Download 4 results 28.06.13

Chapter 4
Results
4. RESULTS
4.1. ASSESSMENT OF IMMUNOMODULATORY ACTIVITY OF TEST
SAMPLES
The test samples were fed orally to BALB/c mice for 14 consecutive days as described in chapter
3 (Materials & Methods). A day after completion of sample dosing i.e. on day 15 of initiation of
dosing, mice were euthanized to isolate their PEC and spleens to evaluate immunomodulatory
activities by investigating the cellular immune response of test sample treated mice and compared
with that of untreated controls. The immune activation of peritoneal macrophages was assessed by
estimating the ROS and NO content and activation of T and B lymphocytes by observing in vitro
lymphocyte proliferation, expression of cell surface CD antigens and production of intracellular
Th1/ Th2 cytokines.
4.1.1. EVALUATION OF IMMUNOMODULATORY ACTIVITIES OF ANNONA
SQUAMOSA CRUDE ETHANOLIC EXTRACT AND FRACTIONS
The crude ethanolic extract and its four fractions [hexane (F1), chloroform (F2), n-butanol (F3)
and aqueous (F4)] prepared from the twigs of AS were assessed in BALB/c mice for their
immunomodulatory activities after administering at 3.0, 10 and 30 mg/kg doses. The observations
made on these test samples are as below4.1.1.1. The crude AS extract and the three fractions stimulate macrophages resulting in to
enhanced ROS and NO production in a dose dependent manner
ROS and NO are ultimate effecter molecules which are detrimental for pathogen and help the
immune system to clear the infection. Profound generation of ROS was observed in the peritoneal
macrophages isolated from mice after treatment with crude ethanolic extract (P <0.01) at all the
three doses in a dose dependent manner. The three fractions F2, F3 and F4 although showed a
dose dependent increase that was highly significant (P <0.01) at higher dose of 10 mg/kg with
highest stimulation at the highest dose tried i.e. 30 mg/kg. In contrast, fraction F1 down-regulated
the oxidative burst (Fig. 4.1 A). Simultaneously the peritoneal macrophages from crude ethanolic
extract and fractions F2, F3 and F4 treated mice also produced enhanced nitric oxide content in a
dose dependent fashion (Fig. 4.1 B). The NO production almost doubled when the cells were
stimulated with LPS overnight (Fig. 4.1 C).
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Fig: 4.1 Reactive Oxygen Species (ROS) (A) and Nitric oxide (B: unstimulated with LPS; C:
stimulated with LPS) released by peritoneal macrophages isolated from mice treated with A.
squamosa ethanolic extract and its Hexane (F1), Chloroform (F2), n-Butanol (F3) and Aqueous
(F4) fractions (3, 10 and 30 mg/ kg). Untreated control mice received vehicle under identical
conditions.
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4.1.1.2. The crude extract and three fractions of AS caused dose dependent increase in the in
vitro proliferation of T and B lymphocytes of mice
Treatment of mice with AS ethanolic extract led to a dose dependent increase in B (P <0.01) (Fig.
4.2 A) and T (P <0.05 to P <0.01) (Fig. 4.2 B) lymphocyte proliferation in vitro in presence of
nonspecific B cell (LPS) and T cell (Con A) mitogens (P <0.01). The fractions F2, F3 and F4 also
showed a dose dependent increase in cellular proliferation (Fig. 4.2 A, B), however, the data was
significant (P <0.01) only at 30 mg/kg. F1 did not promote cellular proliferation in splenic
lymphocytes (Fig. 4.2 A and B). The lymphoproliferative index was much higher in case of the
crude extract while the fractions in spite of exhibiting significant proliferation revealed lower
stimulation index than that of crude extract especially in presence of B cell mitogen, LPS (Fig. 4.2
A).
Fig: 4.2 Effect of A. squamosa extract and its Hexane (F1), Chloroform (F2), n-Butanol (F3) and
Aqueous (F4) fractions (3, 10 and 30 mg/ kg) on in vitro splenic B (A) and T lymphocyte (B)
proliferation.
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4.1.1.3. Crude ethanol extract of AS and the three fractions differentially up-regulate CD4+,
CD8+ and CD19+ splenic lymphocyte population
There was dose dependent increase in CD4+ T cell population in crude extract and F2 treated mice
with the values being significant at the highest dose of 30 mg/kg. F1 and F4 were ineffective while
F3 showed dose dependent effect but the increase was not statistically significant (Fig. 4.3 A).
Although all the fractions including the crude extract demonstrated dose dependent up-regulation
in CD8+ T cell population, all except F1 (P >0.05) brought about significant increase (P <0.01) in
the CD8+ cytotoxic T cell population. The positive modulatory effect was comparatively much
higher in case of fractions instead of the crude extract (Fig. 4.3 B). On the other hand CD19+ cells
(B-cells) demonstrated a significant expansion after treatment of mice with the crude ethanol and
one of the four fractions i.e. F2. The trend was rather decreasing as the dose increased thus higher
stimulation was achieved at the lowest dose (Fig. 4.3 C). The other fractions (F1, F3 and F4) did
not have any noticeable effect on B cells.
Fig: 4.3 Flow cytometric detection of splenic CD4+ (A), CD8+ (B) T cell sub-population and
CD19+ (C) B cell population in mice fed with various doses (3, 10 and 30 mg/kg) of A. squamosa
extract and its Hexane (F1), Chloroform (F2), n-Butanol (F3) and Aqueous (F4) fractions.
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4.1.1.4. Effect on intracellular Th1 and Th2 cytokines (Flow cytometric analysis)
Measurement of intracellular Th1 and Th2 cytokines also acts as an important parameter for
immunomodulation indicating the nature of T cell response whether pro-inflammatory (Th1) or
anti-inflammatory (Th2) type. The proinflammatory Th1 cytokine contents (IL-2 and IFNγ) were
found to be increased intracellularly in the splenic cell population after treatment of mice with
crude extract and F2 fraction in contrast to F3 and F4 fractions (Fig. 4.4 A and B). In case of IL-2,
F3 and F4 fractions significantly down-regulated the cytokine production at all the three doses
while in case of IFNγ, the decrease in cytokine content was not significant. The Th2 (antiinflammatory cytokines IL-4 and IL-10), however, were observed to be raised in crude extract, F2,
F3 and F4 fractions. Nevertheless, these followed a dose dependent decreasing pattern in spite of
being higher than that of untreated control (Fig. 4.4 C and D). The fractions appeared much more
effective than the crude preparation specifically as regards to Th2 cytokine response. Amongst the
fractions, F2 appeared best. Fraction F1 did neither influence Th1 nor Th2 cytokine responses.
Fig: 4.4 Flow cytometric detection of intracellular type 1 cytokines IL-2 (A) and IFNγ (B), and
type 2 cytokines IL-4 (C) and IL-10 (D) in splenocytes of mice fed with various doses (3, 10 and
30 mg/ kg) of A. squamosa extract and fractions Hexane (F1), Chloroform (F2), n-Butanol (F3)
and Aqueous (F4).
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A comparative list of immunological responses in BALB/c mice activated by the AS crude
ethanolic extract and its four chromatographic fractions is presented in Table-1 which depicts the
fold increase in each immune parameter. This table shows that the crude extract along with the
three fractions (F2, F3, F4) stimulate both T and B cell proliferation. All the fractions except F1
stimulate CD8+ T cell. Both the crude extract as well as F2 stimulate the production of both Th1
and Th2 cytokines while F3 and F4 fractions principally induce the production of Th2 cytokines.
Based upon the comparative fold increase in various immunological parameters, the AS
chloroform fraction (F2) was selected and explored further to identify the active molecules
responsible for the immunomodulatory activities.
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Table – 1: Fold Increase in immune parameters of Annona squamosa ethanolic extract and its four fractions (Hexane, Chloroform, nButanol and Aqueous) treated BALB/c mice compared to their respective untreated groups after a 14 days oral dose schedule.
The increases at least two times or more are indicated in bold font.
Sl.
Parameters
No.
Annona squamosa
Hexane Fraction
Chloroform
n-Butanol
Aqueous
Ethanol Extract
(F1)
Fraction (F2) Dose
Fraction (F3) Dose
Fraction (F4) Dose
Dose (mg/kg)
Dose (mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
3
1.
10
ROS synthesis 2.623 3.153
30
3
10
30
3
10
30
3
10
30
3
10
30
3.797
0.511
0.554
0.619
1.969
2.707
3.952
1.484
1.891
3.699
4.038
4.978
6.553
by activated
Macrophages
2.
B Cell
2.114 2.282
2.504
0.854
0.971
1.043
1.305
1.461
2.015
1.133
1.399
1.816
1.375
1.647
2.070
3.
T Cell
1.103 1.322
1.727
0.910
0.971
1.181
1.257
1.597
1.867
0.823
0.975
1.604
0.957
1.247
1.701
4.
CD4
1.13
1.241
1.405
1.003
0.961
1.037
1.135
1.220
1.466
0.969
1.197
1.319
1.024
1.008
1.020
5.
CD8
1.327 1.373
1.478
1.085
1.087
1.165
1.562
1.735
2.082
1.761
2.331
2.871
2.083
2.202
2.525
6.
CD19
1.309 1.287
1.106
1.148
1.083
1.093
1.329
1.107
1.054
1.206
0.987
0.962
1.129
1.217
1.148
7.
IL-2
1.194 4.610
5.549
0.883
0.830
0.874
1.252
1.608
1.836
0.640
0.552
0.495
0.552
0.504
0.486
8.
IL-4
2.124 1.428
0.822
0.848
0.679
0.630
2.121
1.801
1.266
1.371
1.418
1.976
1.514
1.349
1.038
9.
IL-10
1.794 1.103
0.684
0.885
0.683
0.686
2.778
2.499
2.193
1.630
1.712
2.049
2.278
2.269
1.836
10.
IFNγ
1.283 1.954
2.101
1.013
0.923
1.074
0.846
1.634
2.388
1.080
0.657
0.522
0.375
0.737
0.876
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4.1.2. EVALUATION
Results
OF
IMMUNOMODULATORY
ACTIVITIES
OF
PURE
MOLECULES ISOLATED FROM AS CHLOROFORM FRACTION
From the above immunomodulatory activities of extract and fractions of AS (table 1), it was
clear that its chloroform fraction was most promising immunostimulant. This bioactivity in
chloroform fraction pressed us to isolate pure compounds from this fraction and identify the
lead responsible for the immunostimulatory activity. A total of 12 pure compounds were
isolated from chloroform fraction of which five pure compounds Lanuginosine (1), (+) –O–
methylarmepavine (2), (+)–anomuricine (3), Isocorydine (4), and N-methyl-6, 7dimethoxyisoquinolone (5) were assessed for their immunomodulatory activities in the current
study.
4.1.2.1. Compounds 1, 2 and 5 isolated from chloroform fraction of AS up-regulate the
production of ROS and NO in macrophages
Of the five compounds, three (Compound 1, 2 and 5) were found to induce the production of
ROS in peritoneal macrophages in a dose dependent manner. Compound 1 significantly
increased (P <0.05) ROS generation at 1.0 and 3.0 mg/kg while compound 2 significantly
increased (P <0.01) the oxidative burst at 3.0 mg/kg dose. Compound 5 however, caused
highly significant (P <0.001) oxidative burst at both 1.0 and 3.0 mg/kg doses. Treatment with
compounds 3 and 4 did not lead to significant ROS generation and represented more or less
activity similar to untreated controls. The effect of standard immunostimulant compound
Picroliv had comparatively inferior activity at the single dose tried (P >0.05) (Fig. 4.5 A).
Macrophages from compounds 1 and 2 at the dose of 3 mg/kg, compound 5 at 1 and 3 mg/kg
doses and Picroliv at 1 mg/kg dose were found to produce highly significant (P <0.01) amount
of nitric oxide spontaneously and when stimulated with LPS invariably at the highest dose
tried, however, compounds 3 and 4 did not produce significant amount of nitric oxide at all the
doses tried (Fig. 4.5 B and C). Thus two of the compounds viz. 3 and 4 had negligible oxidant
activity.
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Fig: 4.5 Reactive Oxygen Species (ROS) (A) and Nitric oxide (B: without mitogen
stimulation; and C: with LPS stimulation) released by peritoneal macrophages isolated from
mice treated with compounds 1, 2, 3, 4 and 5 (each at 0.3, 1.0 and 3.0 mg/kg doses) of A.
squamosa and Picroliv (Pic; 1.0 mg/kg). Untreated control mice received vehicle under
identical conditions.
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4.1.2.2. Compounds 1, 2 and 5 present in the chloroform fraction of AS enhanced in vitro
lymphocyte proliferation
Treatment of mice with compounds 1, 2 and 5 led to dose dependent increase in splenic B and
T lymphocyte proliferation in vitro (Fig. 4.6 A and B) in presence of nonspecific mitogens LPS
and Con A respectively. Of the three compounds, treatment with compound 1 and 2 enhanced
B cell proliferation (P <0.01) while compound 5 augmented the proliferation of both classes of
lymphocytes and these values were highly significant at the highest dose tried (3.0 mg/kg).
Compounds 3 and 4 did not demonstrate cellular proliferation. The effect of Picroliv was
comparable to that of compound 5 though at a lower dose of 1 mg/kg (Fig. 4.6 A and B).
Fig: 4.6 Effect of A. squamosa compounds 1, 2, 3, 4 and 5 on in vitro splenic B (A) and T (B)
lymphocyte proliferation. Picroliv (Pic) served as reference immunostimulant drug. Untreated
control mice (Ctl) received vehicle under identical conditions.
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4.1.2.3. Compounds 1, 2 and 5 isolated from chloroform fraction of AS up-regulate the Band T-cell population in mouse spleen
A dose dependent increase in the number of CD4+, CD8+ T cells and CD19+ B cells was
observed in groups of mice fed with compounds 2 and 5 except that compound 5 led to highly
significant increase (P <0.01) at all the three doses (Fig. 4.7 A, B and C) while compound 2
showed comparable effect at 3.0 mg dose except CD8+ cells where the difference was highly
significant (P <0.01) at 1 mg dose too. Compound 1 on the other hand, significantly upregulated only CD8+ T cells at 1.0 and 3.0 mg without any noticeable effect on CD4+ or
CD19+ cells (Fig. 4.7 B). Compounds 3 and 4 did not demonstrate any significant expansion in
the lymphocyte population. Picroliv had significant effect in the proliferation of CD4+
population (P <0.01) but not on CD8+ or CD19+ cells. Compound 5 thus appeared to offer
best immunostimulatory effect on T and B cells and was superior to Picroliv.
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Fig: 4.7 Flow cytometric measurements of splenic CD4+ (A) and CD8+ (B) T cell subpopulations and CD19+ (C) B-cell populations in mice fed with various doses (0.3, 1.0 and 3.0
mg/kg) of A. squamosa compounds 1, 2, 3, 4 and 5. Picroliv (Pic) served as reference
immunostimulant drug. Untreated control mice (Ctl) received vehicle under identical
conditions.
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4.1.2.4. Compound 5 significantly up-regulates the production of Th1 cytokine
Treatment with compound 5 predominantly led to a dose dependent increase (P <0.01) in Th1
cytokine production viz. IL-2 and IFNγ (Fig. 4.8 A and B). Compounds 1 and 2 were
ineffective in eliciting any noticeable increase in Th1 or Th2 response (Fig. 4.8 C and D)
though statistically insignificant increase in IL-4 and IL-10 in case of compound 2 and Il-10 in
case of compound 1 could be seen. Picroliv significantly stimulated IFNγ at 1 mg/kg while an
increase noticed in case of IL-10 proved statistically not significant (Fig. 4.8 B).
Fig: 4.8 Flow cytometric detection of intracellular type 1 cytokines IL-2 (A) & IFNγ (B) and
type 2 cytokines IL-4 (C) and IL-10 (D) in splenocytes of mice fed with various doses (0.3, 1.0
and 3.0 mg/kg) of A. squamosa compounds 1, 2, 3, 4 and 5. Picroliv (Pic; 1.0 mg/kg) served as
standard immunostimulant drug. Untreated control mice (Ctl) received vehicle under identical
conditions.
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Of the five compounds of AS evaluated in the current study, only three (compound 1, 2 and 5)
revealed immune modifying properties with one of these (compound 5) demonstrating most
noticeable effect on Th1 immunity. Thus, from the above observations it is clear that the lead
immunostimulatory molecule in AS twigs is compound 5 which in the current study was
evaluated further against human lymphatic filarial parasite B. malayi as immunoprophylactant
and chemotherapy adjunct.
4.1.3. EVALUATION OF IMMUNOMODULATORY ACTIVITIES IN ROOT
EXTRACT OF WS CHEMOTYPE 101R (WS 101R)
Naïve mice were fed with the crude extract of WS chemotype 101R at three doses i.e. 3, 10
and 30 mg/kg for 14 consecutive days. The cells were isolated on day 15 for studying the
effect of sample on immune response of mice.
4.1.3.1. Peritoneal macrophages from WS 101R fed mice produced enhanced ROS and
NO
Peritoneal macrophages isolated from WS 101R fed mice produced considerable amount of
ROS (Fig. 4.9 A) and NO (Fig. 4.9 B) in dose dependent manner and the increase in oxidants
was highest at the highest dose of 30 mg/kg used (P < 0.01). NO production further increased
significantly (P < 0.01) at 10 and 30 mg doses after induction with LPS. The increase in the
content of ROS and NO was however not significant at lower doses (3 and 10 mg in case of
ROS and 3 mg in case of NO) (Fig. 4.9 A and B).
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Fig: 4.9 Reactive Oxygen Species (ROS) (A) and Nitric oxide (B) released by peritoneal
macrophages of mice treated with W. somnifera chemotype (WS 101R) crude aqueous-ethanol
extract. Each histogram represents the value of an individual mouse in that particular group
and value is closer to the mean.
4.1.3.2. WS 101R root extract stimulates T and B cell proliferation in vitro
Treatment of mice with WS 101R root extract led to dose dependent increase in splenic B and
T lymphocyte proliferation in vitro in presence of LPS (Fig. 4.10 A) and Con A (Fig. 4.10 B)
respectively. The values were significant (P <0.05) at 10 mg dose and highly significant at a
higher dose of 30.0 mg/kg. Dose 3 mg/kg was not much effective when compared with that of
vehicle treated control.
Fig: 4.10 Effect of W. somnifera chemotype (WS 101R) root extract on in vitro splenic B (A)
and T (B) lymphocyte proliferation.
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4.1.3.3. WS 101R stimulates splenic T and B cell proliferation in treated mice
Splenocytes isolated from the test sample fed mice showed differential modulation in the
percentage of T and B cells. The upregulation in CD4+ T cells in treated mice was visibly
higher at 10 and 30 mg doses over that of control group (Fig. 4.11 A; P < 0.01). WS 101R
fed mice exhibited higher CD8+ population at all the three doses including 3.0 mg/kg (Fig.
4.11 B; P < 0.01). Thus WS 101R seems to be a potent stimulator of both T helper cells as well
as cytotoxic T cells. The number of antibody producing CD19+ cells (humoral response)
increased in treated group of mice in a dose dependent manner. The increased expansion of
CD19+ cells was even highly significant at the lowest dose of 3.0 mg tried (Fig. 4.11 C; P <
0.01). The cellular stimulation was almost similar at the two doses viz. 10 and 30 mg/kg
suggesting former as the optimal immune stimulatory dose.
Fig: 4.11 Flow cytometric analysis of CD4+ (A) & CD8+ (B) T and CD19+ (C) B lymphocyte
proliferation in mice fed with W. somnifera chemotype (WS 101R) root extract. Each
histogram represents the value of an individual mouse in that particular group and value is
closer to the mean.
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4.1.3.4. WS 101R displayed mixed cytokine response
In the current study the WS chemotype 101R crude extract fed mice showed increased
population of CD4+ cells producing both the Th1 (IL-2 and IFNγ) (Fig. 4.12 A and B) and
Th2 cytokines (IL-4 and IL-10) (Fig. 4.12 C and D) at 10 and 30 mg/kg doses. The increase
was statistically significant at 10 mg and highly significant at 30 mg/kg dose for Th1 cytokines
while Th2 cytokine up-regulation was highly significant at both 10 and 30 mg doses. The
chemotype although stimulated the production of both Th1 and Th2 cytokines, the increase
was not significant at the lower dose of 3 mg. The WS chemotype 101R crude extract thus
revealed a mixed Th1 and Th2 immune response.
Fig: 4.12 Efficacy of W. somnifera chemotype (WS 101R) root extract on intracellular
Th1/Th2 cytokine IL-2 (A), IFNγ (B), IL-4 (C) and IL-10 (D) expression. Each histogram
represents the value of an individual mouse in that particular group and value is closer to the
mean.
4.1.4. EVALUATION OF IMMUNOMODULATORY ACTIVITIES OF PURE
COMPOUNDS ISOLATED FROM WS 101R
Three of the pure molecules isolated from WS 101R were evaluated in BALB/c mice in the
same way as crude extract, however, the doses used were much reduced (0.1, 0.3 and 1 mg/kg)
because of being the pure compound. These three pure molecules were Withaferin A,
Withanolide A and Withanone.
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4.1.4.1. Macrophages from withaferin A fed mice produced significantly enhanced ROS
and NO at a comparatively lower dose
Peritoneal macrophages isolated from mice treated with the three pure molecules enhanced
production of ROS (Fig. 4.13 A) and NO (Fig. 4.13 B). Addition of LPS to the cells led to
more increase in the oxidants at decreased dose of 0.1 mg/kg. The data from the three groups
were more or less similar and the increase in oxidant contents was statistically significant (P <
0.05 to p < 0.01). Interestingly, the enhanced oxidative burst was even evident at a very low
dose of 0.1 mg/kg in case of withaferin A but not in other two compounds.
Fig: 4.13 Reactive Oxygen Species (A) and Nitric oxide (B) released by peritoneal
macrophages of mice treated with withanolides.
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4.1.4.2. Withaferin A isolated from WS 101R stimulates both the T and B cell
proliferation in vitro
Treatment of mice with withaferin A led to increased B and T lymphocyte proliferation in vitro
in presence of LPS (Fig. 4.14 A) and Con A (Fig. 4.14 B), respectively. Withaferin A
significantly augmented the B cell proliferation at 0.3 and 1.0 mg doses (Fig. 4.14 A, P <0.01).
On contrary withanone could induce T cell proliferation only that too at a minimum dose of 0.3
mg/kg (Fig. 4.14 B, P <0.01). Withanolide A had a positive influence only on B cells that too
at the high dose of 1 mg/kg (Fig. 4.14 B, P <0.01). Results thereby suggest withaferin A to be
the best T and B cell stimulator.
Fig: 4.14 Effect of withanolides (isolated from W. somnifera 101R) on in vitro splenic B (A)
and T (B) lymphocyte proliferation.
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4.1.4.3. Withaferin A stimulates splenic T cell sub population and antibody forming B
cells in mice
The three compounds showed variable efficacy on CD4+, CD8+ and CD19+ lymphocytes.
Withanone induced only CD19+ B cell population at the highest dose of 1 mg/kg without
altering T cell populations (Fig. 4.15 A, B and C; P < 0.01). Withanolide A on contrary
induced the T helper (CD4+) and T cytotoxic (CD8+) lymphocytes. Withaferin A conferred
highly significant expansion in all the three cell phenotypes i.e. CD4+, CD8+ and CD19+ cells
at the minimum dose ranging between 0.1 and 0.3 mg/kg. (Fig. 4.15 B; P < 0.01). Thus, data
revealed withaferin A to be the most potent immunostimulatory enhancing both the arms of
immune response.
Fig: 4.15 Flow cytometric analysis of splenocytes for CD4+ (A),
CD8+ (B) T cells and
CD19+ (C) B lymphocytes, isolated from withanolides treated and untreated control mice.
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4.1.4.4. Withaferin A displayed comparatively better mixed cytokine response
Splenocytes isolated from test sample fed mice showed variable mixed cytokine response. All
the three pure molecules in general induced a mixed type of cytokine response enhancing the
intracellular levels of both Th1 and Th2 cytokines at a minimum lowest dose tried (0.1 mg/kg).
Of the three compounds, withaferin enhanced the content of IL-2, IFNγ, IL-4 and IL-10 while
in contrast withanone up-regulated the level of IFNγ, IL-4 and IL-10 while withanolides A
enhanced IL-4 and IFNγ at 0.1 mg/kg and IL-10 at a high dose of 1 mg/kg. Thus, after a
comparison, withaferin A appeared marginally better amongst the three pure compounds
isolated from WS chemotype 101R (Fig. 4.16 A, B, C and D).
Fig: 4.16 Efficacy of withanolides (isolated from W. somnifera 101R) on expression of
intracellular Th1 and Th2 cytokines IL-2 (A), IFNγ (B) IL-4 (C) and IL-10 (D).
4.1.5. EVALUATION OF IMMUNOMODULATORY ACTIVITIES OF WC AND MK
Ethanolic extracts prepared from the leaves of Murraya koenigii (MK) and fruits of Withania
coagulans (WC) were evaluated for their immunomodulatory activities in BALB/c mice. The
extracts were fed orally at various log doses viz. 3.0, 10.0 and 30.0 mg/kg of body weight to
groups of mice for 14 consecutive days as described in chapter 3 (Materials and Methods).
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4.1.5.1. WC induced ROS generation by PECs
The extract derived from WC induced ROS generation in the peritoneal macrophages in a dose
dependent fashion, however, the values were statistically significant only at 30 mg/kg dose
(Fig. 4.17 A, P <0.01). PECs also produced significant amount of NO at all the doses tried
(Fig. 4.17 B, P <0.01). MK did not have any effect on ROS generation at any dose tried (P
>0.05). The standard immunostimulatory compound, Picroliv (positive control) also
represented significant oxidative burst in the peritoneal macrophages (P <0.01) at 1.0 mg/kg
tried (Fig. 4.17 A and B).
Fig: 4.17 Reactive Oxygen Species (A) and Nitric oxide (B) released by peritoneal
macrophages isolated from mice treated with Murraya koenigii (MK) leaf, Withania coagulans
(WC) fruit ethanolic extracts (3, 10 and 30 mg/kg) and Picroliv (1 mg/kg).
4.1.5.2. MK and WC extracts failed to stimulate in vitro lymphoproliferation
The extracts from the plants MK and WC did not demonstrate cellular proliferation at any dose
when compared to that of untreated controls. Picroliv on the other hand significantly (P <0.05)
induced both B (Fig. 4.18 A) and T (Fig .4.18 B) cell proliferation though at a much lower
dose.
Fig: 5.18 Effect of Murraya koenigii (MK) leaf and W. coagulans (WC) fruit ethanolic
extracts (3, 10 and 30 mg/kg) and Picroliv (1 mg/kg) on splenic B- (A) and T-lymphocyte (B)
proliferation in treated animals.
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4.1.5.3. Effect of MK and WC on T-cell sub-population and B cells
None of the two plant extracts exhibited any significant effects on T or B cell population or
their in vitro proliferation at any of the three doses tried. Picroliv on the other hand upregulated the CD4+, CD8+ and CD19+ lymphocyte population at the tested dose of 1 mg/kg
which was significantly higher than those observed in untreated control mice (P < 0.5)The
picroliv treated group represented significant proliferation of CD4 (Fig. 4.19 A), CD8 (Fig.
4.19 B) and CD19 positive cells (P <0.05) (Fig. 4.19 C).
Fig: 4.19 Flow cytometric detection of splenic CD4+ (A), CD8+ (B) T cell sub-populations
and CD19+ B-cell (C) population in untreated control (Ctl), Murraya koenigii (MK) leaf
extract, W. coagulans (WC) fruit extract and Picroliv fed mice.
4.1.5.4. Effect on production of Th1 and Th2 cytokines
The population of CD4+ T cells producing both Th1 (IFNγ) and Th2 (IL-10) type of cytokines
was assessed by FACS. Both pro-inflammatory (IFNγ) and anti-inflammatory (IL-10) classes
of cytokines were up-regulated intracellularly in the spleen cells of Picroliv treated mice. The
extract prepared from MK, however, significantly stimulated the production of Th1 cytokines
IL-2 and IFNy. IL-2 induction was seen at 3 and 10 mg/kg while IFNγ production increased
only at 10 mg/kg dose when compared with that of untreated control animals. WC on the other
hand did not exhibit any immunomodulatory properties in terms of their effect on cytokine
response (Fig. 4.20 A, B, C and D).
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Fig: 4.20 Effect of Murraya koenigii (MK) leaf and W. coagulans (WC) fruit ethanolic
extracts on expression of intracellular Th1/Th2 cytokines IL-2 (A), IFNγ (B), IL-4 (C) and IL10 (D).
4.2. IN VITRO CYTOTOXICITY AND ANTIFILARIAL ACTIVITY OF
TEST SAMPLES
To further ensure that the protective and chemotherapy adjunct activities of test samples are
not due to their cytotoxic effects, assay for checking the cytotoxicity of all the test samples was
performed using fluorescent dye resazurin to evaluate the inhibitory concentration at which
50% of the cells under investigation became dead (CC-50) as described earlier in chapter 3
(materials and methods). All the test samples were safe showing more than 200 μg/ml CC-50
in vitro on vero cell line. Standard antifilarial drug, Ivermectin showed CC-50 values of 63.12
μg/ml.
To ensure the immunoprophylactic and chemotherapy adjunct activities of the test samples
correspond to immunostimulation and not due to their direct antifilarial effects, the crude
extracts were assessed in vitro on adult and L3 stage of B. malayi for their antifilarial efficacy.
All the extracts tested did not show inhibition in worm motility at all the concentrations tried
and the adult and L3 were found active almost comparable to controls (Table – 2).
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Table – 2: In vitro antifilarial efficacy of crude extracts of Annona squamosa, Murraya
koenigii, Withania coagulans and Withania somnifera chemotype 101R. The test samples were
were dispensed at different concentrations (62.5 to 7.8 µg/ ml) into duplicate wells of a 12 well
culture plate containing L3 or adult female Brugia malayi in DMEM medium. The worms were
incubated for 48h at 37 °C in CO2 incubator and observed for motility (where, 4: no reduction;
3: 1-49% reduction; 2: 50-74%; 1: 75-99% reduction and D: 100% reduction).
Sl.
No.
Test samples
Parasite
stage
Concentrations
(µg/ml)
Motility score
(Duplicate)
1.
Control/ media only
Adult
L3
---
4, 4
4, 4
2.
Ivermectin
3.
Annona squamosa
Adult
L3
Adult
10.00
10.00
62.50
31.25
15.60
7.80
1, 2
D, D
4, 3
4, 4
3, 3
4, 4
62.50
31.25
15.60
3, 4
4, 4
4, 4
7.80
4, 4
62.50
31.25
15.60
7.80
3, 3
4, 4
4, 4
4, 3
62.50
31.25
15.60
7.80
3, 4
4, 4
4, 4
3, 4
Adult
62.50
31.25
3, 4
3, 4
L3
15.60
7.80
4, 4
4, 4
L3
4.
Murraya koenigii
Adult
L3
5.
Withania coagulans
Adult
L3
6.
Withania somnifera
101R
Adult
L3
7.
Picroliv
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4.3. ASSESSMENT OF IMMUNOPROPHYLACTIC ACTIVITY OF
ACTIVE TEST SAMPLES AGAINST B. MALAYI
On the basis of immunomodulatory activities exhibited by the WS, AS and their fractions/ pure
compounds, the most active samples were chosen to assess their immunoprophylactic and
chemotherapy adjunct effects (at the optimum immunostimulatory dose) against B. malayi in
susceptible rodent host M. coucha as detailed in chapter 3 (Materials and methods).
4.3.1. WITHAFERIN A FOLLOWED BY CHEMOTYPE 101R AND AS PURE
COMPOUND 5 EXHIBITED THE BEST IMMUNOPROPHYLACTIC EFFICACY IN
MASTOMYS
The appearance of microfilaria started after 90 days post L3 challenge in blood circulation of
mastomys. Monitoring of the course of microfilaraemia was continued till day 180 following
which all animals of each group were euthanized for the recovery of adult worms. The Mf
density was lower (P <0.01) in all the experimental groups during initial monitoring (day 120)
which subsequently increased in all the groups but was comparatively lower than vehicle
control (Fig. 4.21 A). Amongst various treated groups, microfilaraemia remained significantly
low in withaferin and WS 101R fed animals followed by standard immunostimulant, Picroliv
(P <0.01) and compound 5 of AS (P <0.05) when compared with that of vehicle treated control
group. The crude extract and chloroform fraction of AS were not much effective.
In addition to the protective effect on resulting Mf, prophylactically treated mastomys also
demonstrated adverse effect of pretreatment on further establishment of challenged larvae in to
adult parasites. Mastomys fed with withaferin A (P <0.01) and WS 101R followed by AS
compound 5 and Picroliv (P <0.05) were found to harbor significantly reduced number of adult
parasites showing withaferin A to be most effective followed by WS 101R and Picroliv
respectively when compared to that of control (Fig. 4.21 B). In terms of percentage of adult
female worm fecundity, mastomys fed with withaferin and WS 101R (P <0.01) followed by
AS compound 5 (P <0.05) exerted significant adverse effect in the intrauterine embryogenesis.
AS extract and fraction neither had any significant reduction in adult worm establishment nor
in the female worm fecundity (Fig. 4.21 C).
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Fig: 4.21 Microfilaraemea counts (A), adult worm recovery (B), and female worm sterilization
(%) (C) in mastomys fed orally with Annona squamosa crude extract (ASE), AS chloroform
fraction (ASF), AS compound 5 (ASC), Withania somnifera chemotype 101R extract (WSE),
withaferin A (WFN) or Picroliv (Pic) at their optimum immunostimulatory doses and untreated
controls (Ctl).
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4.3.2. ANTIBODY ISOTYPES REVEAL MIXED TH1/ TH2 TYPE OF IMMUNE
RESPONSE
Treatment with withaferin A followed by WS 101R and AS compound 5 led to ample
generation of filaria-specific IgG antibody, whose levels were maintained in all treated groups
until the day of autopsy. The highest level of filaria-specific IgG antibody was recorded in
Withaferin A treated group (Fig. 4.22 A). Serum IgG isotype levels in AS compound 5,
Withaferin A and WS 101R treated animals demonstrated a significant increase in specific
IgG1 and IgG2a levels (Fig. 4.22 B). Feeding with AS extract, fraction or Picroliv did not
exhibit such high IgG levels which were more similar to that of vehicle fed L3 exposed group.
Regarding IgG2b, IgG3, IgM and IgA, these represented more or les similar titres compared
with vehicle treated controls on day of autopsy i.e. on day 181 (Fig. 4.22 C). In general,
compound 5 from AS revealed best antibody generation.
Fig: 4.22 Total IgG content/ titre (A) and generation of filaria specific antibody isotypes (B
and C) in mastomys fed orally with Annona squamosa crude ethanolic extract (ASE, 30
mg/kg), chloroform fraction (ASF, 30 mg/kg), compound 5 (ASC, 3 mg/kg), Withania
somnifera chemotype 101R extract (WSE, 10 mg/kg), withaferin (WFN, 0.3 mg/kg) or Picroliv
(Pic, 1.0 mg/kg) at their respective optimum immunostimulatory doses and untreated controls
(Ctl).
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4.3.3. PERITONEAL MACROPHAGES OF MASTOMYS PRODUCE SIGNIFICANT
AMOUNT OF OXIDANTS WHEN TREATED WITH TEST SAMPLES
Profound up-regulation in ROS production (P <0.05 to P <0.01) was noticed in peritoneal
macrophages isolated from mastomys fed with all the test samples. Among these, compound 5
purified from AS, withaferin A and Picroliv induced highly significant (P <0.01) ROS
generation. The other samples were also efficient ROS inducers (P <0.05) (Fig. 4.23 A). The
nitric oxide production also showed almost similar pattern when estimated by Griess reagent
which increased further after LPS stimulation (Fig. 4.23 B).
Fig: 4.23 Reactive oxygen species (A) and nitric oxide (B) released by peritoneal macrophages
isolated from mastomys fed orally with Annona squamosa crude ethanolic extract (ASE),
chloroform fraction (ASF), compound 5 (ASC), Withania somnifera chemotype 101R extract
(WSE), withaferin (WFN) or Picroliv (Pic) at their respective optimum immunostimulatory
doses and untreated controls (Ctl).
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4.3.4. TREATMENT WITH WITHAFERIN A AND AS COMPOUND 5 LEAD TO
SIGNIFICANT IN VITRO LYMPHOPROLIFERATION
The proliferative response of splenocytes harvested from test sample fed mastomys was tested
against B-cell mitogen LPS (Fig. 4.24 A), T-cell mitogen Con A (Fig. 4.24 B) and adult filarial
somatic antigen (Fig. 4.24 C) and compared with that of vehicle treated mastomys. Withaferin
(P <0.01) and AS compound 5 (P <0.05) exhibited an increased B cell proliferation. Picroliv,
AS extract and WS 101R also exerted B-cell stimulation, however, the stimulation was not
significant (Fig. 4.24 A). Withaferin followed by WS 101R exerted the highest stimulation of
T-cell proliferation over control (Fig. 4.24 B) and Picroliv, AS extract and compound 5 though
significantly induced T cell proliferation (P <0.05), however, proved inferior to withaferin A.
In vivo treatment of animals with all the test samples could bring about significant filariaspecific lymphoproliferation (Fig. 4.24 C).
Fig: 4.24 Effect of Annona squamosa crude ethanolic extract (ASE), chloroform fraction
(ASF), compound 5 (ASC), Withania somnifera chemotype 101R extract (WSE), withaferin
(WFN) and Picroliv (Pic) on in vitro splenic B- (A) and T-lymphocyte (B) proliferation.
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Chapter 4
4.3.5.
TEST
Results
SAMPLES
DIFFERENTIALLY
UP-REGULATE
T
AND
B
LYMPHOCYTE POPULATIONS
A significant up-regulation of CD4+ T helper cell population (P <0.01) was observed in the
spleens of withaferin fed mastomys followed by AS compound 5 and Picroliv (both P <0.05)
fed animals (Fig. 4.25 A). The CD8+ cytotoxic T cell population was also augmented (P
<0.01) in Withaferin A treated animals, however, none of the other test samples could augment
the CD8+ T cell proliferation (Fig. 4.25 B). AS extract and fraction could not exert significant
stimulatory effect either on CD4+ or CD8+ T cell population in treated animals although all
the test samples showed increase in T cell population. Except WS 101R and AS fraction,
treatment with all other test samples resulted in expansion of CD19+ B cells (P <0.05 to P
<0.01). AS compound 5 exerted the maximal stimulation of CD19+ B cells (P <0.01) followed
by Picroliv, AS extract and withaferin (Fig. 4.25 C). The results thus conclude that withaferin
A induced both T helper and T cytotoxic cells along with antibody producing cells.
Fig: 4.25 Flow cytometric detection of CD4+ (A), CD8+ (B) T cell subpopulations and CD19+
B cell (C) population in splenocytes of vehicle treated/ control (Ctl), Annona squamosa crude
ethanolic extract (ASE), chloroform fraction (ASF), compound 5 (ASC), Withania somnifera
chemotype 101R extract (WSE), withaferin (WFN) and Picroliv (Pic) fed animals.
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Chapter 4
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4.3.6. CHEMOTYPE 101R, WITHAFERIN A AND AS EXTRACT GENERATE A
MIXED TH1/TH2 RESPONSE
To examine whether the early T-cell response obtained in lymphoproliferation assay translates
into a functional response, the levels of both pro-inflammatory and anti-inflammatory
cytokines were determined intracellularly in the splenic cell population of test sample fed and
vehicle treated mastomys by FACS. There was an increase in the level of both types of
cytokines viz. IL-2, IFNγ and IL-4, IL-10 in 101R, Withaferin A and AS extract fed animals
when labeled cells measured by flow cytometry indicating production of a mixed Th1/Th2 type
of immune response. Among these, withaferin A appeared to be the best immunostimulator (P
<0.01) followed by WS 101R (P <0.05 to P <0.01). Compound 5 isolated from AS, however,
could induce predominantly the Th1 cytokines IL-2 and IFNγ (P <0.05). The standard
immunomodulator, Picroliv though showed immunostimulation, however, the activity was
much inferior to withaferin and even to WS 101R (Fig. 4.26 A, B, C and D).
Fig: 4.26 Effect of oral feeding of Annona squamosa crude ethanolic extract (ASE), AS
chloroform fraction (ASF), AS compound 5 (ASC), Withania somnifera chemotype 101R
extract (WSE), withaferin (WFN) and Picroliv (Pic) on intracellular Th1/Th2 cytokine
expression of test sample fed and vehicle treated/ control (Ctl) animals.
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4.4. ASSESSMENT OF CHEMOTHERAPY ADJUNCT EFFICACY OF
ACTIVE
TEST
SAMPLES
AGAINST
LYMPHATIC
FILARIAL
PARASITE B. MALAYI IN RODENT MODEL
To assess the chemotherapy adjunct efficacy of immunostimulatory test samples, the samples
were fed orally to infected mastomys showing patent microfilaraemia. The immunostimulants
were fed to these animals alone for 14 consecutive doses (at their optimum immunostimulatory
doses) or simultaneously with standard antifilarial drug, DEC (25 mg/kg) for last five days as
described earlier in chapter 3 (materials and methods). On day 60 of start of dosing, all the
animals were euthanized to assess the parasite killing as well as alteration in various immune
parameters and the results are summarized below:4.4.1. WITHAFERIN A FOLLOWED BY CHEMOTYPE 101R AND AS PURE
COMPOUND 5 EXHIBITED BEST CHEMOTHERAPEUTIC ADJUNCT EFFICACY
IN MASTOMYS
After feeding with withaferin A, WS 101R or AS compound 5, mastomys although
demonstrated progressive increase in microfilaraemia, however, the Mf intensity was lower
than in untreated controls. The microfilarial counts were drastically reduced in mastomys on
day 15 post initiation of treatment when immunostimulants were accompanied with standard
filaricidal drug, DEC (P <0.05 to P <0.01). The initial decrease in Mf on day 15 post treatment
was due to microfilaricidal effect of DEC, however, the Mf counts rose thereafter (Fig. 4.27
A).
Mastomys fed with withaferin A, WS 101R, AS extract, AS compound 5 and Picroliv along
with DEC showed significant (P <0.05) reduction in adult worm recovery on the day of
autopsy. However, AS chloroform fraction alone or with DEC could not exert significant effect
on the adult parasites (Fig. 4.27 B). The adverse effect on female worm fecundity was
observed to be highly significant (P <0.01) in mastomys fed with withaferin A, WS 101R and
AS compound 5 alone which increased further when DEC was also given in combination (P
<0.01). The embryostatic effect was however, comparatively less by AS extract and fraction in
combination with DEC (P <0.05). Picroliv with DEC on the other hand brought about
significant sterilization in adult female worms. The standard antifilarial drug DEC had no
significant effect on adult worm survival or female worm fecundity (Fig. 4.27 C).
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Chapter 4
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Fig: 4.27 Microfilaraemia counts (A), adult worm recovery (B), and female worm sterilization
(%) (C) in mastomys fed orally with Annona squamosa crude ethanolic extract (AS Ext, 30
mg/kg), chloroform fraction (AS Frn, 30 mg/kg), compound 5 (AS Comp 5, 3 mg/kg),
Withania somnifera chemotype 101R extract (WS 101R, 10 mg/kg), withaferin A (0.3 mg/kg)
and Picroliv (1 mg/kg) alone and along with diethyl carbamazine citrate (DEC, 25 mg/kg for
five days only) at their respective optimum immunostimulatory doses.
4.4.2. ANTIBODY ISOTYPES REVEAL DIFFERENTIAL TH1/ TH2 TYPE OF
IMMUNE RESPONSE
Treatment of patent mastomys with DEC did not affect the filaria-specific IgG antibody titre
and it was similar to that of untreated control mastomys. Treatment with all the
immunostimulants led to increase in the IgG titre with withaferin A showing highest increase
(Fig. 4.28 A).
Serum IgG isotype levels in the sera of all the animals fed with Picroliv, AS compound 5 and
withaferin A demonstrated an increase in specific IgG1, IgG2a, IgG2b, IgM and IgA levels
(Fig. 4.28 B to D, F and G). IgG3 antibody levels were although increased in all the treated
86
Chapter 4
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groups compared to untreated controls, remained comparable to DEC (Fig. 4.28 E). The
figures represent the data of pooled sera samples of each mastomys group and therefore SD or
SE values are not shown.
Fig: 4.28 Total IgG (A), IgG1 (B), IgG2a (C), IgG2b (D), IgG3 (E), IgM (F) and IgA (G)
titers in mastomys fed with Annona squamosa crude extract (AS Ext, 30 mg/kg), chloroform
fraction (AS Frn, 30 mg/kg), compound 5 (AS Comp 5, 3 mg/kg), Withania somnifera
chemotype 101R extract (WS 101R, 10 mg/kg), withaferin A (0.3 mg/kg) and Picroliv (1
mg/kg) alone and along with diethyl carbamazine citrate (DEC, 25 mg/kg).
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Chapter 4
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4.4.3. PERITONEAL MACROPHAGES OF TREATED MASTOMYS REVEAL
DIFFERENTIAL LEVEL OF OXIDANTS
Profound up-regulation in ROS production (P <0.05 to P <0.01) was noticed in peritoneal
macrophages isolated from animals after treatment with all the test samples. This increase
further enhanced significantly when DEC was given along with immunostimulants.
Interestingly, DEC alone was able to stimulate substantial ROS production in the macrophages
(Fig. 4.29 A). All the test samples except AS extract induced ROS production similar to that of
DEC treatment.
The results of NO production were quite similar to those of ROS and here also AS extract
stimulated the increased NO production in addition to DEC (P <0.01). Picroliv had better
response than DEC. NO contents were found to be enhanced when the cells were stimulated
with LPS O/N in vitro (Fig. 4.29 B).
Fig: 4.29 Reactive oxygen species (A) and nitric oxide (B) released by peritoneal macrophages
of mastomys fed with Annona squamosa crude ethanolic extract (AS Ext, 30 mg/kg),
chloroform fraction (AS Frn, 30 mg/kg), compound 5 (AS Comp 5, 3 mg/kg), Withania
somnifera chemotype 101R extract (WS 101R, 10 mg/kg), withaferin A (0.3 mg/kg) and
Picroliv (1 mg/kg) alone and along with diethyl carbamazine citrate (DEC, 25 mg/kg).
88
Chapter 4
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4.4.4. TREATMENT WITH WITHAFERIN A AND AS COMPOUND 5 LEAD TO
SIGNIFICANT IN VITRO LYMPHOPROLIFERATION
The proliferative response of splenocytes harvested from test sample fed mastomys was
investigated in presence of B-cell mitogen LPS (Fig. 4.30 A), T-cell mitogen Con A (Fig. 4.30
B) and adult filarial soluble somatic antigen (Fig. 4.30 C). Although all the test samples
increased the proliferative response of splenic lymphocytes, B cell proliferated significantly in
presence of all the test samples. However, The T cell proliferation was evident only in
mastomys fed with pure molecules such as Picroliv, Withaferin A or AS compound 5 (P
<0.01) though the proliferative index was higher as compared to that of Picroliv. Adult B.
malayi antigen also led to cell proliferation and followed the same pattern as in case of T cell
mitogen Con A (P <0.01). Combined treatment of immunostimulators with DEC did not
influence cell proliferation (Fig. 4.30 A, B and C).
Fig: 4.30 Effect of Annona squamosa crude ethanolic extract (AS Ext, 30 mg/kg), chloroform
fraction (AS Frn, 30 mg/kg), compound 5 (AS Comp 5, 3 mg/kg), Withania somnifera
chemotype 101R extract (WS 101R, 10 mg/kg), withaferin A (0.3 mg/kg) and Picroliv (1
mg/kg) alone and along with diethyl carbamazine citrate (DEC, 25 mg/kg) on in vitro splenic
B- (A) and T-lymphocyte (B) proliferation.
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4.4.5. WITHAFERIN A AND AS COMPOUND 5 SIGNIFICANTLY UP-REGULATED
T AND B LYMPHOCYTE POPULATIONS IN INFECTED MASTOMYS
Feeding mastomys with Picroliv, AS compound 5 and withaferin A alone or with DEC upregulated the CD4+, CD8+ T lymphocyte sub-population and CD19+ B cell population (P
<0.05 to P <0.01) (Fig. 4.31 A, B and C). The other test samples such as AS fractions, AS
extract and WS 101R extract did not demonstrate significant improvement in the cell
proliferation.
Fig: 4.31 Flow cytometric detection of CD4+ (A), CD8+ (B) T cell subpopulations and CD19+
B cell (C) population in splenocytes of vehicle treated/ control, DEC (25 mg/kg), Annona
squamosa crude ethanolic extract (AS Ext, 30 mg/kg), chloroform fraction (AS Frn, 30
mg/kg), compound 5 (AS Comp 5, 3 mg/kg), Withania somnifera chemotype 101R extract
(WS 101R, 10 mg/kg), withaferin A (0.3 mg/kg) and Picroliv (1 mg/kg) fed mastomys alone
and along with DEC.
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4.4.5. CHEMOTYPE 101R, WITHAFERIN A AND AS EXTRACT GENERATED A
MIXED TH1/TH2 CYTOKINE RESPONSE
The levels of both pro-inflammatory and anti-inflammatory cytokines were determined
intracellularly in the splenic cell population of test sample fed and vehicle treated mastomys by
FACS. Highly significant (P <0.01) increase in the levels of pro-inflammatory cytokines IL-2,
IFNγ and anti-inflammatory cytokines IL-4 and IL-10 in Picroliv, Withaferin A and AS
compound 5 fed animals were observed when given alone. The administration of these test
samples along with DEC, however, had similar effect (Fig. 4.32 A, B, C and D). Of these pure
compounds, AS compound 5 though up-regulated both Th1 and Th2 cytokines, the increase in
IL-2 production was not statistically significant when compared with that of controls (Fig. 4.32
A).
Fig: 4.32 Effect of oral feeding of Annona squamosa crude ethanolic extract (AS Ext, 30
mg/kg), AS chloroform fraction (AS Frn, 30 mg/kg), AS compound 5 (AS Comp 5, 3 mg/kg),
Withania somnifera chemotype 101R extract (WS 101R, 10 mg/kg), withaferin A (0.3 mg/kg)
and Picroliv (1 mg/kg) alone and along with diethyl carbamazine citrate (DEC, 25 mg/kg) on
expression of intracellular Th1/Th2 cytokine.
91