Synthesis of 4′-thiosemicarbazonegriseofulvin and its effects
on the control of enzymatic browning and postharvest
disease of fruits
Zhi-Zhen Pan
a#
, Yu-Jing Zhua#, Xiao-Jie Yub, Qi-Fan Linb, Rong-feng Xiaoa,
Jian-Yang Tanga, Qing-xi Chenb,* and Liu Boa,*
a
Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Sciences,
Fuzhou 350003, China
b
Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems,
College of Environment and Ecology, School of Life Science, Xiamen University,
Xiamen 361005, China
#
The authors contribute equally in this work.
* Corresponding author: Tel/Fax: +86 591 87864601. E-mail: liubofaas@163.com (B.
Liu), chenqx@xmu.edu.cn (Q.-X. Chen).
1
ABSTRACT
Browning and postharvest disease of fruits lead to large losses of food industry. A
new thiosemicarbazide derivative of griseofulvin (4′-thiosemicarbazonegriseofulvin)
was synthesized and evaluated for its potential on the control of browning and
postharvest disease of fruits. Browning is mainly due to the enzymatic oxidation of
phenolic compounds catalyzed by tyrosinase, 4′-thiosemicarbazonegriseofulvin can
effectively inhibit the activity of tyrosinase and its IC50 value was 37.8 μmol/L. It was
a reversible and noncompetitive inhibitor of tyrosinase and its inhibition constant (KI)
was
determined
to
be
38.42
μmol/L.
The
antifungal
activity
of
4′-thiosemicarbazonegriseofulvin was studied on four fungi (F. oxysporum, F.
moliniformin, F. solani and Colletotrichum) which often caused postharvest disease of
fruits. The results showed the 4′-thiosemicarbazonegriseofulvin can also strongly
inhibited the mycelial growth of four target fungi, the LC50 values were 5.4, 7.0, 15.3
and 1.5 mmol/L, respectively.
Keywords: 4′-thiosemicarbazonegriseofulvin, browning, postharvest disease
2
1. Introduction
Browning and postharvest disease of fruits cause considerable losses to the food
industry (Segovia-Bravo, Jaren-Galan, Garcia-Garcia & Garrido-Fernandez, 2007;
Wan, Tian & Qin, 2003). The mechanical damage and collision during fruit
post-harvest handing and processing will accelerate the browning (Arias, Gonzalez,
Oria & Lopez-Buesa, 2007). It reduces not only the visual quality but also results in
undesirable changes in flavour and nutrient loss. The browning is mainly due to the
enzymatic oxidation of phenolic compounds catalyzed by tyrosinase (EC 1.14.18.1)
(Yan et al., 2009). Tyrosinase can catalyze monophenols into o-diphenols and
o-diphenols into quinones, respectively (Qiu et al., 2009; Song, Chen, Wang, Qiu &
Huang, 2005). Quinones will be converted into brown pigments undergo further
non-enzymatic polymerization (Huang et al., 2009). Thus, the tyrosinase inhibitors
will be effective antibrowning agents, such as Enokitake mushroom extracts (Jang,
Sanada, Ushio, Tanaka & Ohshima, 2002), Artocarpus heterophyllus extracts (Zheng,
Cheng, To, Li & Wang, 2008), oxyresveratrol and Morus alba L, extract (Li, Cheng,
Cho, He & Wang, 2007).
Fruit is easily attacked by several fungal pathogens for the high amount of
nutrients, water content and low pH and fruit have lost most intrinsic resistance to the
fungal pathogens after harvest, therefor postharvest disease of fruit caused great losses
to the food industry (Janisiewicz & Korsten, 2002; Nunes, 2011). Adequate control of
postharvest diseases is a prerequisite for the production of a stable and profitable food
supply. The pathogens that cause the most important postharvest disease are several
species belonging to Alternaria, Aspergillus, Botrytis, Fusarium, Geotrichum,
Gloeosporium, Mucor, Monillinia, Penicillium, Colletotrichum and other genera
(Kobiler, Akerman, Huberman & Prusky, 2011; Kong, Shan, Liu, Wang & Yu, 2010;
Schirra, D'Aquino, Cabras & Angioni, 2011; Sharma, Singh & Singh, 2009).
Griseofulvin is one of the representative antifungal antibiotics and has been widely
used as an antifungal drug. Its antifungal activity has been demonstrated in many
filamentous fungi (Cole & Stricklin, 1989; Gupta, Williams, Zaman & Singh, 2009;
Robinson & Robinson, 1959). However, it has weak effect on antibrowning of fruit.
Our objective in the present study was to prepare a new thiosemicarbazide
derivative of griseofulvin (4′-thiosemicarbazonegriseofulvin) and investigate its
inhibitory effects on tyrosinase and antifungal properties.
3
2. Materials and methods
2.1 Reagents
Griseofulvin (99.9% powder) was purchased from Shanghai Pharmaceuticals Holding
Co., Ltd. (Shanghai, China). Mushroom tyrosinase (EC1.14.18.1) with 6680 U/mg
was purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO)
and L-3,4-dihydroxyphenylalanine (L-DOPA) were obtained from Aldrich (St. Louis,
MO). Fusarium oxysporum (FJAT-3701), Fusarium moliniformin (FJAT-165),
Fusarium solani (FJAT-176) and Colletotrichum (FJAT-763) were from Fujian
Academy of Agricultural Sciences. All other reagents were local products of
analytical grade.
2.2 Synthesis of 4′-thiosemicarbazonegriseofulvin
The 4′-thiosemicarbazonegriseofulvin was prepared by one-step reaction of
griseofulvin and thiosemicarbazide in an acidic solution of ethanol, as previous
described (Zhu et al., 2009). Griseofulvin (10 mmol) and acetic acid (10 ml) were
added to a solution of thiosemicarbazide (10 mmol) in methanol (60 ml). The mixture
was stirred and refluxed for 72 h and cooled to room temperature. Then the reaction
mixture was kept under 4 °C for night and filtrated. The product was purified by
recrystallized in ethanol. The structure of the product was established by
spectroscopic methods (MS and NMR).
2.3 Enzymatic activity assay
The enzyme activity assay was performed as reported by (Guo et al., 2010). In this
investigation, L-DOPA was used as a substrate for the activity assay of tyrosinase. The
reaction media (3 ml) for activity assay contained 0.5 mmol/L L-DOPA in 50 mmol/L
sodium phosphate buffer (pH 6.8). The 4′-thiosemicarbazonegriseofulvin were
dissolved in DMSO and diluted to appropriate concentrations. The final concentration
of DMSO in the test solution was 3.3%. The controls, without inhibitors but
containing 3.3% DMSO in the reaction media, were routinely carried out. The
reaction was carried out at a constant temperature of 30℃. The inhibition type was
assayed by the Lineweaver–Burk plot, and the inhibition constant was determined by
the second plots of the apparent Km/Vm or 1/Vm versus the concentration of the
4
inhibitor. A Beckman UV-650 spectrophotometer was used for absorbance and kinetic
parameters.
2.4 Antifungal activity assay
The antifungal of 4′-thiosemicarbazonegriseofulvin was tested on four
phytoathogenic species fungi: F. oxysporum, F. moliniformin, F. solani and
Colletotrichum. Some proper concentrations of 4′-thiosemicarbazonegriseofulvin in
DMSO were evenly spread on the culture medium PDA, in sterilized Petri dishes and
dried for 2 h under a laminar flow, and then F. oxysporum, F. moliniformin, F. solani
and Colletotrichum were inoculated in the middle with 100 μl fungal conidial
suspension (~1×105 CFU/ml). In parallel, control experiments only with DMSO were
also performed. The Petri dishes were incubated at 28±1 °C and the fungal colony
diameter was measured daily. The inhibition rates of 4′-thiosemicarbazonegriseofulvin
were calculated after 7 days of incubation when mycelium in the control experiments
completely covered the dishes. It was expressed as an average diameter and calculated
using the following equation:
Where Dc and Dt represent mycelium growth diameter in control and treated Petri
plates, respectively. The antifungal effects were measured with three replications.
Lethal concentration 50 (LC50) values on the target fungi were analyzed using Probit
Analysis and considered significantly different if 95% confidence intervals did not
overlap. The statistical analyses were done using the China-DPS program.
2.5 Effect of 4′-thiosemicarbazonegriseofulvin on hyphal morphology
The effects of 4′-thiosemicarbazonegriseofulvin on hyphal growth of F. oxysporum,
F. moliniformin, F. solani and Colletotrichum were studied by using microscope. Ten
milliliters of agar medium inoculated with fresh fungal culture (~1×105 CFU/ml) were
poured into Petri dishes as the upper layer. After solidification, two wells of 8-mm
diameter were created. 16 mmol/L 4′-thiosemicarbazonegriseofulvin solution and
controlled DMSO were deposited in wells. Petri dishes were then incubated at
28±1 °C for 72 h. The hyphae closely around the inhibition zones were collected and
observed by Leica DMI 3000 M Inverted Microscope (Leica Corp.).
5
3 Results and discussion
3.1 Synthesis of 4′-thiosemicarbazonegriseofulvin
The 4′-thiosemicarbazonegriseofulvin (the structure was showed in Fig. 1) was
obtained as a white powder. Yield: 85.4%. 1H NMR (CDCl3, 600 MHz): δ (ppm) 7.25
(HN, 1H, s), 5.61 (CH, 2/3H, s), 5.72 (CH, 1/3H, s), 6.12 (bezene, H, s), 6.35, 8.68
(NH2, 4/3 H, s), 6.32, 8.97 (NH2, 2/3 H, s), 3.98, 4.06 (CH3O-bezene, 6H, s), 3.60
(3′-CH3O, 2H, s), 3.67(3′-CH3O, H, s), 2.72(CH, 2/3H, m), 3.10 (CH, 1/3H, m), 1.26
(CH2, 2H, m), 0.96 (CH3, 3H, m). ESI-MS: m/z 426.2 (M+H+).
3.2 Effect of 4′-thiosemicarbazonegriseofulvin on the activity of mushroom
tyrosinase
We studied the effect of 4′-thiosemicarbazonegriseofulvin on the activity of
mushroom tyrosinase using L-DOPA as substrate. The enzyme activity was monitored
by following the increasing absorbance at 475 nm accompanying the oxidation of
L-DOPA.
From the Figure 2, we found 4′-thiosemicarbazonegriseofulvin could
effectively inhibit the activity of tyrosinase with dose-dependence. When the
concentration of was enhanced to 20 μmol/L, the relative activity of tyrosinase was
only
to
be
64%.
The
50%
inhibitory
concentration
(IC50)
of
4′-thiosemicarbazonegriseofulvin against tyrosinase was estimated to be 37.8 μmol/L,
It was much better than arbutin (IC50=5.32) as previous report (Song, Qiu, Huang &
Chen, 2003), which was well-known tyrosinase inhibitors.
3.3 The inhibition mechanism of 4′-thiosemicarbazonegriseofulvin on the activity
of mushroom tyrosinase
The inhibition mechanism of 4′-thiosemicarbazonegriseofulvin on tyrosinase was
investigated and the result was showed in Fig. 3. The plots of enzyme activity versus
the concentrations of enzyme in the present of different concentrations of
4′-thiosemicarbazonegriseofulvin gave a family of straight lines, which all passed
through the origin indicating that the inhibition of 4′-thiosemicarbazonegriseofulvin
on
tyrosinase
was
a
reversible
reaction
course.
The
present
of
4′-thiosemicarbazonegriseofulvin did not bring down the amount of the efficient
enzyme, but just resulted in the descending of the activity of the enzyme.
6
3.4 The inhibition type of 4′-thiosemicarbazonegriseofulvin on the activity of
mushroom tyrosinase.
The inhibitory type of 4′-thiosemicarbazonegriseofulvin on tyrosinase was
determined by Michaelis-Menten kinetics. As Fig. 4a showed, the plots of 1/ν versus
1/[S] gave a family of lines with different slope and intersect one another in the
X-axis. It meant 4′-thiosemicarbazonegriseofulvin could decrease the value of Vm but
have no effect on the Km of tyrosinase. Above all, we concluded that
4′-thiosemicarbazonegriseofulvin was a noncompetitive inhibitor of tyrosinase, it
could bind with both the free tyrosinase and the enzyme-substrate complex, and the
equilibrium constants are the same. The equilibrium constant (KI) was obtained from
Fig. 4b. The value of KI was 38.42 μmol/L.
3.5 Antifungal activity assay
The antifungal activity of 4′-thiosemicarbazonegriseofulvin against four target
fungi was evaluated from inhibition rates on the mycelium growth. After 7 days of
incubation, the inhibition rates were calculated and the results were reported in Figure
5. The results showed that 4′-thiosemicarbazonegriseofulvin had potential antifungal
activity on all target fungi and its effectiveness decreases with the dilution. The effect
of 4′-thiosemicarbazonegriseofulvin was most efficient on F. moliniformin and
Colletotrichum in the concentration of 0.25 mmol/L with the inhibition rates of 23.0%
and
22.4%,
respectively.
However,
in
the
concentration
of
1
mmol/L,
4′-thiosemicarbazonegriseofulvin showed most efficient on Colletotrichum with the
inhibition rate of 61.9%, which was similar with the concentrations of 4 mmol/L and
16 mmol/L. We estimated LC50 values of 4′-thiosemicarbazonegriseofulvin on F.
oxysporum, F. moliniformin, F. solani and Colletotrichum were 5.4, 7.0, 15.3 and 1.5
mmol/L by Probit analysis. It was similar to griseofulvin. Our findings showed that
the coupling with griseofulvin and thiosemicarbazide did not hurt the antifungal
activity of griseofulvin. The 4′-thiosemicarbazonegriseofulvin could effectively inhibit
the mycelium growth of four target fungi, the inhibitory effect was best on
Colletotrichum.
7
3.6 Effect of 4′-thiosemicarbazonegriseofulvin on hyphal morphology
The alternation of F. oxysporum, F. moliniformin, F. solani and Colletotrichum
hyphae grown on PDA amended with 4′-thiosemicarbazonegriseofulvin was studied
under microscope. The hyphae of four target fungi treated with DMSO as control
treatment were thick, elongated, even and smooth surfaced, whereas degenerative
changes
was
observed
on
the
hyphal
morphology
in
all
4′-thiosemicarbazonegriseofulvin treatments (as showed in Fig. 6). While treated by
16 mmol/L 4′-thiosemicarbazonegriseofulvin, the growth of four target fungi hyphae
were strongly inhibited, as indicated by mycelia sparsity, asymmetry, swelling, curling
and twisting.
4. Conclusion
Browning and postharvest disease were two main issues of fruit industry.
Griseofulvin was a broad-spectrum antifungal drug, it can inhibit fungal mitosis by
disrupting the mitotic spindle through interaction with polymerized microtubules
(Crackower, 1972). However it had a weak effect on antibrowning of fruit. In this
study we coupled a thiosemicarbazide group, which may be the functional group of
the inhibitors of tyrosinase, to griseofulvin to enhance its antibrowning activity. It was
interesting to find that the new compound (4′-thiosemicarbazonegriseofulvin) could
effectively inhibit the activity of tyrosinase with the IC50 value was 37.8 μmol/L,
which was much better than arbutin, a well-known tyrosinase inhibitor. It can also
strongly inhibited the mycelium growth of four target fungi (F. oxysporum, F.
moliniformin, F. solani and Colletotrichum), which can often caused postharvest
disease of fruits. Therefor the coupling with griseofulvin and thiosemicarbazide could
improve the inhibitory activity against tyrosinase but do not hurt the antifungal
activity of griseofulvin. The 4′-thiosemicarbazonegriseofulvin may be served as a
novel compound to control both enzymatic browning and postharvest disease of fruits.
Acknowledgements
The present investigation was supported by the Special Fund for Agro-scientific Rese
arch in the Public Interest (200903049), the Fujian Funds for Distinguished Young Sci
entists (2009J06010).
8
Figure Legends
Figure 1 Chemical structure of 4′-thiosemicarbazonegriseofulvin
Figure 2 Effect of 4′-thiosemicarbazonegriseofulvin on the activity of mushroom
tyrosinase for the oxidation of L-DOPA.
Figure 3 Determination of the inhibitory mechanism of
4′-thiosemicarbazonegriseofulvin on mushroom tyrosinase. The concentrations of
4′-thiosemicarbazonegriseofulvin for curves 0-4 were 0, 20, 40, 60 and 80 mol/L,
respectively.
Figure 4 Determination of the inhibitory type and inhibition constants of
4′-thiosemicarbazonegriseofulvin on mushroom tyrosinase. (a) Lineweaver-Burk plots
for inhibiton of 4′-thiosemicarbazonegriseofulvin on mushroom tyrosinase. The
concentrations of 4′-thiosemicarbazonegriseofulvin for curves 0-4 were 0, 20, 40, 60
and 80 mol/L, respectively. (b) represented the secondary plot of Km versus
concentration of 4′-thiosemicarbazonegriseofulvin to determine the inhibition
constant KI.
Figure 5 Effects of 4′-thiosemicarbazonegriseofulvin on mycelium growth of F.
oxysporum, F. moliniformin, F. solani and Colletotrichum. The bars on each column
show standard error.
Figure 6 Micrograph of F. oxysporum, F. moliniformin, F. solani and Colletotrichum
treated with 4′-thiosemicarbazonegriseofulvin or control. (C1, C2, C3, C4) Displayed
micrograph of F.oxysporum, F.moliniformin, F.solani and Colletotrichum mycelium
incubation in controlled DMSO. (T1,T2,T3,T4) Displayed micrograph of F. oxysporum,
F. moliniformin, F. solani and Colletotrichum mycelium incubation in
4′-thiosemicarbazonegriseofulvin.
9
Figure 1
10
Figure 2
Relative activity (%)
100
80
60
40
20
0
0
20
40
60
[I] ( M)
80
100
11
Figure 3
Enzyme Activity ( M/min)
120
1
100
80
2
60
3
4
5
40
20
0
0.0
2.0
4.0
6.0 8.0 10.0
[E] ( g/ml)
12
Figure 4
4
0.08
0.05
3
0.06
2
0.04
1
0.04
Intercept
1/v (  M/min) -1
5
0.10
0.03
0.02
0.01
0.02
0.00
0
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5
1/[S] (mM
)-1
20
40
60
80
[I] ( M)
13
Fusarium solani
Inhibition rate(%)
Fusarium oxysporum
Inhibition rate(%)
Inhibition rate(%)
Inhibition rate(%)
Figure 5
Fusarium moliniformin
Colletotrichum
4′-thiosemicarbazonegriseofulvin (mmol/L)
14
Figure 6
C1
T1
C2
T2
C3
T3
C4
T4
15
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