Preparation and immunological evaluation of organic acid salts of levamisole

Article abstract of DOI:10.14233/ajchem.2014.15457

Four kinds of organic acid salts of levamisole were prepared from levamisole hydrochloride and organic acids (L-ascorbic acid, tartaric acid, citric acid and ferulic acid) by solvent crystallization method, respectively. The salts were characterized by elemental analysis, UVVIS, FTIR, ESI-MS, DSC-TGA, solubility in water, melting point, optical rotation and pH in water. The immunosuppressed mice models were established by intraperitoneal injection of cyclophosphamide to verify and compare the effects of levamisole hydrochloride and organic acid salts of levamisole on immune functions of immunosuppressed mice. The results showed while significantly increasing the contents of serum IgM and IgG and the immune organ indexes in cyclophosphamide-induced immunosuppressed mice, levamisole ferulate and levamisole tartrate presented higher increases in IgG contents and immune organ indexes than other salts did. Thus, it could be concluded that levamisole ferulate and levamisole tartrate were more effective on immune enhancement than other salts.

Full text of DOI:10.14233/ajchem.2014.15457

Asian Journal of Chemistry; Vol. 26, No. 3 (2014), 634-640
http://dx.doi.org/10.14233/ajchem.2014.15457
Preparation and Immunological Evaluation of Organic Acid Salts of Levamisole
*
XIAOLING FENG, YI YANG and JIANHUA WANG
College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
*Corresponding author: Fax: +86 23 65103031; Tel: +86 13983657810; E-mail: wjh@cqu.edu.cn; f042026@163.com
Received: 9 March 2013;
Accepted: 29 May 2013;
Published online: 30 January 2014;
AJC-14604
Four kinds of organic acid salts of levamisole were prepared from levamisole hydrochloride and organic acids (L-ascorbic acid, tartaric
acid, citric acid and ferulic acid) by solvent crystallization method, respectively. The salts were characterized by elemental analysis, UV-
VIS, FTIR, ESI-MS, DSC-TGA, solubility in water, melting point, optical rotation and pH in water. The immunosuppressed mice models
were established by intraperitoneal injection of cyclophosphamide to verify and compare the effects of levamisole hydrochloride and
organic acid salts of levamisole on immune functions of immunosuppressed mice. The results showed while significantly increasing the
contents of serum IgM and IgG and the immune organ indexes in cyclophosphamide-induced immunosuppressed mice, levamisole
ferulate and levamisole tartrate presented higher increases in IgG contents and immune organ indexes than other salts did. Thus, it could
be concluded that levamisole ferulate and levamisole tartrate were more effective on immune enhancement than other salts.
Keywords: Levamisole, Salt formation, Organic acid salts, Immune enhancement.
a proton, so it’s likely to form stable salts with acids. To the
best of our knowledge there has been no report on the use of
organic acids for the synthesis of the salt form of levamisole.
In this study, a series of organic acid salts of levamisole were
prepared (Scheme-I) and characterized. Meanwhile, immuno-
logical evaluation of the five salts [evamisole hydrochloride
(LH), levamisole ascorbate (LA), levamisole tartrate (LT),
levamisole citrate (LC) and levamisole ferulate (LF)] was carried
out on cyclophosphamide (CTX)-induced immunosuppressed
mice models. The immune organ indexes and contents of serum
IgA, IgG and IgM were measured and compared in an attempt
to find and obtain a more effective salt form of levamisole on
immune enhancement.
INTRODUCTION
Levamisole (LMS), the levo-isomer of tetramisole, is an
anthelminthic belonging to a class of synthetic imidazothiazole
derivatives, as well as an immunomodulator which can enhance
the immune responses1-5. Due to the poor solubility and insta-
bility in water, its hydrochloride (levamisole hydrochloride,
LH) is commonly used in clinical. However, LH still has
problems of low bioavailability and side effects, such as a certain
stimulus to the human body. In the past decade, a large number
of researches have been increasingly reported with respect to
chemical modifications of drugs, such as esterification6,
acylation7, amidation8, alkylation9, anhydride formation10, salt
formation11-17, ring opening and formation18,19. As reported,
chemical modifications have many advantages such as
improving the solubility, stability and bioavailability of drugs,
reducing toxic reactions, enhancing pharmacological effects,
extending drug action, making other drug dosage forms and
delivery systems possible or more excellent, etc.
EXPERIMENTAL
Levamisole hydrochloride (Shaanxi Hanjiang Pharmaceu-
tical Co., Ltd., batch number: 090610); cyclophosphamide
(Jiangsu Hengrui Medicine Co., Ltd., batch number: 090802);
D-tartaric acid (TA) (analytical grade, Nantong Qihai Chemical
Co., Ltd.); citric acid (CA) (analytical grade, Shanghai Pan Ke
Biotechnology Co., Ltd.); ferulic sodium (Livzon Pharmaceutical
Group Inc., batch number: 080307); L-ascorbic acid (VC) (ana-
lytical grade, Chongqing Boyi Chemical Co., Ltd.); IgG, IgM
and IgA kits (Adlitteram Diagnostic Laboratories Inc.); Other
reagents were commercially available analytical grade.
Preparation of organic acid salts of levamisole:
Levamisole hydrochloride (12 g, 0.05 mol) was dissolved in
Salt formation is a simple and effective chemical modifi-
cation, which can influence a number of physicochemical prop-
erties of the parent drug, including solubility, dissolution rate,
stability and hygroscopicity11.As a result, different salts of drugs
may bring about different pharmacokinetic and pharmaco-
dynamic qualities and therefore different clinical qualities;
some even may show intrinsic toxicity14. Levamisole base has
a tertiary nitrogen atom, whose lone pair of electrons can accept

Vol. 26, No. 3 (2014)
Preparation and Immunological Evaluation of Organic Acid Salts of Levamisole 635
O
N
S
O
H
H
OH
H
O
OH
·
HO
HO
N
OH
OH
H
OH
H
OH
O
TA (tartaric acid)
LT (levamisole tartrate)
H
N
H
S
H
OH
· HCl
OH
N
S
HO
H
N
HO
O
·
O
O
O
N
LH
HO
LA (levamisole ascorbate)
OH
HO
OH
OH-
VC (L–ascorbic acid)
O
OH
N
S
N
H
S
O
OH
H
·
+
N
N
O
O
O
O
OH
L–
OH
HO
OH
HO
OH
LMS
LC (levamisole citrate)
CA (citric acid)
O
O
H3CO
N
S
H
OH
H3CO
HO
·
OH
N
HO
LF (levamisole ferulate)
FA (ferulic acid)
H+
O
H3CO
HO
ONa
SF (sodium ferulic)
Scheme-I: General synthesis of organic acid salts of levamisole
50 mL of distilled water under the condition of ice bath. With
magnetically stirring, 50 mL of 1 M NaOH solution was
dropwise added to the LH solution until the pH became 7 and
white turbidity appeared. Dropwise adding was continued until
the pH finally became 12. Following warming the reaction
system to 40 ºC in a water bath, stirring was continued for
another 0.5 h while maintaining the pH at 12. Then, the resul-
tant mixture was filtered to separate levamisole base from the
system. The first part of product was washed to neutral pH
with distilled water and dried under reduced pressure. The
filtrate was extracted three times with dichloromethane. The
extract was dried overnight in anhydrous calcium chloride and
filtered. The resultant filtrate was evaporated to remove
dichloromethane under reduced pressure. The second part of
levamisole base was obtained and combined with the first part.
The total yield was 98.32 %.
priate amount of anhydrous ether and freezing crystallization
for 1h. The resultant mixture was filtered. The filter cake was
washed with a small amount of anhydrous ether and dried in
phosphorus pentoxide. The crude product was recrystallized
with anhydrous methanol-ethyl acetate to obtain more pure
organic acid salts of levamisole.
Characterization of organic acid salts of levamisole:
The solubility in water at 25 ºC (GB/T 21845-2008, China),
melting point (MP), optical rotation ([α]D20) and pH in water
were tested. A UV-VIS spectrophotometer (UV-2450,
Shimadzu, Japan) was used for the spectra measurements of
LMS, LH, LA, LT, LC, LF and the four organic acids in ethanol.
Elemental Analysis was determined by using an elemental
analyzer (CE-440 Elemental Analyzer, EAI, USA). Fourier
transform infrared spectra of LMS, LH, LA, LT, LC, LF, TA,
VC, CA, ferulic acid (FA) and four physical mixtures (LMS and
VC, LMS and TA, LMS and CA, LMS and FA) were obtained
in the range of 4000-400 cm-1 using a fourier transform infrared
spectrometer (Spectrum GX, Perkin-Elmer, USA), according
to the KBr disk method. Electrospray ionization mass spectro-
metry(ESI-MS)wascarriedoutonamassspectrometer(micrOTOF
Q, Bruker, Germany) equipped with an electrospray ionization
All the four salts LF, LA, LC and LT were prepared by
solvent crystallization method as follows: Levamisole base and
respective organic acids (mole ratio 1:1) were dissolved in
absolute ethanol and stirred for a period of time at a suitable
temperature. The reaction mixture was filtered. The filtrate
was chilled to 5-10 ºC, followed by slowly adding an appro-

636 Feng et al.
Asian J. Chem.
source. LA, LT, LC and LF were dissolved in methanol and
diluted to use, respectively. Infusion rates were 3 µL min-1
during sample analysis. DSC and TGA curves of LMS, LH,
LA, LT, LC and LF were recorded on a differential scanning
calorimeter (STA-449C, NETZSCH, Germany). All the
samples were accurately weighed (2-5 mg), put in aluminium
pans and heated at a scanning rate of 10 ºC min-1 from 25-
400 ºC. Nitrogen was used as the purge gas with the flow rate
set at 30 mL min-1. An identical empty pan was used as a
reference.
(thymus, spleen and liver) were obtained and washed with
distilled water, removing the connective tissue and fat on their
surfaces. The organs were weighed after sucking up all the
water with filter paper and observed under a microscope
(ShanghaiYuguang Instrument Co., Ltd.).At last, the immune
organ indexes were calculated using the following equation20:
Immune organ (thymus, spleen or liver) index (mg g-1)
Weight of thymus,spleen or liver (mg)
=
Body weight of the mouse (g)
Statistical method: All data were statistically analyzed
by SPSS17.0 software. All results were presented as mean
Immunological evaluation: Seventy adult Kunming mice
(35 males and 35 females) of clean grade, weighing 19-23 g,
were obtained from the experimental animal center of
Chongqing Academy of Animal Sciences. All experimental
procedures were in accordance with the ethics and regulation
of animal experiments of Chongqing Academy of Animal
Sciences. The mice were housed in cages at a temperature
between 20-25 ºC and relative humidity of 40-60 % under
natural light/dark conditions for 1 week and were given
conventional feed and free drinking water.
standard deviation
Comparisons between groups were
(x s).
carried out using one-way ANOVA, wherein Least-Signifi-
cant Difference (LSD) method was used for homogeneity of
variance and Tamhane’s T2 method was used for herterogeneity
of variance. The differences were statistically significant when
p < 0.05.
RESULTS AND DISCUSSION
Based on advantages of salt formation and combination
principles in pharmaceutical chemistry that structures of two
compounds can be put together in one molecule for the purpose
of reducing poisonous side effects and obtaining the joint
effect, four organic acid salts of LMS (LA, LT, LC and LF)
were prepared in order to find a more effective salt form of
enhancing immune function.
Seventy mice were randomly divided into 7 groups of 5
males and 5 females each. One group was intraperitoneally
injected with saline solution and the remaining groups were
injected with cyclophosphamide (CTX) solution (50 mg kg-1
d-1) for five consecutive days to establish immunosuppressed
models. From the sixth day, five immunosuppressed groups
were intraperitoneally injected with equimolar LH, LA, LT,
LC and LF solutions (12, 19, 17.7, 19.8 and 20 mg kg-1 d-1) for
21 consecutive days, respectively, while other groups were
injected with an equivalent volume of saline solution. The
resultant groups were named LH-CTX group, LA-CTX group,
LT-CTX group, LC-CTX group, LF-CTX group, CTX group
and Control group, respectively. Body weights were measured
on time every day, while recording the clinical performances
of all groups. Following the last administration, all mice were
fasted for 12 h, but had free access to fresh water. Then whole
blood samples were collected in Eppendorf tubes (SorensonTM,
Bio Science, Inc. USA) from the orbits and allowed to clot for
at least 1 h at 4 ºC. Samples were centrifuged at 4 ºC (10 min,
3000 × g) and the serum was separated and stored at -20 ºC
until further analysis.
Characterization of organic acid salts of levamisole:
Four organic acid salts of levamisole were obtained. Levanisole
ascorbate was a light yellow crystalline powder without odor
and bitter taste. Levanisole tartarate and LC were white
crystalline powders without odor and bitter taste, but LF was
a light green crystalline powder with bitter taste and odorless.
They were all soluble in water, the solubility results are listed
in Table-1. Levanisole ascorbate solution was readily oxidized
to turn dark yellow, whereas other three solutions were more
stable. LA, LT and LC were dissociated into white insoluble
precipitate in 0.1 M NaOH solution, which was identified as
LMS by thin layer chromatography method. Levanisole
ferulate was dissociated into white insoluble precipitates both
in 0.1 M HCl solution and 0.1M NaOH solution, which were
identified as FA and LMS by TLC, respectively. Meanwhile,
Determination of serum antibody content:An enzyme
linked immunosorbent assay (ELISA) was carried out to measure
the contents of serum IgG, IgM and IgA using a microplate
reader (ELX808, Baote, USA), according to the kit instruction
(Adlitteram Diagnostic Laboratories Inc.).
elemental analysis was tested, as well as some other physical
20
properties, including melting point, optical rotation ([α]D
)
and pH, which were different from those of LMS (Table-1).
The results of elemental analysis showed that the measured
values were in good accordance with the calculated ones for
the C, H and N contents of organic acid salts of levamisole.
Determination of immune organ indexes: The mice
were killed by cervical dislocation and weighed. Organs
TABLE-1
RESULTS OF ELEMENTAL ANALYSIS AND SOME PHYSICAL
PROPERTIES OF ORGANIC ACID SALTS OF LEVAMISOLE
Element content (%)a
H
Solubility in water
at 25 ºC (mg/mL)b
20
[α]D
Compounds
m.p. (ºC)
pH (H2O)
C
N
Meas. Count Meas. Count Meas. Count
LA
LT
LC
LF
137.19 1.17
151.95 0.44
184.47 0.48
105.25 0.80
175.2-176.9
163.6-165.1
125.4-126.7
141.6-142.7
-23.34
-141.68
-116.52
-98.01
5.93
5.86
5.52
6.15
53.59 53.67
50.73 50.84
51.67 51.51
63.21 63.30
5.36
5.28
5.14
5.48
5.30
5.12
5.09
5.56
7.42
7.85
6.93
7.15
7.36
7.91
7.07
7.03
aMeas. indicates the measured values, count indicates the calculated ones. bSolubility is present as Mean standard deviation, n = 3.

Vol. 26, No. 3 (2014)
Preparation and Immunological Evaluation of Organic Acid Salts of Levamisole 637
3.5
Thus, it may be concluded that levamisole reacted with organic
acids to form salts.
3.0
2.5
2.0
1.5
1.0
0.5
0
UV Spectroscopic analysis: UV spectroscopy was used
to observe the behaviour of the LMS absorption spectra with
the addition of different organic acids. The UV spectra of LMS,
VC, FA, LA, LT, LC and LF are shown in Fig. 1. LMS, LC,
LT, VC and FA exhibited absorption peaks at 208, 214, 214,
265 and 322 nm, respectively. Levanisole ascorbate exhibited
two absorption peaks at 212 and 254 nm. Levanisole ferulate
exhibited two absorption peaks at 212 and 315 nm. However,
TA and CA had no obvious absorption in the wavelength range
of 200-400 nm. As shown in Fig. 1, the absorption peak of
LMS at 208 nm shifted to 214 nm (absorption peaks of LC
and LT), 212 nm (one absorption peak of LA and the absor-
ption peakof LF), respectively. The absorption peakof VC at
265 nm and the absorption peak of FA at 322 nm had hypso-
chromic shifts to 254 nm (one absorption peak of LA) and
315 nm (one absorption peakof LF), respectively. In addition,
the peak shapes of LA, LT, LC and LF were distinguished
from those of LMS and organic acids, as well as those of their
simple summations, respectively. From the above, it could be
concluded that the shifts of absorption peaks and the changes
of peak shapes were due to the formation of organic acid salts
of levamisole.
200
250
300
λ (nm)
350
400
1.2
1.0
0.8
0.6
0.4
0.2
0
f
Mass spectrometry (ESI-MS): The main ions fragments
of mass spectrometry are listed in Table-2. The ions corres-
ponding to LMS (m/z 205), VC (m/z 175), TA (m/z 149), CA
(m/z 191), FA (m/z 193) were seen as strong peaks in the scan
spectra of respective solutions of organic acid salts of
levamisole, indicating that the bonds connecting LMS and
organic acids were weak so that organic acid salts of LMS
were readily subjected to cleavage to form the ions of LMS
and organic acids. The scan spectra also showed peaks at m/z
379, 353, 395 and 397 ions corresponding to the 1:1 comp-
ounds of LMS with VC, TA, CA and FA, respectively. All
these results suggested the formation of organic acid salts of
levamisole.
200
250
300
λ (nm)
350
400
1.2
1.0
0.8
0.6
0.4
0.2
0
g
Fourier transform infrared spectroscopy: FTIR spec-
troscopy was also used to detect the possible molecular inter-
action between LMS and organic acids, since upon the forma-
tion of organic acid salts of LMS shifts or changes in the ab-
sorption spectrum occured. FTIR spectra of all samples are
illustrated in Fig. 2. Characteristic absorption peaks of LMS
such as C=N and C-S stretching vibrations (1465 and 874
cm-1) were observed in the spectra of the four salts. However,
there were some changes that functional groups showed band
shifts, broadening and disappearing in the FTIR spectra of
LA, LT, LC and LF when compared to those of LMS,VC, TA,
200
250
300
λ (nm)
350
400
Fig. 1. UV spectra of LC (a), LT (b), VC (c), LMS (d), LA (e), LF (f) and
FA (g)
TABLE-2
RESULTS OF MASS SPECTROMETRY
Compounds
LA
Molecular formula
C11H12N2S.C6H8O6
Molecular weight
Ionization mode
(Positive) ESI
(Negative) ESI
(Positive) ESI
(Negative) ESI
(Positive) ESI
(Negative) ESI
(Positive) ESI
(Negative) ESI
Peak(m/z)
205[LMS+H]+ 177[VC+H]+
175[VC-H]– 379[LA-H]– 396[2VC-2H+Na]–
205[LMS+H]+ 173[TA+Na]+
149[TA-H]- 353[LT-H]-
380
LT
LC
LF
C11H12N2S.C4H6O6
C11H12N2S.C6H8O7
C11H12N2S.C10H10O4
354
396
398
205[LMS+H]+ 215[CA+Na]+
191[CA-H]– 395[LC-H]–
205[LMS+H]+ 217[FA+Na]+
193[FA-H] – 397[LF-H]–

638 Feng et al.
Asian J. Chem.
peaks at 1363 cm-1 β(O-H), 1136 cm-1 ν(C-O), 1263 cm-1
ν(C-O), 1583 cm-1 νas(C-O) and 1529 cm-1 νs(C-O) and its weak
absorption band in the range of 3000-2500 cm-1 disappeared,
which was the characteristic band of the carboxyl group of
CA. Levanisole ferulate showed peaks at 1594, 853 and 821
cm-1, which could be attributed to the oxygen anion of carboxyl
and the para-substitution and meta-substitution of benzene
ring, respectively, but its broad absorption band between 3400-
2500 cm-1 ν(O-H) disappeared, which could be observed in
the FTIR spectra of FA. Conclusively, FTIR spectra obtained
from various LMS organic acid salts showed peaks which were
not a summation of the characteristic peaks of LMS and organic
acids and thus couldn’t be simply regarded as the superposition
of FTIR spectra of LMS and organic acids, indicating the
interaction of LMS with organic acids.
LMS
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
LA
LT
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
VC
TA
DSC and TGA analyses: DSC can be used to investigate
thermal events such as physical transitions (the glass transi-
tion, crystallization, melting and the vaporization of volatile
compounds) and chemical reactions. The information obtained
characterizes the sample with regard to its thermal behaviour
and composition. TGA is used to measure the change in mass
of a sample as a function of temperature or time. It provides
information on the properties of the sample and its compo-
sition. If the sample decomposes as a result of a chemical
reaction, the mass of the sample often changes in a stepwise
fashion. The temperature at which the step occurs characterizes
the stability of the sample material in the atmosphere used.
For more information, simultaneous DSC is often used to
measure with TGA21.
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
Mixture of LMS and VC
Mixture of LMS and TA
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
LF
LC
Fig. 3 shows the DSC and TGA curves obtained for LT,
LA, LC, LF and LMS. The curves of LT and LC presented
endothermic peaks at 165 and 127 ºC corresponding to
respective melting points and exothermic peaks at 235 and
250 ºC, respectively. The curve of LA presented a sharp
endothermic peak at 176 ºC corresponding to the melting point
and two consecutive exothermic peaks at 210 and 265 ºC
caused by the oxidative decomposition of LA. The curve of
LF presented a sharp endothermic peak at 142 ºC correspon-
ding to the melting point and an exothermic peak at 250 ºC.
But the curve of LMS presented a sharp endothermic peak at
61 ºC and an exothermic peak at 263 ºC corresponding to the
melting and decomposition of LMS, associated with loss of
moisture between 25 and 100 ºC.Apparently, the melting points
of LMS organic acid salts significantly increased, indicating
that new compounds had been formed.
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
FA
CA
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
Mixture of LMS and FA
Mixture of LMS and CA
In the TGA curves shown in Fig. 3, there was no weight
loss due to water evaporation for LA, LT, LC and LF, but a
small weight loss for LMS. LT began to decompose before
melting. Other salts began to decompose in an exothermic
process after melting. This could be seen in the TGA curves
as a weight loss as well as in the DSC curves as exothermic
peak decomposition. Meanwhile, the process of weight loss
of LA, LT, LC and LF was differentiated from that of LMS by
the combination of TGA and DSC curves, which was another
indication of the formation of organic acid salts of LMS.
Immunological evaluation: Since products for immune
enhancement are mainly used by the population with low
immunity at the present, the immunological evaluation performed
on immunosuppressed animal models is reasonable and
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
4000 3000 2000 1500 1000 0.400
Wavenumber (cm–1)
Fig. 2. FTIR spectra of all samples
CA, FA and their respective physical mixtures. The FTIR spec-
trum of LA displayed a broad absorption band at 3420 cm-1,
three O-H stretching vibrations at 3614, 3321 and 3037 cm-1
and two C-O stretching vibrations at 1062 and 1032 cm-1.
Levanisole tartarate showed peaks at 3071 cm-1 ν(O-H), 1759
cm-1 ν(C=O), 1248 cm-1 ν(C-O), 1350 cm-1 β(O-H) and 1101
cm-1 ν(C-O), which could be attributed to the carboxyl group and
secondary alcohol hydroxyl group. Levanisole citrate presented

Vol. 26, No. 3 (2014)
Preparation and Immunological Evaluation of Organic Acid Salts of Levamisole 639
100
80
60
40
20
0
1
100
80
60
40
20
0
(B)
(A)
TABLE-3
CONTENTS OF SERUM IgA, IgG AND IgM OF MICE IN
0
0
-0.5
a
DIFFERENT GROUPS [n = 10,
]
-1
-2
-3
-4
-5
(x s)
IgG (mg/mL)
9.47 1.73de
-1.0
-1.5
-2.0
-2.5
Group
Control
CTX
IgA (mg/mL)
1.04 0.14a
0.94 0.16a
0.98 0.10a
0.93 0.13a
0.95 0.16a
0.95 0.10a
0.99 0.18a
IgM (mg/mL)
0.54 0.07cd
0.35 0.08a
0.47 0.12bc
0.56 0.06d
0.51 0.08bcd
0.53 0.06cd
0.58 0.07d
5.83 1.43a
9.13 1.75cde
10.17 0.93ef
7.92 1.28bc
7.46 1.51b
10.79 1.05f
LH-CTX
LT-CTX
LA-CTX
LC-CTX
LF-CTX
50. 100 150 200 250 300 350 400
Temperature (°C)
50. 100 150 200 250 300 350 400
Temperature (°C)
100
80
60
40
20
0
2
100
80
60
40
20
0
(D)
(C)
1.0
0.5
0
1
0
aDifferent small letters in the same column indicate significant
differences (p < 0.05).
-0.5
-1.0
-1.5
-2.0
-1
-2
-3
As shown in Table-3, serum IgA contents of all groups
were not significantly different from each other (p > 0.05),
but all salts significantly increased the contents of IgG and
IgM in the serum of immunosuppressed mice (p < 0.05). More-
over, serum IgM contents of all groups for levamisole salts
showed no significant difference when compared to that of
Control group. The serum IgG content of LF-CTX group was
significantly higher than those of Control group, LH-CTX
group, LA-CTX group and LC-CTX group (p < 0.05), while
the serum IgG content of LT-CTX group was only higher than
those of LA-CTX group and LC-CTX group (p < 0.05). This
indicated LF may be more effective on improving the antibody
level in vivo, implying that LF could convert to LMS and FA
to produce a joint effect in vivo because FA is an immuno-
potentiator26 as well as LMS. However, for LF and LT there
was no significant difference between their IgG and IgM
contents (p > 0.05).
50. 100 150 200 250 300 350 400
Temperature (°C)
50.
100
150
200 250
Temperature (°C)
300
350
400
0.5
100
(E)
95
90
85
80
75
70
65
60
0
-0.5
-1.0
-1.5
-2.0
-2.5
50. 100 150 200 250 300 350 400
Temperature (°C)
Fig. 3. TGA-DSC curves of LT (A), LA (B), LC (C), LF (D) and LMS (E)
effective. Without activity in vitro, CTX is one of the most
commonly used drugs in the clinical treatment of tumors,
which can be hydrolyzed to glyciphosphoramide with
activation by excess ifosfamide or phosphatase in vivo and
thus suppress both humoral and cell-mediated immune
responses22. Therefore, it has been commonly used to establish
Determination of immune organ indexes: The results
of body weights of all groups are listed in Table-4. Body
weights of all groups increased in varying degrees, wherein
CTX group gained the least amount of weight. The body
weights of LT-CTX group, LF-CTX group and control group
significantly increased when compared to those of other groups
(p < 0.05). The LH-CTX group and CTX group became dispirited
and inactive after injection. However, other groups were
different, wherein especially LT-CTX group and LF-CTX
group with injection after 0.5 h became inactive firstly, but
returned to normal after 0.5 h of excitement and gained weight
gradually.
immunosuppressed animal models23,24
.
Determination of serum antibody content: Immunoglo-
bulins play a major role in humoral immune responses. Serum
IgG, IgA and IgM are the molecular basis of humoral immune
responsesplaying a role in specific immunity responses. In
addition, IgG and IgM can activate the classical pathway of
complement to take part in non-specific immunity responses25.
Table-3 shows that the serum IgG and IgM contents of
CTX group were significantly lower than that of Control group
(p < 0.05), but the IgA content between them had no signifi-
cant difference, indicating that CTX were able to suppress the
generation of IgG and IgM effectively and had no obvious
influence on the generation of IgA in mice. This corresponded
to the report that CTX could inhibit both humoral and cell-
mediated immune responses22, giving information that the
models established in this study were successful.
In recent years, immune organ indexes have been com-
monly applied to the evaluation of immunomodulators on
immune function, which to some extent can reflect the number
and function of immune cells27. The results for immune organ
indexes are presented in Table-4. Compared with control group,
TABLE-4
BODY WEIGHT AND THYMUS, SPLEEN, LIVER INDEXES OF MICE IN DIFFERENT GROUPS [n = 10,
a
]
(x s)
Body weight (g)
Before experiment
Index (mg g-1)
Body weight
change (g)
Group
After experiment
27.79 1.00
24.76 0.98
24.89 1.18
26.49 0.91
24.54 1.03
27.26 1.40
24.83 0.79
Thymus index
4.62 0.29d
2.88 0.55a
3.66 0.53b
4.31 0.43cd
3.54 0.57b
4.56 0.22d
3.03 0.37a
Spleen index
6.44 0.30c
4.95 0.41a
4.86 0.56a
6.06 0.44c
5.25 0.47b
6.45 0.40c
5.57 0.28b
Liver index
Control
CTX
21.50 1.21
21.30 1.12
20.97 1.12
20.77 1.26
20.49 0.90
21.73 0.63
20.85 1.30
6.29 1.70c
3.45 1.69a
3.93 0.68a
5.72 1.20cd
4.05 1.23a
5.53 1.73bc
3.98 0.94a
58.60 4.24ab
57.85 3.87ab
60.04 4.31b
58.90 2.09ab
60.07 2.12b
58.72 2.25ab
59.54 2.10b
LA-CTX
LT-CTX
LC-CTX
LF-CTX
LH-CTX
aDifferent small letters in the same column indicate significant differences (p < 0.05).

640 Feng et al.
Asian J. Chem.
the thymus and spleen indexes of CTX group significantly
decreased (p < 0.05), while the decrease of the liver index was
not significant (p > 0.05), suggesting both the thymus and
spleen had atrophied and thus probably couldn’t perform
immune function normally because of injection of CTX. As
shown in Table-4, liver indexes showed no significant diffe-
rence between any two groups. The thymus indexes of LA-
CTX group, LT-CTX group, LC-CTX group and LF-CTX
group showed significant increases, compared with that of CTX
group (p < 0.05). The spleen indexes of LH-CTX group, LC-
CTX group, LT-CTX group and LF-CTX group were signifi-
cantly higher than that of CTX group (p < 0.05). However,
both the spleen and thymus indexes of LT-CTX group and
LF-CTX group showed no significant difference when com-
pared with those of control group (p > 0.05), whereas they
were significantly higher than the spleen and thymus indexes
of the remaining groups, indicating LF and LT were more
effective on restoration of immune organs than other salts,
which may be attributed to their higer bioavailablity. Further-
more, the spleen and thymus indexes of LF-CTX group were
not significantly different from those of LT-CTX group (p >
0.05), which was in accordance with the above result for their
serum antiboby contents, suggesting that LF and LT had similar
effects on immune enhancement. However, studies on bioavail-
ablity are necessary to further confirm the results, because
demonstration of bioequivalence is generally the most appro-
priate method of substantiating therapeutic equivalence
between medicinal products which are pharmaceutically
equivalent or pharmaceutical alternatives28.
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