Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro

Article abstract of DOI:10.1016/j.carres.2007.11.024

Three different acyl thiourea derivatives of chitosan (CS) were synthesized and their structures were characterized by FT-IR spectroscopy and elemental analysis. The antimicrobial behaviors of CS and its derivatives against four species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina) and four crop-threatening pathogenic fungi (Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides (Penz.) Saec, and Phyllisticta zingiberi) were investigated. The results indicated that the antimicrobial activities of the acyl thiourea derivatives are much better than that of the parent CS. The minimum value of MIC and MBC of the derivatives against E. coli was 15.62 and 62.49 μg/mL, respectively. All of the acyl thiourea derivatives had a significant inhibitory effect on the fungi in concentrations of 50-500 μg/mL; the maximum inhibitory index was 66.67%. The antifungal activities of the chloracetyl thiourea derivatives of CS are noticeably higher than the acetyl and benzoyl thiourea derivatives. The degree of grafting of the acyl thiourea group in the derivatives was related to antifungal activity; higher substitution resulted in stronger antifungal activity.

Full text of DOI:10.1016/j.carres.2007.11.024

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 566–570
Note
Synthesis of acyl thiourea derivatives of chitosan and
their antimicrobial activities in vitro
a,b
a
a,b
a,b
a,b
Zhimei Zhong, Ronge Xing, Song Liu, Lin Wang, Shengbao Cai
a,b,
*
and Pengcheng Li
a
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
b
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Received 26 September 2007; received in revised form 15 November 2007; accepted 25 November 2007
Available online 4 December 2007
Abstract—Three different acyl thiourea derivatives of chitosan (CS) were synthesized and their structures were characterized by
FT-IR spectroscopy and elemental analysis. The antimicrobial behaviors of CS and its derivatives against four species of bacteria
(
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina) and four crop-threatening pathogenic fungi (Alter-
naria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides (Penz.) Saec, and Phyllisticta zingiberi) were
investigated. The results indicated that the antimicrobial activities of the acyl thiourea derivatives are much better than that of
the parent CS. The minimum value of MIC and MBC of the derivatives against E. coli was 15.62 and 62.49 lg/mL, respectively.
All of the acyl thiourea derivatives had a significant inhibitory effect on the fungi in concentrations of 50–500 lg/mL; the maximum
inhibitory index was 66.67%. The antifungal activities of the chloracetyl thiourea derivatives of CS are noticeably higher than the
acetyl and benzoyl thiourea derivatives. The degree of grafting of the acyl thiourea group in the derivatives was related to antifungal
activity; higher substitution resulted in stronger antifungal activity.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Antibacterial activity; Antifungal activity; Chitosan; Acyl thiourea derivatives
Chitosan (CS) is the N-deacetylated product of chitin
and an attractive material because of its properties such
as immunological activity, wound healing, biocompati-
Thioureas have strong antifungal activities that are
comparable to the activity observed for the common
2
0,21
antifungal antibiotic ketoconazole.
Moreover, they
1
–6
22,23
bility, low toxicity, and biodegradability. The antimi-
crobial properties of chitosan and its derivatives have
have antibacterial and insecticidal properties.
Eweis
2
4
et al. have prepared a benzoyl thiourea derivative
of chitosan and studied its antifungal efficacy against
sugar-beet pathogens, and showed that the antifungal
activity of the derivative was much better than that of
native chitosan.
7
–9
been widely explored.
Studies have shown that the
antimicrobial activity of chitosan depends on its mole-
cular weight, deacetylation degree, pH of chitosan solu-
1
0–13
tion, and the target organism.
To improve the
antimicrobial activity of chitosan, other researchers
have prepared derivatives such as N,N-dimethyl-chito-
san, N,N,N-trimethyl-chitosan iodide, and other quater-
nary ammonium salts of chitosan, as well as Schiff-base
Although many chitosan derivatives showed higher
antimicrobial activities than native chitosan, the antimi-
crobial activities of these materials are still lower than
that of the antimicrobial agents currently used. As part
of our search for new types of chitosan derivatives with
much higher antimicrobial activity, we describe here the
preparation of acetyl, chloracetyl, and benzoyl thiourea
derivatives of chitosan (ATUCS, CATUCS, and
BZTUCS, Fig. 1). The antimicrobial activities of these
materials against four bacterial species (Escherichia coli,
1
4–19
and sulfanilamide derivatives of chitosan.
*
Corresponding author. Tel.: +86 532 82898707; fax: +86 532
2968951; e-mail addresses: zhimeihappy@126.com; pcli@ms.qdio.
8
ac.cn
0
008-6215/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.11.024

Z. Zhong et al. / Carbohydrate Research 343 (2008) 566–570
567
R-COCl + NH SCN
R-CO-NCS
O
the parent CS. As for as the FT-IR spectra of ATUCS
and CS are concerned, first, an obvious change between
3500 and 3200 cm due to the O–H and N–H group
4
OH
O
OH
O
ꢀ1
O
+
R-CO-NCS
stretching vibration is observed. In addition, the charac-
NH2
OH
OH
NH
ꢀ1
teristic absorbance of –NH at 1600 cm disappeared;
2
n
n
C=S
NH
C=O
R
these results show that –NH group had reacted with
2
ꢀ1
acetyl thiocyanate. Second, new peaks at ꢁ1736 cm
ATUCS:R= -CH3;
ꢀ1
and 1657 cm appeared in the spectra of ATUCS, which
CATUCS:R= -CH Cl;
2
is the characteristic absorbance of the C@O group in a
secondary acylamide. Third, there are new strong peaks
BZTUCS:R=
.
ꢀ1
ꢀ1
at 1519.97 cm and 1076.13 cm in the ATUCS spec-
tra, which were assigned to the characteristic absorbance
of –NH and C@S. All of the above results support the
structure of ATUCS. In the spectrum of CATUCS, new
Figure 1. Synthesis of acyl thiourea derivatives of chitosan.
Pseudomonas aeruginosa, Staphylococcus aureus, and
Sarcina) and four crop-threatening pathogenic fungi
Alternaria solani, Fusarium oxysporum f. sp. vasinfec-
ꢀ
1
ꢀ1
peaks appeared at 1733 cm
(C@O), 1657 cm
ꢀ
1
ꢀ1
(
(C@O), 1517 cm (N–H), and 1077 cm (C@S), which
tum, Colletotrichum gloeosporioides (Penz.) Saec, and
Phyllisticta zingiberi) are also described.
Figure 2 presents a comparison of the transmission FT-
IR spectra for ATUCS, CATUCS, and BZTUCS with
showed that CATUCS was obtained. For the same rea-
ꢀ
1
ꢀ1
son, new peaks at 1663 cm (C@O), 1517 cm (N–H),
ꢀ
1
ꢀ1
ꢀ1
1065 cm (C@S), 1544 cm (phenyl), and 795 cm
(phenyl) indicated that BZTUCS was formed.
Figure 2. FT-IR spectra of chitosan and its acyl thiourea derivatives.

568
Z. Zhong et al. / Carbohydrate Research 343 (2008) 566–570
Table 1. Yield, elemental analysis, and grafting degree of chitosan
derivatives
rupted the barrier properties of the outer membrane of
Gram-negative bacteria, as chitosan is protonated at
acidic conditions and the carboxyl and phosphate
groups of the bacterial surface are anionic and offer
potential sites for electrostatic binding. The C@O, C@S,
and NH groups in the acyl thiourea derivatives of chito-
san can be protonated under acidic conditions, so they
can react with the carboxyl and phosphate groups of
the bacterial surface and therefore show antibacterial
activity against Gram-negative bacteria. On the other
hand, S. aureus and Sarcina, typical Gram-positive bac-
teria, have cell walls composed solely of peptidoglycan,
which does not allow the formation of a surface layer.
A. solani is the causal agent of early blight disease of
tomato. Epidemics induced by this economically impor-
tant pathogen can cause severe tomato seeding defolia-
tion in areas with high humidity and frequent night
dew. F. oxysporum f. sp. vasinfectum can result in cotton
fusarium wilt, which is highly destructive and economi-
cally limiting to the production of good quality cotton.
C. gloeosporioides (Penz.) Saec can cause anthracnose,
which is a major post-harvest disease that affects many
fruits and crops. P. zingiberi is the causal fungi of black
spot disease in crops. It mainly appears on the leaves of
crops leading to the formation of holes, which in serious
cases can cause death.
Compound Yield (%)
Elemental analysis (%)
Grafting
degree (%)
C
N
H
S
CS
—
75
81
71
44.28
8.52 7.36
—
—
ATUCS
CATUCS
BZTUCS
43.40 10.01 6.22 10.76 89.2
38.92
50.93
9.00 5.26
8.62 5.46
9.83 91.5
8.82 81.0
The results of elemental analysis, yield, and the graft-
ing degree of the derivatives are listed in Table 1. From
Table 1, the yield of ATUCS, CATUCS, and BZTUCS
was 75%, 81%, and 71%, respectively. The elemental
analysis results indicate that the substitution degree of
ATUCS, CATUCS, and BZTUCS is ꢁ89.2%, 91.5%,
and 81.0%. The procedure used in this work for the prep-
aration of BZTUCS is different from that of Eweis
2
4
et al. The toxicity of the solvent used in our experiment
dimethylformamide and acetic acid) was lower than that
(
of their works (acetonitrile). The sulfur content (8.82%)
is slightly higher than that of the BZTUCS (8.25%) pre-
pared by Eweis et al., and so we speculate that the substi-
tution degree of acyl thiourea group is also higher.
The antibacterial activity of ATUCS, CATUCS, and
BZTUCS was compared with that of CS. Table 2 shows
the MIC and MBC of the derivatives against various
microorganisms. The results indicate that all of the
derivatives showed strong antibacterial activity against
the tested bacteria, and the minimum value of MIC
and MBC of the derivatives against E. coli was 15.62
and 62.49 lg/mL, respectively. Moreover, the antibacte-
rial activities of ATUCS, CATUCS, and BZTUCS are
significantly higher than that of CS. In addition, the
antibacterial activity of the derivatives against E. coli
and P. aeruginosa was more effective than against S. aur-
eus and Sarcina, so we can conclude that the derivatives
showed stronger antibacterial activity against Gram-
negative than Gram-positive bacteria.
The antifungal activities of these CS derivatives
against A. solani, F. oxysporum f. sp. vasinfectum, C.
gloeosporioides (Penz.) Saec, and P. zingiberi are shown
in Table 3. These data indicate that the derivatives had
effective activities against these fungi, with inhibitory
indices ranging from 31.23% to 66.67% at 500 lg/mL.
The inhibitory index of all of the compounds enhanced
with an increase in concentration, and the highest anti-
fungal activity was observed at 500 lg/mL. Moreover,
the antifungal activities of CATUCS are noticeably
higher than those of ATUCS and BZTUCS, which
may be due to the presence of the chlorine atom in CA-
TUCS. The chloro-group is used in many fungicides
This observation may be attributed to their different
cell walls. E. coli and P. aeruginosa are typical Gram-
negative bacteria, the cell wall of which is made up of
a thin membrane of peptidoglycan and an outer mem-
brane constituted of lipopolysaccharide, lipoprotein,
2
6
such as pentachloronitrobenzene and chlorothalonil.
However, these fungicides have pronounced toxicities
and their residues in the environment have been serious
problems. When these groups are grafted onto CS, they
might be released slowly and may induce lower pollution
2
5
and phospholipids. Chitosan and its derivatives with
large molecular weight can be expected to coat the cell
surface and prevent the leakage of intracellular compo-
2
7
to the environment. As a final trend, a higher grafting
degree of the acyl thiourea group in these derivatives
resulted in stronger antifungal activity.
1
2
nents. Helander et al. have shown that chitosan dis-
Table 2. MIC and MBC values of chitosan derivatives against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina
Samples
MIC/MBC (lg/mL)
S. aureus
E. coli
P. aeruginosa
Sarcina
CS
62.49/124.99
15.62/62.49
15.62/62.49
15.62/62.49
62.49/249.98
62.49/124.99
15.62/124.99
62.49/124.99
249.98/499.95
62.49/124.99
62.49/124.99
62.49/124.99
124.33/249.98
62.49/124.99
62.49/124.99
62.49/124.99
ATUCS
CATUCS
BZTUCS

Z. Zhong et al. / Carbohydrate Research 343 (2008) 566–570
569
Table 3. Inhibitory indices of the chitosan derivatives against Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides
(
Penz.) Saec, and Phyllisticta zingiberi
Samples
CS
Concentration (lg/mL)
A. solani
F. oxysporum f. sp. vasinfectum
C. gloeosporioides (Penz.) Saec
P. zingiberi
50
0
0
0
5
10
0
0
1
5
00
00
3.57
20.86
19.51
39.02
18.75
ATUCS
50
00
00
14.28
21.43
42.86
31.70
40.78
58.54
20
30
40
16.67
25.33
46.67
1
5
CATUCS
BZTUCS
50
00
00
21.42
27.86
53.57
32.39
46.82
60.98
21.78
48.33
66.67
28.57
33.58
57.89
1
5
50
00
00
13
20
32.14
14.63
19.51
48.78
8.33
21.11
31.23
11.05
24.58
36.84
1
5
1
. Experimental
phase transfer catalyst. After stirring for 2 h at room
temperature, the mixture of products was filtered
through a Buchner funnel under reduced pressure. The
filtrate (acyl thiocyanate) was added to a chitosan solu-
tion in dimethylformamide and acetic acid. The reaction
medium was stirred at 100 °C for 5 h, cooled, and fil-
tered. The yellowish white product was washed with
methanol and dried at 50 °C.
1
.1. Materials and methods
Chitosan was supplied by Qingdao Baicheng Biochemi-
cal Corp. (China). Its deacetylation is 96% and its aver-
age molecular weight is 50 kDa. All other chemicals and
reagents used were of analytical grade, and were used
without further purification. The crop-threatening patho-
genic fungi (A. solani, F. oxysporum f. sp. vasinfectum,
C. gloeosporioides (Penz.) Saec, and P. zingiberi) and
bacteria (E. coli, P. aeruginosa, S. aureus, and Sarcina)
used for the antimicrobial assay were provided by Key
Laboratory of Experimental Marine Biology (Institute
of Oceanology, Chinese Academy of Sciences) and
Qingdao Academy of Agricultural Sciences.
1.4. Antifungal assays
Antifungal assays were performed based on the method
28
of Jasso et al. Briefly, the derivatives were dissolved in
1% (v/v) HCl. Then, a solution of each derivative
(ATUCS, CATUCS, and BZTUCS) was added to the
sterilized potato dextrose agar to give a final concentra-
tion of 50, 100, and 500 lg/mL. After the mixture was
cooled, the mycelium of fungi was transferred to the test
plate and incubated at 29 °C for 3 days. When the mycel-
ium of fungi reached the edges of the control plate
(without the added samples), the inhibitory index was
calculated as follows:
1
.2. Analytical methods
Fourier transform infrared (FT-IR) spectra of the deriv-
atives were measured in the 4000–400 cm
ꢀ
1
regions
using a Nicolet Magna-Avatar 360 FT-IR spectrometer
with samples prepared as KBr disks. Elemental analysis
C, H, N) was performed on a Carlo-Erba 1106 elemen-
tal analyzer and sulfur content was measured on a SC-
32 sulfur meter (LECO). The average viscometric
Inhibitory index ð%Þ ¼ ð1 ꢀ D =D Þ ꢂ 100;
a
b
(
where D is the diameter of the growth zone in the test
a
1
plate and D is the diameter of growth zone in the con-
b
molecular weight of chitosan was estimated from the
intrinsic viscosity determined in the solvent 0.1 M
trol plate. Each experiment was performed three times,
and the data were averaged. The Scheffe method was
used to evaluate the differences in antifungal index in
antifungal tests. Results with P < 0.05 were considered
CH COOH–0.2 M NaCl using the Mark–Houwink
3
parameter a = 0.96, K = 1.424 at 25 °C when the intrin-
g
2
9
sic viscosity is expressed in mL/g.
statistically significant.
1
.3. Preparation of ATUCS, CATUCS, BZTUCS
1.5. Antibacterial assays
Dry ammonium thiocyanate (NH SCN, 0.67 mol) was
The minimum inhibition concentration (MIC) of CS
and its acyl thiourea derivatives was determined by a
turbidimetric method. In this method, a number of test
tubes each containing 5.0 mL Muller–Hinton broth
(MHB, Difco, England) were autoclaved for 20 min at
4
dissolved in CH Cl (30 mL), then a CH Cl solution
2
2
2
2
3
0
containing 0.04 mol acetyl chloride, chloracetyl chlo-
ride, or benzoyl chloride was added. Polyethylene
glycol-400 (PEG-400, 0.8 mL) was added dropwise as

5
70
Z. Zhong et al. / Carbohydrate Research 343 (2008) 566–570
6. No, H. K.; Park, N. Y.; Lee, S. H.; Meyers, S. P. Int. J.
1
21 °C. The samples were accurately quantified and
Food Microbiol. 2002, 74, 65–72.
added to 1% HCl. To the first tube, 5.0 mL of chitosan
or ATUCS (CATUCS or BZTUCS) (1 mg/mL) was
added. After mixing, 5.0 mL of the mixture was trans-
ferred to the second tube, and similar transformations
were repeated. Hence, each tube contained a test sample
solution with half of the concentration of the previous
one. The tubes were inoculated under aseptic conditions
with 50 lL of the freshly prepared bacterial suspension.
The blank control tubes were incubated at 37 °C for
7
8
. Lim, S. H.; Hudson, S. M. Carbohydr. Polym. 2004, 56,
2
27–234.
. Rabea, E. I.; Badawy, M. E. T.; Sterens, C. V.; Smagge,
G.; Steurbaut, W. Biomacromolecules 2003, 4, 1457–1465.
9. Shahidi, F.; Arachchi, J. K. V.; Jeon, Y.-J. Trends Food
Sci. Technol. 1999, 10, 37–51.
1
1
0. Jeon, Y. J.; Park, P. J.; Kim, S. K. Carbohydr. Polym.
2
001, 44, 71–76.
1. Liu, N.; Chen, X. G.; Park, H. J.; Liu, C. G.; Liu, C. S.;
Meng, X. H.; Yu, L. J. Carbohydr. Polym. 2006, 64, 60–65.
12. Helander, I. M.; Nurmiaho-Lassila, E. L.; Ahvenainen,
2
4 h. The tubes were then studied for the visible signs
R.; Rhoades, J.; Roller, S. Int. J. Food Microbiol. 2001, 71,
of growth or turbidity. The lowest concentration of
chitosan or its acyl thiourea derivatives that inhibited
the growth of bacteria was considered as the minimum
inhibitory concentration or MIC.
The minimum bactericidal concentration (MBC), or
the lowest concentration of the samples that kills
2
35–244.
1
3. Liu, X. F.; Guan, Y. L.; Yang, D. Z.; Li, Z.; Yao, K. D. J.
Appl. Polym. Sci. 2001, 79, 1324–1335.
14. Muzzarelli, R. A. A.; Tanfani, F. Carbohydr. Polym. 1985,
5, 297–307.
1
5. Domand, A.; Rinaudo, M.; Terrassin, C. Int. J. Biol.
Macromol. 1986, 8, 105–107.
9
9.9% of the bacteria, was determined by assaying the
1
6. Lee, M. K.; Chun, S. K.; Choi, W. J.; Kim, J. K.; Choi, S.
H.; Kim, A. Biomaterials 2005, 26, 2147–2156.
live organisms from those tubes that, in the MIC test,
3
1
showed no growth. A loopful from each of those tubes
was inoculated on EMB (Eosin–Methylene Blue) agar,
and signs of growth were monitored. The growth of bac-
teria demonstrates the presence of these germs in the ori-
ginal tube. In contrast, if no growth was observed, the
original tube contained no live bacteria and the material
was considered bactericidal at that concentration.
17. Jia, Z. S.; Shen, D. F.; Xu, W. L. Carbohydr. Res. 2001,
333, 1–6.
1
8. Guo, Z. Y.; Xing, R.; Liu, S.; Zhong, Z. M.; Ji, X.; Wang,
L.; Li, P. C. Carbohydr. Res. 2007, 342, 1329–1332.
9. Zhong, Z. M.; Chen, R.; Xing, R.; Chen, X. L.; Liu, S.;
Guo, Z. Y.; Ji, X.; Wang, L.; Li, P. C. Carbohydr. Res.
2007, 342, 2390–2395.
1
20. Phuong, T.; Khac-Minh, T.; Van Hal, N. T.; Phuong, H.
T. Bioorg. Med. Chem. Lett. 2004, 14, 653–656.
2
1. Huehr, O. F.; Marsh, J. L.; Mizzoni, R. H.; Mull, R. P.;
Schroeder, D. C.; Troxell, H. A.; Scholz, C. R. J. Am.
Chem. Soc. 1953, 75, 2274–2275.
Acknowledgment
2
2
2. Ramadus, K.; Suresh, G.; Janarthanan, N.; Masilamani,
S. Pestic. Sci. 1998, 52, 145–151.
3. Criado, J. J.; Rodr ´ı guez-Fem a´ ndez, E.; Garc ´ı a, E.;
Hermosa, M. R.; Monte, E. J. Inorg. Biochem. 1998, 69,
113–119.
This work was financially supported by the Innovational
Foundation of Chinese Academy of Sciences (KZX-2-
YW-209).
2
2
2
4. Eweis, M.; Elkholy, S. S.; Elsabee, M. Z. Int. J. Biol.
Macromol. 2006, 38, 1–8.
5. Ramos, A. C. S.; Dantas Neto, A. A.; Castro Dantas, T.
N. C. Braz. J. Chem. Eng. 1997, 14, 159–165.
6. Nester, E. W.; Anderson, D. G.; Roberts, E.; Pearshall, N.
N.; Nester, M. T. Microbiology; McGraw-Hill: Boston,
MA, 2003; pp 518–524.
References
1
. Muzzarelli, R. A. A.; Guerrieri, M.; Goteri, G.; Muzzar-
elli, C.; Armeni, T.; Ghiselli, R.; Cornelissen, M. Bioma-
terials 2005, 26, 5844–5854.
. Jayakumar, R.; New, N.; Tokura, S.; Tamura, H. Int. J.
Biol. Macromol. 2007, 40, 175–181.
. Balakrishnan, B.; Mohanty, M.; Umashankar, P. R.;
Jayakrishnana, A. Biomaterials 2005, 26, 6335–6342.
. Sinha, V. R.; Singla, A. K.; Wadhawan, S.; Kaushik, R.;
Kumria, R.; Bansal, K.; Dhawan, S. Int. J. Pharm. 2004,
2
7. Avadi, M. R.; Sadeghi, A. M. M.; Tahzibi, A.; Bayati, K.
H.; Ponladzadeh, M.; Zohuriaan-Mehr, M. J.; Rafiee-
Tehrani, M. Euro. Polym. J. 2004, 40, 1355–1361.
8. Sun, L. P.; Du, Y. M.; Fan, L. H.; Chen, X.; Yang, J. H.
Polymer 2006, 47, 1796–1804.
2
3
4
2
2
3
3
9. Sha, J. J. Handbooks of Chemical Product, 3rd ed.;
Chemical Industry: Beijing, 1994; pp 190–192.
0. Guo, Z. Y.; Chen, R.; Xing, R.; Liu, S.; Yu, H. H.; Wang,
P. B.; Li, P. C. Carbohydr. Res. 2006, 341, 351–354.
1. Jasso de Rodr ´ı guez, D.; Hern a´ ndez-Castillo, D.; Rodr ´ı -
guez-Garc ´ı a, R.; Angulo-S a´ nchez, J. L. Ind. Crop. Prod.
2
74, 1–33.
5
. Muzzarelli, R. A. A.; Mattioli-Belmonte, M.; Pugnaloni,
A.; Biagini, G. In Chitin and Chitinases; Jolles, P.,
Muzzarelli, R. A. A., Eds.; Birkhauser: Basel, Switzerland,
2
005, 21, 81–87.
1999; pp 251–264.