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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). 53 Chapter 4 Results 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. 54 Chapter 4 Results 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. 55 Chapter 4 Results 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. 56 Chapter 4 Results 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). 57 Chapter 4 Results 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. 58 Chapter 4 Results 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 59 Chapter 4 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. 60 Chapter 4 Results 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. 61 Chapter 4 Results 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. 62 Chapter 4 Results 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. 63 Chapter 4 Results 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. 64 Chapter 4 Results 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. 65 Chapter 4 Results 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). 66 Chapter 4 Results 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. 67 Chapter 4 Results 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. 68 Chapter 4 Results 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. 69 Chapter 4 Results 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. 70 Chapter 4 Results 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. 71 Chapter 4 Results 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. 72 Chapter 4 Results 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). 73 Chapter 4 Results 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. 74 Chapter 4 Results 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). 75 Chapter 4 Results 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). 76 Chapter 4 Results 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 77 Chapter 4 Results 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). 78 Chapter 4 Results 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). 79 Chapter 4 Results 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). 80 Chapter 4 Results 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). 81 Chapter 4 Results 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. 82 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. 83 Chapter 4 Results 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. 84 Chapter 4 Results 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). 85 Chapter 4 Results 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 Results 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). 87 Chapter 4 Results 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 Results 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. 89 Chapter 4 Results 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. 90 Chapter 4 Results 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