Enhancement of invitro fungicidal activity

of fuberidazole to Botrytis cinerea by

cucurbiturils
Nail Saleh, Suad M.Ajeb, Arjun Sham &
Synan F.AbuQamar

Journal of Inclusion Phenomena and

Macrocyclic Chemistry
and Macrocyclic Chemistry
ISSN 1388-3127
Volume 79
Combined 3-4
J Incl Phenom Macrocycl Chem (2014)
79:301-309
DOI 10.1007/s10847-013-0352-8

1 23

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J Incl Phenom Macrocycl Chem (2014) 79:301309
DOI 10.1007/s10847-013-0352-8

ORIGINAL ARTICLE

Enhancement of in vitro fungicidal activity of fuberidazole

to Botrytis cinerea by cucurbiturils
Nail Saleh Suad M. Ajeb Arjun Sham
Synan F. AbuQamar

Received: 19 March 2013 / Accepted: 1 July 2013 / Published online: 11 July 2013
Springer Science+Business Media Dordrecht 2013

Abstract The hostguest interaction of fuberidazole

(FBZ) fungicide with cucurbituril (CB) macromolecules
was characterized in pure water using UVvis spectrophotometric and NMR techniques. The in vitro applications
(at pH 5.5 in pure water) of hostguest complexes were
conducted against Botrytis cinerea. While addition of CB5
to FBZ had no significant effect in vitro, mixing CB7 or
CB8 with FBZ in a 1:2 ratio improved fungal growth
inhibition at least threefold, when compared to the corresponding concentration of the unbound fungicide molecules. Empty CB hosts were completely inactive.
Furthermore, the inhibitory activity to B. cinerea was relatively maintained down to a concentration of 5:10 lM of
the CB7/8@FBZ complexes, relative to any of controls.
Complexation by CB7/8 further improved the photostability of the fungicides with photostabilization factors of 7
and 3, respectively. CB7/8 bound the protonated forms of
these guests very strongly but their neutral forms were
significantly weaker, which reflects a complexationinduced increase of their pKa values by 3.8 units with CB7
and 1.4 units with CB8. The present investigation

Electronic supplementary material The online version of this

article (doi:10.1007/s10847-013-0352-8) contains supplementary
material, which is available to authorized users.
N. Saleh (&)
Department of Chemistry, College of Science,
United Arab Emirates University, P.O. Box 15551, Al-Ain,
United Arab Emirates
e-mail: n.saleh@uaeu.ac.ae
S. M. Ajeb  A. Sham  S. F. AbuQamar (&)
Department of Biology, College of Science,
United Arab Emirates University, P.O. Box 15551, Al-Ain,
United Arab Emirates
e-mail: sabuqamar@uaeu.ac.ae

constitutes an innovative, nonclassical, approach to

enhance fungicides efficacy utilizing macromolecules with
a potential application in crop protection technology.
Keywords

Botrytis cinerea  Cucurbiturils  Fuberidazole

Introduction
Non-covalent/reversible interactions are the basis of the
most impressive functions of living systems and life sciences. The preparation of hostguest complexes based on
such recognitions has been the focus of many supramolecular chemists ever since the pioneer work of Cram and Cram
[1]. Such hostguest approaches have been utilized in many
biological and medicinal applications, such as drug delivery
[2, 3], pharmaceutical (pre)formulations [4], sensing/detections of bioactive analytes [57], diagnostic tools [8], and in
agricultural uses [9]. The microcapsule formulation technology, i.e., Warrior II insecticide with zeon, is a lambdacyhalothrin insecticide that has been widely used against a
broad spectrum of primary and secondary insects on crops
such as corn, cotton, soybean and tomato (Syngenta Crop
Protection Inc.). Encapsulation has been used in the formulation of pesticides including fungicides. A recent report
demonstrated, for instance, the effect of CB8 on carboxins
activity against Rhizoctonia solani in aqueous solution [9].
The antifungal activity of this systemic anilide fungicide
towards Rhizoctonia solani significantly improved upon
complex formation with CB8 in vitro.
Recent examples of such macromolecules, cucurbit[n]uril (CB) macrocycles in Fig. 1 [10], are readily
synthesized on a gram-to-kilogram scale by the condensation reaction of glycouril with formaldehyde under
acidic conditions. CB hosts possess negligible in vitro

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In the United States of America, Botrytis cinerea

(Pers.:Fr) is considered among the most important disease
of tomato, after Alternaria solani, Phytophthora infestans,
and Pseudomonas sp. This fungal pathogen causes the grey
mold disease over a broad host range [2123]. Applications
of chemical fungicides are common control strategies to
control B. cinerea. Previous reports have shown that this
fungus has developed resistance against benzimidazole
(BZ) and dicarboximide fungicides [24, 25]. New chemical
classes of fungicides, such as fluopyram, fenhexamid, boscalid, fludioxonil, and strobilurins, have been labeled for
control against B. cinerea [26]. Therefore, the principal
means of disease management is often the use of
fungicides.
In the present work, hostguest approaches using CB
were implemented to enhance the activity of aqueous
solution of the fungicide fuberidazole, FBZ (see chemical
structures in Fig. 1) [27] towards the model fungus
B. cinerea at submicromolar levels. The test on FBZ,
which belongs to BZ family of compounds, was performed
in vitro using B. cinerea as a pathogen in combination with
cucurbituril (CB) containers CB5, CB7 and CB8. Interestingly, the observed enhancement on FBZ activity
against B. cinerea in the present study was achieved using
a non-conventional approach that is based on supramolecular interactions.

cytotoxicity and in vivo toxicity and can be isolated in

different sizes (n = 5, 6, 7, 8, and 10) [11, 12]. Among
these containers, CB5 and CB7 have excellent solubility in
water [13].
Recently, the use of CB macrocycles in biomedical fields
has attracted attention due to their distinct properties when
compared to other classes of delivery vehicles such as dendrimers, nanoparticles, or carbon nanotubes [2]. For example,
CB vehicles have the ability to embed the drug within their
cavity, maintaining the drug stability and in a manner where
the drug retention and release can be tuned. While liposomes
and micelles are known to sequester drugs within their
structure, they are unable to control drug release and retention.
Furthermore, CB stand out among other macromolecules such as cyclodextrins (CD), calixarenes, cryptands,
crown-ethers, etc., particularly in the protection and
transport of therapeutic agents, due to their high guestloading capacity (K values up to 1015; [14] as well as their
ability to significantly increase the pKa value of the
encapsulated ionized drug molecules (from 2 to *5 units)
[5, 1518]. While the induced pKa shifts were utilized in
enhancing the solubility, chemical stability, and penetration effects of some commercial drugs [15, 17, 18], the
high binding affinities motivated others to functionalize CB
with the goal of improving its selectivity towards the biological targets in living systems [19, 20].

Fig. 1 Chemical structures of

the examined fungicide FBZ
and host macrocycles CB5, CB7
and CB8

NO
N

O
NO

N
N

O
N

O
N

ON

N O

N
O

NN

N
O

N
N

NO N

N
O

N
N

O
N

N
ON

N
N
N
N

N
O

N N

N
O

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N
N

N
N

O
N N
O

NN
N

N
O

N
H

3'

5'

N
6

CB5

CB8

N NN

O
N

CB7
O

O
ON

FBZ

4'

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(a)

Absorption spectroscopy and determination

of hostguest binding affinities

0.6

Upon addition of CB7 (up to 300 lM) or CB8 (up to

30 lM) host molecules to the aqueous solutions of FBZ at
pH 5.5 (Fig. 2); and CB8 at pH 2.0 (Supplementary
Fig. S1A), characteristic changes in the UVvis absorption
spectra were observed with the occurrence of several
isosbestic points, indicating the formation of a 1:1 binding
stoichiometry. These changes can be attributed to preferential inclusion of benzimidazole (BZ) moiety inside the
cavity of the host, in agreement with our previous findings
on the interactions of CB7 with FBZ in its protonated and
neutral forms [17]; also see NMR measurements below at
pH 2.0 in Fig. 3. However, no changes in the absorbance of
neutral FBZ (i.e., at pH 8.5) upon CB8 addition were
noticed, suggesting the absence of significant binding
(Supplementary Fig. S1B). The corresponding binding
constants between FBZ and CBn (n = 7 or 8) at a given
pH were derived directly from the optical titration plot at
332 nm (Table 1). We used the non-linear formula (in
Sigma Plot software), which was previously reported in the
fitting procedure, assuming a 1:1 binding model [5]. For
the neutral FBZ with CB8, the binding constant was estimated indirectly from the observed pKa values (see the
induced-pKa shifts below) through the conditions imposed
by the thermodynamic cycle in Fig. 4 using the relation
[17, 18]:
 0
Ka
KFBZ KFBZH 
1
Ka

0.4

OD@332nm

Results

303

0.4
0.3
0.2

where KFBZ and KFBZH are the binding constants of the

host molecule with the neutral and protonated guests; and
Ka, and Ka0 are the acidity constants of the free and complexed guest (Table 1). The monitored protonation-deprotonation process in this experiment occurs at the nitrogen-3
(Fig. 1); pKa of FBZ = 4.8 [17]. Equation 1 provides a
quantitative value for the binding constant (KFBZ), whose
unprotonated hostguest complexation equilibrium has
resulted from the combination of the three other thermodynamic equilibria in Fig. 4. These are the: (i) protonationdeprotonation equilibrium in the uncomplexed guest (Ka)
(ii) the protonated hostguest complexation equilibrium
(KFBZH ), and (iii) the protonation-deprotonation equilibrium in the complexed guest (Ka0 ).
Noteworthy, while CB5 showed no effects on the optical
profiles of FBZ, the very low solubility of CB6 (30 lM)
prevented accurate monitoring of hostguest recognitions
with FBZ in agreement with a previous report [17]. Note
that the solubility in water for empty CB8 was reported to
be 100 lM, which limits the maximum concentration that

OD

0.1
0.0
0

100

200

300

[CB7]/M

0.2

0.0
250

300

350

400

450

/nm
OD@332

(b)

OD

0.30

0.06

0.25

0.04

0.20

0.02
0.00

0.15

10

20
CB8/M

30

0.10
0.05
0.00
250

300

350

400

450

/nm

Fig. 2 UVvis hostguest titrations of FBZ (25 lM for CB7 and

10 lM for CB8) at pH = 5.5 with CB7 (a) and CB8 (b). The insets
show the nonlinear fitting according to a 1:1 binding model

can be added of CB8. Furthermore, all measurements were

performed in pure water and at room temperature to avoid
pKa tuning by heat or salts [16].
1

H NMR measurements

H-NMR spectra were measured to study the type/mode of

interactions between FBZ molecules and CB8 host. In
general, the observed complexation-induced shifts in the
present work was rationalized based on literature precedent
on the interaction of FBZ with CB7 host in the sense that
both hosts possess a cavity with similar shielding effect on
the protons of the guest molecule [17]. The spectra of the
guest molecule (0.25 mM) were measured in D2O at
pD * 2.0 in the absence, and presence of CB8 container in
excess amount. Addition of CB8 to the free guests in D2O
resulted in disappearing of the uncomplexed guest proton
signals and a new set of the aromatic proton signals
appeared broad and upfield shifted (Fig. 3). Also, the
protons attributed to the furanyl ring were almost not

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Fig. 3 1H-NMR spectra of

FBZ (0.25 mM) with CB8
(2 mM) in D2O at pD 1.0.
Arrows indicate the
complexation-induced shift of
the imidazolium and furanyl
proton signals

0.24

Table 1 Binding constants of FBZ with CB7 and CB8 in their

protonated and neutral forms and complexation-induced pKa shifts

CB7

5.0 9 10

CB8

1.5 9 105

-1
K1
FBZH (M )

KFBZ (M-1)

3.2 9 10
c

4.6 9 106

DpKa

KpH

5.5

0.21

(M-1)b

0.18

3.8

5.8 9 10

1.5e

4.1 9 104

Literature values

Binding constant with protonated/neutral FBZ determined by

nonlinear least-squares fitting of the UVvis hostguest titrations
according to a 1:1 complexation model; error 10 %, cf. Fig. 2
c

Binding constant with neutral FBZ estimated from the thermodynamic cycle in Fig. 4, see text
d

Binding constant with protonated FBZ determined directly from

the optical titration plot, cf. Fig. S1A

0.15

ODrel
@330

Host

0.12

pKa= 1.4

0.09
0.06
0.03
0.00
2

pH

Obtained from fitting the pH titrations, cf. Fig. 5. The pKa value of
free FBZ is 4.8 ( 0.2)

KFBZH+

FBZH+

+ H+

+ CB n

FBZH+ CBn

+ H+

pKa

pKa'

KFBZ

FBZ + CBn

FBZ CB n

Fig. 4 Four-state complexation model of neutral and protonated FBZ

with CBn (n = 7 or 8)

shifted. The appearance of such two independent signals

and the broadening of the complexed guest protons with
the addition of CB8 suggest that the exchange of the
complexed and uncomplexed guests is average to fast on
the 1H-NMR timescale [28].

123

Fig. 5 pH titrations of FBZ (10 lM), monitored by UVvis absorption spectrophotometry in the absence (filled circles) and presence of
100 lM CB8 (empty circles). The changes in the UVvis spectra are
shown in the Supplementary Fig. S2

CB-induced pKa shifts

It has been previously reported that the difference in the
binding constant values of protonated and neutral FBZ
molecules (or other guests) with CB7 host molecules (or
other hosts) was reflected in an increase in the pKa values
of the included guests inside the host cavity when compared to water [17, 18]. Indeed, fitting the UVvis
absorption titration data to the known sigmoidal formula
mentioned above for the absorbance of CB8 hostguest
complexes with FBZ (Supplementary Fig. S2) yielded a
shift of 1.4 units in the pKa values of FBZ (Fig. 5).
Photostabilization
Encapsulation in CB8 also enhanced the photostability of
FBZ. Under intense UV irradiation at pH 3.0, FBZ

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0.30

305

decomposed three times more slowly in their complexes

with CB8 than in their free forms (Fig. 6). In comparison,
the photostability of FBZ by CB7 was increased by a factor
of 7 in a precedent report.

0.6
0.5

0.25

0.4

X3

0.3

OD

0.20

0.2
0.1

0.15

In vitro assays

0.0
0

200

400

600

800

Time/secs

0.10

0.05

0.00
250

300

350

400

450

500

/nm

Fig. 6 Photodecomposition of FBZ in the absence (dashed lines)

and presence of CB8 (100 lM, solid lines), followed through the
decrease of the UV absorption band with increasing time of
photoirradiation by UV lamps (300 nm) in a photoreactor (40 min).
In the inset, the characteristic photodecomposition decay function,
f = log [(10Ao - 1)/(10A - 1)], is plotted versus time in seconds
and from the slope the relative photostability was determined by
taking into consideration the molar absorptivity of the CB8-bound
and free FBZ at the excited wavelength before irradiations

Fig. 7 Inhibitory growth effect of inclusion complexes on Botrytis

cinerea. B. cinerea inhibition growth using the inclusion complex
CB7@FBZ (a) and CB8@FBZ (c). Growth inhibition (Y %) on B.
cinerea using inclusion complex solutions CB7@FBZ (b) and

The inhibitory effect of FBZ and its inclusion complex

solutions with CB5, CB7 and CB8 on the mycelia growth
of B. cinerea after 5 days of incubation was illustrated in
Fig. 7 and Supplementary Fig. S3. At pH 5.5, distilled
water (H2O) was used as a negative control treatment (no
FBZ, no CB), the upper panel (right) was the host CB5/7/8
control at 100 lM (Fig. 7a, c; Supplementary Fig. S3a),
and the lower panel (left) was the guest positive control,
FBZ, at 200 lM. The lower panel (right) represents those
treated with CB5/7/8-guest inclusion complex solutions
with micromolar ratios of CB5/7/8@FBZ of 100:200
(Supplementary Fig. S3a; Fig. 7a, c). The inclusion complex of CB5/7/8-guest solutions with micromolar ratios of
CB5/7@FBZ of 5:10, 100:50, 100:100 and 100:200 or
CB8@FBZ of 5:10, 25:50, 50:100, 100:200, 100:100 and
100:50 were tested after 72 h of incubation with B. cinerea

CB8@FBZ (d). In a and c, photos were taken 5 days after

inoculation. In b and d, data were collected 72 h after inoculation.
CB7/8, 100 lM CB7/8; FBZ, 200 lM FBZ; CB7/8:FBZ, 100:200
lM CB7/8:FBZ

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Table 2 pH effect of FBZ on growth Botrytis cinerea. Growth

inhibition (Y %) of FBZ solution (200 lM)
pH

Solution (lM)

Y (%)

3.0

H2O

FBZ

6.36

5.5

H2O

FBZ

6.01

(Supplementary Fig. S3b; Fig. 7b, d). The fungus clearly

grew covering almost the entire surface of the 2xV8
medium in the negative control and any of the containers
(Fig. 7; Supplementary Fig. S3). In the positive control,
although fungal growth could be observed, changes were
minimal and dose-dependent, where a clear fungal semicircle was observed on the plates as the concentration of
the FBZ increased from 10 to 200 lM, and the inhibition
rate increased as the FBZ concentration increased. Treatments of the mixtures of CB7 or CB8 containers, but not
CB5, with FBZ at any of the micromolar ratios increased
growth inhibition rate of the fungus (Fig. 7; Supplementary
Fig. S3). The growth inhibition for each of the administered treatments in pure water at pH 5.5 was tested. While
no enhancement in inhibition activity of FBZ of B. cinerea
was observed upon CB5 addition (Supplementary Fig. S3),
growth inhibition was increased up to 1618 % with the
addition of other hosts, CB7 and CB8 (Fig. 7b, d). The
CB7/CB8-bound FBZ inhibited the growth of B. cinerea at
least three times when compared with that of the same
concentration of the unbound fungicides (Fig. 7). Not
surprisingly, the empty CB containers themselves were
completely inactive. It was also observed that the inhibition
activity was dependent on the administrated micromolar
ratio of the host:guest molecules with the highest inhibition
rate at 1:2 ratio compared to that at the 2:1 and 1:1 ratio in
both CB8@FBZ and CB7@FBZ systems, as compared
with their corresponding empty containers or fungicide
controls alone (Fig. 7). In addition, lowering the concentration of either hosts, CB7 or CB8 container, and the guest
fungicide (FBZ) by 20 times from 200:100 to 10:5 lM
lowered the activity of the fungicide composite by a factor
of 0.75 only. Altogether, this suggests that the growth
inhibitory effect of FBZ increased in a dose-dependent
manner when FBZ is coupled with either CB7 or CB8
container, but not CB5.
To determine the pH effect of FBZ on B. cinerea
growth, we tested the maximum dose of the fungicide
(200 lM) used in previous experiments (Methods) at pH
3.0 and 5.5 (Table 2). The negative controls were also
tested at the same pH values towards B. cinerea growth. As
expected, the negative control at any of the pH values
showed a complete mycelium-covered area to the greatest
extent compared with the positive control samples

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(Table 2). Also, there was no significant inhibition effect of

FBZ treatments at either of the pH tested. Altogether, this
suggests that while the inhibitory effect of FBZ on
B. cinerea is dose-dependent, it is not pH-dependent.
Noteworthy, the change of pH from 3.0 to 5.5 means that
the FBZ fungicide has changed its structure from protonated to neutral forms (pKa of FBZ = 4.8, Table 1 and
Fig. 5). Consequently, the similar inhibition effect at the
two selected pH also means that changing the acidic-basic
properties has no influence on the fungicide activity against
B. cinerea at least in the meaningful pH range.

Discussion
In the present study, we have employed the CB hostguest
approach in vitro. Our in vitro findings demonstrated that
the antifungal activity toward B. cinerea of CB7@FBZ
and CB8@FBZ were quite similar and enhanced up to
three times with the formation of CB7/CB8 hostguest
complexes at pH 5.5 (and pH 3.0) when compared to the
free FBZ. Furthermore, the inhibition activity was found to
depend on the administrated molar ratio of the guest and
host molecules. Noteworthy, lowering the concentration by
20 times from 200:100 to 10:5 micromolar ratio of the
CB7/CB8@FBZ complexes had negligible effects on the
activity of the composite.
From the measured UVvis titration data (see Results
section), the CB8@FBZ system has a 1:1 stoichiometry
and not other ratios. This could be attributed to the size
complementary of the guests to the cavity volume of CB8
[27]. Other things being equal, the binding constants
become highest when the ratios of the volumes to the
guests with the inner cavity volume of CB are close to
0.5529 [27]. In addition, it has also been previously
reported [27, 28] that the release of (high energy) water
molecules from the cavity of CB plays a particularly
important role for the binding of guest molecule. The
results in Table 2 also demonstrated that the protonated
and neutral forms of FBZ have similar activity towards
B. cinerea. Thus, the induced pKa shifts do not seem to
rationalize the trends in the observed data (Fig. 7 and
Supplemetary Fig. S3). For example, the population of the
protonated FBZ is manifested in the degree of ionization,
which is calculated at pH 5.5 to be 99 % and 86 % for FBZ
in its complexed forms with CB7 and CB8, respectively,
taking into consideration the pKa values for the free and
complexed FBZ (Table 1) and using the relation:
a

1
1 10pHpKa

Although the precise mechanism of action of the

FBZ@CB7/8 complexes needs further experiments, one

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possibility is that the macrocyclic complex actually binds

more tightly to the biological targets (particularly proteins)
[2]. Nau and co-workers [7], has shown that the binding
affinities of a drug molecule with the target site can be
enhanced (with a concomitant increase in the target
specificity) in the presence of a CB macrocycle. The
binding events were monitored through changes in the
fluorescence intensity and lifetime of the drug, providing a
proof-of-principle for the formation of suprabiomolecular
ternary complexes that might potentially be useful in the
context of targeted drug delivery and drug specifity. Note that,
CB7 and CB8 bind FBZ with comparable affinities under the
in vitro condition (pH 5.5; Table 1), which explain their
similar activity against B. cinerea. Yet, in vivo experiments
on this or other systems are needed to prove the formation of a
stable ternary complex under the in vivo condition (pH
7.07.5), which are ongoing in our laboratories.
We have used B. cinerea as a model pathogen in our
study, which is a fungus with a broad host range. This
economic pathogen reduces crop quality and productivity under diverse production conditions. Application of
chemical fungicides is a common control strategy against
B. cinerea. Noteworthy, B. cinerea develops resistance to
some fungicides, which severely limits chemical control
options [24, 25]. Nowadays, fluopyram, iprodione, fenhexamid, boscalid, fludioxonil, various triazoles and strobilurins, are widely used fungicides to control B. cinerea
[26]. The selected BZ derivative in this work (e.g. FBZ) is
broadly used as efficient fungicides particularly as pre- or
post-harvest fungicides to control plant diseases on a wide
variety of fruits, vegetables or other field crops in Asia
[29]. We have demonstrated that the CB-complexation of
FBZ enhances the solubility (e.g., three times with CB7)
and increases the photostability by seven and three times in
the current results with CB7 and CB8, respectively [17].
The present results are, thus, important to BZ antifungal
action as the active concentration (10:5 lM) should be less
harmful to human health than the current uses in crop
treatment. Moreover, the enhanced photostability furbishes
another evidence for the hostguest complex formation and
confirms that the new non-conventional methods have
additional benefits to crop protections.
CB hosts bind the FBZ guest molecules via hydrophobic
and iondipole interactions. The glycoluril core at the CB
equator provides a hydrophobic environment for the binding of neutral forms, while the iondipole interactions
occur between the ionizable hydrogen atoms on the nitrogen-3 of the FBZ guests and the oxygens at the portals of
the CB [17]. Recently, it has been reported that the release
of water molecules from the cavity of CB plays a significant role in the binding of guest molecules [30]. In addition, the size complementary of the guests with the cavity

307

volume of CB is manifested in the absolute binding constants of the CB@guest complexes [31]. Such noncovalent
encapsulation inside the nonpolar cavity of CB was
reported to modify the physical and chemical properties of
the guest molecules due to the isolation and confinement of
the guest molecules from the surrounding water medium.
For example, it was reported that CB7 can induce deaggregation and photostabilization of some fluorescent dyes
[32]. In addition to the modification of the physical properties of the guest molecules, CB was demonstrated to
modify the chemical reactivities of the guest molecules for
different applications. As an example, CB can modify the
protonation-deprotonation equilibria of the guest molecules. The carbonyl at the ureidyl portals of CB biases its
negative dipoles towards the positive charges of the guest
over neutral species through the iondipole interactions.
Thus, CB hosts, preferentially, bind the protonated species
of a guest molecule with respect to its neutral form and,
consequently, shift the protonationdeprotonation equilibria towards higher pH, the so-called CBs-induced pKa
shifts. While the induced pKa shifts were utilized in
enhancing the solubility, chemical stability, and penetration
effects of some commercial drugs [2], the present studies
demonstrated no influence of the induced pKa shifts on the
FBZ activity against the fungus in vitro. Utilization the
CB-induced pKa shifts was planned from the beginning, as
it may be desirable to trigger the release of drugs by
shifting the equilibrium towards the uncomplexed drug in
dependence on a certain stimulus in a spatially and/or
temporally controlled manner. As the neutral forms of the
FBZ bind much more weakly with CB than their corresponding protonated forms, a change in the pH of the
medium from below pKa0 (the pKa value of the complex) to
above pKa0 effectively decreases the binding constants of
the drugs and the subsequent rapid release of the encapsulated drugs shifts the chemical equilibrium toward the
uncomplexed guest (and host). Note that this concept can
be advantageously employed at different stages of FBZ
drug delivery to plant cells. For example, FBZ@CB
complexes which have pKa0 values between 6 and 9 could
be manufactured below pH 4 (because of the higher binding constants of the protonated forms and related higher
degree of complexation) while the physiological pH itself
(e.g., pH 7.07.5 for the cytoplasm of higher plant cells)
could subsequently favor the efficient release of the drug
molecules into the biological targets. On the contrary, the
CB-induced pKa shifts and the accompanied tuning in the
CB7/8 affinity towards FBZ in its neutral state at alkaline
pH (Supplementary Fig. S1 lower panel) might become
disadvantageous in the context of the need for highly stable
hostguest CB@FBZ complexes that could sustain competitive displacements by the cytoplasmic compartments.

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308

Therefore, research is undergoing to understand the

relationship between tomato-B. cinerea and the inclusion
complex in vivo, yet this new suggested crop treatment using
a novel hostguest approach in our in vitro findings should
have a lot of potential in the agrochemical fungicide business. In particular, CB hosts have unique supramolecular
recognition properties, which offer numerous advantages
over the use of conventional macrocycles. These include
ultrahigh-affinity binding, slow and tunable complexation
kinetics, pKa shifts in favor of the binding of the protonated
forms of guests, and an increased photochemical and
chemical stability of encapsulated guests. While these
properties are well recognized, their implementation in drug
delivery systems has rapidly unfolded during the past
10 years. Among the different supramolecular host molecules, CD [33] have been most widely used for the formulation of therapeutic and diagnostic agents in pharmacology.
However, their use in clinical conditions is generally limited
to oral and topical drug delivery forms, since they are
nephrotoxic if they enter the body in a nonmetabolized state
[34]. In addition, the poor selectivity and low binding
affinities (Kbinding \ 104 M-1) [35] of nonderivatized (natural) CD towards guest molecules in general, and drug
molecules in particular, requires excess concentrations of
CD to form quantitative CD@guest complexes. Several
alternative macrocyclic host molecules have been suggested
in the literature to have potential as drug delivery vehicles,
for example derivatized calixarenes [36], but not has yet
found actual applications. Thus, CB hosts hold already now
more prospects for drug delivery than calixarenes, and they
are expected to rival CD in many areas.

Conclusions
This study presents an innovative way to enhance the
activity of fungicides against microbes, utilizing the host
guest non-covalent interactions. Inclusion complexes of
CB7@FBZ and CB8@FBZ enhanced the inhibitory effect
of FBZ on B. cinerea. Although this antifungal effect of
FBZ was improved when mixed with the CB containers
in vitro, additional research is required to elucidate the
specific mode of action and to understand the conditions
that facilitate the performance of these inclusion complexes
and their efficacy.

Experimental section
Chemicals and equipment
CB5, CB7 and CB8 (purity [99.9 %) were purchased and
used as recommended (Sigma-Aldrich). The FBZ was also

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J Incl Phenom Macrocycl Chem (2014) 79:301309

purchased from Sigma-Aldrich and used without further

purification. Millipore water was used for dilution. The
UVvis spectra were measured on Cary-50 instrument
(Varian). All NMR spectra were performed on a Varian
400 MHz spectrometer in D2O. All 1H-NMR spectra are
referenced in ppm with respect to a TMS standard. The pH
values of the solutions were adjusted (0.2 units) by
adding adequate amounts of HCl (DCl) or NaOH (NaOD)
and recorded using a pH meter (WTW 330i equipped with
a WTW SenTix Mic glass electrode). The photostabilization of FBZ was monitored using the change in the
absorbance of the free and complexed fungicides at pH 3.0
upon irradiation in a Luzchem LZC-4V photoreactor
equipped with UVB lamps (kex = 300 nm). Also, the
average power intensity was kept at 12 mW cm-2 during
irradiation.
Fungal culture and antifungal activity determination
Botrytis cinerea strain BO5-10 was grown on 2xV8 agar
(36 % V8 juice, 0.2 % CaCO3, 2 % Bacto-agar, Becton,
Dickinson and Company, Sparks, MD USA). Fungal cultures were initiated and subcultured by transferring pieces
of agar containing mycelium to fresh 2xV8 agar and
incubated at 23 2 C as described [37].
The in vitro fungicidal activities against B. cinerea were
tested according to [9] with some modifications. An
aqueous solution of FBZ was prepared with a concentration of 10, 50, 100, or 200 lM. The aqueous solution of
CB5, CB7, or CB8 was prepared with a concentration of 5,
25, 50 or 100 lM. Solutions of CB mixed with FBZ in a
ratio of 1:2, 1:1 and 2:1 were prepared in pure water at pH
of 5.5. Inocula of a diameter of 8-mm were removed from
the margins of actively growing colonies of mycelium, and
placed on one side of a Petri dish on 2xV8 medium as
described above. Filter papers (6 mm in diameter) were
placed on the opposite side of the dish, and soaked onto the
solutions of CB5, CB7, CB8, FBZ (15 lL) or each of the
previously prepared inclusion complex (sterilized water
was used as a negative control). Plates were then covered,
sealed with parafilm, and incubated for 72 h in a 25 C
incubator. Each treatment was prepared in four replicates.
The radius of the mycelium was measured at 72 h. The
growth inhibition rate was calculated according to the
growth inhibition (Y) formula developed previously [38].
Acknowledgments The authors would like to acknowledge the
Office of Research Support and Sponsored Projects at UAEU for their
support under the grant numbers 31S075 to S.AQ. and 31S074 to
N.S., within the framework of National Research Foundation (NRF)
funding program, the Interdisciplinary fund number 31S035 to S.AQ.
We would also like to thank Prof. Dr. Werner Nau and his research
group for their significant contributions in previous joint accomplishments on the recognitions of benzimidazole fungicides by CB in

Author's personal copy

J Incl Phenom Macrocycl Chem (2014) 79:301309
aqueous solutions. We thank Ms. Zeinab A. Abdalla for her contribution in the initiation of this study.

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