3. RESULTS AND DISCUSSION

Transcription

1 3. RESULTS AND DISCUSSION 3.1. PHYTOCHEMICAL INVESTIGATIONS OF F.RACEMOSA BARK The course powder of Ficus racemosa was sequentially extracted by Pet. Ether, Ethyl acetate along with Alcohol by Soxhlet apparatus & Maceration with Water. The results are described in Table No The Petroleum Ether F. racemosa bark extract was waxy and pale yellow, Ethyl acetate extract was powder and dark green, Alcohol extract was powder and dark brown and Water extract was powder and dark brown. Table No. 3.1: Percentage extractives values and physical characteristics of Different extracts of Ficus racemosa bark Extracts Alcoholic Petroleum ether ( C) Ethyl acetate Alcohol Water % dry weight in gm. Colour Odour Consistency 15.6 Dark brown Characteristic Powder SUCCESSIVE EXTRACTION 2.70 Pale yellow Characteristic Waxy 0.61 Dark green Characteristic Sticky 3.35 Dark brown Characteristic Powder 2.08 Dark brown Characteristic Powder The qualitative chemical tests were passed out to know incidence of different class of phyto-constituents in each extract of Ficus racemosa bark and results are discussed below in Table No. 3.2: 163

2 Table No. 3.2: Phyto-constituent Chemical Identification Phyto - Alcoholic SUCCESSIVE EXTRACTION Constituent Extract PE EA AL AQ Carbohydrates Glycosides Phytosterol Steroids Triterpenoids Tannins & Phenolic Group Flavonoids Key words: + = Present, = Absent PE = Petroleum ether extract ( C) AL = Alcoholic extract EA = Ethyl acetate extract AQ = Aqueous extract (by maceration) Qualitative chemical examinations of Ficus racemosa exposed existence of Carbohydrates, Steroids, Glycosides, Flavonoids, triterpinoids, Tannins as well as Phenolics. Physical Parameters of F. racemosa bark Table No. 3.3: Physical parameters of Ficus racemosa Sr. No. Physical Parameters Obtained values in % w/w 1 Total ash % 2 Acid-insoluble ash % 3 Water soluble ash 3.75 % 4 Alcohol soluble extractive % 5 Water soluble extractive % 164

3 In preliminary study, Ficus racemosa showed Total ash (13.34 %), Acid-insoluble ash (0.934 %) and Water soluble ash (3.75 %) PHYTOCHEMICAL INVESTIGATIONS OF AVICENNIA MARINA LEAF The course powder of Avicennia marina was sequentially extracted by Pet. Ether, chloroform as well as Alcohol by soxhlet equipment & Maceration with Water. The results are described in Table No Table No. 3.4: Percentage extractives values and physical characteristics of Different extracts Extracts Alcoholic of Avicennia marina leaf % dry weight in gm. Consistency Odour 11.6 Powder Aromatic SUCCESSIVE EXTRACTION Petroleum ether 3.10 Waxy Aromatic ( C) Chloroform 2.11 Powder Aromatic Alcohol Powder Aromatic Water Powder Aromatic The qualitative chemical tests were conceded to recognize the existence of different class of phyto-constituents in each extract of Avicennia marina leaf Phyto - Constituent Table No. 3.5: Phyto-constituent Chemical Identification Alcoholic Extract Successive Extraction PE CH AL AQ Carbohydrates Glycosides Phytosterol Steroids Triterpenoids Tannins & Phenolic Group Flavonoids

4 Key words: + = Present, = Absent PE = Petroleum ether extract ( C) AL = Alcoholic extract CH = Chloroform extract AQ = Aqueous extract (by maceration) Qualitative chemical examinations of Avicennia marina discovered the existence of Carbohydrates, Steroids, Glycosides, Flavonoids, triterpinoids, Tannins along with Phenolics. Physical Parameters of Avicennia marina leaf Table No. 3.6: Physical parameter of Avicennia marina Sr. No. Physical Parameters Obtained values in % w/w 1 Total ash % 2 Acid-insoluble ash 0.24 % 3 Water soluble ash 9.65 % 4 Alcohol soluble extractive % 5 Water soluble extractive % In preliminary study, Avicennia marina showed Total ash (12.42 %), Acidinsoluble ash (0.24 %) and Water soluble ash (9.65 %) CHARACTERIZATION OF CELL LINES AND CULTURE MEDIA Characterization of cell lines was performed for recognition of microbial as well as cross-contamination. Cell lines worn in these experiments are complimentary from several variety of microbial otherwise fungal contamination (Table No. 3.7), which in essential in order to continue our screening experiments. 166

5 Cell line Table No. 3.7: Results for Characterization of cell lines % Viability Stock After PDT (hrs) Microbial contamination Cross contamination NCI H No contamination No contamination 7.5 HL No contamination No contamination 7.5 Hep G No contamination No contamination 7.5 HEK 293T No contamination No contamination 7.0 ph Culture media were also tested for microbial contaminations. To prevent microbial contamination, 2.5 % Amphotericin B (µg/ml) was supplemented to media which act as working concentration. Bacterial contamination was prevented by addition of 1 % of Antibiotic, 100 X (10000 U/ml Penicillin G, µg/ml Streptomycin) into culture medial. All subculturing activities were done under class II Biosafety cabinet. (Esco, Singapore) Cross contamination of cell line was tested by direct observation of particular cell line under inverted microscope. PDT for specific cell line was determined. From viability studies and PDT, we have concluded that the celllines consequential as of NCCS, Pune be originally free from cross-contamination. To prevent the cross contamination of cell lines during our experiments work, separate pipettes and plastic tips were used for individual cell line. Along with that, particular cell line was used at the time under Class II Biosafety cabinet. These were proving to be valid steps to prevent cross contamination of cell lines throughout the experiment. CELL VIABILITY, DENSITY AND POPULATION DOUBLING TIME 1. HEK 293T cell line At the time of Subculture, density of HEK 293T cell line derived from NCCS, Pune was around cells / flask and viability was %, which was not suitable for cytotoxicity study, considering requirement of cell viability greater than 90 %. So as to augment viability plus cell density of HEK 293T cellline, subculturing was completed via total media furthermore supplementary 5 % FBS along with BSS. As 167

6 a consequence, on the fourth day morning cell density was increased up to and viability was approximately % which was appropriate for cytotoxicities test. PDT for HEK 293T was 19.3 hrs. Table No. 3.8 represents result for Subculture of HEK 293T cell. Table No. 3.8: Result for Subculture of HEK 293T cell line Days 1 st 2 nd 3 rd 4 th Viable cell count Non-viable cell count % viability Cells/ml Total cells in flask (50ml) Viable cells in flask (50ml) ph PDT 19.3 hrs Average PDT for HEK 293T was found to be 19.3 hrs. As the population of cells in the flask increase, more amounts of media were consumed by cells for growth purpose and this lead to acidic ph of the media, which requires continues addition of media for maintainers of ph and nutritional requirements. Subculturing was performed every 3 rd or 4 th day i.e. twice in week. 2. NCI H23 cell line Table No. 3.9: Result for Subculture of NCI H23 cell line Days 1 st 3 rd 4 th Viable cell count Non-viable cell count % viability Cells/ml Total cells in flask (50ml) Viable cells in flask (50ml) ph PDT 36.9 hrs NCI H23 is adherent cell line, viability of which was around % and density was In order to boost cell viability plus cell density, subculturing was performed by diluting cells in to fresh medium in new culture flask % cell 168

7 viability was achieved after 48 hrs of incubation and at that time cell density was found cells / flask. PDT was found to be around 36.9 hrs, so subculturing was done after every 5 th day. Table No. 3.9 represents Result for Subculture of NCI H23 cell line. 3. HL-60 cell line: Table No. 3.10: Result for Subculture of HL-60 cell line Days 1 st 3 rd 4 th Viable cell count Non-viable cell count % viability Cells/ml Total cells in flask (50ml) Viable cells in flask (50ml) ph PDT 35.2 hrs HL-60 is suspension cell line, viability of which was around % and density was In order to boost cell viability plus cell density, subculturing was performed by diluting cells in to fresh medium in new culture flask % cell viability was achieved after 48 hrs of incubation and at that time cell density was found cells / flask. PDT was found to be around 35.2 hrs, so subculturing was done after every 5 th day. Table No represents Result for Subculture of HL-60 cell line. 4. Hep G2 cell line: Table No. 3.11: Result for Subculture of Hep G2 cell line Days 1 st 3 rd 4 th Viable cell count Non-viable cell count % viability Cells/ml Total cells in flask (50ml) Viable cells in flask (50ml) ph PDT 28.7 hrs 169

8 Hep G2 is adherent cell line, viability of which was around % and density was In order to boost cell viability plus cell density, subculturing was performed by diluting cells in to fresh medium in new culture flask % cell viability was achieved after 48 hrs of incubation and at that time cell density was found cells / flask. PDT was found to be around 36.9 hrs, so subculturing was done after every 5 th day. Table No represents Result for Subculture of Hep G2 cell line. Total bacterial and fungal calculate Assessment of test along with control broths subsequent to 14days incubation confirmed the nonappearance of turbidity. Nonappearance of turbidity in test-broth denotes that there was no confirmation of bacteria, fungal as well as crosscontamination ANTIFUNGAL SCREENING BY NCCLS ASSAY Antifungal activity of Methanolic extract of Ficus racemosa The effect of Methanolic extract of Ficus racemosa was studied on five different fungal strains by NCCLS assay antifungal screening method. DRCs constructed between the ranges of µg/ml for plant extracts (test). For NCCLS (antifungal screening method) DRC was prepared flanked by series of µg/ ml and µM for Amphotericin-B. Calculation of IC 50 and R² values has been consequent by curve fitting techniques with Graph-Pad Prism like stastatical application (Ver.5.02). The vulnerability of cells to extract exposure was categorized by IC 50 values. Graph was plotted by observance log concentration on X-axis plus % cell growth inhibition or % cytotoxicity on Y axis. Results designate that antifungal consequences make stronger with augment in the concentration of extract. 170

9 Table No (A): % Inhibition of Methanolic extract of Ficus racemosa Conc. (µg/ml) Log Conc (ng/ml) A.Flavus A.Fumigatus I.Orientalis Table No (B): IC 50 & R 2 values for Methanolic extract of Ficus racemosa Conc. A. A. Cry. Cry. I. (µg/ml) Flavus Fumigatus Albidus Layrentii Orientalis IC >1000 > R² Fig. 3.1: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa 171

10 From the Table No. 3.12, we see that maximum activity of Methanolic extract have established against I.Orientalis and A.fumigatus having IC and respectively. Figure 3.1 shows the regression values of the fungal strains. We can see the methanolic extract against I.Orientalis shows linearity upto the concentration of 33.33µg/ml and achieves IC 50 value within that range. Upto the % inhibition of 50% to 60 % there is a deviation which then goes vertically towards the y-axis. There is no significant activity against C.laurentii & C.Albidus. (IC 50 values more than 1000µg/ml) After evaluation of the activity of methanolic extract of Ficus racemosa, against the five strains, the best was seen against I.orientalis, A.fumigatus and A.flavus in terms of IC 50 and regression Antifungal activity of Methanolic extract of Avicennia marina Table No (A): % Inhibition of Methanolic extract of Avicennia marina Conc. (µg/ml) Log Conc (ng/ml) Cry.Albidus Cry.Layrentii I.Orientalis Table No (B): IC 50 & R 2 values for Methanolic extract of Avicennia marina Conc. (µg/ml) A. Flavus A. Fumigatus Cry. Albidus Cry. Layrentii I. Orientalis IC 50 >1000 > R²

11 Fig. 3.2: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina From the Table No. 3.13, we see that highest activity of methanolic extract have found against I.orientalis, C.laurentii & C.albidus having IC , and respectively. The curve for other strains not having any co-relation between % inhibition & log concentration. There is no significant activity against the A.fumigatus and A.flavus. (IC 50 values more than 1000 µg/ml) Figure 3.2, in case of methanolic extract shows that co-relation in case of I.orientalis. I.orientalis shows maximum linearity of the eight strains at value being C.laurentii and C.albidus also shows linearity pattern as compared with the I.orientalis. The %inhibition is rising with augment in the concentration linearly. 173

12 Antifungal activity of Aqueous extract of Avicennia marina Table No (A): % Inhibition of Aqueous extract of Avicennia marina Conc. (µg/ml) Log Conc (ng/ml) Cry.Albidus Cry.Layrentii I.Orientalis Table No (B): IC 50 & R 2 values for Aqueous extract of Avicennia marina Conc. A. A. Cry. Cry. I. (µg/ml) Flavus Fumigatus Albidus Layrentii Orientalis IC 50 >1000 > R² Fig. 3.3: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina 174

13 From the Table No. 3.14, we can depicts that highest activity of aqueous extract have found against I.orientalis, C.albidus and C.laurentii having IC , and respectively with profound R 2 values 0.916, and respectively. Lower activities were found in other following stains. Figure: 3.3, I.orientalis shows maximum linearity of the five strains at value being The %inhibition is rising with augment in the concentration linearly. The curve for other strains not having any co-relation between % inhibition & log concentration. There is no significant activity against the A.fumigatus and A.flavus. (IC 50 values more than 1000 µg/ml) Antifungal activity of standard drug Amphotericin B Table No (A): % Inhibition of standard drug Amphotericin B Conc. Log Conc. A.Flavus A.Fumigatus I.Orientalis Cry.Albidus Cry.Layrentii Table No (B): IC 50 & R 2 values for standard drug Amphotericin B Conc. (µg/ml) A. Flavus A. Fumigatus Cry. Albidus Cry. Layrentii I. Orientalis IC 50 R²

14 Fig. 3.4: % Inhibition v/s log conc (ng/ml) of standard compound Amphotericin B 176

15 The IC 50 value for the standard compound Amphotericin-B is given in the Table No The IC 50 value against the corresponding fungal strains in descending order of activity is as follows. The strain most susceptible was A.flavus and A.fumigatus with the IC 50 value of & respectively. Lesser activity was seen against C.laurentii, I.orientalis & C.albidus with the IC 50 value of , & respectively. Figure: 3.4, for Amphotericin B, show the regression value. On comparing the IC 50 value of standard to the test extract we can make out following observation. The Ficus racemosa extracts shows significant results compared to the standard against I.orientalis & A.fumigatus. Although the IC 50 values of these extracts were less than the standard but the methanolic extract was more active with better IC 50 values than the other selected extract. The Avicennia marina extracts also depicts significant results compared to the standard against I.orientalis & C.laurentii. Although the IC 50 values of both these extracts were less than the standard but the methanolic extract was more active with better IC 50 values than the other aqueous and other extract. Both the plants are active against the above mentioned fungal strains. 177

16 3.5. CYTOTOXIC SCREENING BY MTT ASSAY All the extracts were evaluated in-vitro against HepG2, HL-60, NCI-H23 and HEK-293T (Human erythrocyte kidney (normal) cellline) by MTT assay. Here, each cell lines were inoculated and preincubated for 24 hrs in 96 well micro-titer plate. Then test compounds were added in 3 fold dilution manner, to make test compound s concentration ranges from 100 µm to µm. Plates were further incubated for 24 hrs. End point determination was performed with use of MTT assay. Results for every test samples were descripted as the % of growth inhibition (IC 50 ) Anticancer evaluation of Methanolic extract of Ficus racemosa Table No. 3.16: IC 50 & R 2 values of Methanolic extract of Ficus racemosa Conc n (µg/ml) HL-60 HepG2 NCI-H23 HEK293 IC > R² Fig. 3.5: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HL

17 Fig. 3.6: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HepG2 Form the Table No. 3.16, we can see that maximum activity of methanolic extract have establish beside HepG2 along with HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and NCI-H23 (near to 1000 µm; can be neglected). Figure: 3.5 & 3.6 for methanolic extract show the dose effect correlation with utmost linearity lest of HepG2 and HL-60 of the six cell lines at R 2 value being and respectively. The %inhibition is rising with augment in the concentration linearly. The graphical correlation for NCI-H23 is non-linear. The trendline for other cell lines is not significant with aberrations. After evaluation, out of the four cell lines, HepG2 and HL-60 cell line showed best results in terms of IC 50 and regression. 179

18 Anticancer evaluation of Methanolic extract of Avicennia marina Table No. 3.17: IC 50 & R 2 values of Methanolic extract of Avicennia marina Conc n (µg/ml) HL-60 NCI-H23 HepG2 HEK293 IC > R² Fig. 3.7: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on HL60 180

19 Fig. 3.8: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on NCI-H23 Form the Table No. 3.17, we can see that maximum activity of methanolic extract have establish beside NCI-H23 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and HepG2 (near to 1000 µm; can be neglected). The figure: 3.7 & 3.8 for methanolic extract show the dose effect correlation with utmost linearity lest of NCI-H23 and HL-60 of the six celllines at R 2 value being and respectively. The graphical correlation for HepG2 is non-linear. The other strains demonstrate irrelevant regression with non-linearity in values of modify of % inhibition with the increase in concentration. After evaluation, out of the four cell lines, NCI-H23 and HL-60 cell line showed best results in terms of IC 50 and regression. 181

20 Anticancer evaluation of Aqueous extract of Avicennia marina Table No. 3.18: IC 50 & R 2 values of Aqueous extract of Avicennia marina Conc n (µg/ml) HepG2 HL-60 NCI-H23 HEK293 IC 50 > R² Fig. 3.9: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on NCI-H23 182

21 Fig. 3.10: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on HL-60 Form the Table No. 3.18, we can see that maximum activity of aqueous extract have establish beside NCI-H23 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and HepG2 (near to 1000 µm; can be neglected). The figure: 3.9 & 3.10 for aqueous extract show the dose effect correlation with utmost linearity lest of NCI-H23 and HL-60 of the six celllines at R 2 value being and respectively. The graphical correlation for HepG2 is non-linear. The other strains show insignificant regression with non linearity in the values of change of % inhibition with the increase in concentration. After evaluation, out of the four celllines, NCI-H23 and HL-60 cell line showed best results in terms of IC 50 and regression. 183

22 3.6. CYTOTOXIC SCREENING BY XTT ASSAY The effect of methanolic extract of Ficus racemosa was executed on 4 diverse celllines via XTT test. DRCs created for XTT method flanked by variety of µg/ml for plant extracts (test) and µM for Doxorubicin, typical drug. Calculation of IC 50 and R² values was completed by graphs produced with Graph-Pad Prism like stastatical application (Ver.5.02). The vulnerability of cells to the extract revelation was differentiating by IC 50 values. Results designates that anti-proliferative consequence reinforces with augment in concentration of extract Anticancer evaluation of Methanolic extract of Ficus racemosa Table No. 3.19: IC 50 & R 2 values of Methanolic extract of Ficus racemosa Conc n (µg/ml) HEK293 HL-60 HepG2 NCI-H23 IC >1000 R² Fig. 3.11: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HL

23 Fig. 3.12: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HEPG2 Form the Table No. 3.19, we can see that maximum activity of methanolic extract have establish beside HepG2 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and NCI-H23 (near to 1000 µm; can be neglected). The figure: 3.11 & 3.12 for methanolic extract show the dose effect correlation with utmost linearity lest of HepG2 and HL-60 of the six celllines at R 2 value being and respectively. The % inhibition is rising with augment in concentration. The graphical correlation for NCI-H23 is non-linear. The trendline for other cell lines is not significant with aberrations. The linearity shown by the HEK-293T cell line is linear upto certain extent & shown aberration afterwards. After evaluation, out of the four cell lines, HepG2 and HL-60 cell line showed best results in terms of IC 50 and regression. 185

24 Anticancer evaluation of Methanolic extract of Avicennia marina Table No. 3.20: IC 50 & R 2 values of Methanolic extract of Avicennia marina Conc n (µg/ml) HEK293 HL-60 HepG2 NCI-H23 IC > R² Fig. 3.13: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on NCI-H23 186

25 Fig. 3.14: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on HL-60 Form the Table No. 3.20, we can see that maximum activity of methanolic extract have establish beside NCI-H23 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and HepG2 (near to 1000 µm; can be negligible or lower activity). The figure: 3.13 & 3.14 for methanolic extract show the dose effect correlation with utmost linearity lest of NCI-H23 and HL-60 of the six celllines at R 2 value being and respectively. The graphical correlation for HepG2 is nonlinear. Other strains show irrelevant regression with non-linearity in the values of alteration of % inhibition with the increase in concentration. After evaluation, out of the four cell lines, NCI-H23 and HL-60 cell line showed best results in terms of IC 50 and regression. 187

26 Anticancer evaluation of Aqueous extract of Avicennia marina Table No. 3.21: IC 50 & R 2 values of Aqueous extract of Avicennia marina Conc n (µg/ml) HEK293 HL-60 HepG2 NCI-H23 IC > R² Fig. 3.15: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on NCI-H23 188

27 Fig. 3.16: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on HL-60 Form the Table No. 3.21, we can see that maximum activity of aqueous extract have establish beside NCI-H23 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and HepG2 (near to 1000 µm; can be neglected). The figure: 3.15 & 3.16 for aqueous extract show the dose effect correlation with utmost linearity lest of NCI-H23 and HL-60 of the six celllines at R 2 value being and respectively. The graphical correlation for HepG2 is non-linear. The other strains show irrelevant regression with non-linearity in the values of alteration of % inhibition with the increase in concentration. After evaluation, out of the four cell lines, NCI-H23 and HL-60 cell line showed best results in terms of IC 50 and regression. 189

28 3.7 CYTOTOXIC SCREENING BY SRB ASSAY The effect of methanolic extract of Ficus racemosa was executed on 4 diverse celllines by SRB assay. DRCs created for SRB technique flanked by the variety of µg/ml for plant extracts (test) and µM for Doxorubicin, typical drug. Calculation of IC 50 and R² values was completed by graphs produced with Graph-Pad Prism like stastatical application (Ver.5.02). The vulnerability of cells to the extract revelation was differentiating by IC 50 values. Results designates that anti-proliferative consequence reinforces with augment in concentration of extract Anticancer evaluation of Methanolic extract of Ficus racemosa Table No. 3.22: IC 50 & R 2 values of Methanolic extract of Ficus racemosa Conc n (µg/ml) HEK293 HL-60 HepG2 NCI-H23 IC >1000 R² Fig. 3.17: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HEPG2 190

29 Fig. 3.18: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HL-60 Form the Table No. 3.22, we can see that maximum activity of methanolic extract have establish beside HepG2 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and NCI-H23 (near to 1000 µm; can be neglected). The figure: 3.17 & 3.18 for methanolic extract show the dose effect correlation with utmost linearity lest of HepG2 plus HL-60 of the six celllines at R 2 value being and respectively. The % inhibition is increasing with increase in the concentration. Graphical correlation for NCI-H23 is non-linear. The trendline for other cell lines is not significant with aberrations. The linearity shown by the HEK-293T cell line is linear upto certain extent & shown aberration afterwards. After evaluation, out of the four cell lines, HepG2 and HL-60 cell line showed best results in terms of IC 50 and regression. 191

30 Anticancer evaluation of Methanolic extract of Avicennia marina Table No. 3.23: IC 50 & R 2 values of Methanolic extract of Avicennia marina Conc n (µg/ml) HEK293 HL-60 HepG2 NCI-H23 IC > R² Fig. 3.19: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on HL

31 Fig. 3.20: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on NCI-H23 Form the Table No. 3.23, we can see that maximum activity of methanolic extract have establish beside NCI-H23 and HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and HepG2 (near to 1000 µm; can be negligible or lower activity). The figure: 3.19 & 3.20 for methanolic extract show the dose effect correlation with utmost linearity lest of NCI-H23 plus HL-60 of the six celllines at R 2 value being and respectively. The graphical correlation for HepG2 is nonlinear. The other strains show irrelevant regression with non-linearity in the values of change of %inhibition with the increase in concentration. After evaluation, out of the four cell lines, NCI-H23 and HL-60 cell line showed best results in terms of IC 50 and regression. 193

32 Anticancer evaluation of Aqueous extract of Avicennia marina Table No. 3.24: IC 50 & R 2 values of Aqueous extract of Avicennia marina Conc n (µg/ml) HEK293 HL-60 HepG2 NCI-H23 IC > R² Fig. 3.21: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on NCI-H23 194

33 Fig. 3.22: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on HL-60 Form the Table No. 3.24, we can see that maximum activity of aqueous extract have establish beside NCI-H23 plus HL-60 having IC 50 : and respectively. But none of extract showed activity against HEK-293T and HepG2 (near to 1000 µm; can be neglected). The figure: 3.21 & 3.22 for aqueous extract show the dose effect correlation with utmost linearity lest of NCI-H23 and HL-60 of the six cell lines at R 2 value being and respectively. The graphical correlation for HepG2 is non-linear. Other strain shows irrelevant regression with non-linearity in the values of change of % inhibition with the increase in concentration. After evaluation, out of the four cell lines, NCI-H23 and HL-60 cell line showed best results in terms of IC 50 and regression. 195

34 3.8. IC 50 OF DOXORUBICIN BY MTT, XTT AND SRB ASSAY Table No. 3.25: IC 50 values of doxorubicin by MTT, XTT and SRB assay Cell lines MTT Assay XTT Assay SRB Assay HL HEPG NCI-H Doxorubicin has good IC 50 value against all the panel tumor cell line where as in case of HEK 293T, Doxorubicin was found inactive. (Table No. 3.25) For the confirmation purpose, Doxorubicin was again screened by XTT assay against the same panels of cell lines. In case of XTT assay, Doxorubicin was found active against all tumor cell line while found inactive against HEK 293T cell line, which confirm the cytotoxicity and inactiveness of Doxorubicin against tumor cell lines and HEK 293T cell line (normal) respectively. Fig. 3.23: % Inhibition v/s log conc (ng/ml) of Doxorubicin by MTT Assay 196

35 Fig. 3.24: % Inhibition v/s log conc (ng/ml) of Doxorubicin by XTT Assay 197

36 Fig. 3.25: % Inhibition v/s log conc (ng/ml) of Doxorubicin by SRB Assay Here particular extracts of Ficus racemosa and Avicennia marina were found Cytotoxic against certain cancer cell lines but found inactive or negligible against normal cell line, HEK-293T. It may because of the reason that extracts are targeting only the rapidly proliferating cells. But this reason is not enough to justify why the compounds are Cytotoxic only against fast proliferating cancer cell lines. Justification behind this thing is that, compounds may have specific molecular targets in tumor cell which may not be present in normal (HEK-293T) cell, and hence compounds are inactive against HEK-293T cells. For assessment of these specific molecular targets, specific molecular targeting assay should be performed. 198

37 Here different IC 50 values of Doxorubicin against tumor cells by various methodologies were observed which is due to different sensitivity of different methods. Both MTT and XTT assay works on similar principle, the only differences between two is that MTT give water insoluble formazan product where as XTT give water soluble formazan product. And SRB assay work on upon the quantitative staining of cellular proteins by sulforhodamine B, which provides a sensitive linear response and stable color development. 199