Immunomodulator effect of picroliv and its potential in treatment against resistant Plasmodium yoelii (MDR) infection in mice.
ABSTRACT The present study was envisaged to evaluate potential of combination therapy comprising of immunomodulator picroliv and antimalarial chloroquine against drug resistant Plasmodium yoelii (P. yoelii) infection in BALB/c mice.
The immunomodulatory potential of picroliv was established by immunizing animals with model antigen along with picroliv. Immune response was assessed using T-cell proliferation assay and also by determining the antibody isotype-profile induced in the immunized mice. In the next set of experiment, prophylactic potential of picroliv to strengthen antimalarial properties of chloroquine against P. yoelii (MDR) infection in BALB/c mice was assessed.
T-cell proliferation as well as antibody production study reveals that picroliv helps in evoking strong immuno-potentiating response against model antigen in the immunized mice. Co-administration of picroliv enhances efficacy of CHQ against experimental murine malaria.
The activation of host immune system can increase the efficacy of chloroquine for suppression of drug resistant malaria infection in BALB/c mice.
- [show abstract] [hide abstract]
ABSTRACT: The co-administration of immunomodulators and antibiotics has been proved very successful for treatment of opportunistic infectious diseases. In the present study, we evaluated the combination of liposomal amphotericin B (lip-Amp B) and immunomodulator tuftsin to cure Cryptococcus neoformans infection in BALB/c mice. Mice infected with C. neoformans were treated with Amp B deoxycholate and tuftsin free or tuftsin-loaded Amp B liposomes. The results of the present study demonstrated higher efficacy of tuftsin-loaded Amp B liposomes against experimental murine cryptococcosis, in terms of enhanced survival rate and reduced fungal burden in organs (lungs and brain) of the treated mice. Interestingly, pre-treatment of mice with liposomal tuftsin before challenging them with the C. neoformans infection resulted in 100% survival of the treated animals followed by treatment with lip-Amp B. Immunomodulator-based therapy seems likely to be more beneficial for treatment of fungal infectious diseases.Journal of Drug Targeting 09/2005; 13(7):423-9. · 2.77 Impact Factor
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ABSTRACT: In the present study, we investigated the immunopotentiating activity of the immunomodulator tuftsin for the treatment of dose-dependent susceptible Candida albicans infection in a murine model. Our results demonstrated that tuftsin increases the susceptibility of C. albicans to phagocytosis by activating murine macrophages. Fluconazole used for the treatment of mice infected with C. albicans showed less in vivo efficacy and proved to be ineffective in the elimination of the infection from leukopenic mice even at higher doses. However, the antifungal activity of fluconazole against the same isolate of C. albicans significantly increased in tuftsin-pretreated mice and resulted in an improved survival rate in mice. The treated mice also showed less severity of infection as supported by a reduced fungal burden in their kidneys. This study indicates that the use of immunopotentiating substances can enhance the therapeutic efficacy of azole antifungal agents and thus can effectively combat azole-resistant fungal pathogens under conditions of immunosuppression.FEMS Immunology & Medical Microbiology 03/2006; 46(1):63-9. · 2.68 Impact Factor
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ABSTRACT: An important aspect of malaria vaccine development is the identification of an appropriate adjuvant which is both capable of stimulating a protective immune response and safe for use by humans. Here, we investigated the feasibility of using novel immunostimulatory molecules as adjuvants combined with a crude antigen preparation and coadsorbed to aluminum hydroxide (alum) as a vaccine against blood-stage Plasmodium chabaudi AS malaria. Prior to challenge infection, immunization of genetically susceptible A/J mice with the combination of malaria antigen plus recombinant interleukin-12 (IL-12) in alum induced a Th1 immune response with production of high levels of gamma interferon (IFN-gamma) and diminished IL-4 levels by spleen cells stimulated in vitro with parasite antigen compared to mice immunized with antigen alone, antigen in alum, or antigen plus IL-12. Mice immunized with malaria antigen plus recombinant IL-12 in alum had high levels of total malaria-specific antibody and immunoglobulin G2a. Compared to unimmunized mice, immunization with antigen plus IL-12 in alum induced the highest level of protective immunity against challenge infection with P. chabaudi AS, which was evident as a significantly decreased peak parasitemia level and 100% survival. Protective immunity was dependent on CD4(+) T cells, IFN-gamma, and B cells and was long-lasting. Replacement of IL-12 as an adjuvant by synthetic oligodeoxynucleotides (ODN) containing CpG motifs induced a similar level of vaccine-induced protection against challenge infection with P. chabaudi AS. These results illustrate that it is possible to enhance the potency of a crude malaria antigen preparation delivered in alum by inclusion of immunostimulatory molecules, such as IL-12 or CpG-ODN.Infection and Immunity 10/2003; 71(9):5178-87. · 4.07 Impact Factor
Immunomodulator Effect of Picroliv and its Potential in Treatment
Against Resistant Plasmodium yoelii (MDR) Infection in Mice
Varun Dwivedi,1Arif Khan,2Azevedo Vasco,3Nishat Fatima,2Vishal Kumar Soni,4Anil Dangi,4
Shailja Misra-Bhattacharya,4and Mohammad Owais2,5
Received November 27, 2007; accepted May 13, 2008; published online June 13, 2008
Purpose. The present study was envisaged to evaluate potential of combination therapy comprising of
immunomodulator picroliv and antimalarial chloroquine against drug resistant Plasmodium yoelii (P.
yoelii) infection in BALB/c mice.
Methods. The immunomodulatory potential of picroliv was established by immunizing animals with
model antigen along with picroliv. Immune response was assessed using T-cell proliferation assay and
also by determining the antibody isotype-profile induced in the immunized mice. In the next set of
experiment, prophylactic potential of picroliv to strengthen antimalarial properties of chloroquine against
P. yoelii (MDR) infection in BALB/c mice was assessed.
Results. T-cell proliferation as well as antibody production study reveals that picroliv helps in evoking
strong immuno-potentiating response against model antigen in the immunized mice. Co-administration of
picroliv enhances efficacy of CHQ against experimental murine malaria.
Conclusion. The activation of host immune system can increase the efficacy of chloroquine for
suppression of drug resistant malaria infection in BALB/c mice.
KEY WORDS: chloroquine; immunomodulator; malaria; picroliv.
While anti-parasitic agents used against the treatment of
infectious diseases prevent rapid multiplication and hamper
vital physiological activities of the parasite, it is basically the
immune system of the host that plays major role in complete
suppression/elimination of the pathogens. In fact parasites
weaken immune armory as a strategy to establish themselves
in the host. It can be speculated that activation/rejuvenation
of the host immune system could be an effective way to
successfully combat various infectious diseases (1–4).
In spite of global efforts to develop a suitable cure,
malaria is still considered as one of the most prevalent and
devastating disease worldwide. Over three billion people live
under the threat of malaria while it kills over a million each
year, mostly children (5). Unfortunately, the development of
multiple drug resistant isolates of Plasmodium spp. and the
increased resistance of its vector, the Anopheles mosquito, to
DDT underscore the importance of developing new chemo-
therapeutic means to control the spread of malaria. In this
regard, it has always remained imperative to enhance anti-
parasitic efficacy of already existing anti-malarial agents. In
this regard, chloroquine which was supposed to be a most
potent anti-malarial agent, and now suffered a set back
because of its non-effectiveness against drug resistant isolates
of malaria parasite (6) can be used as model to test our
hypothesis. To further enhance the efficacy of chloroquine
against less susceptible isolates of Plasmodium, concomitant
usage of chloroquine in combination with some potent
immunomodulators capable of activating host immune system
has been envisaged.
Picroliv, a standardized fraction isolated from the ethanol
extract of the root and the rhizome of Picrorhiza kurroa
(Family: Scrophulariaceae; general name kutki) contains
iridoid glycosides, and is well known for its protective action
against liver damage caused by various hepatotoxins (7,8).
The compound was also found to be effective to correct
liver damage induced by Plasmodium berghei infection in
Mastomys natalensis (9). It was also reported to possess strong
immunostimulant activity and showed significant protection
against challenge with Leishmania donovani (L. donovani)
promastigotes in experimental golden hamsters (10).
Ironically, no detailed information regarding immuno-
modulating role of picroliv is available till date. In the present
0724-8741/08/1000-2312/0#2008 Springer Science + Business Media, LLC
Pharmaceutical Research, Vol. 25, No. 10, October 2008 (#2008)
1Department of Biochemistry, J. N. Medical College, Aligarh Muslim
University, Aligarh, India.
2Inter-Disciplinary Biotechnology Unit, Aligarh Muslim University,
Aligarh, 202002, India.
3Depto. de Biologia Geral, ICB/UFMG, Av. AntonioCarlos, Belo
Horizonte, 6627, Brazil.
4Department of Parasitology, Central Drug Research Institute,
5To whom correspondence should be addressed. (e-mail: owais_
ABBREVIATIONS: CHQ, chloroquine; IFA, incomplete Freunds
adjuvant; μg, microgram; MDR, multi drug resistant; OVA,
ovalbumin; Pic, picroliv; P. yoelii, Plasmodium yoelii.
study, we performed elaborated studies to evaluate immuno-
modulatory effect of picroliv in model animals to establish its
practical suitability in treatment of infectious diseases. We
also evaluated potential of combination therapy involving
picroliv and chloroquine to combat Multi drug resistant
isolate of Plasmodium yoelii in model animals.
MATERIALS AND METHODS
Chloroquine diphosphate was purchased from the Sigma
Chemical Company St Louis, MO, USA. Picroliv was isolated
according to the protocol of Dwivedi et al. (11). [3H]-
thymidine was bought from Bhabha Atomic Research
Center Mumbai, India. Monoclonal anti mouse CD80 and
CD86 FITC conjugates were from Sigma Immuno Chemical,
St. Louis, USA.
Picrorhiza kurroa grows abundantly in Himalayan and
sub Himalayan regions at the height above the sea level
3,300–3,400 m. Roots and rhizomes of the plant were
collected during autumn (August–September), dried, pow-
dered and finally extracted with alcoholic cold percolation.
The extract was evaporated in vacuo below 50°C. The solid
residue was dissolved in mixture of methanol and water (1:1)
and further washed with chloroform. Chloroform phase was
discarded, while aqueous methanolic phase was further
extracted with ethyl acetate and butanol. Both the ethyl
acetate soluble as well as butanol soluble fractions were com-
bined and evaporated to dryness in vacuo to get picroliv. As
revealed by HPLC and TLC (7A), picroliv contained about
60% of Kutkoside and Picroside in the ratio of 1:1.5, the
remainder 40% being a mixture of iridoid as well as cucur-
bitacin glycosides and some still unidentified substances (11).
Animals and Parasite
In bred female BALB/c mice (8- to 10-week-old), of 18±
2 g weight, were obtained from the institute’s Animal House
Facility. The Plasmodium yoelii nigeriensis MDR (Multidrug
resistant) strain was obtained from Division of Parasitology,
Central Drug Research Institute Lucknow. The techniques
used for bleeding, injection, as well as sacrifice of animals
were strictly performed following mandates approved by the
Animal Ethics Committee (Committee for the purpose of
control and supervision of experiments on Animals, Govern-
ment of India).
The parasitized erythrocytes were obtained from the
blood of highly infected mice (average, parasitemia 38%).
The suspension was diluted in 0.9% sodium chloride to get
the stock of 5×107parasitized red blood cells per milliliter.
Animals were inoculated intraperitoneally with 1×107-
parasitized erythrocytes in 0.2 ml of normal saline.
To assess its immuno-modulatory potential, animals were
pretreated with picroliv before immunization with model
antigen. Our pilot studies suggest that pretreatment (at a dose
of 1 mg/kg body weight) with picroliv for 14 consecutive days
before immunization elicit effective immune response. The
picroliv-pretreated animals (1 mg/kg body weight) were
subsequently immunized with free OVA (100 μg/100 μl
normal saline) or OVA emulsified with equal volume of IFA
(100 μg OVA/100 μl of normal saline; emulsion was prepared
by mixing equal volumes of free OVA with IFA). Control
animals (no picroliv treatment) received saline. On day 7 post
immunization, T-cells were isolated from the spleen of
immunized animals using published procedure as standard-
ized in our lab (11). The splenic tissue was macerated and
erythrocytes were lysed with the hemolytic Gey’s solution
(3 ml per spleen) by incubating the cell suspension for 10 min
on ice. The macrophages were removed by panning method,
while B cells were eliminated by incubating the non-adherent
cells with nylon wool (1×5 cm column) as described
elsewhere (12). The cells eluted from the column, were
incubated with cocktail of anti-Mac2, anti IAdand anti IgM
Ab at 4°C for 45 min (The antibodies recognize the molecules
present on surface of specific murine cell population and help
in complement mediated lysis of the target cells). The cells
were washed and then treated with baby rabbit complement
for 30 min at 37°C. Finally; the cells were again washed with
RPMI-1640 and used as an enriched source of T-cell
The BALB/c mice were inoculated with 2–3 ml of
thioglycolate (3%). Four days later peritoneal exudates cells
(PEC) were isolated from peritoneal lavage. The cells were
washed with cold HBSS. The macrophages were obtained by
incubating the cell suspension for 1 h at 37°C on plastic Petri
dishes followed by several washing with cold HBSS.
T-cell Proliferation Assay
The T-cell proliferation assay was performed following
published procedure as standardized in our lab (12). Briefly,
T-cells (2×104cells/well) obtained from spleens of various
groups of mice were cultured in 96 wells plate (triplicate
wells). The cells were incubated with Mitomycin C treated
macrophages (6×104cells/well) followed by exposure with
increasing doses (0.001–100 μg/ml) of ovalbumin. The
Mitomycin C (50 μg/ml) treated macrophages do not
proliferate, thus the observed uptake of [3H] thymidine can
be co-related with the proliferation of T-cells population only.
The cultures were further incubated for 72 h at 37°C/7%
CO2. The cells were pulsed with 1.0 μCi [3H]-thymidine for
16 h before harvesting by automatic cell harvester (Skatron,
Tranby, Norway). The [3H]-thymidine incorporation was
measured by standard liquid scintillation counting method.
The results were expressed as mean counts per minute of
The expression of CD80 and CD86 was detected on the
surface of stimulated macrophages isolated from group of
picroliv pretreated animals that were subsequently immu-
nized with model antigen OVA as described elsewhere (13).
The macrophages were isolated from splenic tissues of the
immunized mice by their ability to adhere to plastic Petri
2313 Immunomodulatory Effects of Picroliv Against Malarial Infection
plates. The recovered adherent cells were consisted of >99%
macrophages as judged by histochemistry and FACS analysis
using labeled anti F4/80 antibodies. Subsequently, cells were
incubated with Fc block followed by further incubation with
FITC conjugated hamster anti mouse CD80 antibody diluted
in FACS buffer. In another set, cells were incubated with
FITC conjugated hamster anti mouse CD86 antibody diluted
in FACS buffer. The stained cells were acquired on FACScan
and analyzed on macrophage/ monocyte zone using CELL-
QUEST software. The exclusion of cell-debris and lympho-
cytes were executed by suitable gating that allowed analysis
of scattering events consistent with macrophage size range.
The analysis of mean fluorescence intensity (MFI) was
performed on histograms in which the abscissa and ordi-
nate denote log FITC fluorescence and relative cell counts
Picroliv Induces Reactive Oxygen/Nitrogen Species
in the Immunized Animals
In an attempt to assess the intracellular redox state of
macrophages in the picroliv treated animals, we used cell-
permeable redox sensitive CM-H2DCFDA dye (14). The
generation of ROI in stimulated macrophages oxidizes
H2DCFDA. Upon oxidation, the reduced form of the dye
(non-fluorescent) converts to the oxidized (fluorescent) form
and can be detected by fluorescent microscopy. For setting up
this experiment, macrophages were isolated from peritoneal
cavity of picroliv treated animals on day 7 post immunization
and seeded on to multi well glass slides at the equal density
(1×106). The cells were incubated with model antigen OVA
(10 μg/ml) for 24 h at 37°C, followed by addition of
H2DCFDA (10 μM) for 30 min. Number of fluorescence
acquiring macrophages were assessed with the help of
Determination of Antigen Specific IgG Isotypes by ELISA
The production of OVA specific antibodies was mea-
sured in the sera of immunized groups of mice as described
elsewhere (12). The animals were injected with two doses of
free antigen, (100 μg/animal) on days 0 and 7 and bled 7 days
later to monitor the presence of antibodies. The ninety six-
well microtiter plates (Costar, Boston. MA. USA) were
coated overnight with 50 μl of antigen (25 μg/ml) in
carbonate–bicarbonate buffer (0.05 M, pH 9.6) at 4°C. The
antigen-coated plate was blocked with 3% skimmed milk and
then incubated with log 2 dilutions of test and control sera of
animals belonging to the immunized animals. The reaction
was allowed to proceed at 37°C for two hours. The micro-titer
plates were washed and 50 μl biotinylated goat anti-mouse
IgG1 and IgG2a antibodies were added. After usual steps of
washings, 50 μl of streptavidin-HRP was added in each well
and the plates were incubated at 37°C for 1 h. The plates
were washed again before adding 50 μl of orthrophenylene
diamine dihydrochloride (OPD 0.2 M HCl) and were finally
incubated at 37°C for 20 min. The reaction was terminated by
the addition of 50 μl of 7% H2SO4. The absorbance of the
colored complex was read at 495 nm with microplate reader
(Eurogenetics, Torino, Italy). Antibody titers were expressed
as the absorbance of the colored complex determined for
different serum samples at 1:4,000 dilutions.
Effect of Picroliv Treatment on Efficacy of Chloroquine
Against Plasmodium yoelii Infection
The efficacy of picroliv pretreatment to improve antima-
larial action of chloroquine was determined against drug
resistant Plasmodium yoelii infection in mouse model. The
potential of combination therapy was assessed on the basis of
survival rate and parasitic load in red blood cells of infected
mice. In pilot study, various doses of chloroquine (i. p.) were
evaluated to achieve best dosage regimen for treatment of
Plasmodium infected BALB/c mice. Finally, a suboptimal
dose of 8 mg/kg body weight of chloroquine was selected for
evaluating the efficacy of picroliv–CHQ combination.
The animals were divided into following four groups.
Each group consisted of 10 animals.
Group: I saline (No drug treatment)
Group: II picroliv treatment (Picroliv at the dose of
1 mg/kg body weight was given orally for 14 days prior
to challenge with P. yoelii infection)
Group: III chloroquine treatment (Chloroquine (i.p.) at
the dose of 8 mg/kg body weight was administered for
threeconsecutivedays(days1,2,3)postP. yoelii infection)
Group: IV treatment with chloroquine in picroliv pre-
treated animals (Picroliv at the dose of 1 mg/kg body
weight wasgiven orally for14 dayspriorto challengewith
P. yoelii infection and chloroquine (i.p.) at the dose of
8 mg/kg body weight was administered for three
consecutive days (days 1, 2, 3) post P. yoelii infection)
The statistical analysis of the data was performed using
SPSS/10.0 software. Multiple groups at the same time points
were compared using ANOVA followed by Dunnett’s post
hoc test. Statistical significance (P value) of parasitic load and
survival data was ascertained by performing t tests. P values
<0.05 were considered statistically significant.
Picroliv Augments Proliferation of OVA-Specific T-cells
in BALB/c Mice
The immunomodulatory potential of picroliv was
assessed by immunizing picroliv pretreated animals with
model antigen OVA. The picroliv pretreatment induced
significantly higher T-cell proliferation in comparison to the
animals that were not pretreated with picroliv. T-cell response
to antigen was observed in a dose dependent manner (data
not shown). The stimulation index (S.I.) value obtained in
animals pretreated with picroliv prior to their exposure with
OVA (Picroliv–OVA–IFA) was around 5.9±0.6, while no
picroliv pretreatment could induce only 1.85±0.6 S.I. in the
immunized mice (Fig. 1). The control groups did not induce
substantial T-cell proliferation.
2314Dwivedi et al.
Picroliv Up-regulates Expression of CD80/86 Molecules
on Antigen Presenting Cells
The minimum requirements for activation of CD4+T
cells include presentation of processed peptide antigen on
class II MHC, and its subsequent recognition by T-cell
receptor (TCR) of the effecter cells. The interaction of
MHC II-peptide complex with TCR of effector cell should
be accompanied with expression of the appropriate co
stimulatory surface markers (CD80 and CD86) on the
surface of macrophages and dendritic cells (15). The data of
present study clearly revealed that macrophages isolated from
animals immunized with Pic–OVA–IFA showed significantly
higher expression of both CD80 (69.50%) and CD86
(101.73%) on their surface. In contrast, macrophages
isolated from animals vaccinated with OVA–IFA expressed
relatively low levels of CD80 (57.20%) and CD86 (78.96%)
co-stimulatory molecules on the surface of antigen presenting
cells (Fig. 2).
Induction of ROI in Picroliv Treated Animals
The picroliv mediated activation of macrophages was
also established by their ability to produce reactive oxygen/
NO species that help in killing of parasite. The free radical
has the ability to oxidize CM-H2DCFDA that gets converted
to fluorescent oxidized form (DCFDA). The fluorescent
micrograph clearly demonstrates that macrophages treated
with Pic–OVA–IFA acquired more intense fluorescence than
those treated with OVA–IFA (Fig. 3). This can be attributed
to the picroliv-mediated higher production of ROI in host
Picroliv Treatment Increases the Secretion of IgG2a Isotype
Antibodies in the Host
ELISA assessed the level of OVA-specific IgG present in
the sera of various groups of immunized animals. Antibody
titers were expressed as absorbance (A492) of the colored
complex developed in the immunosorbent assay. As evident
from Fig. 4 the administration of antigen in picroliv (P<
0.001) pretreated animals, elicited strong immunological
response in terms of antibody production, while
immunization of animals that were not pretreated with
picroliv could induce moderate level of antibodies. We also
determined isotypes of antibody generated in picroliv-
pretreated animals upon their subsequent immunization
with antigen. The control animals immunized with saline or
free OVA could not induce any detectable level of IgG
isotypes (Fig. 5). Interestingly, significant increase in ratio of
IgG2a/IgG1 type of antibodies was detected in the sera of
animals that were primed with picroliv. While low levels of
IgG1 and IgG2a isotypes were found in control animals that
were not pretreated with picroliv (P<0.001) (Fig. 5).
Effect of Co-administration of Picroliv in Combination
with Chloroquine Against Drug Resistant Isolate of P. yoelii
in BALB/c Mice
Finally, we evaluated effect of picroliv pretreatment on
efficacy of chloroquine against drug resistant Plasmodium
yoelii infection in BALB/c mice. The treatment with sub
optimal 8 mg/kg dose of chloroquine alone could not
completely suppress resistant isolates of Plasmodium yoelii.
The animals treated with chloroquine alone (same dose) died
by day 12 only (Fig. 6). Treatment with same dose in animals
that were pretreated with picroliv (1 mg/kg) ensued in 100%
survival up to day 16 post infection. The parasitic burden in
treated animals was monitored after 3 days post challenge to
infection by preparing blood smears of infected animals.
Interestingly there were no parasitized RBCs in case of
picroliv treated animals till day 9th post infection. While in
the group treated with free chloroquine, there was signifi-
cantly higher parasite load (15.8%, P<0.001) on day 9 post
infection. Further, parasitic load shot up to 56.12% on day 12.
In contrast, in the group treated with picroliv and chloroquine
combination, the parasite load was 1.9% (P<0.001) on 12th
day post infection (Fig. 7).
The recent trend of emergence of drug resistant isolates
of various pathogens including Plasmodium spp. has promp-
ted us to develop alternative strategy to surmount this
problem. In general, the pathogen specific host immune
components play active role in elimination of pathogen, their
non-effectiveness in doing so ensued in full-blown infection.
This becomes more pertinent in case of malaria infection as
parasite deactivates immune response of host via autocoids,
down regulation of T lymphocytes and also suppression of
cytokine release by peripheral mononuclear cells (16–18).
It is well evident that modulation of host immune system
may be of great importance in containing infection. In fact,
the effective immune response to various stages of Plasmo-
Fig. 1. Effect of picroliv treatment on proliferation of CD4+ T-cells in
BALB/c. Pretreatment with picroliv prior to immunization with
model antigen ensued in augmented proliferation of OVA-specific
T-cells. T-cells (2×104) were isolated from the groups of five animals
immunized with various formulation of OVA and cultured with OVA
pulsed macrophages (6×104cells/well). After 72 h,
was added, and its incorporation was measured 16 h later by liquid
scintillation spectroscopy. The stimulation index (S.I.) was calculated
as mean cpm values of stimulated culture/mean cpm values of un-
stimulated culture. Control cultures containing cells obtained from
PBS, picroliv followed by no antigen treatment, IFA without antigen
immunized animals or the groups immunized with PBS, gave
background levels of <2,000 cpm of
The data represents mean S.I.±SD of three determinations.
2315Immunomodulatory Effects of Picroliv Against Malarial Infection
Fig. 3. ROI determination of macrophages binding with DCFDA. Macrophages were isolated from OVA–
IFA or Pic–OVA–IFA immunized animals. For the ROI determination macrophages were incubated with
model antigen OVA for 24 h at 37°C followed by the addition of H2DCFDA for 30 min and observed for
the presence of DCFDA using fluorescent microscope.
Fig. 2. Expression of co-stimulatory molecules (CD80 and CD86) on macrophages upon picroliv treatment.
Co-expression of CD80 and CD86 on macrophages of animals immunized with IFA–OVA and Pic–OVA–
IFA was evaluated by flow cytometry as described in the “Materials and Methods” section. The
macrophages were first incubated with Fc block and subsequently stained with FITC conjugated hamster
anti mouse CD80/86 antibodies diluted in FACS buffer.
2316 Dwivedi et al.
dium parasite demands simultaneous activation of both
humoral as well as cell mediated arms of the immune system
of the host. For example, protection against erythrocytic
stages of the P. yoelii relies on activation of CD4+T cells,
B-cells as well as type I cytokines etc. (19–21). Earlier
investigations have suggested that immunity against blood
stage malaria is complex and requires both Th2 and Th1
cellular responses (22–28).
In the present study, we evaluated picroliv for its poten-
tial to induce humoral (antibody production) as well as cell
mediated immunity (Tcell proliferation) in the host. As evident
from data of the present study, pretreatment with picroliv helps
in induction of strong immune response in the animals upon
their subsequent exposure to model antigen OVA.
Picroliv was found to modulate immune components of
the host at both target as well as effector cell level. It up
regulates expression of co-stimulatory molecules CD80/86 on
the surface of macrophages (Fig. 2). Besides, it also activates
macrophages for production of ROS and reactive nitrogen
species in the immunized animals. The released super-oxides
may be of significant help in killing of the pathogens. The
immunomodulator was also found to induce higher T cell
Fig. 5. Effect of picroliv pretreatment on induction of IgG isotype
response against OVA in BALB/c mice. IgG isotype responses in
various groups of animals immunized with ovalbumin and ovalbumin
emulsified with IFA with or without treatment of picroliv were
determined as described in materials and method section. Sera
(1:4,000 dilutions) obtained from normal and experimental animals
were analyzed for the presence of ovalbumin-specific IgG isotype by
ELISA method as described in Materials and Methods. The level of
IgG isotype were expressed as absorbance (A492) of the colored
complex developed in the immunosorbent assay.
Fig. 4. Picroliv pretreatment helps in induction of higher level
antigen specific antibodies in immunized mice. Antibody levels were
obtained in sera of BALB/c mice immunized with ovalbumin
pretreated with picroliv. The level of OVA-specific IgG present in
the sera of all immunized as well as control animals was assessed by
ELISA. Results are expressed as the mean of antibody titre of five
mice in each group±SD. There was a robust increase in total IgG
levels among animals immunized with Pic–OVA–IFA than the
animals immunized without prior treatment of picroliv.
Fig. 6. The effect of Picroliv pretreatment on survival of P. yoelii
animals. The animals were pretreated with picroliv (1 mg/kg body
weight for 14 days) and subsequently challenged with 106parasitized
RBC (lethal Plasmodium yoelii) followed by treatment with
chloroquine. Percent survival of the treated animals was determined
in various groups of experimental animals. Day 8, P<0.001 picroliv vs
chloroquine vs Pic–CHQ; day 10, P<0.001 picroliv vs chloroquine vs
Pic–CHQ; day 14, P<0.001 chloroquine vs Pic–CHQ.
Fig. 7. The effect of picroliv pretreatment on blood parasitic load of
P. yoelii infection. Groups of BALB/c mice (n=5) were pretreated
with picroliv (1 mg/kg for 14 days prior to infection) followed by
challenging with P. yoelii infected erythrocytes (106parasitized
RBCs).The animals were subsequently treated with chloroquine
(8 mg/kg body weight) such as its free form with no prior treatment
with picroliv, picroliv pre-treatment followed by treatment with
chloroquine etc. Control animals (n=5) were given PBS only.
Parasitemia was estimated by preparing thin blood smears, stained
with Giemsa strain. Day 8, P<0.001 picroliv vs chloroquine and Pic–
CHQ; day 10, P<0.001 chloroquine vs Pic–CHQ; day 16, P<0.001
chloroquine vs Pic–CHQ.
2317Immunomodulatory Effects of Picroliv Against Malarial Infection
proliferation as well as antibody production in the immunized
animals (Figs. 1, 4 and 5). Interestingly, picroliv induced
higher expression of IgG2a as compared to IgG1 isotype in
the immunized animals. This is very interesting finding and
indirectly suggests that the immunomodulator helps in skew-
ing of immune response in favor of Th1 subtype of T-helper
cells. Others and we have earlier shown that Th1 subtype of T
cells as well as IgG2a subtypes of antibodies is of great
importance in containing P. yoelii infection in model animals
(29). In general, blood-induced P. yoelii infection in BALB/c
mice led to fulminate infection that peaks to 60% to 80%
parasitemia in 6 to 8 days. In concordance with this fact, in
the present study, the control animals that did not receive
drug treatment succumb to death, while the animals that were
treated with chloroquine were able to with stand infection for
longer duration. Among various experimental groups, the
animals that received combination of CHQ with picroliv were
successful in suppressing Plasmodium infection by day 16 post
infection, while all animals treated with chloroquine alone
succumbed to death by day 12 post infection (Fig. 7).
Further, the protection studies suggest that picroliv
mediated activation of host immune system was effectively
translated to suppress malaria parasite ensuing in enhanced
activity of chloroquine (30).
The data of the present study advocates above fact, as
picroliv-mediated replenishment of type I cytokines helps in
elimination of parasite. It seems, picroliv educate immune cells
(B-cells and T helper cells) for production of IgG2a type of the
antibodies against P. yoelii antigens that are released by lysis of
parasite. Beside the developed antibodies seems to successfully
inhibit released parasite for attacking fresh erythrocytes.
To rule out the possibility that picroliv has any intrinsic
antimalarial role; we evaluated its in vitro anti-plasmodial
activity. Our preliminary studies suggest that picroliv does not
possess anti-plasmodial activity in in vitro condition (data not
shown). As picroliv has been reported to correct liver
functioning of the animals, it seems, besides increasing the
efficacy of chloroquine against Plasmodium infection, the
picroliv is also likely to nullify various drug induced
malfunction of the host liver (31).
The concept of using combination therapy involving
potential immunomodulator and an effective antimicrobial
agent is not new. The muramyl dipeptide as well as PGG
glucan has been shown to help in activation of host immune
system (32,33). The former has been successfully exploited
against fungal infection while PGG glucan was found to
enhance efficacy of antibiotics against drug resistant bacteria
(32,33). In fact, we too have previously demonstrated that co
administration of immunomodulator tuftsin in combination
with chemotherapy could be an effective strategy in suppres-
sion of fungal, bacterial and protozoan pathogens (2–4). The
data of the present study suggest that in a manner similar to
other potent immunomodulators, picroliv activates host
immune system and thereby increase potency of anti-malarial
chloroquine against drug resistant isolates of malaria parasite.
The activation of host immune system can increase the
efficacy of chloroquine for suppression of drug resistant
malaria infection in BALB/c mice.
The authors express their gratitude to Dr. C.M. Gupta,
Director, CDRI, Lucknow for allowing us to avail of the
institute’s research facilities.
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