa,and Klebsiella pneumoniae. The
colouration on dilution indicates the isolates were obtained from microbiology
presence of tannins (Sofowora,1984). laboratory of Murtala Mohammad Specialist
• Test for reducing sugar Hospital (MMSH), Kano, Nigeria. They
One gram of each extract was weighed and were further confirmed using standard
diluted with 2ml distilled water. Fehling’s biochemical test as described by
solution(A and B) were added and the Cheesbrough (2002).The isolates were
mixture warmed. A brick- red precipitate at maintained on freshly prepared nutrient agar
the bottom of the test tube indicates reducing (oxoid) slants and kept in a refrigerator at
sugars (Brain and Turner, 1975). 4oc until required for use.
• Test for flavonoids Preparation of extract concentrations
Two grams of the petroleum ether extract This was carried out using standard method
was weighed and placed in a test tube, described by Cheesbrough, (2002). Stock
followed by the addition of 10ml of DMSO. solution of the petroleum ether crude extract,
The mixture was heated, followed by the chloroform crude extract and methanol
addition of magnesium metal and six drops crude extract were prepared by weighing
of concentrated hydrochloric acid. The 0.008g of each and dissolved in 2ml of
appearance of red colour was indicatives of dimethlysulfoxide (DMSO) in glass vial
the presence of flavonoids. Same procedure bottles.
was repeated with chloroform and methanol This gave an extract concentration of
extracts respectively (Sofowora, 1993). 8000µg/ml (stock solution).Four varied
• Test for resins: extracts concentrations (4000µg, 2000µg,
A 2.0g of each extract was dissolved in 10ml 1000µg and 500µg) were prepared from the
of ethanioc acid anhydride. One drop of stock solution (8000µg) using double serial
concentrated sulphuric acid was added. The dilution for each extract.
appearance of purple colour which rapidly Preparation of antibiotic dilution
changes to violet indicates the presence of (Standard)
resins (Evans, 1995). The antibiotic chloramphenicol was
• Test for antraquinone purchased at a registered Pharmaceutical
Two mills of petroleum ether extractwere store in Kano State, Nigeria and was
treated with 5ml of benzene. This gave two reconstituted by dissolving 0.3 mg of
layers. The clear colorless upper layer was powder in a 100 ml of distilled water so as to
pipette and the organic layer treated with get a concentration of 30μg/ml. The
3ml of 10% aqueous ammonia. Change of prepared dilution of the antibiotic was used
Reducing sugar + + +
Tannins - - +
Steroids - - -
Flavonoids + - +
Resins + - -
Saponins + - -
Anthraquinone - - -
Phlabactannins - - -
Alkaloids + + +
Key: Present (+), Absent (-)
P. miravilis 15 17 19 21 13 16 17 19 17 19 20 23
E. coli 08 10 14 15 00 00 00 00 00 00 00 00
S. aureus 14 16 17 19 12 15 16 20 13 14 15 17
P. earuginosa 07 08 09 11 00 00 00 00 00 00 00 00
K. pneumonia 08 08 09 15 08 15 20 21 00 00 00 00
P. miravilis 14 0
E. coli 16 0
S. aureus 19 0
P. earuginosa 14 0
K. pneumonia 9 0
Table 5, shows the Minimum Inhibitory 62.5μg/ml while E. coli and P. aeruginosa
Concentration (MIC) of different fractions were resistant. Moreover, all the isolates
of A. indica extracts against the selected tested with methanolic extracts of flower of
clinical isolates. The results showed that all A. indica also have MIC value of 62.5μg/ml
the test organisms have the same MIC of with the exception of E.coli,P. earuginosa
62.5μg/ml in the petroleum ether extract and K. pneumoniae which all of them
while P. miravilis, S. aureus and K. showed resistance to methanol flower
pneumonia tested with Chloroform flower extract.
extracts have also the same MIC of
Table 5: Minimum Inhibitory Concentration of Petroleum ether, Chloroform and Methanol
flower extracts of A. indica
Extracts concentration(μg/ml)
PFE CFE MFE
Isolates 250 125 62.5 31.3 15.6 250 125 62.5 31.3 15.6 250 125 62.5 31.3 15.6
P. mirabilis - - - + + - - - + + - - - + +
E. coli - - - + + + + + + + + + + + +
S. aureus - - - + + - - - + + - - - + +
P. aureginosa - - - + + + + + + + + + + + +
K. pneumoniae - - - + + - - - + + + + + + +
Key: + = Growth not inhibited; - Growth inhibited
P. miravilis + + + - + + - - -
E. coli - - + + + + + + +
S. aureus + + + + + + - + +
P. aeruginosa - - + + + + + + +
K .pneumoniae - - + + + + + + +
The zones of growth inhibition recorded for activity of the extracts against the
the methanol and acetone extracts by De and organisms. In this study, P. mirabilis, had
Ifeoma (2002) were also smaller in size than the lowest MBC value (125μg/ml) in
those obtained in this study. Several factors methanolic flower extracts (MFE) (Table 6).
are known to influence yield and biological The quantity of the active ingredients
activities of plant based products, including required to effect complete kill may not
the age of the plant, time of harvest, drying matter since medicinal plants have been
and processing of the materials, methods of reported to have little or no side effects
extraction and the solvents used (Hassain-Eshrat, 2002; Ogbonnia et al.,
The quantitative measure of the in-vitro 2008). The MIC and MBC values for neem
activity of antibiotics and non-antibiotic leaves against some fungal isolates were
antibacterial agents including those agents of reported to be 250μg/ml by Natarajan et al.,
plant origin with antibacterial potentials are (2003) which are in accordance with the
the MIC and MBC. The minimum inhibitory MBC values of P. mirabilis, in Petroleum
concentration was defined as the lowest ether neem flower extract and S.aureus in
concentration of the compound to inhibit the MFE obtained in this study. De and Ifeoma
growth of microorganisms .Table 5 shows (2002) reported that at a concentration of 10
the minimum inhibitory concentration (MIC) mg/ml their crude extracts were unable to
of A. indica flower extracts against the inhibit the growth of some bacteria,
selected clinical isolates and the results particularly P. aeruginosa and E. coli.
showed that all the test organisms have the However, the result of this study showed
same MIC of 62.5μg/ml in PFE, while in that PFE of A. indica inhibited the growth of
CFE P. mirabilis, S. aureus and P. aeruginosa and E. coli even at lower
K.pneumoniae had the same MIC of concentration of 125μg/ml.
62.5μg/ml. In MFE P. mirabilis, and S. The varied zone of inhibition of MBE on E.
aureus had the same MIC values of coli at concentrations of 500, 1000, 2000
62.5μg/ml . The study showed that E. coli and 4000μg in table3 disagreed with reports
and P. aeruginosa had no MIC value in CFE by Yagoub et al.,(2007) who in their
while E. coli, P. earuginosa and K. preliminary screening for anti-microbial
pneumonia also have no MIC values in MFE activity of different plants against different
at all concentrations, meaning that higher organisms, methanolic extracts of A.indica
concentration of the extracts are required to produced zero zone of inhibition against E.
inhibit the growth of these bacteria. The coli.
MBC of the PFE extracts was 125μg/ml Conclusion
against E. coli, P. aeruginosa and K. Based on the pharmacological results of the
pneumonia respectively while P. mirabilis study, it could be confirmed that the extracts
and S. aureus have no MBC across all the contain chemical constituents of
concentration. In CFE only P. mirabilis had pharmacological significance. The
MBC value of 250μg/ml while the observation that the extracts were effective
remaining test organisms have no MBC against the test bacteria suggests the use of
across all concentrations. Meanwhile in crude extract of flower of A. indica against
MFE P. mirabilis had MBC value of infection caused by clinical isolates like P.
62.5μg/ml while, S. aureus had MBC value mirabilis, Escherichia coli, S. aureus,P.
of 250μg/ml while E. coli, P. aeruginosa aeruginosa, and K. pneumonia.It is therefore
and K. pneumoniae had no MBC values at recommended for the isolation and
all the concentrations used. The higher the purification of bioactive compounds in
MBC values is the lower the susceptibility Neem tree responsible for the antimicrobial
of microorganism to the crude extracts and activity.
the lower the MBC values is the higher the
68
UMYU Journal of Microbiology Research
UJMR, Volume 1 Number 1 December, 2016 ISSN: 2616 - 0668
REFERENCES rats. African Journal of Biomedicine.
Ahmad I, Memood, Z. and Mohammad, F. Res. 8:101-104.
(1998): Screening of some Indian Cheesborough, M. (2002): Biochemical tests
medicinal plants for their to identify bacteria in laboratory
antimicrobial properties. Journal of practice in tropical countries.
Ethnopharmacol. 62:183-193. Cheesborough M. (ed).Cambridge
Alade, P.I., and Irobi, O.N. (1993): Edition. pp. 63-87.
Antimicrobial activities of crude leaf Cheesborough, M. (2012): Summary of the
extracts of Acalyphawilkensian. Clinical and Laboratory features of
Journal of Ethnopharmacology 39, microorganisms. Cheesbrough
171–174. M.(ed).cambridge edition pp.157-
Baker C.N and Thormsberg C.H (1983): 233.
Inoculum standardization in Ciulie, I. (1984):Methadology for the
antimicrobial susceptibility tests: analysis of vegetables and drugs.
Evaluation of overnight age culture. Chemical Industry Division, UNIDO
Journal of Clinical Microbiology, Romania. Pp57-58.
17: 140-457. Clinical and Laboratory Standards Institute
Biswas, K.I; Chattopadhyay, A and (2006): Methods for Dilution El-
Banerijee Y,A. (2002): Biological Mahmoodet al.,1421 of
activities and Medicinal properties of Antimicrobial Susceptibility Tests
neem (Azadirachtaindica). for bacteria that grow
Bandopadhyay, U, Curr Sci., aerobically.Approved
82:1336-1345. Standards,7thedn MG7-A7
Brain, K.R. and Tuner, T.D. (1975):The Cunha, B.A. (2001):Antibiotic side effects.
practical evaluation of Med. Clinics North Am. 85:149-185.
phytopharmacheuticals. Wright Davis, J. (1994): Inactivation of antibiotic
Scientectica Publishers, and the dissemination of resistance
Bristol.Pp57-58. genes. Science 264, 375–382.
Boakye-Yiadom K, Fiagbe, N and Ayim ,S. De, N and Ifeoma, E. (2002): Antimicrobial
(1987): Antimicrobial properties of effect of components of the bark
some West African medicinal plants extracts of the neem
IV. Antimicrobial of xylopic acid (Azadirachtaindica A. juss). J.
and constituents of the fruits of Technol.Dev.,8:23-28.
Xylopia athiopica (Anuonaceae). Diamond,R.D. (1993): The growing problem
Lloydia40: 543-545. of mycoses in patients infested with
Chattopadhyay R.R, Chattopadhyay R.N., human immunodeficiency virus.
and Maitra, S, K .(1993).Possible Review of Infectious Diseases 13,
mechanism of anti-inflammatory 480–486.
activity of Azadirachtaindicaleaf EL-Mahmood, A.M, Doughari J.H, and
extract.Indian J. Pharmacol. Ladan, N. (2008): Antimicrobial
25(2):99-100. screening of stem bark extracts
Chattopadhyay, R.R. (1996). Possible ofVitellariaparadoxa against some
mechanism of anti-inflammatory enteric pathogenic microorganisms.
activity of Azadirachtaindicaleaf African Journal of
extract: Part IV. Gen. Pharmacol. Pharmacology.2(5): 089-094.
27(3):431-434. El Olemy, M.M; AL-Muhtadi, F.J.andAfif,
Chattopadhyay,R.R, and Bandyopadhyay M A.A. (1994):Experimental
(2005). Effect of Phytochemistry. A laboratory
Azadirachtaindicaleaf extract on Manual. King Saud University
serum lipid profile changes in normal press.8-9
and streptozotocin induced diabetic
UMYU Journal of Microbiology Research
69
UJMR, Volume 1 Number 1 December, 2016 ISSN: 2616 - 0668
Ergene, A.Guler, P.Tan, S; Mirici, S; C.A. (2008): Phytochemical
Hamzaoglu, E. and Duran, A. evaluation and antibacterial profile of
(2006): Antibacterial and Antifungal TreculiaAfricanaDecne bark extract
Activity of Heracleum spondylium on gastrointestinal bacterial
subsp. Artvinense.African Journal pathogrns.mAfr. J. Biotechnol.,
ofBiotechology. 5(11): 1087-1089. 7(10): 1385-1389.
Evans, M.W. (1995): Textbook on Piddock, K.J.V and Wise, R. (1989):
Pharmacognocy. 13th Mechanisms of resistance to
Edition.Bailiere-Tindal,London. quinolones and clinical perspective.
Ganguli, S. (2002). “Neem: A therapeutic Journal of Antimicrobial
for all seasons”. Current Science Chemotherapy 23, 475–483
82(11):1304. Rao DVR, Sing K, Chopra, P, Chabra P, and
Harborne, J. B. (1973):Phytochemical Ramanujahi G (1986):In-vitro
Methods: A guide to Modern bactericidal activity of neem oil.
Techniques of Plant Analysis. Indian J. Med. Res., 84: 314-316
1stedition. Chapman and Hall Ltd. Rinaldi, M.G. (1991): Problems in the
London.160 diagnosis of invasive fungal diseases.
Hassain-Eshrat, H.M.A. (2002): Review of Infectious Diseases 13,
Hypoglycaemic, hypolipidemic and 493–495.
antioxidant properties of curcumin Robin, E.H ; Anril, W; Alexander, M;
from Curcuma longa, Linn. And Loeto, M and Keith, K. (1998):
partially purified product from Nasopharyngeal carriage and
Abroma auguta,Linn, in antimicrobial resistance in isolates of
streptozotocin induced diabetes. Streptococcus pneumoniae and
Indian J. Clin. Biochem. 17(2): 33-43 Heamophilus influenzaeType b in
Hugo, W.B; Russell A.D. children under 5 years of age in
(1983):Pharmaceutical Micrbiology Botswana. International Journal of
3rd Edition.Blackwell scientific Infectious Diseases 3 (1), 18–25.
publications. pp. 140-163. Saham, D.F, and Washington D.A. (1990):
Ketkar,A.Y and Ketkar CM (1995): Various Antimicrobial susceptibility test
uses of neem products: Medicinal dilution methods: In manuals of
uses including pharmacology in clinical of microbiology Lennette E.
Asia, in H. Schmutterer (Ed). pp. H. 5th edition, America-Society for
518-525. Microbiology Washington D. C, pp.
Mulligen, M.E., Murry-Leisure, K.A., 1105-1106.
Ribner, B.S., Standiford, H.C., John, Singh, M; Chaudhry, M.A; Yadava, J.N.S;
J.F., Karvick, J.A., Kauffman, C.A., and Sanyal, S.C.(1992): The
Yu, V.L., (1993): Methicillin spectrum of antibiotic resistance in
resistant Staphylococcus aureus. human and veterinary isolates of
Natarajan, V; Veugopal, P.V and Menon, T. Escherichia coli collected from
(2003): Effect of 1984–1986 in Northern India.
Azadirachtaindica(neem) on the Journal of Antimicrobial
growth pattern of dermatophytes. Chemotherapy 29, 159–168.
Indian J. Med.Microbiol., 21: 98-101. Sofowora, S.A. (1984):Medicinal plants and
Odugbemi, T. (2006). Medicinal Plants as Traditional Medicine in Africa.
Antimicrobials In: Outline and Spectrum books Ltd book ltd. 1st
pictures of medicinal plants from edition. 150-151 and 162-172.
Nigeria. University of Lagos Press. pp. Sofowora, A. (1993): Medicinal plants and
53-64 Traditional Medicine in Africa.
Ogbonnia, S.O, Enwuru N.V, Onyemenen Spectrum book Limited, Ibadan,
E.U, Oyedele G.A, and Enwuru, Nigeria.Pp385-388.
UMYU Journal of Microbiology Research
70
UJMR, Volume 1 Number 1 December, 2016 ISSN: 2616 - 0668
71
UMYU Journal of Microbiology Research