Antifungal activity of different neem leaf extracts and the nimonol against some important human pathogens

Abstract

This study was conducted to evaluate the effect of aqueous, ethanolic and ethyl acetate extracts from neem leaves on growth of some human pathogens (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Candida albicans and Microsporum gypseum) in vitro. Different concentrations (5, 10, 15 and 20%) prepared from these extracts inhibited the growth of the test pathogens and the effect gradually increased with concentration. The 20% ethyl acetate extract gave the strongest inhibition compared with the activity obtained by the same concentration of the other extracts. High Performance Liquid Chromatography (HPLC) analysis of ethyl acetate extract showed the presence of a main component (nimonol) which was purified and chemically confirmed by Nuclear Magnetic Resonance (NMR) spectroscopic analysis. The 20% ethyl acetate extract lost a part of its antifungal effect after pooling out the nimonol and this loss in activity was variable on test pathogens. The purified nimonol as a separate compound did not show any antifungal activity when assayed against all the six fungal pathogens.

Full text

1007
Brazilian Journal of Microbiology (2011) 42: 1007-1016
ISSN 1517-8382
ANTIFUNGAL ACTIVITY OF DIFFERENT NEEM LEAF EXTRACTS AND THE NIMONOL AGAINST SOME
IMPORTANT
HUMAN PATHOGENS
Mahmoud, D.A.*;Hassanein, N.M.; Youssef, K.A.; Abou Zeid, M.A.
Department of Microbiology, Faculty of science, Ain-Shams University, 11566, Abbassia, Cairo, Egypt.
Submitted: May 22, 2010; Returned to authors for corrections: August 23, 2010; Approved: January 13, 2011.
ABSTRACT
This study was conducted to evaluate the effect of aqueous, ethanolic and ethyl acetate extracts from neem
leaves on growth of some human pathogens (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger,
Aspergillus terreus, Candida albicans and Microsporum gypseum) in vitro. Different concentrations (5, 10,
15 and 20%) prepared from these extracts inhibited the growth of the test pathogens and the effect gradually
increased with concentration. The 20% ethyl acetate extract gave the strongest inhibition compared with the
activity obtained by the same concentration of the other extracts. High Performance Liquid Chromatography
(HPLC) analysis of ethyl acetate extract showed the presence of a main component (nimonol) which was
purified and chemically confirmed by Nuclear Magnetic Resonance (NMR) spectroscopic analysis. The 20%
ethyl acetate extract lost a part of its antifungal effect after pooling out the nimonol and this loss in activity
was variable on test pathogens. The purified nimonol as a separate compound did not show any antifungal
activity when assayed against all the six fungal pathogens.
Key words: Azadirachta indica, Aqueous and organic extracts, HPLC, Fungal inhibitory effect
INTRODUCTION
Neem (Azadirachta indica) tree has attracted worldwide
prominence owing to its wide range of medicinal properties.
Neem leaf and its constituents have been demonstrated to
exhibit immunomodulatory, anti-inflammatory,
antihyperglycaemic, antiulcer, antimalarial, antifungal,
antibacterial, antioxidant, antimutagenic and anticarcinogenic
properties (26).
Leaf and seed extracts of A. indica were tested for
antidermatophytic activity and found effective against some
dermatophytes such as Trichophyton rubrum, T. violaceaum,
Microsporum nanum and Epidermophyton floccosum by the
tube dilution technique (16) and on C. albicans (17).
The minimum inhibitory concentration (MIC) and
minimum fungicidal concentration (MFC) for extracts from
leaves and seeds of neem were evaluated (17) against various
dermatophytes. The authors found that changes in the growth
curve of the treated dermatophytes were statistically significant
with reference to the untreated fungi. The MIC of extracts from
neem leaves and seeds were 31 and 15 µg/ml, respectively and
which was sufficient to destroy Trichophyton rubrum, T.
*Corresponding Author. Mailing address: Department of Microbiology, Faculty of Science, University of Ain Shams, Elkhalifa Elmamoun street,
11566,Abbassia, Cairo,Egypt.; Tel.: +20103887237, Fax: +20226842123.; E-mail: drdaliaali@yahoo.com

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
mentagrophytes and Microsporum nanum.
Neem seed oil is used to treat certain chronic skin
diseases, ulcers, different types of metritis, leprosy, gum and
dental troubles and the seed oil is said to be non-mutagenic.
However, neem seed oil is toxigenic when given orally and
further studies might throw light on the systemic toxicity of the
solvent extracts of the neem seed (3).
Neem elaborates a vast array of biologically active
compounds that are chemically diverse and structurally
variable with more than 140 compounds isolated from different
parts of the tree (26). Quercetin and ß-sitosterol, were the first
polyphenolic flavonoids purified from neem fresh leaves and
were known to have antibacterial and antifungal properties (9).
The same authors purified the active fractions of neem organic
extracts using HPLC and found that their content of major
compounds such as 6-deacetylnimbin, azadiradione, nimbin,
salannin and epoxy-azadiradione were with appreciable active
when bioassayed on many pathogenic fungi (9). Trish et al.
(28) determined the chemopreventive potential of A. indica leaf
extract in murine carcinogenesis model system of 7-week-old
Swiss albino mice and reported that tumor incidence was
reduced by doses of neem leaf extract. The results revealed that
the Indian neem tree contained at least 35 biologically active
principles.
The present investigation aimed to (1) compare the
antifungal activity of aqueous and organic (ethanole and ethyl
acetate) extracts from neem leaves against six important human
pathogens (A. flavus, A. fumigatus, A. niger, A. terreus, C.
albicans and M. gypseum); (2) to analyze the contents of the
most active extract using HPLC and determine the antifungal
activity of the main component against the same test
pathogens.
MATERIALS AND METHODS
Neem leaves
Neem leaves (Azadirachta indica A. Juss) were collected
from 10-12 years old trees from Kalyoub Administry of
Agriculture, Kalyoub city, Egypt.
Pathogenic fungi
The test fungal pathogens (Aspergillus flavus, A.
fumigatus, A. niger, A. terreus, Candida albicans and
Microsporum gypseum) were obtained from the
Microbiological Resource Center (MIRCEN), Faculty of
Agriculture, Ain Shams University, Cairo, Egypt.
Preparation of different leaf extracts
The preparation of aqueous neem leaf extract was carried
out according to the method described by (23). Whereas
organic extracts were prepared following the method of (19).
Antifungal activity of different extracts of neem leaves
The antifungal effect of aqueous and organic extracts of
neem leaves was assessed by measuring radial growth of the
test pathogens following the technique described by (5).
HPLC analyses and chromatographic purification of
nimonol
Analysis of different components present in the mother
organic extract in ethyl acetate obtained from neem leaves was
performed according to the method (8). The organic extract
was fractionated by HPLC apparatus (Perkin-Elmer, Norwalk,
CT, USA) consisted of the following: A 410 LC pump
equipped with a LC 90 UV spectrophotometric detector and a
LCI 100 integrator at 230 nm using acherey-nagel 100 C-18
columns (20 mm x 25 cm, 215 nm). The mobile phase included
methanol (Carlo Erba, Milan, Italy) and Ultra pure water
purified in a Milli-Q system (Millipore, Bedford, MA, USA).
The chromatographic run which lasted for 2 h was carried out
for samples (20 µl for each) containing 3 mg EtoAc extract
dissolved in 1 ml methanol at a flow rate of 20 ml/min through
a stepwise gradient solvents in the following order: methanol :
water (70:30) for 40 minutes; methanol : water (80: 20) for
another 40 minutes and finally methanol : water (90: 10%) for
20 min before a final column wash after run completion with

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
methanol in order to remove the non-polar components. Three
successive injections were carried out for the neem leaf organic
extract and for pure authentic samples of the following:
azadirachtins (Sigma-Aldrich (St. Louis, MO, USA). The main
component contained in peak no. 7 was separated at 63 min. by
HPLC for spectroscopic analyses (
1
H- and
13
C-NMR).
Spectral analysis of nimonol
A solution of nimonol (2.5 mg) in pyridine (0.25 ml) and
Ac
2
O (0.25 ml) was kept at room temperature for 24 hours. A
chromatographic purification using analytical silica gel
chromatographic plates (Kieselgel 60, F
254
, Merck, Germany)
and elution was carried out using 10% EtoAc in n-hexane.
NMR spectral analysis
1
H-NMR and
13
C-NMR spectral analyses were recorded in
CD3OD at 200 and 400 MHz on Bruker Spectrometeres using
the same solvent as internal standard. Chemical shifts are given
at  value while coupling constants (J) are in Hz, carbon
multiplicities were determined by distortion enhancement by
polarization transfer “DEPT”.
Antifungal activity of nimonol
The purified nimonol was tested for antifungal activity
against all tested pathogens in comparison to the following:
mother organic extract in ethyl acetate 20% (A), (A) free from
nimonol (B) as described by (5).
Statistical analysis
The data were analyzed by using a completely randomized
factorial design (21). Significant differences between treatment
means were determined using Costal Program. Biological
results were analyzed by One Way ANOVA.
RESULTS
Activity of different neem leaf extracts against some human
pathogenic fungi
In general, two of the tested Aspergelli (A. flavus and A.
niger) were highly sensitive during assays whereas C. albicans
and M. gypseum were the weakest. The 20% concentration of
ethyl acetate extract gave the highest inhibition activity against
all test pathogens in all used concentrations compared with the
same concentrations from other extracts.
Activity of aqueous extract
The 5 % aqueous leaf extract of neem caused an inhibition
in growth of the six test fungal pathogens. The highest one
(35.22%) was recorded on A. niger while the lowest (4.00%),
was on C. albicans (fig.1-A). A concentration of 10%
moderately inhibited the growth of all test fungi with the
highest value (49.55%) recorded on A. niger and the lowest
(11.53 %) on C. albicans (fig.1-B). An inhibition by 86.22%
was recorded on the growth of A. niger compared with 38.44%
in the growth of M. gypseum (fig.1-C), when the neem leaf
aqueous extraxt was assayed at a concentration of 15%. These
ratios of inhibition jumped to 100 % and 53.66% in the growth
of A. niger and M. gypseum (fig.1-D) during the assay with the
20% concentration.
Activity of organic extracts
In assays using extracts in ethanol, the 5% concentration
gave 44.22% inhibition of A. flavus and 20.30% of C. albicans
(fig.1-A) whereas the 10% scored higher values recorded for A.
flavus (47.44%) and C. albicans (26.92%) (fig.1-B). By
increasing the extract concentration, the 15% (fig.1-C) and
20% (fig.1-D) highly suppressed the mycelia growth of all
tested pathogenic fungi.
When the antifungal activity was measured for all the six
fungal pathogens using the extracts in ethyl acetate, values of
inhibition in their growth significantly differed. The first
concentration (5%) affected A. flavus by 54.33% and C.
albicans by 9.84 % (fig.1-A). Fig.1-B is showing a marked
growth inhibition in growth of test fungi and the concentration
of 15% characteristically inhibited both A. flavus (91.11%) and
A. niger (88.50%), whereas M. gypseum was the least affected

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
(64.88%) (fig.1-C). A maximum inhibition in growth which
reached 100% was recorded for the first time on two of the
pathogenic fungi (A. flavus and A. niger), when a concentration of
20% from the same organic extract was used (fig.1-D).
Figure 1. (A): Effect of different concentrations (5%) of aqueous (Aq.), ethanolic (Et.) and ethylacetate (EtoAc) leaf extracts of neem on growth
of pathogenic fungi on solid media. (B): Effect of different concentrations (10%) of aqueous (Aq.), ethanolic (Et.) and ethylacetate (EtoAc) leaf
extracts of neem on growth of pathogenic fungi on solid media. (C): Effect of different concentrations (15%) of aqueous (Aq.), ethanolic (Et.)
and ethylacetate (EtoAc) leaf extracts of neem on growth of pathogenic fungi on solid media. (D): Effect of different concentrations (20%) of
aqueous (Aq.), ethanolic (Et.) and ethylacetate (EtoAc) leaf extracts of neem on growth of pathogenic fungi on solid media.

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
HPLC analyses and chromatographic separation of
nimonol
The HPLC diagram of the highly active organic extract
(A) showed ten well defined chromatographic peaks (Table 1
and Figure 3). These peaks were eluted at different retention
time (r
t
). The first peak was eluted after 9 min. and contained
three Azadirachtins (A, B, and C) at ratios of 7%, 5% and 8%,
respectively. The third peak was evident at r
t
of 22 min and
contained the azadirachtins A, B, D, H and I at ratios of 11%,
10%, 4%, 9%, and 7%, respectively. The 4
th
Peak was eluted
after 34 min and was identified as 6 de-acetyl nimbin of 39 %
purity. The peaks no. 2 and 5 (eluted at r
t
16 and 39 min,
respectively) yielded very small amounts of non-pure material
and were not enough for accurate identification. Peak no. 6 was
eluted at 54 min and contained mainly the azadiradione (51 %)
whereas at 63 min, peak no. 7 was eluted and contained the
nimonol with the highest purity level (82 %), followed by peak
no. 8 at 68.6 min which contained the epoxyazadiradione as a
major constituent (43 %). The last two peaks (9 and 10) were
eluted after 76 and 90 min, respectively and contained also
small amounts of non-pure materials especially the last one
which seems containing at least three components (Table 1).
Table 1. HPLC pattern of neem leaf ethylacetate (EtoAc) extract.
Band no. r
t
(min.) Total peak area etected (%) ID
Band 1 9 69.8% Azadirachtins: A (7%), B (5%), C (8%)
Band 2* 16 21.6% Not identified
Band 3 22 56.4 % Azadirachtins: A (11%), B (10%), D (4%),H (9%), I (7%)
Band 4 34 32.7% 6 De-acetyl nimbin (39%)
Band 5* 39 40.0% Not identified
Band 6 54 66.0% Azadiradione (51%)
Band 7 63 74.0% Nimonol (82%)
Band 8 68.6 30.0% Epoxyazadiradione (43%)
Band 9* 76 32.0% Not identified
Band10* 90 25.0% Not identified
Peaks analyzed by preparative HPLC using the same solvent system and found to be complex mixtures; quantities of pure material collected following their
purification were very small to be considered for further investigation.
Figure 3. High performance liquid chromatographic
pattern of neem leaf ethylacetate extract.

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
Purification and spectral identification of nimonol
The used HPLC linear gradient solvent which consisted of
a mixture from methanol and water permitted the separation of
the pure compound contained in peak no. 7 and which was
subjected to NMR spectral analysis for the complete
identification.
The
1
H-NMR signals of nimonol were at  7.38 m; 7.26 m;
6.29 m (-substituted furan); 7.12 d, J= 10.06 Hz; 5.90 d, J=
10.06 Hz (-CH= CH-CO-); 5.42 dd, J = 1.82, 2.84 Hz (C= CH-
CH
2
), 2.05 (CH
3
CO). The
1
HNMR spectrum showed also three
methane protons at  2.21 d; J = 11.65 Hz (-CH-); 4.38 dd, J =
11.65Hz and 2.37 (-CHOH) and 5.35d, J= 2.37 Hz (-CHOAc-).
Coupling of the last three methane protons was recorded in
correlated spectroscopy (COSY) where the proton at  2.21 had
cross peaks with that at  4.38 whereas the proton at  4.38 had
cross peaks with those at  2.21,  5.31 in addition to the
proton at  5.36 with that at  4.38. The COSY spectrum also
showed the presence of the characteristic group –CH-CHOH-
CHOAc on a cyclohexane ring system.
The
13
C-NMR spectrum of nimonol revealed the presence of
an important signal which corresponds to the olefinic bond
between C-14 at 158.51 ppm and C-15 at 119.54 ppm and which
are characteristic for this class of compounds. The complete
1
H-
13
C-NMR spectral data are represented in (Table 2).
Table 2.
1
H and
13
C-NMR spectral data* of the purified nimonol
Detected
Protons

signal
J
(Hz) Detectedcarbons

H-1 7.12 d 10.06 C-1 157.63
H-2 5.9 d 10.06 C-2 126.33
H-5 2.21 d 11.68 C-3 204.98
H-6 4.38 d d 1.62,2.37 C-4 40.08
H-7 5.36 d 2.37 C-5 50.36
H-15 5.42 d d 1.82,2.84 C-6 68.42
H-17 2.83 d d C-7 78.83
H-21 7.26 M C-8 44.46
H-22 6.29 m C-9 38.09
H-23 7.39 m C-10 42.59
Oac 2.05 C-11 16.52
OH n.d. C-12 32.77
C-methyls 0.82 C-13 46.66
OAc 119.55
C-methyls 33.65
Chemical shifts are recorded in () values (ppm) in CDcl
3
and CD
3
OD , respectively.
Antifungal activity of nimonol
Data represented in Fig. 2 shows the antifungal activity of
the purified nimonol when tested separately against the six test
human pathogens. Results revealed that the pure nimonol alone
as a compound has no inhibitory effect on the growth of all test
fungi when assayed at concentration of 20%. Values recorded
indicates 2-3 times-higher inhibition values for the extract (A)
over that of (B) against the tested pathogens. For extract (B), it
was found that the inhibition percentages of the test pathogens
were lowered after pooling out nimonol (peak no. 7) from the
mother extract (A) at all concentrations. Values of inhibition
for the extract (A) when assayed at a concentration of 20%
showed the highest inhibition percentages against A. flavus
(55.28%) and M. gypseum (42.12%).

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens












Figure 2. Percentage of inhibition of different ethylacetate extracts (Aand B) and pure nimonol on the growth of pathogenic fungi.
DISCUSSION
Results obtained during assay with aqueous and organic
extracts from neem leaves showed their inhibitory effect at all
used concentrations against the sex pathogenic fungi. These
human pathogens included four Aspergillus species (A. niger,
A. flavus, A. terrues and A. fumigatus) which are known to
cause aspergilloses, in addition to Microsporum gypseum (a
dermatophyte) and Candida albicans, the causal agent of
dermatophytosis and candidiases. All concentrations of the
aqueous extract effectively suppressed the mycelial growth of
these fungi and this effect was found to increase with
concentration where a maximum activity was reached using the
last one (20%). These results are in agreement with Dube and
Tripathi (6) who showed that the aqueous extracts of A. indica
obtained from bark and leaf, inhibited both spore germination
and mycelial growth of Epidermophyton floccosum,
Microsporum canis and Trichophyton mentagrophytes. They
also found that this antifungal toxic effect was also retained in
organic extracts using ethanol. The 20 % aqueous neem leaf
extract had a toxic effect on 19 out of 22 tested moulds
including Aspergillus flavus (10).
Also, different types of extracts from neem leaves were
found to have inhibitory effect on Candida albicans (15).
The complete inhibition (100%) in the growth of A. niger
obtained in assay with 20% concentration of aqueous leaf
extract of neem agrees with the results of Bohra and Purohit(2)
who mentioned that the aqueous extracts of A. indica gave the
highest inhibition of A. flavus growth.
The inhibition in growth of the six test fungi by organic
extracts were higher than those recorded by the aqueous ones.
It was found that all concentrations of organic extracts (in
ethanol and ethyl acetate) effectively suppressed the mycelial
growth and the recorded values were increasing gradually with
concentration and reaching the highest ones with 20%. This
concentration gave 88.77% on A. flavus growth and 100% in
the growth of A. flavus and A. niger.
Khan et al. (12) reported that some leaf extracts including
those from neem had a characteristic effect on dermatophytes
especially for low polar extracts over the high polar ones. The
authors suggested that one possible explanation for this is the
flavonoid quercetin contained in the extracts. Shivpuri et al.
(24) noticed that the extracts in ethanol of A. indica had
fungitoxic properties against five pathogenic fungi when tested
under laboratory conditions at concentrations ranging between
500 and 1000 µg ml
-1
. The results obtained during assay with
organic extracts were also in accordance with those recorded
by Verma et al. (30) who found that a purified fraction (ethyl
acetate : chloroform, 3:1) of extracts in methanol from neem
seed coat showed strong antifungal activity against A. niger
and Curvularia lunata with MIC of 250 ppm. They found also
that the extracts in petroleum ether from the neem leaves were

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
highly active at a lower MIC (100 ppm) against the same
pathogens. In recent study, Kishore et al. (13) reported that
ethanolic leaf extracts of A.indica inhibited the conidial
germination of Phaeoisariopsis personata by > 90% to control
late leaf spot of groundnut.
Upasana et al. (29) found that neem seed extract in
methanol was effective against Aspergillus niger, Fusarium
oxysporum and Trichoderma resii and that both dried and fresh
organic extracts from leaves were effective only against
Trichoderma resii.
Leaf extracts of neem were found to have a potent
antidermatophytic activity against Trichophyton rubrum, T.
violaceaum, Microsporum nanum and Epidrmophyton
floccosum (16). The same extracts were found to have
interesting inhibitory action on a wider spectrum of
microorganisms, including C. albicans, C. tropicalis, Neisseria
gonorrhea, multi-drug resistant Staphylococcus aureus, urinary
tract E. coli, Herpes simplex-2 and HIV with safety of the used
formulations and acceptability (26). The kill kinetics of A.
indica was determined by Okemo et al. (18) on different
pathogenic microorganisms including Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Candida
albicans. They concluded that the killing ability of A. indica
extracts is time and concentration dependent and cell wall
related.
Singh et al. (25) owed the fungicidal and bactericidal
properties of extracts from neem leaves either in vitro or in
vivo trials to the presence of several antimicrobial active
ingredients in leaves of neem tree such as desactylimbin,
quercetin and sitosterol. Whereas other researchers explained
this activity by the presence of active ingredients like
triterpenes or the limonoids such as meliantriol, azadirachtin,
desactylimpin, quercetin, sitosterol, nimbin, nimbinin,
nimbidin, nimbosterol and margisine (1) and/or to different
bitter substances such as alkaloids, phenols, resins, glycocides,
terpenes and gums (7, 11). Lyer and Williamson (14) attributed
antifungal properties of neem extracts to the inhibition in
protease activity of dermatophytes induced by the neem
organic extract.
The HPLC analysis of the most active organic extract (in
ethyl acetate) showed 4 peaks containing mainly the
triterpenoids, among these are the famous group of
Azadirachtins A, B, C, D, H, and I which don’t possess any
antifungal activity as proved by Govindachari et al. (8). A
similar analytical method was applied by Sharma et al. (22)
who purified three major constituents with only nematicidal
activity from the neem seed kernels on reversed phase
medium-pressure liquid chromatography and allowed to
separate the main component in a pure form which was
identified later by NMR spectroscopic techniques as nimonol.
1
H-NMR and
13
C-NMR showed characteristic signals of -
substituted furan and chemical shifts of an olefinic bond
between n C-14 and C-15 which confirm the chemical structure
of nimonol. The COSY spectrum also showed the presence of
the characteristic group (–CH-CHOH-CHOAc) on a cyclo
hexane ring system. Nimonol is a naturally occurring limonoid
(tetranortriterpinoid) with -methyl group at C-13 (27).
It was noticed that a loss (40-50%) occurs in the
antifungal activity for the four used concentrations of the
organic extract (in ethyl acetate) when the nimonol was pooled
even if this compound proved to be inactive against the test
fungi when separately assayed at the highest concentration
(20%). This reflects a possible potent synergism for the
different constituents present in this organic extract which is
together responsible for the characteristic antifungal activity
recorded during this study. This conclusion was illustrated in a
previous results by Govindachari et al. (9) who showed that a
mixture of fractions eluted from the HPLC was more effective
when assayed for antifungal activity than the purified
constituents. The authors attributed the lowering in antifungal
activity to an important fact that the triterpenoids when
purified, loose effect whereas in combination, an additive
synergism occurs between them and give the excellent activity
recorded for the extract.

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Mahmoud, D.A. et al. Neem leaf extracts against human pathogens
The inhibition in the growth of dermatophytes was
explained by changes in hydrophobicity of candidal cells
during assays in yeast adhesion to hydrocarbons (20). This
anti-adhesive mechanism was confirmed later by De Rezende
Ramos et al. (4) who established the effect of neem extracts on
cell surface hydrophobicity and biofilm formation, which affect
the colonization by C. albicans.
In conclusion, the mixture of fractions eluted by HPLC
was more effective than the pure nimonol, the low antifungal
activity even at the highest concentration (20%) may be
explained by a fact that these triterpenoids, in combination
exhibit an additive effect which produce the excellent
antifungal activity recorded in this study for extracts from
neem leaves.
ACKNOWLEDGMENT
The authors wish to thank Center of Statistical Analyses.
Department of Mathematics, Faculty of Scince, El-Monoufya
University for technical support during experiments. The
authors acknowledge the assistance of Dr. Anna Andolfi
(DISPA, Italy) for support during the identification of
nimonol.

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