Download EVALUATION OF SOME MEDICINAL PLANTS FOR

Chapter 5
Discussion
5. DISCUSSION
Immune system is a set of organization which acts as a shield to infectious agents or a buffer
able to maintain homoeostasis. However, there exists malfunctioning of the immune system
that may arise due to the suppression or over-expression of the immune components or due to
the infection of a pathogen. The impaired immune system may be corrected by using either an
immune stimulant or suppressant drug, as the case demands. Immunomodulation is an
effective means of altering the immune system in favour of the host either by stimulating the
immune cells for better performance or by suppressing their response in case of auto-immune
disorder and tissue transplantation. Immune activation of major immune cells including T and
B lymphocytes by immunostimulatory agents is likely to restore immune balance necessitating
the discovery of such agents from synthetic or natural source. A burgeoning area of research is
the development or discovery of immunomodulatory agents from traditional medicinal plants
that are free from toxic side effects and can be used for a long duration, thus resulting in
continuous immune-activation (Fulzele et al., 2002). Ayurveda, one of the oldest medical
prescriptions, has recommended several plants and their products as ethno medicines and
assumes them to contain properties of drug (Rasayan) with characteristics of restraining many
diseases by modulation of immune responses (Ziauddin et al., 1996). Previous research carried
out on Rasayan herbs have validated that they activate immune functionality without causing
an imbalance in overall physiology (Vayalil et al., 2002). Appropriate modulation of biological
homeostasis in order to boost the ability to fight infection, counteract diseases, prevent cancer,
and so forth, is the primary focus of Rasayan therapy (Ali et al., 2009). Immunomodulators
derived from medicinal plants are critically being considered as an alternative and/or
adjunct approach to conventional treatment against various diseases. Extracts or molecules
derived from plants or by chemical synthesis based on leads obtained from natural
products are opening new vistas for modern therapeutics by positively modulating the
immune system against infections. In view of the relatively high cost and considerable side
effects of many of the allopathic medicines, researchers are now focusing on re-examining the
traditional formulations used in Ayurveda and Homeopathy possessing minimal side effects.
Rasayan herbs are generally regarded as non-toxic even at high doses. Lack of cytotoxicity is
an important attribute of Rasayan drug (Fulzele et al., 2002).
Thus, keeping in view of the tremendous medicinal properties of the traditional plants, the vast
flora of India and lack of validation of medicinal claim of these plants prompted exploitation of
this treasure in the proposed thesis where four medicinal plants namely, Annona squamosa,
Murraya koenigii, Withania somnifera chemotype 101R and Withania coagulans were selected
92
Chapter 5
Discussion
on the basis of their traditional medicinal claim as described earlier in chapter 1 (introduction).
The immunomodulatory efficacy of these plants was explored by analyzing the humoral and
cellular immune responses, Th1/ Th2 cytokines and activation of antigen presenting cells after
administering these test samples in BALB/c mice. Those test samples which demonstrated
immunostimulatory efficacy in mice were finally selected and further assessed for their
immunoprophylactic and chemotherapy adjunct efficacy in rodent host, Mastomys coucha
experimentally infected with human lymphatic filarial parasite, Brugia malayi. The probable
mechanism of action of the active samples is also explored. The present findings demonstrate
that the one of the five pure molecules (compound 5) isolated from Annona squamosa (AS)
and one of the three withanolides (withaferin A) purified from chemotype 101R of Withania
somnifera (WS) exhibited excellent immunostimulatory activity and these two molecules also
demonstrated considerable prophylactic and chemotherapy adjunct effects. We shall first
discuss here the immunomodulatory efficacy of extracts/ fractions and pure compounds of test
samples one by one starting from AS extract followed by their cytotoxicity,
immunoprophylactic and chemotherapy adjunct efficacy evaluation of the active test samples.
Annona squamosa, a traditional plant has various medicinal uses against epilepsy, cardiac
problems, constipation, ulcers, hysteria, fainting spells, cancer and hyperthyroidism. Since the
fruit of this plant contains high sugar content, the twig part of this plant is reported to contain a
large number of alkaloids, it was chosen by us to seek its medicinal properties (Yang et al.,
2008; Yadav et al., 2011). The twigs of this plant was extracted in ethanol and its further
bioactivity guided fractionation yielded 4 fractions; the hexane, chloroform, n-butanol and
aqueous fractions which were subsequently exploited for immunomodulatory activity in vivo in
BALB/c mice. Since the cellular immune response plays an important role in the host defense
machinery. We have evaluated the effect of AS extract on various cellular immune parameters
of BALB/c mice. Among the immune cells, macrophages are believed to be the first line of
cell mediated defense playing a key role in the non specific defense mechanism against host
infection and killing of tumor cells. They act as Antigen presenting cells (APC) and interact
with T cells to present the components of phagocytosed pathogens. Macrophages are also
involved in the process of intracellular parasite killing via increased production of ROS, NO
and other inflammatory mediators. The AS extract and its three fractions [chloroform (F2), nbutanol (F3) and aqueous (F4)] increased the oxidative burst and nitric oxide production in
peritoneal macrophages thereby indicating AS to be a potent stimulant of naïve immune
response. Evaluation of the immune cells especially the splenocytes proliferation is always
used to screen the potential immunomodulatory effect of a substance. This is because spleen is
93
Chapter 5
Discussion
a major secondary lymphoid organ for T cells where they respond to antigens. Therefore, the
regulation of splenocyte proliferation which is closely related to maintaining immune
homeostasis can be considered as an important marker for immune response control. In the
present study we have evaluated the effect of test samples on the proliferative response of
treated mice. A marginal increase in the splenic CD4+ T cells by the crude extract and
chloroform fraction shows the augmentation of T helper cell activity. CD8+ or T cytotoxic
(Tc) cells were stimulated by all the three active fractions. Tc cells are responsible for killing a
tumor cell or a virus infected cell. In the present study, significant up regulation of the CD4,
CD8 and CD19 positive cell populations was observed after crude AS extract and fraction F2
treatment. This also indicates that the AS extract and F2 can inhibit cancerous cell proliferation
as these may act against the altered self cells. CD19 is a cell surface marker of B cells which
are responsible for humoral immune response against the foreign antigens. Humoral immune
response is mediated by antibodies secreted by plasma cells (effector B cells) which function
by neutralizing extracellular microbial toxins (Goldsby et al., 2000). The increased population
of CD19+ cells after treatment with the crude ethanol extract and its chloroform fraction thus
may be correlated with the increased humoral activity in the host. The fractions F1, F3 and F4
could not stimulate the CD4+ and CD19+ cells significantly, however, F2, F3 and F4
significantly up-regulated the CD8+ T cells indicating their support for humoral immune
response. After activation, CD4+ T cells can be subdivided into either Th1-type cells that
secrete IL-2 or IFNγ in mice or Th2-type cells that secrete IL-4 or IL-10 etc. Lymphocyte
proliferation is a complex event involving the participation of IL-2 (one of the major
regulatory cytokine of the immune system) and IL-2 receptor expression. In the current study
AS and F2 significantly stimulated the production of pro-inflammatory cytokines IL-2 and
IFNγ at increasing doses and anti inflammatory cytokines IL-4 and IL-10 at decreasing doses
demonstrating induction of both Th1 and Th2 TH cell sub populations and thus, might be useful
for treating various Th1 or Th2 dominant pathological disorders. It was interesting to observe
that maximum stimulation of Th2 cytokines was observed at the lowest dose tried i.e. 3 mg/ kg
and the doses higher than 3 mg led to continuous and sustained decrease in the Th2 cytokine
levels be it IL-4 or IL-10 and this phenomenon correlated well with the CD19+ population
where similar trend was noticed as regards to doses. It appears that doses lower than 3 mg/ kg,
if used, might have given still higher Th2 stimulation. These results demonstrate that the
selection of dose of immunostimulant is crucial in mounting desired Th1- or Th2 arm of helper
immune response. Thus the findings indicate that AS contains molecules which act via both
Th1 and/ or Th2 biased pathway and the dose selection would be the determining factor for
94
Chapter 5
Discussion
polarization of the type of cytokine secretion. Fractions F3 and F4 up-regulated Th2 cytokine
response with concomitant down-regulation of the Th1 cytokines supporting the presence of
both Th1 and Th2 stimulating as well as down-regulating active principles in Annona
squamosa twigs. The hexane fraction did not possess any immune regulatory properties that
could be due to absence of immunomodulatory constituents in this fraction. However, Yadav et
al. (2011) have shown the chloroform and hexane fraction of AS imparts gastroprotection via
inhibition of H+ K+-ATPase (proton pump) activity and simultaneous strengthening of
mucosal defense mechanism but we could not get the immune potentiating activity in this
fraction may be due to the dose level tried was not optimal. Based on the comparative fold
increase data (table – 1), it may be inferred that the immunomodulatory activity is distributed
among the active fractions and F2 appears to be the most effective fraction as it stimulated
almost all the immunological responses included in the present study.
Thus the findings indicate that ethanolic extract of Annona squamosa twigs and its three
fractions (chloroform, butanol and aqueous) to be highly efficient in augmenting both cellular
and humoral immune responses by stimulating the proliferation of T and B lymphocytes in the
form of increased T and B cell population including increased CD4, CD8 and CD19 positive
cells and also stimulate peritoneal macrophages to produce greater amount of ROS. They also
regulate the Th1 and Th2 cytokines based upon the dose. One of the four fractions, hexane did
not show any immune involvement and all the immune parameters studied remained
unaffected by this fraction. The crude ethanol extract demonstrated immunostimulating
characteristics closer to chloroform fraction. The immunostimulation appeared to be mostly
dose dependent. The highest oxidative burst was caused by aqueous fraction which was dose
dependent followed by chloroform and crude ethanol extract. All the three fractions along with
the crude extract possessed lymphocyte proliferative capability in presence of non specific
mitogens. The present investigation supports the traditional claim of ethno medicinal use of AS
and suggests that ethanolic extract and its three fractions (F2 – F3) derived from the twigs have
therapeutic potential and could serve as an effective immunomodulatory candidate and may
stimulate both cellular and humoral immune responses and therefore, molecules responsible
for the above activity were further investigated. The localization of bioactivity in the pure
molecules and the molecular mechanism of immunostimulation were established in order to
identify their therapeutic potential for the prevention of immune disorders and infectious/
parasitic diseases where infections are often associated with immunosuppression.
95
Chapter 5
Discussion
In continuation to the identification of best immunostimulatory fraction of AS (i.e. chloroform
fraction), the chloroform fraction was further fractionated and yielded 12 pure molecules, of
which 5 were taken in the current investigation to exploit their immune modifying activities in
BALB/c mice. The stimulatory effects of these compounds on the production of the ROS and
nitric oxide were also examined. Of these, only three chemical entities, compounds 1, 2 and 5
had significant effect on the immune response of mouse, these compounds stimulated ROS and
NO production in murine peritoneal macrophages in a dose dependent manner with compound
5 displayed the utmost effect. The present findings indicate that all the three compounds may
provide a strong barrier against intracellular organisms as higher production of ROS may cause
their killing. The stimulation of murine macrophages results in the expression of an inducible
nitric oxide synthase (iNOS), which catalyzes the production of a large amount of NO from Larginine and molecular oxygen (Hibbs et al., 1987). Since, AS compound 5 best augments the
abilitity of LPS to increase macrophage activity, it could provide a second signal for
synergistic induction of NO synthesis in macrophages, and may act synergistically with other
mitogens in host defense system’s against microbial infection. Compound 5 activated both T
and B lymphocytes signifying its merit as the most active contributor of immunostimulation
thus supporting the worthiness of AS plant especially twigs to extract chemical entities that can
counter immune deficiencies. It is well known that T cells are involved in cell mediated
immunity, whereas B cells are primarily responsible for humoral immunity through production
of antigen specific antibodies that function by neutralizing extracellular microbial toxins thus
maintaining homoeostasis in the body. CD19 is expressed at relatively constant levels
throughout B cell development. The pure compounds 1, 2 and 5 of AS affected both T and B
lymphocytes via up-regulated expression of CD4, CD8 and CD19 surface markers wherein
Compound 5 exercised the uppermost efficacy at all the three doses followed by compound 2
at only 3 mg/kg and compound 1 at 1 mg/kg dose. Further investigations were made to dissect
the type of helper immune response developed, by measuring the production of cytokines,
whether Th1 or Th2 type. Th1 cytokines promote cellular immune response against
intracellular infections such as viruses, bacteria, protozoa and cancer cells while Th2 cytokines
are supportive of humoral immune response (Goldsby et al., 2000). In current investigation,
the three pure molecules extracted from AS twig stimulated the production of proinflammatory cytokines IL-2 and IFNγ, however, the increased levels were statistically
significant with only compound 5. The Th2 Cytokine IL-4 was found to be up-regulated in
mice fed with only compound 2 though statistically the difference was not significant. All the
three compounds showed up-regulated expression of another Th2 cytokine IL-10, although the
96
Chapter 5
Discussion
increase was not significant when compared with that of the controls. Compounds 3 and 4
derived from AS, on the other hand, remained ineffective and showed all the immune
parameters tested more or less comparable to control.
It may thus be summarised that AS twigs’ ethanol-extract contains active molecules that act
via Th1 and Th2 helper immune pathways. The three compounds had differential
immunomodulatory effects. It was also interesting to observe that at higher doses a compound
in fact down-regulated the immune response while at lower dose the same molecule exerted
good immunostimulation. The most pharmacologically active compound was identified to be
compound 5 and had been selected for its further evaluation as immunoprophylactant or as
chemotherapy adjunct in the current study against Brugia malay.
Withania somnifera (Ashwagandha) is a well known ethnomedicinal plant documented in India
as Ayurvedic medicine. The plant has long been applied on human beings as an anti
inflammatory, chondroprotective, cardio- protective, anti cancer, hypoglycaemic, anti
oxidant,
hypolipidaemic,
immunomodulatory
hepatoprotectant,
anti
stress, neuroprotective
or
agent (Uddin et al., 2012). However, the predominant class of its
phytochemicals i.e. withanolides possessed by the variants in the wild type and the
cultivars appear to be the same. The variations with respect to discrete sets of
individual members of withanolide group define the distinct chemotypes. Since it is well
known that the active constituents vary in the plant during different seasons and geographical
variation also has impact on the compound composition, differences in composition and
concentration of constituents along with minor modifications in chemical entities
in
various chemotypes may have led to differences in immunomodulatory properties.
Thus, it was worth investigating the pharmacological profile of chemotypes of this plant and
amongst several chemotypes received from CSIR-CIMAP, Lucknow, one of the chemotypes
i.e. 101R was found in preliminary investigations to be the most immunostimulatory variety
since it exhibited highest hemagglutinating antibody titre, plaque forming cells and delayed
type hypersensitivity reaction showing foot pad swelling in BALB/c mice after immunization
with sheep red blood cells (data not shown). The present investigation reports on
immunomodulatory efficacy of aqueous-ethanol extract from the roots of its chemotype, WS
101R raised at the experimental farm of CSIR-CIMAP at Lucknow, India, following standard
agronomic practices.
The immunomodulatory activity of WS 101R was assessed at various log doses viz. 3, 10 and
30 mg/kg and the immunostimulation was observed to be dose dependent. WS 101R induced
97
Chapter 5
Discussion
increased generation of ROS and NO in macrophages which is suggestive of a strong primary
immune response. It may be inferred that the increase in ROS and NO generation may be due
to the presence of one or more active constituents or the combined effect of some other
chemical constituents. The macrophage activation by WS chemotype 101R was further
augmented by an increased IFNγ production. WS 101R also increased the proliferative
response of splenic T and B cells which corresponded with the significant up regulation in
CD4+ TH and CD8+ TC cells and CD19+ B cells denoting their ability to stimulate both the T
and B cell populations, the major immune regulatory cells. The WS 101R was found to up
regulate both Th1 (IL-2 and IFNγ) and Th2 (IL-4 and IL-10) cytokines thereby conferring a
mixed helper immune response suggesting the presence of both Th1 and Th2 stimulating
constituents. The above response exhibited by WS 101R may be due to the predominant
presence of 17- hydroxy withaferin A and 3-hydroxy withanone which might have augmented
such responses in treated animals.
Based upon the immunostimulatory activity of WS 101R, further fractionation was undertaken
to isolate major active constituents, of which three compounds withanone, withanolide A and
withaferin A were isolated and selected for pharmacological evaluation depending upon their
yield and literature survey. These compounds were assessed for the immunomodulatory
activities in the similar way as WS 101R. Although all the three WS compounds stimulated
oxidant production, withaferin A was comparatively more effective in generating the oxidative
burst at lower dose. Macrophages play an important role in the defense mechanism against host
infection and the killing of tumor cells. Phagocytosis by activated macrophages is
accompanied by the production of O2− and H2O2 as well as the release of lysosomal enzymes
such as acid phosphatase, these products are involved in killing and digesting microbial
pathogens. The increased amount of oxidative burst represented by these compounds indirectly
indicates the probable increased intracellular organism killing activity (Pathak et al., 2011).
Among the three WS compounds, withaferin A significantly stimulated both T and B
lymphocyte proliferation at an optimum dose of 10 mg/kg while withanolide A could only
stimulate B cells and withanone only the T cell proliferative response. The CD4+ T cells
recognize antigens presented by the major histocompatibility complex class II (MHC II)
proteins and mediate both cellular and humoral immune responses through Th1 and Th2
cytokines respectively while CD8+ T cells recognize antigens presented by MHC I molecules
and mediate cellular immune responses through cytotoxic T cells. In the present study,
significant up regulation of the CD4+ and CD8+ T cell population and CD19+ B cell
population after withaferin A treatment was observed. T and B cells are considered the cardinal
98
Chapter 5
Discussion
immune cells. Their increased proliferation corresponds to activation of both the cellular and
humoral arms of the immune system and, therefore, increased protective efficacy. The type of
immune response determines the way of mounting the immune action by immune system. In
the present study, though all the three compounds incited the generation of Th1 and Th2 type
of immune response, however, withaferin A demonstrated comparatively better cytokine
stimulation. The cytokine level was found to be higher at lower doses of withanone and
opposite effect was seen as the dose increased. It is quite likely that it works better at lower
concentration. These results showed that withaferin targets both the Th1 and Th2 cells and thus
might be useful for treating pathological disorders where both pro-inflammatory and antiinflammatory immune arms are compromised. Withaferin A, thus, may be considered to be the
best immunostimulant among the three WS compounds and could have been the major
constituent responsible for immunostimulatory action of WS.
Withania coagulans, another species of medicinally important genera Withani,a commonly
known as Indian cheese maker is prevalent in dry parts of India. Its various parts have been
ascribed to possess diverse biological activities including anti-hyperglycemic activity. WC is
known to contain withanolides (withaferin-A), phenolic tannin, flavonoids and flavonols
(Hemalatha et al., 2008; Maurya, 2010). In spite of innumerable reports on the various
pharmacological properties of WC, the immunomodulatory activity in the fruit ethanolic
extract has not been investigated. The other plant Murraya koenigii, commonly known as
curry-leaf tree also falls under traditional medicine plants. This plant is found almost
everywhere in the Indian subcontinent, excluding the higher levels of the Himalayas and leaves
of this (MK) plant find wide use in kitchen as a spice and condiment in tropical countries
including India. The leaves, bark and the root are used intensively in indigenous medicine from
ancient time, as a tonic for stomachache, stimulant and carminative (Shah et al., 2008). Thus
from the literature it becomes apparent that in spite of extensive traditional use of these two
plants (i.e. WC and MK), their effect on the immune response of host especially in the
vegetative parts has not been seriously investigated which may contain different chemical
constituents extractable in different solvents.
In the present study leaves of MK and fruits of WC have been evaluated for their
immunomodulatory activities in BALB/c mice as above. MK did not reveal any noticeable
effect while WC significantly up-regulated the ROS production in peritoneal macrophages at
higher dose (30 mg/kg) which was almost comparable to that of standard immunostimulant
drug, Picroliv. However, Picroliv at 1 mg/kg, MK at 10 mg/kg and WC at all the dosed tried,
99
Chapter 5
Discussion
significantly up-regulated the NO production by the APC suggesting the strong primary
immune response in the treated animals. MK and WC could neither influence the in vitro
lymphoproliferation, nor CD4+, CD8+ or CD19+ cells. MK induced the production of Th1
cytokines IL-2 and IFNγ while WC had no apparent effect on intracellular cytokine production.
Since MK and WC extracts in preliminary evaluation did not induce noticeable immune
response at the doses tried, these two preparations were not followed further for fractionation
or their effects on filarial infection either as prophylactants or as chemotherapy adjuncts. It is
difficult to say whether these two plants lacked adequate immunomodulatory properties or it
was because of absence of active chemical moieties in the part of the plant used for evaluation
or the nature of chemical constituents present due to the type of solvent used in extraction
process. Shah and Juvekar reported inactivity of methanolic extract of MK leaves on
macrophage function including oxidant activity while aqueous leaf extract caused macrophage
activation, nitric oxide up-regulation and antibody augmentation (Shah et al., 2008) suggesting
the presence of variable pharmacological activity depending on the type of solvent used for
extraction.
Thus, among the four plants selected for immunomodulatory activity assessment in BALB/c
mice, AS and WS were found to possess strong immune stimulating properties. In recent years
use of plants or plant products as immunomodulators has gained momentum for developing
novel therapeutics against various immune disorders. Being derived from plant, these
immunomodulators are inexpensive, help in maintaining immune system homoeostasis and are
safe during prolong treatment schedules. Since the immunostimulants find its use as
immunoprophylactant or as chemotherapy adjunct to treat diseases accompanied with
immunosuppression
such
as
AIDS,
leishmaniasis,
tuberculosis
or
filariasis,
the
immunostimulatory plants such as AS and WS therefore might have the potential to stimulate
required immune functions and suppress unnecessary functions. These two plants were
therefore chosen for assessment of immunoprophylactic and chemotherapy adjunct efficacy
against a multicellular chronic helminth infection of filarial parasite Brugia malayi. Since
active B. malayi infection is accompanied with parasite driven predominant Th2 immune
response with down regulation of Th1 immune response, the mixed Th1 and Th2
immunostimulatory profile of AS and WS would complement the suppressed proinflammatory responses of B. malayi infected host as discussed in the chapter 1.
100
Chapter 5
Discussion
Filariasis, a neglected tropical disease, is a nematode borne infection. Long-lived helminth
parasites have evolved highly effective strategies to evade host immunity (Maizels, 2009).
Although highly effective microfilaricidal drugs are available to treat the infection, drugs
principally acting on adult parasites are lacking. Therefore, there is an urgent need to develop
therapeutics which may complement this activity or enhance the action of existing
microfilaricides such as DEC or ivermectin. Since L3 of filarial parasite initiates infection in
the vertebrate host, the infection can be prevented by killing L3 at the time of entry in to the
human host. The infection can thus be prevented by the use of an immunostimulatory agent
before the infection sets in. Secondly, the efficacy of drug may not be up to the mark when
immune system of the host is compromised especially those drugs which mediate their action
through the immune system of the host. Under such condition, the antifilarial drug may be
administered along with an immunostimulant as adjunct to antifilarial chemotherapy.
Mastomys coucha, is highly susceptible to a sub-periodic strain of human B. malayi. Mastomys
when administered orally with selected immunostimulatory test samples viz. withaferin A, WS
101R, compound 5 of AS or Picroliv for 14 consecutive days and challenged with B. malayi L3
showed reduced parasitic burden posing immunoprophylactic efficacy and resisted
development of filarial larvae to varying degrees. Among the immunostimulatory test samples,
WS 101R provided considerable degree of protection followed by Picroliv. The pure
withanolide i.e. withaferin A offered better protection than the crude chemotype extract of WS
101R at a very low dose of 0.3 mg/kg. To ensure that the protection offered by test samples
was because of immunostimulatory activity and not due to direct killing effect on L3, in vitro
antifilarial efficacy of the test samples were carried out to observe their lethal effects, if any on
L3 as well as on adult worms. It was thought necessary since the test samples were
administered continuously for 14 days covering both pre- and post-L3 inoculation period. All
the test samples did not show any lethal effect on L3 and adult parasites in vitro, thereby
confirming that the reduced parasite load in treated animals was due to immunostimulation
rather than in vivo antiparasitic activity of withaferin A.
Pre administration of immunostimulatory test samples led to reduced Mf load in the blood of
treated animals, which could have either been due to lower adult filarial parasite burden or due
to adverse effects of heightened immune response on embryogenesis of female parasites or Mf
release. Likewise, adult worm burden was also lowest (P <0.01) in case of withaferin A
followed by WS 101R, compound 5 of AS and Picroliv (P <0.05). Withaferin A, WS 101R and
compound 5 of AS also caused significant adult female worm sterilization with withaferin
101
Chapter 5
Discussion
demonstrating highest embryostatic effect amongst all. AS extract and its chloroform fraction
had no prophylactic effect.
The parasite-specific IgG antibody was noticed in all the treatment groups once the larval
progression to adult parasites was complete and maintained till the end of experiment. All the
test samples stimulated secretion of high amount of serum IgG titers with withaferin A
producing highest antibody level. All the samples demonstrated elevated titers of IgG1, IgG2a,
IgG2b and IgG3 antibodies, however, reduced titers of IgM and IgA antibodies. Antibodies are
characteristically induced in many parasitic infections. The class and subclass of the antibody
response is instrumental because of the distinct biological function of each isotype. It is thus
crucially important for an infected individual to mount the most appropriate secondary
antibody response that is the response that has the best chance of clearing the infection and/or
controlling disease. The role of secretory IgA is not much worked out in helminthes including
filarial infections; however, a recent study reveals that putatively immune subjects from
endemic areas contain high levels of IgA while the microfilariae carriers were deficient
indicating the role of IgA as protective antibody in human filariasis (Sahu et al., 2008). Nitric
oxide is a potent macrophage-derived effector molecule against a variety of bacteria, parasites,
and tumors (Nathan and Hibbs 1991). It is known that macrophages produce some reactive
oxygen species (ROS) during the phagocytic process, and NO is considered important for the
killing of foreign organisms (Roszell and Rice, 1998).
Significant ROS generation was
observed in all the test sample treated groups, however, NO production was increased
significantly in all groups except AS extract and fraction treated groups where no
immunoprophylactic effects were noticed. Macrophage activation was further assisted by
increased IFNγ secretion in compound 5 of AS, WS 101R and withaferin A treated groups that
might have contributed towards protection. Deficiency of IFN-γ may promote the tumor
formation. IFN-γ plays an important role to inhibit chemical carcinogenesis and to inhibit
lymphomas (Dranoff, 2004). In the present study, AS compound 5 and withaferin A have
stimulated the production of these intermediates by activated macrophages, therefore, we may
conclude that treatment with AS compound 5 and withaferin might augment innate immune
response and provide protection against parasitic infection.
Lymphocyte hypo-responsiveness during filarial infections is a well-known phenomenon
associated with both B- and T-cells (Harnett and Harnett, 2006). In the present study,
withaferin A and compound 5 of AS enhanced both the B and T cell lymphoproliferation
suggesting the accentuation of cellular immune response by these samples. It has been well
102
Chapter 5
Discussion
established that the mutant mice strains lacking T lymphocytes alone or in combination with B
cells fail to reject worms and permit them to persist to maturity (Spencer et al., 2003)
indicating the role of both T and B cells in imparting resistance to filarial parasite
establishment. B-cells are also known to play a significant role in providing resistance to the
early phases of experimental murine filariasis (Babu et al., 1999). The immune parameters
indicated significant up regulation in CD4+ T cell population in Picroliv, compound 5 of AS
and withaferin A treated groups demonstrating the ability of these samples to stimulate major
immune regulatory T helper cells. Withaferin A, being the pure withanolide was highly
effective and showed promising immune stimulation at a dose that was much less than the
crude extracts. In addition, there was also a significant increase in CD8+ cytotoxic T cells in
mastomys treated with withaferin A. The increased pool of T helper cell and cytotoxic T cell
might be a crucial factor for partial protection in mastomys since T cell hypo-responsiveness is
a well established phenomenon in filarial infections. All the test samples (except AS fraction
and WS 101R) also stimulated the CD19+ B cells representing the increased humoral response
in the treated groups. The test sample treated animals were found to develop a mixed Th1/Th2
response as evidenced by the up-regulated levels of Th1 cytokines IL-2, IFNγ and Th2
cytokines IL-4 and IL-10 along with increased titers of both IgG1 and IgG2a in serum. The
response generated by chemotype 101R could be attributed to the presence of withaferin A and
its various derivatives like 17-hydroxy- and 27-deoxy withaferin A in this chemotype
(Kushwaha et al., 2011).
Our findings thus suggest that stimulation of both Th1 and Th2 helper immune response
provide better degree of protection than either a Th1 or Th2 biased stimulation.
Immunosuppression has been earlier reported to be accompanied with invasion of infective
filarial larvae facilitating their establishment in the vertebrate host. The immunostimulation
provided by the test samples during infection mounted sufficient resistance or at least
neutralized to some extent the immunosuppression brought about by the invading larvae
interfering with its establishment, further growth and development. The administration of test
samples was done before and after L3 challenge in order to elicit host immune system for
substantial period of time since filarial parasites have a long developmental cycle. Down
regulation of immune system has been attributed to the induction of
regulatory T cells and
alternatively activated macrophages, which are able to suppress both Th1 and Th2 responses.
This might possibly be the reason of adequate protective immune response imparted by
compound 5, WS 101R and withaferin A via generation of mixed Th1/Th2 response. Taken
103
Chapter 5
Discussion
together, our findings are highly pertinent and demonstrate the usefulness of WS chemotype
101R, withaferin A, compound 5 of AS and Picroliv as immunoprophylactant.
Using immunostimulants as adjunct to antiparasitic chemotherapy may efficiently enhance the
efficacy of drugs. The plant-derived adjuvants have lesser side effects and stimulate different
arms of the immune system. Further, since the action of such immunomodulators is mediated
via the host cells, their use in chemotherapy poses a much lesser problem of side effects. In the
present study, the test samples (AS and WS) showing best immunostimulatory activities in
BALB/c mice were assessed further for their probable efficacy as adjunct to anti-filarial
chemotherapy.
Findings of the present study suggest Compound 5 of AS, crude extract of WS 101R as well as
its pure molecule withaferin A to be the best DEC adjuncts as these significantly killed adult
filarial worms and also posed sterility in good proportion of the adult female parasites.
However, the microfilaricidal effect of DEC could not be enhanced when these test samples
were used as adjunct to chemotherapy. The findings are significant in view of enhanced
adulticidal effects of DEC in combination with immunostimulants since DEC is primarily a
microfilaricidal drug. The increased embryostatic effect of DEC with immunostimulants was
also encouraging.
Interactions among immune cells and those of immune cells with the other tissues in the body
are highly diversified and mostly unexplored. The ability to counteract infections mainly
involves a three-tiered functionality: humoral immunity, cellular immunity and the regulators
of immune system, such as cytokines. In the present study, augmentation of the humoral
response was evidenced by increased antibody production (total IgG titre) in sera of mastomys
treated with compound 5 of AS and WS samples which was marginally enhanced in
combination with DEC. The enhanced responsiveness is indicative of up-regulation of
macrophages and dendritic cells involved in antibody synthesis. All the antibody isotypes such
as IgG1, IgG2a, IgG2b, IgM and IgA were increased in concentration except IgG3. Picroliv
also showed augmented level of IgM antibody when fed along with DEC, suggesting these
samples exerted adjunct efficacy not only towards parasite killing but also towards initial
humoral immune subsets. Nitric oxide and ROS production in filaria affected individuals can
actively limit the development of potentially pathological inflammatory responses via
induction of macrophage or T-cell apoptosis (Okuda et al, 1996). In the present study
significantly increased level of NO (in Picroliv and AS extract fed animals) and ROS in
immunostimulatory sample treated animals was observed which may contribute to the
104
Chapter 5
Discussion
inhibition of filarial parasite development in the infected animals. Activation of B- and Tlymphocytes is also considered an important attribute of immunostimulators (Fulzele et al.,
2002). All the test samples when fed alone stimulated the proliferation of B- and Tlymphocytes (except AS extract and fraction and WS 101R, which only stimulated B cell
proliferation) on exposure to mitogens or adult-filarial antigen, however, their combination
with DEC inversely affected the T- and B- cell proliferative responses. The function and state
of activation of different cell types can be determined by the expression or presence of CD
antigens on the cell surface. The antigen presenting cells lead to a Th1 1 or Th2 predominant
immune response. The intensity and duration of T-cell receptor signaling is also important in
influencing the T-cell subset polarization. To determine if the immune response generated by
these test samples, involves T-helper or cytotoxic cells, the splenocytes of treated mastomys
were labelled with fluorochrome-conjugated monoclonal antibodies to CD4 and CD8 cell
surface markers. The results of the present study revealed that the treatment with Picroliv,
compound 5 of AS and withaferin led to significantly (P <0.05) increased level of MHC class
II restricted CD4+ T-helper, CD8+ T-cytotoxic and CD19+ B-cells expansion and correspond to
lymphoproliferative responses. The expansion of T and B cells was augmented further when
test samples were given in combination with DEC. An early immune response to filarial
parasite involves both type 1 and type 2 cytokine secretion and complete clearance of B.
malayi is delayed in the absence of both pathways (Babu et al., 2007). In the present study,
Picroliv and withaferin fed animals demonstrated increased levels of both Th1 and Th2 type of
cytokines indicating a mixed cytokine response.
The immune response to a specific antigen must induce an appropriate set of effector functions
that can eliminate the particular pathogen involved in the infection. The chemotherapy adjunct
assay performed in the present study indicate that the immunostimulatory test samples could
revert the parasite mediated immune suppression in infected animals and potentiated humoral
as well as cell mediated immunity as evident by increased antibody titre, mitogen induced Band T- cell lymphoproliferation, increase in T and B cell population, generation of ROS and
NO by peritoneal macrophages and production of Th1 and Th2 cytokines. These test samples
strengthen the immune cells when combined with DEC and thus, enhance the parasite killing
and sterilization effects of DEC or combination of DEC mounts inhibitory effects (parasitekilling) and sterilization efficacies.
105