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

 

Venkateswarlu G1*, Raja sundararajan2

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

2Department of Chemistry, GITAM University, India.

*Corresponding Author E-mail: venkateswarlugunji@gmail.com, rsundara@gitam.edu

 

ABSTRACT:

Introduction: Plants have a special place in the treatment of cancer. It is estimated that plant-derived compounds constitute more than 50% of anticancer agents. 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. Material method: I have collected the plants, dried very well and extracted with methanol crude 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 analysed and The Methanol extract of Limnophila repens extract showed comparable activity to the standard compound, i.e., Adriamycin on Ishikawa and SCC-29B 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: Limnophila repens, Argyeia cymosa, Anticancer Activity Srb Assay.

 

 


INTRODUCTION: 

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 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.2,3 Now people have started realizing the importance of natural bioactive substances found in fruits, vegetables, and herbs, as antioxidants and functional foods.4-6

 

Some of these substances are believed to be potential chemo preventive or therapeutic agents for cancer Therefore, the induction of apoptosis in tumour cells has become an indicator of the tumour-treating ability of naturally derived bioactive substances.7,8

 

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.10, 11 Apoptosis itself also plays an important role in the development of various diseases including cancer.12 Apoptosis is triggered by activation of the death receptor (extrinsic) and mitochondrial (intrinsic) pathways, results from activation of members of cysteine protease family called caspases .13,14Mitochondria are involved in a variety of key events, including the release of caspase activators, changes in electron transport etc. 15In this manner, released cytochrome C interacts with Apaf-1 and pro-caspase-9 to form the apoptosome. Then caspase-9 cleaves and activates caspase-3, the executioner caspase, which cleaves poly (ADP-ribose) polymerase (PARP) and activates endonucleases leading to DNA fragmentation .16, 17

 

In addition to monitoring caspase activity, some of the biochemical features of apoptosis such as loss of membrane phospholipid asymmetry and DNA fragmentation can also be used to identify apoptosis. which are major sources of phytochemicals and micronutrients, may reduce the risk of developing cancer.. It has been used for psoriasis, leukaemia and diseases of the liver, gall bladder, spleen and stomach. 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. 18, 19

 

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 4017 and 5504, 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 The powder (1 kg) it was extracted by way of petroleum after that It was air-dried and then macerated by way of methanol, strained and then concentrated at 45oC used for activity.

 

Anticancer Activity of Limnophila repens and Argyeia cymosa By Using SRB Assay:

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:

The experiment data were estimated using linear regression method of plots of the cell viability against the molar drug concentration of tested compounds.


 

RESULTS:

 

Table no 1: Drug Concentrations (µg/ml) and Percentage of growth inhibition on Ishikawa Cell lines

Extracts

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

Extracts

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

 


Anticancer Activity of Argyreia cymosa and Limnophila repens

 

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

 

Figure 2: Effect of Methanolic extract of Argyreia cymose and Limnophila repens on SCC-29B Cell line groth curve

 

Table 3: Drug concentration of calculated from Graph

 

Drug concentrations (µg/ml) calculated from the graph on Ishikawa Cell Lines

Drug concentrations (µg/ml) calculated from graph on SCC-29B Cell Lines

Compound

Cell line

LC50

TGI

GI50

Cell line

LC50

TGI

GI50

MEAC

Ishikawa

NE

NE

>80

SCC-29B

NE

NE

NE

MELR

Ishikawa

NE

38.9

<10

SCC-29B

58.12

<10

<10

Adriamycin

Ishikawa

NE

11.6

<10

SCC-29B

NE

<10

<10

LC50 = Con of drug causing 50% cell kill GI50 = Con of drug causing 50% inhibition of cell growth

TGI = Con of drug causing total inhibition of cell growth NE = Non- evaluable data.

DISCUSSION:

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 good . 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 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.

 

REFERENCES:

1.     Gladys BlockBlossom Patterson &Amy Subar(1992) Nature and cancer.18(1) https://doi.org/10.1080/01635589209514201

2.     Earnshaw, W. C. (1995). Nuclear changes in apoptosis. Current opinion in cell biology, 7(3), 337-343. DOI: 10.1016/0955-0674(95)80088-3

3.     Kok, M., & Pechčre, J.-C. (2012). Nature and pathogenicity of microorganisms. Infectious Diseases. 2010: 3–29. doi: 10.1016/B978-0-323-04579-7.00001-0

4.     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. DOI: 10.1023/a: 1007078414639.

5.     Lee Lee, J.-C., & Lim, K.-T. (2001). 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, 34(5), 463-471. DOI: 10.1023/a: 1007078414639

6.     Wang, H., Cao, G., & Prior, R. L. (1997). Oxygen radical absorbing capacity of anthocyanins. Journal of agricultural and Food Chemistry, 45(2), 304-309. doi.org/10.1021/jf960421t

7.     Paschka, A. G., Butler, R., & Young, C. Y.-F. (1998). Induction of apoptosis in prostate cancer cell lines by the green tea component,(−)-epigallocatechin-3-gallate. Cancer letters, 130(1-2), 1-7. DOI: 10.1016/s0304-3835(98)00084-6

8.     Smets, L. A. (1994). Programmed cell death (apoptosis) and response to anti-cancer drugs. Anti-cancer drugs, 5(1), 3-9. DOI: 10.1016/s0304-3835(98)00084-6

9.     Rumani Singh, Anthony Letai, and Kristopher Sarosiek and Nat Rev Mol Cell Biol. 2019 Mar; 20(3): 175–193. doi: 10.1038/s41580-018-0089-8

10.  M Hill , A Norman , D Cunningham , M Findlay , M Watson , V NicolsonA Webb , G Middleton , F Ahmed , T Hickish Impact of protracted venous infusion fluorouracil with or without interferon alfa-2b on tumor response, survival, and quality of life in advanced colorectal cancer. Journal of Clinical Oncology 13, no. 9 (September 01, 1995) 2317-23. DOI: 10.1200/JCO.1995.13.9.2317

11.  Panchal RG. Novel therapeutic strategies to selectively kill cancer cells. Biochem Pharmacol. 1998 Feb 1; 55(3):247-52.DOI: 10.1016/s0006-2952(97)00240-2

12.  David J. McConkey, Sangkyou Lee, Woonyoung Choi, Mai Tran, Tadeusz Majewski, Sooyong Lee, Arlene Siefker-Radtke, Colin Dinney, and Bogdan Czerniak Molecular genetics of bladder cancer: Emerging mechanisms of tumor initiation and progression Urol Oncol. 2010 Jul–Aug; 28(4): 429–440. DOI: 10.1016/j.urolonc.2010.04.008

13.  Miller RW1, Rabkin CS.Merkel cell carcinoma and melanoma: etiological similarities and differences. Cancer Epidemiol Biomarkers Prev. 1999 Feb;8(2):153-8.

14.  IGH Schmidt-Wolf, S Finke, B Trojaneck, A Denkena, P Lefterova, N Schwella, H-G Heuft, G Prange, M Korte, M Takeya, T Dorbic, A Neubauer, B Wittig and D Huhn1Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and Lymphoma British Journal of Cancer (1999) 81(6), 1009–1016.DOI: 10.1038/sj.bjc.6690800

15.  Kroemer G, Petit P, Zamzami N, Vayssie`re JL, Mignotte B. The biochemistry of programmed cell death. FASEB J. 1995; 9:1277–1287.DOI: 10.1096/fasebj.9.13.7557017

16.  Roberta A. Gottlieb; Minireview Mitochondria: execution central FEBS Letters 482 (2000) 6-12.https://doi.org/10.1016/S0014-5793 (00)02010-X

17.  Green D1, Kroemer G. The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol. 1998 Jul;8(7):267-71. DOI: 10.1016/s0962-8924(98)01273-2

18.  Kerr JF, Wyllie AH, Currie AR.Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug; 26(4):239-57. DOI: 10.1038/bjc.1972.33

19.  Wlodkowic D, Telford W, Skommer J, Darzynkiewicz Z. Apoptosis and beyond: Cytometry in studies of programmed cell death. Meth Cell Biol. 2011; 103:55–98. doi: 10.1016/B978-0-12-385493-3.00004-8

 

 

 

 

 

Received on 25.04.2020             Modified on 17.07.2021

Accepted on 12.10.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1459-1462.

DOI: 10.52711/0974-360X.2023.00240