3. results and discussion - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/27220/12/12_chapter...

RESULTS AND DISCUSSION

PH.D. THESIS CHAPTER 3

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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. 3.1. 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 % dry weight

in gm. Colour Odour Consistency

Alcoholic 15.6 Dark brown Characteristic Powder

SUCCESSIVE EXTRACTION Petroleum

ether (40-600C)

2.70 Pale yellow Characteristic Waxy

Ethyl acetate 0.61 Dark green Characteristic Sticky

Alcohol 3.35 Dark brown Characteristic Powder

Water 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:



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Table No. 3.2: Phyto-constituent Chemical Identification

Phyto - Constituent

Alcoholic Extract

SUCCESSIVE EXTRACTION PE EA AL AQ

Carbohydrates + – + + + Glycosides + – + + + Phytosterol

Steroids + + + + –

Triterpenoids + + + + – Tannins & Phenolic Group

+ – – + +

Flavonoids + – + + + Key words:

+ = Present, – = Absent

PE = Petroleum ether extract (40-600C)

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 13.34 %

2 Acid-insoluble ash 0.934 %

3 Water soluble ash 3.75 %

4 Alcohol soluble extractive 6.112 %

5 Water soluble extractive 8.986 %



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In preliminary study, Ficus racemosa showed Total ash (13.34 %), Acid-insoluble

ash (0.934 %) and Water soluble ash (3.75 %).

3.2. 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. 3.4.

Table No. 3.4: Percentage extractives values and physical characteristics of Different extracts

of Avicennia marina leaf

Extracts % dry weight

in gm. Consistency Odour

Alcoholic 11.6 Powder Aromatic

SUCCESSIVE EXTRACTION Petroleum

ether (40-600C)

3.10 Waxy Aromatic

Chloroform 2.11 Powder Aromatic

Alcohol 13.35 Powder Aromatic

Water 19.48 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

Table No. 3.5: Phyto-constituent Chemical Identification

Phyto - Constituent

Alcoholic Extract

Successive Extraction

PE CH AL AQ

Carbohydrates + – + + + Glycosides + – + + – Phytosterol

Steroids + + + + –

Triterpenoids + + + + – Tannins & Phenolic Group

+ – – + +

Flavonoids + – – + +



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Key words:

+ = Present , – = Absent

PE = Petroleum ether extract (40-600C)

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 12.42 %

2 Acid-insoluble ash 0.24 %

3 Water soluble ash 9.65 %

4 Alcohol soluble extractive 11.12 %

5 Water soluble extractive 18.06 %

In preliminary study, Avicennia marina showed Total ash (12.42 %), Acid-

insoluble ash (0.24 %) and Water soluble ash (9.65 %).

3.3. CHARACTERIZATION OF CELL LINES AND CULTURE MED IA

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.



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Table No. 3.7: Results for Characterization of cell lines

Cell line % Viability PDT

(hrs) Microbial

contamination Cross

contamination pH

Stock After

NCI – H23 64.54 81.91 36.9 No contamination No contamination 7.5

HL-60 61.19 86.53 35.2 No contamination No contamination 7.5

Hep G2 63.63 88.77 28.7 No contamination No contamination 7.5

HEK 293T 61.81 87.98 19.3 No contamination No contamination 7.0

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, 10000 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 TIM E

1. HEK 293T cell line

At the time of Subculture, density of HEK 293T cell line derived from NCCS, Pune

was around 1.8 × 107 cells / flask and viability was 61.81 %, 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



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a consequence, on the fourth day morning cell density was increased up to 6.4 ×

107 and viability was approximately 86.91 % 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 1st 2nd 3rd 4th

Viable cell count 34 43 70 77

Non-viable cell count 21 18 17 11

% viability 61.81 70.79 80.16 86.91

Cells/ml 3.6×105 5.4×105 9×105 12.8×105

Total cells in flask (50ml) 1.8×107 2.7×107 4.5×107 6.4×107

Viable cells in flask (50ml) 9.81×106 16.95×106 30.2×106 46 ×106

pH 7.5 5.0 4.5 5.0

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 3rd or 4th day i.e. twice in week.

2. NCI – H23 cell line

Table No. 3.9: Result for Subculture of NCI – H23 cell line

Days 1st 3rd 4th

Viable cell count 43 56 86

Non-viable cell count 24 23 18

% viability 64.54 71.23 81.91

Cells/ml 5.2×105 10.4×105 15×105

Total cells in flask (50ml) 2.6×107 5.2×107 7.5×107

Viable cells in flask (50ml) 15×106 37×106 65×106

pH 7.5-8.0 7.5 7

PDT 36.9 hrs

NCI – H23 is adherent cell line, viability of which was around 64.54 % and density

was 2.6 × 107. In order to boost cell viability plus cell density, subculturing was

performed by diluting cells in to fresh medium in new culture flask. 81.91 % cell



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viability was achieved after 48 hrs of incubation and at that time cell density was

found 7.5 × 107 cells / flask. PDT was found to be around 36.9 hrs, so subculturing

was done after every 5th 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 1st 3rd 4th



% viability 61.19 74.68 86.53

Cells/ml 6.15×105 13.4×105 16×105



pH 7.5-8.0 7.5 7

PDT 35.2 hrs

HL-60 is suspension cell line, viability of which was around 61.19 % and density






HL-60 cell line.

4. Hep G2 cell line:

Table No. 3.11: Result for Subculture of Hep G2 cell line

Days 1st 3rd 4th



% viability 63.63 73.56 88.77

Cells/ml 5.8×105 12.3×105 15.8×105



pH 7.5-8.0 7.5 7

PDT 28.7 hrs



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Hep G2 is adherent cell line, viability of which was around 63.63 % and density






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 cross-

contamination.

3.4. ANTIFUNGAL SCREENING BY NCCLS ASSAY

3.4.1. 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 0.005-100µg/ml for plant extracts (test). For NCCLS

(antifungal screening method) DRC was prepared flanked by series of 0.005 -

100µg/ ml and 0.005 - 100µM for Amphotericin-B. Calculation of IC50 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 IC50 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.



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Table No. 3.12 (A): % Inhibition of Methanolic extract of Ficus racemosa Conc. (µg/ml) Log Conc (ng/ml) A.Flavus A.Fumigatus I.Orientalis

100 5 55.42 54.99 62.08 33.3 4.518514 54.78 38.03 56.66 11.1 4.041393 52.96 31.98 55.32 3.7 3.568202 38.74 22.36 40.52

1.23 3.089905 31.26 19.24 22.57 0.41 2.613842 19.32 17.69 7.43 0.13 2.136721 11.27 14.75 4.50 0.04 2.732394 7.52 8.62 1.19

0.015 1.176091 2.20 2.64 0.62 0.005 0.69897 1.38 0.58 0.17

Table No. 3.12 (B): IC50 & R 2 values for Methanolic extract of Ficus racemosa

Fig. 3.1: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa

Conc.

(µg/ml)

A.

Flavus

A.

Fumigatus

Cry.

Albidus

Cry.

Layrentii

I.

Orientalis

IC 50 476.072 359.536 >1000 >1000 208.129

R² 0.985 0.974 0.951 0.968 0.952



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From the Table No. 3.12, we see that maximum activity of Methanolic extract have

established against I.Orientalis and A.fumigatus having IC50 208.129 and 359.536

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 IC50 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. (IC50 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 IC50 and regression.

3.4.2. Antifungal activity of Methanolic extract of Avicennia marina

Table No. 3.13 (A): % Inhibition of Methanolic extract of Avicennia marina Conc. (µg/ml) Log Conc (ng/ml) Cry.Albidus Cry.Layrentii I.Orientalis

100 5 44.25 45.92 65.22 33.3 4.518514 38.08 32.51 58.76 11.1 4.041393 23.93 25.96 56.42 3.7 3.568202 21.67 19.13 41.50

1.23 3.089905 17.01 15.22 25.18 0.41 2.613842 13.06 11.87 19.59 0.13 2.136721 11.53 8.46 5.68 0.04 2.732394 9.44 5.56 2.05

0.015 1.176091 5.34 2.30 1.46 0.005 0.69897 1.34 0.08 1.40

Table No. 3.13 (B): IC50 & 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 >1000 675.231 599.442 412.131

R² 0.978 0.932 0.902 0.919 0.985



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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 IC50 412.131, 599.442 and

675.231 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. (IC50 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 0.985.

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.



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3.4.3. Antifungal activity of Aqueous extract of Avicennia marina

Table No. 3.14 (A): % Inhibition of Aqueous extract of Avicennia marina Conc. (µg/ml) Log Conc (ng/ml) Cry.Albidus Cry.Layrentii I.Orientalis

100 5 49.56 51.89 60.69 33.3 4.518514 43.74 46.73 52.95 11.1 4.041393 42.96 37.45 41.32 3.7 3.568202 31.90 36.76 18.42

1.23 3.089905 23.77 36.42 17.79 0.41 2.613842 17.54 22.32 10.50 0.13 2.136721 11.68 16.53 7.48 0.04 2.732394 9.06 10.2 3.77

0.015 1.176091 5.02 3.57 1.65 0.005 0.69897 3.92 1.97 0.28

Table No. 3.14 (B): IC50 & R 2 values for Aqueous extract of Avicennia marina

Fig. 3.3: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina

Conc.

(µg/ml)

A.

Flavus

A.

Fumigatus

Cry.

Albidus

Cry.

Layrentii

I.

Orientalis

IC 50 >1000 >1000 617.129 602.120 347.179

R² 0.918 0.926 0.909 0.975 0.916



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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 IC50 347.179,

617.129 and 602.120 respectively with profound R2 values 0.916, 0.909 and 0.975

respectively. Lower activities were found in other following stains.

Figure: 3.3, I.orientalis shows maximum linearity of the five strains at value being

0.916. 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.

(IC50 values more than 1000 µg/ml)

3.4.4. Antifungal activity of standard drug Amphotericin B

Table No. 3.15 (A): % Inhibition of standard drug Amphotericin B Conc. Log

Conc. A.Flavus A.Fumigatus I.Orientalis Cry.Albidus Cry.Layrentii

100 5 67.26 78.56 62.05 74.19 68.08 33.3 4.518514 61.01 68.76 49.67 55.65 59.65 11.1 4.041393 55.72 55.75 45.03 49.67 34.65 3.7 3.568202 45.35 47.76 37.88 38.60 32.55

1.23 3.089905 39.86 38.16 32.23 34.57 28.94 0.41 2.613842 33.95 27.05 20.50 27.54 23.76 0.13 2.136721 25.15 19.87 18.96 23.53 18.56 0.04 2.732394 18.09 10.26 13.66 19.45 15.84

0.015 1.176091 15.23 4.67 9.49 12.43 9.56 0.005 0.69897 7.37 2.57 7.68 6.56 5.65

Table No. 3.15 (B): IC50 & R 2 values for standard drug Amphotericin B

Conc.

(µg/ml)

A.

Flavus

A.

Fumigatus

Cry.

Albidus

Cry.

Layrentii

I.

Orientalis

IC 50 4.365 6.025 16.456 22.387 27.542 R² 0.985 0.992 0.913 0.897 0.967



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



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The IC50 value for the standard compound Amphotericin-B is given in the Table

No. 3.15.

The IC50 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 IC50 value of 4.365 & 6.025 respectively. Lesser activity was seen against

C.laurentii, I.orientalis & C.albidus with the IC50 value of 22.387, 27.542 &

16.456 respectively.

Figure: 3.4, for Amphotericin–B, show the regression value. On comparing the IC50

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 IC50 values of these extracts were less

than the standard but the methanolic extract was more active with better IC50 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 IC50 values of both these extracts were less than the standard but the

methanolic extract was more active with better IC50 values than the other aqueous

and other extract.

Both the plants are active against the above mentioned fungal strains.



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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 0.005 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

(IC50).

3.5.1. Anticancer evaluation of Methanolic extract of Ficus racemosa

Table No. 3.16: IC50 & R 2 values of Methanolic extract of Ficus racemosa

Conc’n (µg/ml) HL-60 HepG2 NCI-H23 HEK293

IC 50 276.85 362.95 >1000 775.65

R² 0.9707 0.9843 0.9624 0.9834

Fig. 3.5: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HL-60



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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 IC50: 362.95 and 276.85

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 R2 value being

0.9843 and 0.9707 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 IC50 and regression.



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3.5.2. Anticancer evaluation of Methanolic extract of Avicennia marina

Table No. 3.17: IC50 & R 2 values of Methanolic extract of Avicennia marina

Conc’n (µg/ml) HL-60 NCI-H23 HepG2 HEK293

IC 50 297.934 210.987 >1000 762.980

R² 0.9452 0.9610 0.9712 0.9870

Fig. 3.7: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on HL60



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Fig. 3.8: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on NCI-H23


have establish beside NCI-H23 plus HL-60 having IC50: 210.987 and 297.934

respectively. But none of extract showed activity against HEK-293T and HepG2


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 R2 value being

0.9610 and 0.9452 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 IC50 and regression.



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3.5.3. Anticancer evaluation of Aqueous extract of Avicennia marina

Table No. 3.18: IC50 & R 2 values of Aqueous extract of Avicennia marina

Conc’n (µg/ml) HepG2 HL-60 NCI-H23 HEK293

IC 50 >1000 281.175 220.127 717.125

R² 0.8089 0.9927 0.9863 0.9523

Fig. 3.9: % Inhibition v/s log conc (ng/ml) of Aqueous extract of Avicennia marina on NCI-H23



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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




The figure: 3.9 & 3.10 for aqueous extract show the dose effect correlation with



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




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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 0.005-

100 µg/ml for plant extracts (test) and 0.005 - 100µM for Doxorubicin, typical

drug. Calculation of IC50 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 IC50 values. Results designates that

anti-proliferative consequence reinforces with augment in concentration of extract.



Conc’n (µg/ml) HEK293 HL-60 HepG2 NCI-H23

IC 50 812.177 287.126 321.742 >1000

R² 0.9807 0.9723 0.9857 0.9846




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Fig. 3.12: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HEPG2


have establish beside HepG2 plus HL-60 having IC50: 321.742 and 287.126



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 R2 value being

0.9857 and 0.9723 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.





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IC 50 757.981 277.129 >1000 221.173

R² 0.9076 0.9727 0.9709 0.9565




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Fig. 3.14: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on HL-60




(near to 1000 µM; can be negligible or lower activity).


with utmost linearity lest of NCI-H23 and HL-60 of the six celllines at R2 value

being 0.9525 and 0.9727 respectively. The graphical correlation for HepG2 is non-

linear. Other strains show irrelevant regression with non-linearity in the values of

alteration of % inhibition with the increase in concentration.





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IC 50 794.792 291.773 >1000 237.179

R² 0.9870 0.9149 0.9034 0.9645




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The other strains show irrelevant regression with non-linearity in the values of

alteration of % inhibition with the increase in concentration.





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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

0.005-100 µg/ml for plant extracts (test) and 0.005 - 100µM for Doxorubicin,

typical drug. Calculation of IC50 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 IC50 values. Results designates

that anti-proliferative consequence reinforces with augment in concentration of

extract.




IC 50 701.271 290.175 312.765 >1000

R² 0.9478 0.9618 0.9188 0.9802

Fig. 3.17: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Ficus racemosa on HEPG2



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have establish beside HepG2 plus HL-60 having IC50: 312.765 and 290.175




with utmost linearity lest of HepG2 plus HL-60 of the six celllines at R2 value

being 0.9188 and 0.9618 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.





192192192192




IC 50 751.272 312.147 >1000 293.177

R² 0.9774 0.9865 0.9872 0.9112

Fig. 3.19: % Inhibition v/s log conc (ng/ml) of Methanolic extract of Avicennia marina on HL-60



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have establish beside NCI-H23 and HL-60 having IC50: 293.177 and 312.147


(near to 1000 µM; can be negligible or lower activity).


with utmost linearity lest of NCI-H23 plus HL-60 of the six celllines at R2 value

being 0.9112 and 0.9865 respectively. The graphical correlation for HepG2 is non-

linear. The other strains show irrelevant regression with non-linearity in the values

of change of %inhibition with the increase in concentration.





194194194194




IC 50 771.492 302.156 >1000 275.917

R² 0.9893 0.9417 0.9980 0.9709




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utmost linearity lest of NCI-H23 and HL-60 of the six cell lines at R2 value being


Other strain shows irrelevant regression with non-linearity in the values of change

of % inhibition with the increase in concentration.





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3.8. IC50 OF DOXORUBICIN BY MTT, XTT AND SRB ASSAY

Table No. 3.25: IC50 values of doxorubicin by MTT, XTT and SRB assay

Cell lines MTT Assay XTT Assay SRB Assay

HL-60 28.39 29.46 27.83

HEPG2 21.19 25.73 23.89

NCI-H23 26.76 25.43 25.80

Doxorubicin has good IC50 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



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



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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.



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Here different IC50 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.

3. results and discussion - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/27220/12/12_chapter...

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