5.1 Organism Chosen for the Study 5.2 Results of crude extracts Antibacterial Activity MIC Activity on Human PBMC

Transcription

1 Results

2 5. Results 5.1 Organism Chosen for the Study A total of 1 marine cyanobacterial strains viz., Nostoc calcicola, Oscillatoria boryana, Oscillatoria chlorina, Oscillatoria formosa, Oscillatoria laete-virens, Phormidium valderianum, Oscillatoria willei, Pseudoanabaena shmidlei, Synechocystis pelvalikii and Spirulina subsalsa were chosen and tested for their bioactive potentials (Plate 1a & 1b). 5.2 Results of crude extracts Antibacterial Activity All the ten marine cyanobacterial strains chosen were tested for their antibacterial activity against seven clinical pathogens that includes Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeroginosa, Salmonella typhi, Streptococcus faecalis and Staphylococcus aureus. Results revealed that Phormidium valderianum was found to be highly active against Klebsiella pneumoniae, Salmonella typhi, and Staphylococcus aureus. Oscillatoria willei was found to be highly active against Pseudomonas aeroginosa, Salmonella typhi, Staphylococcus aureus and Streptococcus faecalis. Synechocystis pelvalikii was found to be highly active against Salmonella typhi and Staphylococcus aureus (Table 1). Results based on zone of inhibition showed that, Phormidium valderianum had a maximum zone of inhibition of 16 mm against Salmonella typhi, followed by Staphylococcus aureus (15 mm) and Klebsiella pneumoniae (14 mm). In case of Oscillatoria willei the zone of inhibition was found to be maximum in Streptococcus faecalis (16 mm), followed by Salmonella typhi, (15 mm), Staphylococcus aureus (14 mm) and Pseudomonas aeroginosa (13 mm). Synechocystis pelvalikii had a zone of inhibition of 15 mm against Staphylococcus aureus and 14 mm against Salmonella typhi (Table 2) (Plate 2 11) MIC MIC was found to vary with the nature of extract and the clinical strain. For almost all the cyanobacterial crude extracts MIC was found to be between 5 µg to 4 µg against most of the bacterial strains (Table 3) Activity on Human PBMC When the cyanobacerial extracts were tested on human peripheral blood mononuclear cells and asscessed by dye exclusion method it was observed that Nostoc calcicola, 66

3 Plate 1a Microphotograph of marine cyanobacterial strains choosen Nostoc calcicola Spirulina subsalsa Pseudoanabaena shmidlei Phormidium valderianum Synechocystis pelvalikii

4 Plate 1b Microphotograph of marine cyanobacterial strains choosen Oscillatoria willei Oscillatoria formosa Oscillatoria boryana Oscillatoria laete-virens Oscillatoria chlorina

5 Table 1: Antibacterial activity of Cyanobacterial extracts on clinical pathogens in terms of sensitivity Bacterial pathogens and activity S. No Extract E. coli K. pneumoniae P. mirabilis P. aeroginosa S. typhi S. aureus S. faecalis 1. N. calcicola O. boryana O. chlorina O. formosa O. laete - virens P. valderianum O. willei Pseudo. shmidlei Spirulina subsalsa Synechocystis pelvalikii Highly sensitive ++ Moderately Sensitive + Sensitive - Resistant

6 Table 2: Antibacterial activity of cyanobacterial extracts on clinical pathogens in terms of inhibition zone Bacterial pathogens and activity (Zone of inhibition in mm) S. No Extract E. coli K. pneumoniae P. mirabilis P. aeroginosa S. typhi S. aureus S. faecalis 1. N. calcicola mm - 2. O. boryana mm O. chlorine mm 4. O. formosa mm O. laete - virens mm mm - 6. P. valderianum - 14 mm mm 15 mm - 7. O. willei mm 15mm 14 mm 16 mm 8. Pseudo. shmidlei - 6 mm mm - 9. Spirulina subsalsa mm - 1. Synechocystis pelvalikii mm 15 mm - - Resistant

7 Plate 2 Antibacterial activity of Oscillatoria willei against clinical pathogens Pseudomonas aeroginosa Streptococcus faecalis Salmonella typhi Staphylococcus aureus + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

8 Plate 3 Antibacterial activity of Oscillatoria laete-virens against clinical pathogens Klebsiella pneumoniae Proteus mirabilis Salmonella typhi Staphylococcus aureus + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

9 Plate 4 Antibacterial activity of Nostoc calcicola against clinical pathogens Escherichia coli Proteus mirabilis Streptococcus faecalis Salmonella typhi + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

10 Plate 5 Antibacterial activity of Oscillatoria boryana against clinical pathogens Escherichia coli Proteus mirabilis Streptococcus faecalis Salmonella typhi + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

11 Plate 6 Antibacterial activity of Oscillatoria chlorina against clinical pathogens Escherichia coli Proteus mirabilis Streptococcus faecalis + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

12 Plate 7 Antibacterial activity of Oscillatoria formosa against clinical pathogens Escherichia coli Proteus mirabis Pseudomonas aeroginosa Staphylococcus aureus + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

13 Plate 8 Antibacterial activity of Phormidium valderianum against clinical pathogens Escherichia coli Klebsiella pneumoniae Salmonella typhi Staphylococcus aureus + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

14 Plate 9 Antibacterial activity of Pseudoanabena shmidlei against clinical pathogens Escherichia coli Klebsiella pneumoniae Proteus mirabilis Staphylococcus aureus + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

15 Plate 1 Antibacterial activity of Spirulina subsalsa against clinical pathogens Escherichia coli Proteus mirabilis Salmonella typhi + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

16 Plate 11 Antibacterial activity of Synechocystis pelvalikii against clinical pathogens Salmonella typhi Proteus mirabilis Staphylococcus aureus + Positive control - Tetracycline - Negative control - Methanol: DMSO: Water (1:1:1) 1-5 µg of the extract / disc 2-1 µg of the extract / disc 3-2 µg of the extract / disc 4-4 µg of the extract / disc

17 Table 3: Antibacterial activity of cyanobacterial extracts on clinical pathogens in terms of Minimum Inhibitory Concentration (MIC) Bacterial Pathogens & Minimum Inhibitory Concentration (MIC) S. No Extract E. coli K. pneumoniae P. mirabilis P. aeroginosa S. typhi S. aureus S. faecalis 1. N. calcicola O. boryana ± O. chlorina ± O. formosa O. laete - virens P. valderianum - 9 ± ± O. willei Pseudo. shmidlei ± ± ± ± Spirulina subsalsa Synechocystis pelvalikii ± ± ± ± ± ± ± ± ± Positive Control - Tetracycline, Negative Control Methanol: DMSO: Water (1:1:1) SD Standard Deviation, Mean Value of 1 assay with different Bacterial Pathogens.

18 Oscillatoria boryana, Oscillatoria formosa, Pseudoanabaena shmidlei and Spirulina subsalsa were found to be cytotoxic starting from 4 µg concentration onwards up to 32 µg. Oscillatoria chlorina and Oscillatoria laete virens were found to be cytopathic i.e. the morphology of the cells alone was affected. Whereas Phormidium valderianum, Oscillatoria willei and Synechocystis pelvalikii were found to enhance the proliferation of cells from 4 µg and above (Table 4 & 5) (Figure 1 1) (Plate 12 14). When the activity of the cyanobacterial extracts was asscessed on Human PBMC by SRB method, it was found that Nostoc calcicola, Oscillatoria boryana, Oscillatoria chlorina, Oscillatoria formosa, Pseudoanabaena shmidlei and Spirulina subsalsa, were cytotoxic from 4 µg irrespectively. Whereas Oscillatoria laete virens and Synechocystis pelvalikii were found to be nontoxic. Phormidium valderianum enhanced the growth of PBMC from 2 µg and Oscillatoria willei enhanced the growth of PBMC from 4 µg (Figure 11-2). When MTT assay was performed for all the ten cyanobacterial crude extracts, the results revealed that Nostoc calcicola, Oscillatoria boryana, Oscillatoria chlorina, Pseudoanabaena shmidlei and Spirulina subsalsa were found to be cytotoxic. Oscillatoria formosa, Oscillatoria late virens, Phormidium valderianum and Synechocystis pelvalkii were found to be nontoxic. Oscillatoria willei alone was found to enhance the growth of PBMC and acted as immunopotentiator at 4 µg (Figure 21-3) Activity on cancer cell lines Jurkat and Raji Activity of the cyanobacterial crude extracts on Jurkat, a T cell cancer cell line, when analysed the effect by DEM, the results revealed that Nostoc calcicola, Oscillatoria boryana, Oscillatoria formosa, and Spirulina subsalsa were found to be cytotoxic at different concentrations. Rest of the cyanobacterial strains Pseudoanabaena shmidlei, Oscillatoria chlorina, Oscillatoria laete virens, Phormidium valderianum, Oscillatoria willei and Synechocystis pelvalikii were nontoxic (Table 6) (Figure 31 4). By SRB most of the cyanobacterial strains neither down regulated nor up regulated Jukat cell line except Oscillatoria willei which down regulated Jurkat cell line from 2 µg concentrations (Figure 11 2). Similar kind of results were obtained when MTT assay was performed, where Oscillatoria willei alone down regulated Jurkat cell line from 2 µg concentration (Figure 21 3). On Raji, a B cancer cell line, by DEM cyanobacterial strains Nostoc calcicola, Oscillatoria boryana and Spirulina subsalsa were found to be cytotoxic. Rest of the cyanobacterial strains Oscillatoria formosa, Pseudoanabaena shmidlei, Oscillatoria chlorina, 67

19 Table 5: Effect of marine Cyanobacterial crude extract on human PBMC by DEM S.No Cyanobacterial Extract Activity of the extract on PBMC NT CP CT IP 1. Nostoc calcicola Oscillatoria boryana Oscillatoria chlorina Oscillatoria formosa Oscillatoria laete virens Phormidium valderianum Oscillatoria willei Pseudoanabena Shmidlei Spirulina subsalsa Synechocystis pelvalikii CP- Cytopathic CT- Cytotoxic IP- Immunopotentiator N- Non toxic + Positive - Negative

20 Table 6: Effect of marine cyanobacterial extract on Jurkat by DEM S.No Extract from the cyanobacteria Concentration of the extract in mg & activity Nostoc calcicola N N N CT CT CT CT 2. Oscillatoria boryana N N N CT CT CT CT 3. Oscillatoria chlorina N N N N N N N 4. Oscillatoria formosa N N N CT CT CT CT 5. Oscillatoria laete virens N N N N N N N 6. Phormidium valderianum N N N N N N N 7. Oscillatoria willei N N N N N N N 8. Pseudoanabena Shmidlei N N N N N N N 9. Spirulina subsalsa N N N CT CT CT CT 1 Synechocystis pelvalikii N N N N N N N N- Non toxic CT- Cytotoxic

21 Figure 1: Effect of Nostoc calcicola crude extract on human PBMC by DEM Figure 2: Effect of Oscillatoria boryana crude extract on human PBMC by DEM

22 Figure 3: Effect of Oscillatoria chlorina crude extract on human PBMC by DEM Figure 4: Effect of Oscillatoria formosa crude extract on human PBMC by DEM

23 Figure 5: Effect of Oscillatoria laete virens crude extract on human PBMC by DEM Figure 6: Effect of Phormidium valderianum crude extract on human PBMC by DEM

24 Figure 7: Effect of Oscillatoria willei crude extract on human PBMC by DEM Figure 8: Effect of Pseudoanabena shmidlei crude extract on human PBMC by DEM

25 Figure 9: Effect of Spirulina subsalsa crude extract on human PBMC by DEM Figure 1: Effect of Synechocystis pelvalikii crude extract on human PBMC by DEM

26 Plate 12 Effect of P. valderianum on Human PBMC Fig A. Human PBMC viewed under light microscope Fig B. Immunopotentiation effect of P. valderianum on PBMC after 24 hrs. Fig C. Immunopotentiation effect of P. valderianum on PBMC after 48 hrs.

27 Plate 13 Effect of O.willei on Human PBMC Figure A. Human PBMC viewed under light microscope Figure B. Immunopotentiation effect of O. willei on PBMC after 24 hrs. Figure C. Immunopotentiation effect of O. willei on PBMC after 48 hrs.

28 Plate 14 Effect of S. pelvalikii on Human PBMC Figure A. Human PBMC viewed under light microscope Figure B. Immunopotentiation effect of S. pelvalikii on PBMC after 24 hrs. Figure C. Immunopotentiation effect of S. pelvalikii on PBMC after 48 hrs.

29 Figure 11: Effect of N.calcicola crude extract on PBMC, Jurkat and Raji by SRB.6.5 % Cytotoxicity PBMC Jurkat Raji Concentration of the extract in µg Figure 12: Effect of O.boryana extract on PBMC, Jurkat and Raji by SRB % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji

30 Figure 13: Effect of O.chlorina crude extract on PBMC, Jurkat and Raji by SRB.6.5 % Cytotoxicity PBMC Jurkat Raji Concentration of the extract in µg Figure 14: Effect of O.formosa crude extract on PBMC, Jurkat and Raji by SRB.6.5 % Cytotoxicity PBMC Jurkat Raji Concentration of the extract in µg

31 Figure 15: Effect of O. l. virens crude extract on PBMC, Jurkat and Raji by SRB.7.6 % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji Figure 16: Effect of P. valderianum crude extract on PBMC, Jurkat and Raji by SRB % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji

32 Figure 17: Effect of Oscillatoria willei extract on PBMC, Jurkat and Raji by SRB % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji Figure 18: Effect of P. shmidlei on PBMC, Jurkat and Raji by SRB.6.5 % Cytotoxicity PBMC Jurkat Raji Concentration of the extract in µg

33 Figure 19: Effect of S. subsalsa extract on PBMC, Jurkat and Raji by SRB % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji Figure 2: Effect of S. pelvelkii extract on PBMC, Jurkat and Raji by SRB.7.6 % Cytotoxicity PBMC Jurkat Raji Concentration of the extract in µg

34 Figure 21: Effect of Nostoc calcicola extract on PBMC, Jurkat and Raji by MTT.12.1 % Cytotoxicity PBMC Jurkat Raji Concentration of the extract in µg Figure 22: Effect of O. boryana extract on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji

35 Figure 23: Effect of O. chlorina extract on PBMC, Jurkat and Raji by MTT.1 % Cytotoxicity PBMC Jurkat Raji Concentration of the extraction in µg Figure 24: Effect of O. formosa extract on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji

36 Figure 25: Effect of O. l. virens extract on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji Figure 26: Effect of P. valderium extract on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji

37 Figure 27: Effect of O. willei extract on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji Figure 28: Effect of P. schemedii on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat raji

38 Figure 29: Effect of S. subsalsa extract on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the extract in µg PBMC Jurkat Raji Figure 3: Effect of Synechocystis pelvelkii extract on PBMC, Jurkat and Raji by SRB % Cytotoxicity Concentration of the extract on µg PBMC Jurkat Raji

39 Table 6: Effect of marine cyanobacterial extract on Jurkat by DEM S.No Extract from the cyanobacteria Concentration of the extract in mg & activity Nostoc calcicola N N N CT CT CT CT 2. Oscillatoria boryana N N N CT CT CT CT 3. Oscillatoria chlorina N N N N N N N 4. Oscillatoria formosa N N N CT CT CT CT 5. Oscillatoria laete virens N N N N N N N 6. Phormidium valderianum N N N N N N N 7. Oscillatoria willei N N N N N N N 8. Pseudoanabena Shmidlei N N N N N N N 9. Spirulina subsalsa N N N CT CT CT CT 1 Synechocystis pelvalikii N N N N N N N N- Non toxic CT- Cytotoxic

40 Figure 31: Effect of Nostoc calcicola extract on Jurkat by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 32: Effect of Oscillatoria boryana extract on Jurkat by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

41 Figure 33: Effect of Oscillatoria chlorina extract on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 34: Effect of Oscillatoria formosa extract on Jurkat by DEM 12 1 % Viable Cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

42 Figure 35: Effect of Oscillatoria laete-virens extract on Jurkat by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 36: Effect of Phormidium valderianum extract on Jurkat by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

43 Figure 37: Effect of Oscillatoria willei extract on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 38: Effect of Pseudo. shmidlei extract on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

44 Figure 39: Effect of Spirulina subsalsa extract on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 4: Effect of Synechocystis pelvelkii extract on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

45 Oscillatoria laete virens, Phormidium valderianum, Oscillatoria willei and Synechocystis pelvalikii were nontoxic when the concentration and time duration was increased (Table 7) (Figure 41 5). SRB and MTT assay showed that Oscillatoria willei alone down regulated Raji cancer cell line effectively from 4 µg by both the methods. All the other cyanobacterial strains not showed any impact on Raji cell line by both the methods (Figure 11 2) (Figure 21 3). 5.3 Activity of different fractions of Oscillatoria willei Antibacterial activity, sensitivity and MIC All the six fractions of Oscillatoria willei when tested for antibacterial activity against seven clinical pathogens, viz Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeroginosa, Salmonella typhi, Streptococcus faecalis and Staphylococcus aureus methanol and ethyl acetate fraction were found to be highly active against Pseudomonas aeroginosa, Salmonella typhi, Streptococcus faecalis and Staphylococcus aureus (Table 8). MIC was found to vary with the nature of fraction and the clinical strain. For P. aeroginosa it was found to be between 1 µg for methanol fraction, 2 µg for water fraction. MIC for S. typhi ranges from 2 µg /ml to 4 µg /ml. For S. aureus it ranges between 12 µg / ml for methanol fraction and for S. faecalis 4 µg /ml of ethyl acetate fraction (Table 9) Activity on Human PBMC When different fraction of Oscillatoria willei tested on human PBMC by DEM, the results revealed that all five fractions viz., hexane, benzene, chloroform, methanol and water were found to be nontoxic except ethyl acetate fraction which was found to enhance the growth from 2 µg onwards (Table 1) (Figure 51 56). Exactly similar results were obtained by SRB and MTT assays also on PBMC and it was related to concentration and time duration (Figure 57 62) (Figure 63 68) Activity on cancer cell lines On cancer cell lines Jurkat and Raji, methanol fraction and ethyl acetate fraction were found to be an immunomodulator by DEM and it was proportional to the concentration of the fraction and time duration (Table 11) (Table 12) (Figure 69 74) (Figure 75 8). When the six fractions were tested by SRB assay ethyl acetate fraction down regulated both Jurkat and 68

46 Table 7: Effect of marine cyanobacterial extract on Raji by DEM S.No Extract from the cyanobacteria Concentration of the extract in mg and activity Nostoc calcicola N N N CT CT CT CT 2. Oscillatoria boryana N N N CT CT CT CT 3. Oscillatoria chlorina N N N N N N N 4. Oscillatoria formosa N N N N N N N 5. Oscillatoria laete virens N N N N N N N 6. Phormidium valderianum N N N N N N N 7. Oscillatoria willei N N N N N N N 8. Pseudoanabena Shmidlei N N N N N N N 9. Spirulina subsalsa N N N CT CT CT CT 1 Synechocystis pelvalikii N N N N N N N N- Non toxic CT- Cytotoxic

47 Figure 41: Effect of Nostoc calcicola extract on Raji by DEM % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 42: Effect of Oscillatoria boryana extract on Raji by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

48 Figure 43: Effect of Oscillatoria chlorina extract on Raji by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 44: Effect of Oscillatoria formosa extract on Raji by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

49 Figure 45: Effect of Oscillatoria laete - virens extract on Raji by DEM % Viable cells Concentration of the extract on µg hr 24 hrs 48 hrs 72 hrs Figure 46: Effect of Phormidium valderinum extract on Raji by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

50 Figure 47: Effect of Oscillatoria willei extract on Raji by DEM % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 48: Effect of Pseudo. shmidlei extract on Raji by DEM % Viable cells hr 24 hrs 48 hrs 72 hrs concentration of the extract on µg

51 Figure 49: Effect of Spirulina subsalsa extract on Raji by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the extract on µg Figure 5: Effect of Synechocystis pelvelkii on Raji by DEM % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the extract on µg

52 Table 8: Antibacterial activity of Oscillatoria willei fractions on clinical pathogens in terms of Sensitivity S.No Fractions Bacterial pathogens & activity P. aeroginosa S. typhi S. aureus S. faecalis 1 Hexane Benzene Chloroform Ethyl acetate Methanol Water Highly sensitive ++ Moderately Sensitive + Sensitive - Resistant Table 9: Antibacterial activity of Oscillatoria willei fractions on clinical pathogens in terms of Minimum Inhibitory Concentration (MIC) S.No Fractions 1 Hexane 2 Benzene 3 Chloroform 4 Ethyl acetate 5 Methanol 6 Water Bacterial pathogens & MIC P. aeroginosa S. typhi S. aureus S. faecalis ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 25. Positive Control - Tetracycline, Negative Control Methanol: DMSO: Water (1:1:1) SD Standard Deviation, Mean Value of 1 assay with different Bacterial Pathogens.

53 Table 1: Effect of different fractions of O. willei on PBMC by DEM S.No Fractions Concentration of the fraction in mg & activity Hexane fraction of O. willei N N N N N N N 2. Benzene fraction of O. willei N N N N N N N 3. Chlorofom fraction of O. willei N N N N N N N 4. Ethyl acetate fraction of O. willei N N N IM IM IM IM 5. Methanol fraction of O. willei N N N N N N N 6. Water fraction of O. willei N N N N N N N N Non- Toxic IM - Immunomodulator

54 Figure 51: Hexane fraction of Oscillatoria willei on PBMC by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg Figure 52: Benzene fraction of Oscillatoria willei on PBMC by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg

55 Figure 53: Chloroform fraction of Oscillatoria willei on PBMC by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg Figure 54: Ethyl Acetate fraction of Oscillatoria willei on PBMC by DEM 12 1 % Viable cells Concentration of the fraction on µg hr 24 hrs 48 hrs 72 hrs

56 Figure 55: Methanol fraction of Oscillatoria willei on PBMC by DEM % Viable cells Concentration of the fraction on µg hrs 24 hrs 48 hrs 72 hrs Figure 56: Water fraction of Oscillatoria willei on PBMC by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of fraction of µg

57 Figure 57: Hexane fraction of O.willei on PBMC, Jurkat and Raji by SRB.6.5 % cytotoxicity PBMC Jurkat Raji Concentration of the fraction in µg Figure 58: Benzene fraction of O.willei on PBMC, Jurkat and Raji by SRB.7.6 % cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji

58 Figure 59: Chloroform fraction of O.willei on PBMC, Jurkat and Raji by SRB % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji Figure 6: Ethyl Acetate fraction of O.willei on PBMC, Jurkat and Raji by SRB.7.6 % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji

59 Figure 61: Methanol fraction of O.willei on PBMC, Jurkat and Raji by SRB.7.6 % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji Figure 62: Water fraction of O.willei on PBMC, Jurkat and Raji by SRB.6.5 % Cytotoxicity PBMC Jurkat Raji Concentration of the fraction in µg

60 Figure 63: Hexane Fraction of O.willei on PBMC, Jurkat and Raji by MTT.7.6 % Cytotoxicity PBMC Jurkat Raji Concentration of the fraction in µg Figure 64: Benzene fraction of O.willei on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji

61 Figure 65: Chloroform fraction of O.willei on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji Figure 66: Ethyl Acetate fraction of O.willei on PBMC, Jurkat and Raji by MTT % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji

62 Figure 67: Methanol fraction of O.willei on PBMC, Jurkat and Raji by MTT.7.6 % Cytotoxicity Concentration of the fraction in µg PBMC Jurkat Raji Figure 68: Water fraction of O.willei on PBMC, Jurkat and Raji by MTT.7.6 % Inhibition Concentration of the fraction in µg PBMC Jurkat Raji

63 Table 11: Effect of different fractions of O. willei on Jurkat by DEM S.No Fractions Concentration of the extract in mg & activity Hexane fraction of O. willei N N N N N N N 2. Benzene fraction of O. willei N N N N N N N 3. Chloroform fraction of O. willei N N N N N N N 4. Ethyl acetate fraction of O. willei N N N IM IM IM IM 5. Methanol fraction of O. willei N N N IM IM IM IM 6. Water fraction of O. willei N N N N N N N N- Non Toxic IM - Immunomodulator

64 Table 12: Effect of different fractions of O. willei on Raji by DEM S.No Fractions Concentration of the extract in mg & activity Hexane fraction of O. willei N N N N N N N 2. Benzene fraction of O. willei N N N N N N N 3. Chloroform fraction of O. willei N N N N N N N 4. Ethyl acetate fraction of O. willei N N N IM IM IM IM 5. Methanol fraction of O. willei N N N IM IM IM IM 6. Water fraction of O. willei N N N N N N N N- Non Toxic IM - Immunomodulator

65 Figure 69: Hexane fraction of Oscillatoria willei on Jurkat by DEM 12 1 % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg Figure 7: Benzene fraction of Oscillatoria willei on Jurkat by DEM % Viable cells Concentration of the fraction on µg hr 24 hrs 48 hrs 72 hrs

66 Figure 71: Chloroform fraction of Oscillatoria willei on Jurkat by DEM 12 1 % viable cells o hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg Figure 72: Ethyl acetate fraction of Oscillatoria willei on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg

67 Figure 73: Methanol fraction of Oscillatora willei on Jurkat by DEM % Viale cells Concentration of the fraction on µg hrs 24 hrs 48 hrs 72 hrs Figure 74: Water fraction of Oscillatoria willei on Jurkat by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg

68 Figure 75: Hexane fraction of Oscillatoria willei on Raji by DEM % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg Figure 76: Benzene fraction of Oscillatoria willei on Raji by DEM % Viable cells hrs 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg

69 Figure 77: Chloroform fraction of Oscillatoria willei on Raji by DEM 12 1 % Viable Cells hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg Figure 78: Ethyi acetate fraction of Oscillatoria willei on Raji by DEM 12 1 % Viable cells hr 24 hrs 48 hrs 72 hrs Concentration of the fraction on µg

70 Figure 79: Methanol fraction of Oscillatoria willei on Raji by DEM % Viable cells Concentration of the fraction on µg hrs 24 hrs 48 hrs 72 hrs Figure 8: Water fraction of Oscillatoria willei on Raji by DEM % Viable cells Concentration of the fraction on µg hr 24 hrs 48 hrs 72 hrs

71 Raji cell lines (Figure 57 62). By MTT assay also ethyl acetate fraction down regulated both the cancer cell lines (Figure 63 68). 5.4 Purified compound identification GCMS analysis As the ethyl acetate fraction of O.willei was active against bacterial pathogens, non toxic & down regulated 2 cancer cell lines it was subjected for purification by column chromatography & identified. Analysis of the purified compound of Oscillatoria willei (GC-MS analysis) indicates the presence of one major compound i.e. 2,6-di-tetra-butyl phenol and four more minor compounds (Plate 15a). The structural formula of the major compound is C1 4 H 22 O and the molecular weight is 26 (Plate 15b). This major compound was used for our further study to rule out Cytotoxicity. 5.5 Animal Study General Health Condition (Morphological observation) General health condition revealed that the animals were hale and healthy. There were no abnormal symptoms like hair falling, itching, colour change, tumor formation, over activity and lethargy. There was no death in control and the animal group given with the partially pure compound (Table 13) Morphometry (Body weight) When the weights of the animals were analyzed it was found that both the control and test group were found to be almost similar on day as well as on 45 th day (Figure 81) Feed Consumption Feed consumption was found to be almost similar in both the control and test group but day wise comparison revealed that both in test and control it was low at 45 th day to that of day one (Figure 82) Water Consumption Water consumption was found to be less on 45 th day in both test and control group when compared to day one (Figure 83). 69

72 Plate 15a GC-MS Analysis of the partially pure compound of O. willei

73 Plate 15b GC-MS Analysis of the partially pure compound of O. willei - Spectrum

74 Table 13: General Health Condition S.No Parameter Analyzed Control Category Test 1. Hair falling Nil Nil 2. Itching Nil Nil 3. Colour change Nil Nil 4. Tumor formation Nil Nil 5. Overactive Nil Nil 6. Lethargy Nil Nil 7. Death Nil Nil

75 Figure 81: Morphometry Total body weight in g Duration in days Control Test Figure 82: Feed Consumption Food consumption in g Duration in days Control Test

76 Figure 83: Water Consumption Water consumption in ml Duration in days Control Test

77 5.5.5 Excreta Quantity Much difference was not found in excreta quantity of both test and control group RBC Count The respiratory pigment haemoglobin is carried throughout the body by RBC. For this reason RBC was calculated. The overall healthy conditions of the treated animals were equivalent to the control was indicated by RBC level (Figure 84) WBC Count WBC happens to be the index of the host defense system. The foreign material given to the system will be faced by the WBC. This is the reason for counting WBC in this study. As shown in figure (Figure 85), on 45 th day WBC count of the test group was higher which indicates the triggering of the host defense system by the compound given. When the haemoglobin level was analyzed in this study, not much variation was observed (Figure 86). When we compared the RBC count and haemoglobin percentage of the test and control, they were parallel, in the sense, the RBC count and haemoglobin level of the test group was more or less similar to the control group Gravimetric Analysis Gravimetric analysis of various organs like liver, kidney, lymphoid organs and reproductive organs were found to be almost similar in both the control and test groups when analyzed on different time durations of day 1, 2, after 4 days, 8 days, 12 days, 24 days, 36 days and after 45 days (Table 14) SGOT & SGPT There was not much variation in the levels of SGOT & SGPT of the treated animal when we compared to control (Figure 87 & Figure 88) Bilurubin Total bilurubin & direct bilurubin of the test group showed a fall. This may be due to inflammation of the liver which needs to be proved by liver histology (Figure 89 & Figure 9). 7

78 Figure 84: Erythrocyte Count No. of RBC X Duration in days Control Test Figure 85: Leukocyte Count No. of cells/mm Duration in days Control Test

79 Figure 86: Hemoglobin Content Haemoglobin in g/1ml Control Test 45 Duration in days Figure 87 SGOT Level SGOT level in units/ml Control Test 45 Duration in days

80 Table 14: Gravimetric Analysis Different organ weight in grams % Treatment Duration in days Liver Kidney Lymphoid organs Reproductive organs Control Test Control Test Control Test Control Test

81 Figure 88: SGPT Level SGPT level in units /ml Control Test 45 Duration in days Figure 89:Total Bilirubin Bilirubin level in mg/1 ml Duration in days Control Test

82 Figure 9: Direct Bilirubin Bilirubin level in mg/1ml Control Test 45 Duration in days Figure 91: Creatinine Creatinine Level in mg/1 ml Duration in days Control Test

83 Creatinine 45 th day test group animal showed creatinine level equivalent to that of control (Figure 91), which indicates the normal functioning of kidney. This is again is revealed by the histology of the kidney Histology of organs Histological investigation of vital organs like liver, kidney and spleen will reveal the abnormalities & toxic side effects. The present study did not reveal any serious abnormalities in any of the animals treated with the compound extracted from O. willei (Plate 16). 5.6 Reproductive System Sperm analysis Male reproductive system seems to be normal. No visible changes were observed here as in the case of earlier studies conducted in this lab. The gravimetric analysis revealed that the seminal vesicle and the ventral prostrate increased significantly over the control. Sperm count from cauda epididymis region was found decreased in the pure compound treated group. On the other hand the duration of the sperm motility in the treated mice is significantly lesser than that in the control Sperm abnormalities Various abnormalities were found in sperm of treated animal. A sperm with top shaped head, another sperm with cytoplasmic droplet, with double tail (Plate 17), multiple agglutination, abnormal head (Plate 18), abnormal middle piece and a sperm with abnormal head (Plate 19) (Figure 92) were found. In treated group roughly around 6% were with cytoplasmic droplet which is an alarming observation. Hence we can say that the effect of the compound was found to be on spermeogenesis. In the normal circumstances, particularly the cytoplasmic droplet should have been shed during the epididymal transit. Presence of cytoplasmic droplet is a weight on the sperm and thus can inhibit the sperm motility Sperm Count, Motility and Viability Compared to the control the sperm count was reduced by 5% in test animal (Figure 93). Sperm motility (Figure 94) and sperm viability (Figure 95) was also found to be less in test animal than that of the control. 71

84 Plate 16: Histology of Various Organs Liver Control Test a) b) Spleen VS VS WP WP c) RP 1x X 4x d) RP 1x X 4x Kidney DT DT e) C P 1x X 4x f) C P 1x X 4x Histopathology of various tissues of Control & Test group shows the normal cellular architecture and there was no abnormality. The tissues of test group showed no difference in histology. Notes: N : Nucleus CV : Central Vein H : Hepatocyte VS : Venous Sinus WP : White Pulp RP : Red Pulp C : Capsular space P : Podocytes DT : Distal Tubules

85 Male Reproductive System Plate 17: Sperm Abnormalities 1x X 4x i. A sperm with top shaped head. Another sperm with cytoplasmic droplet. (Giemsa stain, 4X). 1x X 4x ii. Sperm with double tail. Another sperm with cytoplasmic droplet. (Giemsa stain, 4X).

86 Plate 18: Sperm Abnormalities i. Sperm with multiple agglutination (Giemsa stain, 4X). 1x X 4x 1x X 4x ii. A sperm with abnormal head and cytoplasmic droplet. Another sperm also with cytoplasmic droplet (Giemsa stain, 4X).

87 Plate 19: Sperm Abnormalities i. A sperm with abnormal middle piece (Giemsa stain, 4X). 1x X 4x 1x X 4x ii. A sperm with abnormal head, a peculiar nature. Neighbouring sperms with cytoplasmic droplets and double tail (Giemsa stain, 4X).

88 Figure 92: Sperm Abnormalities a. Control 3% 2% 4% 8% 7% 13% 63% b. Test 8% 8% 2% 6% 4% 13% 59% Normal Head alone Modified midpiece Top shaped head With Cytoplasmic Droplet Double tail Other abnormalities

89 Figure 93: Sperm Count Motility time in minutes Treatment Duration in Days Control Test Figure 94: Sperm Motility Motility Time in minutes Treatment Duration in days Control Test

90 Figure 95:Sperm Viability No. of Sperms x Treatment Duration in Days Control Test