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Anticancer Activity of Methanol extract of Limnophila repens and Argyeia cymosa by

Using SRB Assay
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Manuscript Number:

Full Title: Anticancer Activity of Methanol extract of Limnophila repens and Argyeia cymosa by
Using SRB Assay

Article Type: Research Article

Section/Category: Author-initiated submission

Keywords: Limnophila Repens, Argyeia Cymosa, Anticancer Activity Srb Assay

Corresponding Author: GUNJI VENKATESWARLU

A M Reddy Memorial College Of Pharmacy,
Narasaraopeta, Andhraparadesh INDIA

Corresponding Author's Institution: A M Reddy Memorial College Of Pharmacy,

First Author: GUNJI VENKATESWARLU

Order of Authors: GUNJI VENKATESWARLU

S Ganapaty

Order of Authors Secondary Information:

Manuscript Region of Origin: INDIA

Abstract: Introduction: Plant-derived compounds constitute more than 50% of anticancer agents.
Cell viability assays can be combined with apoptosis assays to provide more
information about mechanisms of cell death through multiplexing assays on a single. I
attempted an experiment to find out the anti cancer activity of selected plants
Limnophila repens and Argyeia cymosa by using SRB Assay. Material method:
Methanol extract of the both plants tested for its anti cancer activity. Results and
disscussionThe anticancer activity of methanolic extracts of Argyreia cymosa and
Limnophila repens was determined by sulforhodamine B colorimetric assay. The
results of the cytotoxicity of extracts from both plant extracts analysedand  The
Methanol extract of Limnophila repens extract showed comparable activity to the
standard compound, i.e., Adriamycin on Ishikawa (human endometrial
adenocarcinoma) and SCC-29B (human oral cancer) cell lines, respectively. This
extract showed TGI, and GI 50 was 38.9 and <10 µg/ ml on Ishikawa cell lines and
58.12, <10 and <10 µg/ ml of LC 50 , TGI and GI 50 activity on SCC-29B cell lines
respectively.The Methanol extract of Argyeia cymosa showed GI 50 was >80 µg/ ml
on Ishikawa cell lines and <10 and <10 µg/ ml of, TGI and GI 50 activity on SCC-29B
cell lines .Estimations based on GI 50 values shows that MELR was more active
against Ishikawa and SCC-29B cell lines than MEAC on Ishikawa (human endometrial
adenocarcinoma) and SCC-29B (human oral cancer) cell lines

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Title:

Anticancer Activity of Methanol extract of Limnophila repens and

Argyeia cymosa by Using SRB Assay

Venkateswarlu G *1, Ganapaty S 2

1
Department of Pharmacognosy and Phytochemistry,
A. M. Reddy Memorial College of Pharmacy, India.
2
Department of Pharmacognosy and Phytochemistry, Gitam University, India

Corresponding author:
G Venkateswarlu,M.Pharm
Faculty of pharmacy,
Department of Pharmacognosy and Phytochemistry,
A. M. Reddy Memorial College of Pharmacy, India.
Email:venkateswarlugunji@gmail.com
Ph.No:9000079873
Abstract

Anticancer Activity of Methanol extract of Limnophila repens and

Argyeia cymosa by Using SRB Assay

Abstract:

Introduction: Plant-derived compounds constitute more than 50% of anticancer agents. Cell
viability assays can be combined with apoptosis assays to provide more information about
mechanisms of cell death through multiplexing assays on a single. I attempted an experiment to
find out the anti cancer activity of selected plants Limnophila repens and Argyeia cymosa by
using SRB Assay. Material method: Methanol extract of the both plants tested for its anti cancer
activity. Results and disscussionThe anticancer activity of methanolic extracts of Argyreia
cymosa and Limnophila repens was determined by sulforhodamine B colorimetric assay. The
results of the cytotoxicity of extracts from both plant extracts analysedand The Methanol extract
of Limnophila repens extract showed comparable activity to the standard compound, i.e.,
Adriamycin on Ishikawa (human endometrial adenocarcinoma) and SCC-29B (human oral
cancer) cell lines, respectively. This extract showed TGI, and GI50 was 38.9 and <10 µg/ ml on
Ishikawa cell lines and 58.12, <10 and <10 µg/ ml of LC50, TGI and GI50 activity on SCC-29B
cell lines respectively.The Methanol extract of Argyeia cymosa showed GI50 was >80 µg/ ml on
Ishikawa cell lines and <10 and <10 µg/ ml of, TGI and GI50 activity on SCC-29B cell lines
.Estimations based on GI50 values shows that MELR was more active against Ishikawa and SCC-
29B cell lines than MEAC on Ishikawa (human endometrial adenocarcinoma) and SCC-29B
(human oral cancer) cell lines
Keywords Click here to access/download;Keywords;key words.docx

Key words: Limnophila Repens, Argyeia Cymosa, Anticancer Activity Srb Assay
Manuscript Body

Anticancer Activity of Methanol extract of Limnophila repens and

Argyeia cymosa by Using SRB Assay

INTRODUCTION

ANTICANCER

Cancer is one of the most life-threatening diseases with more than 100 different types. Due to the
lack of effective drugs, expensive cost of chemotherapeutic agents and side effects of anticancer
drugs, cancer can be a cause of death. Cell death can occur through several different
mechanisms, of which the most widely described are apoptosis and necrosis.[1,2] A significant
physiological consequence of cell death by apoptosis is that the apoptotic cells are immediately
phagocytosed by macrophages. Therefore, the release of intracellular molecules that cause
secondary disturbance to the surrounding tissue is limited to a low level compared with necrosis,
which causes further tissue destruction and inflammation .[3,4]Now people have started realizing
the importance of natural bioactive substances found in fruits, vegetables, and herbs, as
.[5,6]
antioxidants and functional foods Some of these substances are believed to be potential
[7,8]
chemopreventive or therapeutic agents for cancer Most of these substances exert their
chemotherapeutic activity by blocking the cell cycle progression and triggering apoptotic cell
death. Therefore, the induction of apoptosis in tumour cells has become an indicator of the
tumour-treating ability of naturally derived bioactive.[9,10] Apoptosis or programmed cell death is
a highly organized physiological process to eliminate damaged or abnormal cells. It also plays a
major role in embryogenesis where normal cells undergo apoptosis. It is involved in maintaining
homeostasis in multicellular organisms.During the past decades, the induction of apoptosis in
cells has been recognized as a novel strategy for the identification of anticancer drugs
.[10]Apoptosis itself also plays an important role in the development of various diseases including
cancer .Apoptosis is triggered by activation of the death receptor (extrinsic) and mitochondrial
(intrinsic) pathways, results from activation of members of cysteine protease family called
[11]
caspases. Recent studies on tumour inhibitory compounds of plant origin have yielded an
impressive array of novel structures. Epidemiological studies suggest that consumption of diets
containing fruits and vegetables, which are major sources of phytochemicals and micronutrients,
may reduce the risk of developing cancer. Certain products from plants are known to induce
[12]
apoptosis in neoplastic cells but not in normal cells. In this present study I attempted an
experiment to find out the anti cancer activity of selected plants Limnophila repens and Argyeia
cymosa by using SRB Assay

Materials and Methods

Plant Collection and Authentication

The herb, Argyreia cymosa, was collected at Tirupati during September 2017. The
examined herb was recognised and verified by the botanist Dr .K. Madhava Chetty. A specimen
of the herb, with the voucher number 1568, was deposited at A. M. Reddy memorial college of
Pharmacy, Narsaraopet, Andhra Pradesh.

Preparation of Extract

The freshly gathered herbs (two plants) were shade dried and pulverized. The powder (1 kg) was
extracted by way of petroleum ether meant for removing fatty and waxy materials. It was air-
dried and then macerated by way of methanol, strained and then concentrated at 45oC in Buchi
rotavapor. Finally, the weight of methanolic extract acquired was 75g (7.5% w/w yield).This
crude extract used for evaluation of anti cancer activity.

ANTICANCER ACTIVITY OF Limnophila repens AND Argyeia cymosa BY USING SRB

ASSAY

The anticancer activity of isolated constituents of A Limnophila repens and Argyeia cymosa was
performed on Ishikawa SCC-29B and cancer cell lines by the Advanced Centre for Treatment
Research and Education in Cancer (ACTREC) Mumbai, India. The cell viability was measured
using the SRB assay. All the environmental conditions were maintained throughout the
experiment for all the groups. The assay was performed in triplicate for each of the extracts. The
growth curve was plotted against the molar drug concentration of isolated constituents and %
control growth.

Experimental procedure or SRB assay [13, 14, 15]

The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM
L-glutamine. For the present screening experiment, cells were inoculated into 96 well microtiter
plates in 100 µL at plating densities as shown in the study details above, depending on the
doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated
at 37°C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h before addition of experimental
drugs.

Experimental drugs were initially solubilized in dimethyl sulfoxide at 100mg/ml and diluted to
1mg/ml using water and stored frozen before use. At the time of drug addition, an aliquot of
frozen concentrate (1mg/ml) was thawed and diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and
800 μg/ml with complete medium containing test article. Aliquots of 10 µl of these different drug
dilutions were added to the appropriate microtiter wells already containing 90 µl of the medium,
resulting in the required final drug concentrations i.e.10 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml.

After compound addition, plates were incubated at standard conditions for 48 hours, and the
assay was terminated by the inclusion of cold TCA. Cells had been fixed in situ by the mild
addition of 50 µl of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 1
hr at 4°C. The supernatant had been discarded; the plates had been rinsed 5 times with tap water
and air-dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid had
been put into each one of the wells, and plates had been incubated for 20 minutes at room
temperature. After staining, the unbound dye was retrieved, and the residual dye had been
eliminated through washing five times with 1 % acetic acid. The plates were air dried. The bound
stain had been consequently eluted with 10 mM trizma base, and the absorbance was read on a
plate reader at a wavelength of 540 nm with 690 nm reference wavelength.

Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells.
Percentage Growth was expressed as the ratio of average absorbance of the test well to the
average absorbance of the control wells X 100.

Using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in
the presence of drug at the four concentration levels (Ti)]; the percentage growth was calculated
at each of the drug concentration levels.

Percentage growth inhibition= For concentrations for which Ti>/=Tz (Ti-Tz) positive or zero =
[(Ti-Tz)/(C-Tz)] × 100
For concentrations for which Ti >/=Tz (Ti-Tz) positive or zero = [(Ti-Tz)/(C-Tz)] × 100
For concentrations for which Ti < Tz (Ti-Tz) negative = [(Ti-Tz)/(C-Tz)] × 100
Growth inhibition of 50%
GI50 = [(Ti-Tz)/(C-Tz)] × 100
GI50 is that value of the drug concentration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in control cells during the drug incubation. The drug
concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 is
the drug concentration resulting in a 50% reduction in the measured protein at the end of the
drug treatment as compared to that at the beginning. During this there is a net loss of 50% cells
following treatment is calculated from [(Ti-Tz)/Tz] × 100 = -50.

Statistical analysis

Values were calculated for each of these three parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded, the values for that parameter were
expressed as greater or less than the maximum or minimum concentration tested. The experiment
data were estimated using linear regression method of plots of the cell viability against the molar
drug concentration of tested compounds.

RESULTS AND DISSCUSSION

ANTICANCER ACTIVITY OF Argyreia cymosa and Limnophila repens

In the present investigation, anticancer activity (in vitro) of methanolic extracts of Argyreia
cymosa and Limnophila repens were carried out on Ishikawa (human endometrial
adenocarcinoma cell lines) and SCC-29B (Human oral cancer cell lines) by SRB assay. After
completion of the experimentation, the absorbance was read on an Elisa plate reader at a
wavelength of 540 nm and values were plotted on graph and LC50, TGI and GI50 were then
calculated from the graph (Figure 1 & 2). Along with adriamycin and extracts treated cells also
showed karyolysis, apoptosis, rounding of the cell. GI50 means the drug concentration resulting
in a 50% reduction in the net protein increase as compared to control cells. TGI is the drug
concentration resulting in total growth inhibition. The LC50 is the drug concentration resulting in
a 50% reduction in the measured protein at the end as compared to the beginning. The MELR
showed TGI, and GI50 was 38.9 and <10 µg/ ml on Ishikawa cell lines and 58.12, <10 and <10
µg/ ml of LC50, TGI and GI50 activity on SCC-29B cell lines respectively .The MEAC showed
GI50 was >80 µg/ ml on Ishikawa cell lines and no activity on SCC-29B cell lines respectively
(Table 1 – 4).

ANTICANCER ACTIVITY OF ISOLATED CONSTITUENTS FROM MEAC AND

MELR

The anticancer activity of methanolic extracts of Argyreia cymosa and Limnophila repens
was determined by sulforhodamine B colorimetric assay. The results of the cytotoxicity of
extracts from both plant extracts are shown in Table 3. The MELR extract showed comparable
activity to the standard compound, i.e., Adriamycin on Ishikawa (human endometrial
adenocarcinoma) and SCC-29B (human oral cancer) cell lines, respectively. The MELR showed
TGI, and GI50 was 38.9 and <10 µg/ ml on Ishikawa cell lines and 58.12, <10 and <10 µg/ ml of
LC50, TGI and GI50 activity on SCC-29B cell lines respectively.The MEAC showed GI50 was
>80 µg/ ml on Ishikawa cell lines and <10 and <10 µg/ ml of, TGI and GI50 activity on SCC-29B
cell lines respectively (Table 5.25 – 5.28).

Estimations based on GI50 values shows that MELR was more active against Ishikawa
and SCC-29B cell lines than MEAC on Ishikawa (human endometrial adenocarcinoma) and
SCC-29B (human oral cancer) cell lines.

CONCLUSION

These earlier research reveal this plant has many phytochemicals which could possess possible
anticancer activity, possibly singly or in combination. The current outcomes, along with more
previous scientific studies, claim that Argyreia cymosa and Limnophila repens have shown
anticancer activity.

ACKNOWLEDGEMENT

We the authors are thankful to the management and staff of GITAM University,

Visakapatnam and A M Reddy Memorial college of Pharmacy,Department of pharmacognosy

for providing facilities and encouragement.

CONFLICT OFR INTERST

The authors have no conflict of intrest

REFERENCES:

1.George, S., Bhalerao, S. V., Lidstone, E. A., Ahmad, I. S., Abbasi, A., Cunningham, B. T., &
Watkin, K. L. Cytotoxicity screening of Bangladeshi medicinal plant extracts on pancreatic
cancer cells. BMC Complementary and Alternative medicine, 2010,10(1), 52.

2.Sengupta, A., Ghosh, S., & Bhattacharjee, S.. Allium vegetables in cancer prevention: an
overview. Asian Pacific Journal of Cancer Prevention, 2004,5(3), 237-245.

3.Earnshaw, W. C.. Nuclear changes in apoptosis. Current opinion in cell biology, 1995,7(3),
337-343.

4.Kok, M., & Pechère, J.-C. (2004). Nature and pathogenicity of microorganisms.

5. Kitts, D. D., Wijewickreme, A. N., & Hu, C. (2000). Antioxidant properties of a North
American ginseng extract. Molecular and cellular biochemistry, 203(1-2), 1-10.

6. Lee, J.-C., & Lim, K.-T.. Inhibitory effects of the ethanol extract of Ulmus davidiana on
apoptosis induced by glucose-glucose oxidase and cytokine production in cultured mouse
primary immune cells. BMB Reports, 2001,34(5), 463-471.

7. Christou, L., Hatzimichael, E., Chaidos, A., Tsiara, S., & Bourantas, K. L.. Treatment of
plasma cell leukemia with vincristine, liposomal doxorubicin and dexamethasone. European
journal of haematology, 2001,67(1), 51-53.

8. Mukherjee, D., Nissen, S. E., & Topol, E. J.. Risk of cardiovascular events associated with
selective COX-2 inhibitors. Jama, 2001.,286(8), 954-959

9. Paschka, A. G., Butler, R., & Young, C. Y.-F.. Induction of apoptosis in prostate cancer cell
lines by the green tea component,(−)-epigallocatechin-3-gallate. Cancer letters, 1998130(1-2), 1-
7.
10. Smets, L. A.. Programmed cell death (apoptosis) and response to anti-cancer drugs. Anti-
cancer drugs, (1994) 5(1), 3-9.

11. Scott, D. L., Wolfe, F., & Huizinga, T. W. J. Rheumatoid arthritis. The Lancet, (2010).
376(9746), 1094-1108. doi: https://doi.org/10.1016/S0140-6736(10)60826-4

12. Gacche, R., Shaikh, R., Pund, M., & Deshmukh, R,Cyclooxygenase Inhibitory, Cytotoxicity
and Free Radical Scavenging Activities of Selected Medicinal Plants Used in Indian Traditional
Medicine. Pharmacognosy Journal,2011. 3(19).

13.Sulforhodamine B colorimetric assay for cytotoxicity screening. Vichai V, Kirtikara K Nat Protoc.
2006; 1(3):1112-6.

14, A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer.

Kasinski AL, Kelnar K, Stahlhut C, Orellana E, Zhao J, Shimer E, Dysart S, Chen X, Bader AG,
Slack FJ .Oncogene. 2015 Jul; 34(27):3547-55.

15. New colorimetric cytotoxicity assay for anticancer-drug screening. Skehan P, Storeng R,
Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR J
Natl Cancer Inst. 1990 Jul 4; 82(13):1107-12.
Table

Table 1: Drug Concentrations (µg/ml) and Percentage of growth inhibition on Ishikawa Cell lines

Human Endometrial Adenocarcinoma Cell Line Ishikawa

% Control Growth
Drug Concentrations (µg/ml)
Experiment 1 Experiment 2 Experiment 3 Average Values
10 20 40 80 10 20 40 80 10 20 40 80 10 20 40 80
MEAC 123.3 174.3 170.1 138.5 110.4 118.4 118.9 122.6 91.5 106.8 104.5 132.4 108.4 133.1 131.2 131.6

MELR 116.2 18.1 6.9 -10.6 56.0 -34.3 -55.5 -26.8 43.8 -54.7 -62.5 -31.6 72 -23.6 -37.03 -23

Adriamycin 3.6 -4.3 -30.8 -42.3 7.2 1.6 -21.3 -37.1 2.6 -8.2 -32.1 -39.0 4.46 -3.6 -28.1 -39.5
Table

Table 2: Drug Concentrations (µg/ml) and Percentage of growth inhibition on SCC-29B Cell lines

Human Oral Cancer Cell Line SCC-29B

% Control Growth
Drug Concentrations (µg/ml)
Experiment 1 Experiment 2 Experiment 3 Average Values
10 20 40 80 10 20 40 80 10 20 40 80 10 20 40 80
MEAC 92.3 96.2 98.6 118.4 79.4 90.2 93.3 104.0 84.6 102.6 112.4 112.3 85.4 96.33 101.43 111.5

MELR 1.8 -28.6 -78.9 -65.9 -4.4 -30.9 -58.3 -64.8 -32.0 -32.9 -42.4 -47.8 -11.5 -30.8 -59.8 -59.5

Adriamycin -75.9 -80.2 -79.8 -64.1 -78.6 -77.7 -74.7 -69.3 -73.9 -70.4 -80.3 -63.0 -76.13 -76.1 -78.2 -65.6
Table

Table 3: Drug concentrations (µg/ml) calculated from the graph on Ishikawa Cell Lines
Compound Cell line LC50 TGI GI50

MEAC SCC-29B NE NE NE

MELR SCC-29B 58.12 <10 <10

Adriamycin SCC-29B NE <10 <10

LC50 = Concentration of drug causing 50% cell kill

GI50 = Concentration of drug causing 50% inhibition of cell growth

TGI = Concentration of drug causing total inhibition of cell growth

NE = Non- evaluable data. The experiment needs to be repeated using the different set of drug
concentrations.
Table

Table 4: Drug concentrations (µg/ml) calculated from graph on SCC-29B Cell Lines
Compound Cell line LC50 TGI GI50

MEAC SCC-29B NE NE NE

MELR SCC-29B 58.12 <10 <10

Adriamycin SCC-29B NE <10 <10

LC50 = Concentration of drug causing 50% cell kill

GI50 = Concentration of drug causing 50% inhibition of cell growth

TGI = Concentration of drug causing total inhibition of cell growth

NE = Non- evaluable data. The experiment needs to be repeated using the different set of drug
concentrations.
Figure1Effect of Methanolic extractselected plantson Ishikawa cell
line growth curve

Figure 1: Effect of Methanolic extract of Argyreia cymosa and Limnophila repens on

Ishikawa cell line growth curve

Growth Curve: Huan endometrial

adrenocarcinoma cell lines (Ishikawa)
150
MEAC
% Control Growth

MELR
100
Adriamycin

50

0
10

20

40

80
-50
Drug Concentration
FigureEffect of Methanolic extractselected plants SCC-29B cell
line growth curve

Figure 2: Effect of Methanolic extract of Argyreia cymosa and Limnophila repens on SCC-
29B cell line growth curve

Growth Curve: Human oral cell lines (SCC-29B)

150
MEAC
% Control Growth

100 MELR
Adriamycin
50

0
10

20

40

80
-50

-100 Drug Concentration

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