Synthesis, characterization, and antifungal activity of novel inulin derivatives with chlorinated benzene

Article abstract of DOI:10.1016/j.carbpol.2013.08.044

A group of novel inulin derivatives containing benzene or chlorinated benzene were synthesized by reaction of chloracetyl inulin (CAIL) with the Schiff bases of 4-amino-pyridine, including (2-pyridyl)acetyl inulin chloride (PAIL), 2-[4-(2-chlorobenzylideneamino)-pyridyl]acetyl inulin chloride (2CPAIL), 2-[4-(4-chlorobenzylideneamino)-pyridyl]acetyl inulin chloride (4CPAIL), and 2-[4-(2,4-dichlorobenzylideneamino)-pyridyl]acetyl inulin chloride (2,4DCPAIL). Their antifungal activity against three kinds of phytopathogens was estimated by hypha measurement in vitro. Of all the synthesized chitosan derivatives, 2,4DCPAIL inhibited the growth of the tested phytopathogens with inhibitory indices of 67%, 47%, and 43% against Colletotrichum lagenarium (Pass) Ell.et halst, Phomopsis asparagi (Sacc.) Bubak and Fusarium oxysporum (schl.) F.sp. niveum (F. oxysporum) respectively at 1.0 mg/mL. The results indicate that all the inulin derivatives have better antifungal activity than inulin, and the inhibitory index is affected by the chlorine atom grafted to the inulin derivatives.

Full text of DOI:10.1016/j.carbpol.2013.08.044

Carbohydrate Polymers 99 (2014) 469–473
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Synthesis, characterization, and antifungal activity of novel inulin
derivatives with chlorinated benzene
Zhanyong Guoa,∗, Qing Lia, Gang Wanga, Fang Donga, Haoyuan Zhoua,b, Jing Zhangc
a Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003,
China
b Graduate School of Chinese Academy of Sciences, Beijing 100039, China
c Laishan Forestry Bureau, Yantai, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2013
Received in revised form 15 July 2013
Accepted 18 August 2013
Available online 25 August 2013
A group of novel inulin derivatives containing benzene or chlorinated benzene were synthesized
by reaction of chloracetyl inulin (CAIL) with the Schiff bases of 4-amino-pyridine, including (2-
pyridyl)acetyl inulin chloride (PAIL), 2-[4-(2-chlorobenzylideneamino)-pyridyl]acetyl inulin chloride
(2CPAIL), 2-[4-(4-chlorobenzylideneamino)-pyridyl]acetyl inulin chloride (4CPAIL), and 2-[4-(2,4-
dichlorobenzylideneamino)-pyridyl]acetyl inulin chloride (2,4DCPAIL). Their antifungal activity against
three kinds of phytopathogens was estimated by hypha measurement in vitro. Of all the synthesized chi-
tosan derivatives, 2,4DCPAIL inhibited the growth of the tested phytopathogens with inhibitory indices
of 67%, 47%, and 43% against Colletotrichum lagenarium (Pass) Ell.et halst, Phomopsis asparagi (Sacc.) Bubak
and Fusarium oxysporum (schl.) F.sp. niveum (F. oxysporum) respectively at 1.0 mg/mL. The results indi-
cate that all the inulin derivatives have better antifungal activity than inulin, and the inhibitory index is
affected by the chlorine atom grafted to the inulin derivatives.
Keywords:
Inulin derivatives
Antifungal activity
Chlorine
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and antifungal activity of inulin derivatives (Ren, Liu, Dong, & Guo,
2011; Ren et al., 2012), which proved the chemical modification
As a kind of abundant natural polysaccharide, inulin is mainly
extracted from such low-requirement crops as Helianthus tubero-
sus, chicory, and yacon. Inulin consists primarily of ˇ-fructosyl
fructose units – always presented in furanose form – usually with
a glucopyranose unit reducing end (GFn) (Rogge & Stevens, 2004).
Inulin has unique range of molecular weight with the degree of
polymerization (DP) varies from 2 to 70, which is rare as a kind of
polysaccharide. This polysaccharide has exhibited some interest-
ing properties such as beneficial nutritional attributes for human
health, moderate average degree of polymerization and readi-
ness of being obtained (Beylot, 2006; Causey, Feirtag, Gallaher,
Tungland, & Slavin, 2000). As a kind of source of renewable,
biodegradable and environmentally benign polysaccharide, inulin
is a promising candidate to meet the continuously increasing
demand for biodegradable and environmentally benign polymeric
material. This structural singularity raises its interesting proper-
ties like beneficial nutritional attributes, which in turn excites
the chemical modification of inulin in recent years (Beylot, 2006;
Habibi & Dufresne, 2008). We have reported the antioxidant activity
could improve the bioactivity of inulin.
Many reports have demonstrated that aromatic nucleus Schiff
bases had good bioactivities (Karthikeyan et al., 2006). It is also
reported that salicylaldehyde derivatives, with one or more halo-
atoms in the aromatic ring, showed a variety of biological activities
(Li, Guo, & Jiang, 2010). Meanwhile, as antifungal group, chlorinated
benzenes are used in many fungicides such as pentachloronitroben-
zene and chlorothalonil which have five or four chlorine atoms
respectively (Sha, 1994). Chemical fungicides with chlorine atoms
have good fungicidal activity and used widely years ago, but their
toxicities and residues give rise to serious problems for environ-
ment and human health. When these groups are grafted onto inulin,
they should be released slowly and may meet the requirements of
environmental safety.
In this paper, we reported the synthesis and antifungal proper-
ties of a group of inulin derivatives with benzene or chlorinated
benzene as substituent including PAIL, 2CPAIL, 4CPAIL, and
2,4DCPAIL. The chloracetyl inulin (CAIL) was first synthesized by
reaction between the C-6 hydroxyl of inulin and chloracetyl chlo-
ride. CAIL was an excellent intermediate of the project as the
chlorine of CAIL can easily attack pyridine to give N-alkypyridinium
salts (Li et al., 2013; Sajomsang, 2010). Subsequently the benzene
or chlorinated benzene groups were introduced into inulin through
the reaction between Schiff bases of 4-amino-pyridine and CAIL.
∗
Corresponding author. Tel.: +86 535 2109171; fax: +86 535 2109000.
E-mail addresses: zhanyongguo@gmail.com, zhanyongguo@hotmail.com
(Z. Guo).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.08.044

470
Z. Guo et al. / Carbohydrate Polymers 99 (2014) 469–473
Cl-
+
ClCH2COO
N
CH2COO
O
O
OH
HO
O
OH
ClCH2COCl
rt
OH
O
Py
O
O
DMF
CH2
CH2
CH2
n
n
OH
n
OH
OH
CAIL
PAIL
Cl-
+
N CH2COO
RCH=N-
O
OH
EtOH
O
CAIL
DMF
RCHO
NH2
N
N=CHR
N
reflux
CH2
n
OH
Cl
Cl
2,4CPAIL:R=
4CPAIL:R=
Cl
2CPAIL:R=
Cl
;
;
Scheme 1. Synthetic routes for inulin derivatives.
The aimed inulin derivatives designed in this way were expected
to have advantageous features such as high antifungal activity and
good water solubility. Apart from these advantages, the reaction
generates Schiff bases functional groups, which is also helpful for
the constructions of bioactive inulin derivatives (Guo, Ren, Dong,
Wang, & Li, 2013). The chemical structures of the derivatives were
characterized by FT-IR and 1H NMR.
Plant pathogenic fungi negatively affect a large number of
important vegetables and fruits. The losses caused by plant
pathogenic fungi have attracted wide attention throughout the
world. For example, Phomopsis asparagi can cause stem blight of
asparagus and subsequent yield loss; the premature plant death
caused by Colletotrichum Lagenarium can lead to a yield loss of
6–48% (Chen & Dai, 2012; Elena, 2006). Therefore, three common
plants-threatening fungi, C. Lagenarium, P. asparagi, and Fusarium
oxysporum, were selected to evaluate the antifungal properties of
the derivatives by hypha measurement in vitro. Control of these
plants-threatening fungi could benefit the production of related
vegetables and fruits.
2.3. The synthesis of chloracetyl inulin
Chloracetyl inulin (CAIL) was synthesized as follows (Li, Guo, &
Jiang, 2010). 1.61 g inulin was dissolved in 100 mL H2O at room tem-
perature (r.t.), and 0.02 mol chloracetyl chloride was added drop
wise. After stirring for 12 h at r.t., the solution was concentrated
under reduced pressure. The concentrated solution was precipi-
tated by the addition of excess acetone and the precipitate was
filtered. The products were washed with ether dried at 60 ◦C for
6 h.
2.4. The synthesis of inulin derivatives
A solution of 0.3 g CAIL and 0.5 mL pyridine or the Schiff bases
of 4-amino-pyridine in 20 mL N, N-dimethylformamide (DMF) was
stirred for 24 h at 60 ◦C. The solution was precipitated with excess
acetone and the precipitate was filtered. The unreacted pyridine,
amine, and other outgrowth were extracted in a Soxhlet apparatus
with ethanol for two days. The inulin derivatives were obtained
after drying at 60 ◦C for 6 h (Li, Guo & Jiang, 2010).
2. Experimental
2.1. Materials and analytical methods
2.5. Antifungal assays
Inulin was purchased from E. Merck (Darmstadt, Germany).
Antifungal assays were performed by following the plate growth
rate method (Jasso de Rodríguez, Hernández-Castillo, Rodríguez-
García, & Angulo-Sánchez, 2005; Li, Guo & Jiang, 2010). Briefly, the
compounds were dissolved in distilled water at a concentration
of 5.0 mg/mL. Then, each sample (inulin, CAIL and inulin deriva-
tives) solution was added to sterilized potato dextrose agar to give
a final concentration of 0.1, 0.5, and 1.0 mg/mL. After the mixture
was cooled in the plate (6.0 cm diameter), the mycelium of fungi of
a 5.0 mm diameter was transferred to the test plate and incubated
at 27 ◦C. When the mycelium of fungi reached the edges of the con-
trol plate (without the presence of samples), the antifungal index
was calculated as follows:
2-Chlorobenzaldehyde,
4-chlorobenzaldehyde,
and
2,4-
dichlorobenzaldehyde were purchased from the Sigma–Aldrich
Chemical Corp. The other reagents are all of analytical grade and
used without further purification. FT-IR spectra were recorded on
a Jasco-4100 Fourier Transform Infrared Spectrometer (Japan, pro-
vided by JASCO Co., Ltd Shanghai, China) with KBr disks. 1H nuclear
magnetic resonance (1H NMR) spectra were measured with a
Bruker AVIII-500 Spectrometer (Switzerland, provided by Bruker
Tech. and Serv. Co., Ltd. Beijing, China.). Three plants-threatening
fungi C. lagenarium (Pass) Ell.et halst, P. asparagi (Sacc.) Bubak and
F. oxysporum (schl.) F.sp. niveum (F. oxysporum) were provided by
Qingdao Academy of Agricultural Sciences.
Da
Antifungal index (%) = 1 −
× 100
Db
2.2. The synthesis of the Schiff bases of 4-amino-pyridine
As shown in Scheme 1,
a solution of 0.01 mol alde-
where Da is the diameter of the growth zone in the test plates and
Db is the diameter of the growth zone in the control plate. Each
experiment was performed three times, and the data were shown
with mean SD. The Scheffe method was used to evaluate the dif-
ferences in antifungal index in the antifungal tests. Results with
P < 0.05 were considered statistically significant (Jasso de Rodríguez
et al., 2005).
hydes (2-chlorobenzaldehyde, 4-chlorobenzaldehyde or 2,4-
dichlorobenzaldehyde) and 0.01 mol 4-amino-pyridine were
refluxedin 30 mL ethanol for 4 h. Then the solvent was concentrated
under reduced pressure. The crude products were purified by crys-
tallization from ethanol. (Kaboudin, Afsharinezhad, & Mohammad,
2010).

Z. Guo et al. / Carbohydrate Polymers 99 (2014) 469–473
471
3. Results and discussion
3.1. Structure of the inulin derivatives
Each step of the synthesis was followed by FT-IR and 1H NMR
spectroscopy. The FT-IR spectra of inulin, CAIL, PAIL, 2CPAIL,
4CPAIL, and 2,4DCPAIL are shown in Fig. 1. The FT-IR spectrum
of inulin shows peaks of saccharide at 848 cm−1 and 1029 cm−1
(Ren et al., 2012). As far as the FT-IR spectra of CAIL and inulin
are concerned, the characteristic peak of carbonyl (C O) vibration
deformation appears at 1747 cm−1. Another new peak at 771 cm−1
is assigned to the C Cl group which indicates that CAIL is synthe-
sized successful. After pyridine grafted onto CAIL, the absorbance
of C Cl at 771 cm−1 is weakened and new peaks appear at 1488
and 1538 cm−1 which are assigned to the absorbance of pyridine in
the spectrum of PAIL (Panicker et al., 2006; Li, Guo & Jiang, 2010).
In the spectra of 2CPAIL, 4CPAIL and 2,4DCPAIL, new peaks appear
at about 1535, 1465, 830 cm−1, which are attributed to the char-
acteristic absorption of benzene and pyridine. Another prominent
peak at about 1654 cm−1 was assigned to the C N of Schiff bases
group.
Fig. 2 presented the 1H NMR spectra of inulin, CAIL, PAIL,
2CPAIL, 4CPAIL and 2,4DCPAIL. It is known that all the signals
at 3.0–5.4 ppm are assigned to the protons of inulin (Ren et al.,
2012). After reacted with chloracetyl chloride, new signals appear
at 1.1–1.3 ppm in CAIL, which are related to the proton of CH2Cl.
New signals appear at 7.9–8.9 ppm (proton of pyridine) in PAIL,
and corresponding signals including proton of benzene and pyri-
dine move to high field at about 6.7–8.0 ppm in 2CPAIL, 4CPAIL,
and 2,4DCPAIL, which should be assigned to the conjugate struc-
ture. Above results further confirmed the success of the preparation
of inulin derivatives.
Fig. 1. FT-IR spectra of inulin, CAIL, PAIL, 2CPAIL, 4CPAIL, and 2,4DCPAIL.
Fig. 2. 1H NMR spectra of inulin, CAIL, PAIL, 2CPAIL, 4CPAIL, and 2,4DCPAIL.

472
Z. Guo et al. / Carbohydrate Polymers 99 (2014) 469–473
the antifungal activity of inulin derivatives. Meanwhile, as shown
in three figures, the antifungal indices had the same order as fol-
low: 2,4DCPAIL > 4CPAIL > 2CPAIL > CAIL > PAIL > inulin. It is easily
to find that the order of antifungal indices is in accord with the num-
ber of chlorine atom in those compounds. 2,4DCPAIL, which has
two chlorine atoms, had the best inhibitory activity among all of the
measured samples. And the inhibitory indices of 2,4DCPAIL against
C. lagenarium, P. asparagi, and F. oxysporum are 67%, 47%, and 43%,
respectively at 1.0 mg/mL. The results further confirm that chlorine
grafted in the synthesized inulin derivatives contribute a lot to the
antifungal action and consequently increase the antifungal activity
of them.
4. Conclusions
Fig. 3. The antifungal activity of inulin, CAIL, PAIL, 2CPAIL, 4CPAIL, and 2,4DCPAIL
against C. lagenarium.
The development of fungicides has attracted people’s attention
along with the food safety. And environmental problems become
serious caused by the chemical pesticides. More and more effective
fungicides have been prohibited in recent years, and the require-
ment for “green pesticide” is urgent. In this paper, four inulin
derivatives containing benzene or chlorinated benzene with the
Schiff bases of 4-amino-pyridine were designed and synthesized,
and their antifungal activity against three kinds of phytopathogens
was estimated by hypha measurement in vitro. All the inulin deriva-
tives have good solubility in water, and exhibit higher inhibition
indices than inulin. These data demonstrate that the chemical
modification of inulin with chlorinated benzene would lead to
an enhancement of the biological activity against some plant
pathogenic fungi. In addition, the antifungal activity of these inulin
derivatives is influenced by the number of chlorine grafted on to
the periphery of molecular chains. Further study will be carried out
to ascertain this hypothesis.
Fig. 4. The antifungal activity of inulin, CAIL, PAIL, 2CPAIL, 4CPAIL, and 2,4DCPAIL
against P. asparagi.
50
100 µg/mL
500 µg/mL
Acknowledgments
40
30
20
10
0
1000 µg/mL
We thank the project of the 12th Five-Year Science and Tech-
nology Plan for Agriculture (2012BAD32B09), the Science and
Technology Project of Yantai (20121321) and the Taishan Scholar
Program of Shandong Province for financial support of this work.
References
Beylot, M. (2006). Effects of inulin-type fructans on lipid metabolism in man and in
animal models. British Journal of Nutrition, 93(Suppl. 1), S163–S168.
Causey, J. L., Feirtag, J. M., Gallaher, D. D., Tungland, B. C., & Slavin, J. L. (2000).
Effects of dietary inulin on serum lipids, blood glucose and the gastrointestinal,
environment in hypercholesterolekmic men. Nutrition Research, 20(2), 191–201.
Chen, Y. J., & Dai, G. H. (2012). Antifungal activity of plant extracts against Col-
letotrichum lagenarium, the causal agent of anthracnose in cucumber. Journal
of the Science of Food and Agriculture, 92(9), 1937–1943.
Inulin
CAIL
PAIL
2CPAIL
4CPAIL
2,4DCPAIL
Fig. 5. The antifungal activity of inulin, CAIL, PAIL, 2CPAIL, 4CPAIL, and 2,4DCPAIL
against F. oxysporum.
3.2. Antifungal activity
Elena, K. (2006). First report of Phomopsis asparagi causing stem blight of asparagus
in Greece. Plant Pathology, 55, 300.
The antifungal activities of inulin, CAIL, PAIL, 2CPAIL, 4CPAIL,
and 2,4DCPAIL. against C. lagenarium, P. asparagi, and F. oxysporum
were tested and the results are shown in Figs. 3–5. As shown in
those figures, inulin did not inhibit the growth of all the measured
fungi under the tested concentration (1.0–0.1 mg/mL), which is in
accord with the earlier report (Ren et al., 2012). All the inulin deriva-
tives show inhibitory activity against three fungi and the antifungal
indices of them enhanced with increasing concentration. After the
reaction of inulin and chloracetyl chloride, the antifungal ability of
CAIL increases and the antifungal indices at 1.0 mg/mL are 22%, 17%,
and 12% against C. lagenarium, P. asparagi, and F. oxysporum, respec-
tively. This is possible due to the introducing of chloracetyl group in
CAIL. As shown in Figs. 3–5, PAIL has lower antifungal ability than
CAIL on all the measured fungi. It would be reasonable to presume
that chlorine atom should be the important factor which influences
Guo, Z. Y., Ren, J. M., Dong, F., Wang, G., & Li, P. C. (2013). Comparative study of the
influence of active groups of chitosan derivatives on antifungal activity. Journal
of Applied Polymer Science, 127(4), 2553–2556.
Habibi, Y., & Dufresne, A. (2008). Highly filled bionanocomposites from functional-
ized polysaccharide nanocrystals. Biomacromolecules, 9(7), 1974–1980.
Jasso de Rodríguez, D., Hernández-Castillo, D., Rodríguez-García, R., & Angulo-
Sánchez, J. L. (2005). Antifungal activity in vitro of Aloe vera pulp and liquid
fraction against plant pathogenic fungi. Industrial Crops and Products, 21(1),
81–87.
Kaboudin, B., Afsharinezhad, & Mohammad, B. (2010). Synthesis of a novel class
of ␤-lactam derivatives of 1-aminophosphonates by staudinger ketene-imine
[2+2]-cycloaddition reaction. Synthesis, 20, 3504–3508.
Karthikeyan, M. S., Prasad, D. J., Poojary, B., Bhat, K. S., Holla, B. S., & Kumari, N. S.
(2006). Synthesis and biological activity of Schiff and Mannich bases bearing
2,4-dichloro-5-fluorophenyl moiety. Bioorganic & Medicinal Chemistry, 14(22),
7482–7489.
Li, Q., Ren, J. M., Dong, F., Feng, Y., Gu, G. D., & Guo, Z. Y. (2013). Synthesis and anti-
fungal activity of thiadiazole-functionalized chitosan derivatives. Carbohydrate
Research, 373(0), 103–107.

Z. Guo et al. / Carbohydrate Polymers 99 (2014) 469–473
473
Li, R. C., Guo, Z. Y., & Jiang, P. A. (2010). Synthesis, characterization, and antifun-
gal activity of novel quaternary chitosan derivatives. Carbohydrate Research,
345(13), 1896–1900.
Panicker, C. Y., Varghese, H. T., Philip, D., & Nogueira, H. I. S. (2006). FT-IR, FT-
Raman and SERS spectra of pyridine-3-sulfonic acid. Spectrochimica Acta, Part
A: Molecular and Biomolecular Spectroscopy, 64(3), 744–747.
for facile chemical modifications of inulin. Carbohydrate Polymers, 87(2),
1744–1748.
Rogge, T. M., & Stevens, C. V. (2004). Facilitated synthesis of inulin esters by trans-
esterification. Biomacromolecules, 5(5), 1799–1803.
Sha, J. J. (1994). Handbook of chemical product (3rd ed., pp. 190–192). Beijing: Chem-
ical Industry.
Ren, J. M., Liu, J. L., Dong, F., & Guo, Z. Y. (2011). Synthesis and hydroxyl radicals scav-
enging activity of N-(aminoethyl)inulin. Carbohydrate Polymers, 85(1), 268–271.
Ren, J. M., Wang, P. B., Dong, F., Feng, Y., Peng, D. J., & Guo, Z. Y. (2012). Synthe-
sis and antifungal properties of 6-amino-6-deoxyinulin, a kind of precursors
Sajomsang, W. (2010). Synthetic methods and applications of chitosan containing
pyridylmethyl moiety and its quaternized derivatives. Carbohydrate Polymers,
80(3), 631–647.