In-vitro Anticancer Activity of Ethanol and Aqueous Extract of Leaves and Stem of Cissus woodrowii (Stapf Ex T. Cooke) Santapau against HepG2 Liver Cancer Cell Line

Pallavi N. Patil*^1,2, Ritu M. Gilhotra¹, Shweta Sharma¹, Prashant K. Dhakd, Kiran A. Wadkar³

¹School of Pharmacy, Suresh Gyan Vihar University, Mahal Jagatpura, Jaipur, Rajasthan India. 302017.

²Department of Pharmaceutical Chemistry, Dr. J.J. Magdum Trust’s, Dr. J.J. Magdum Pharmacy College, Jaysingpur, Dist - Kolhapur, Maharashtra, India, 416101.

³Department of Pharmacognosy, Teerthankar Education Society’s, Dr. Shivajirao Kadam College of Pharmacy, Kasabe Digraj, Taluka - Miraj, Dist - Sangli, Maharashtra, India.416305.

*Corresponding Author E-mail: reachpallu58@gmail.com

ABSTRACT:

Background and purpose: Researchers looked at the HepG2 liver cancer cell line to see whether ethanol and an aqueous extract of Cissuswoodrowii leaves and stems had any anticancer effects. Experimental approach: Using the soxhlet equipment, ethanol and water-based plant extracts were produced for this study. Using the MTT assay, trypan blue, and brine shrimp lethality assay, in-vitro anticancer action of certain plant extracts was investigated at doses ranged from 10 to 100µg/ml against designated cell line. Findings/Results: The anticancer action was achieved with the usage of 5-flurouracil. In comparison to the water-based stem and leaf extracts of CissuswoodrowiiSantapau, ethanolic stem extract had the greatest % of inhibition against the HepG2 cell line, along with effective IC50 values. Conclusion and implication: In conclusion, our results suggest that the 50% ethanol-aqueous stem and leaves extract of Cissuswoodrowii (Stapf Ex T. Cooke) Santapu showed a clear cytotoxic and brine shrimp lethality inductive impact on the human hepatoma (HepG2) cell line, suggesting an anticancer activity.

KEYWORDS: Cytotoxicity, MTT assay, Hep G2 cell lines, Brine shrimp lethality assay, Cissuswoodrowii.

INTRODUCTION:

When it comes to global mortality, cancer takes the cake. Worldwide, cancer rates are on the rise, with dietary factors, environmental factors, and diseases caused by carcinogenic viruses being major contributors, according to cancer studies released by WHO and World Cancer Research Fund.

Overweight, chronic infections, environmental and occupational hazards, such as exposure to radiation (both ionizing and non-ionizing), and poor dietary habits account for more than 30% of cancer cases. Patients with cancer often get conservative medication regimens in hospitals. Still, studies on therapeutic plants and cancer have progressed due to phytochemicals' less harmful and toxic effects^1-3. Many areas of laboratory research rely on cell lines, for cancer studies. Among their many benefits is the fact that they are simple to work with and provide an endless, self-replicating source that may be expanded to nearly infinite proportions. Moreover, they are rather homogeneous and may be readily replenished from frozen supplies in the event of contaminated loss^4-6. Researchers can employ cell line studies to check for potential molecules that control medication resistance. In order to develop more accurate drug resistance models and investigate the variations among the drug resistant sublines chosen by various treatments^7-9.

Based on the results of the DPPH assay, which involved reducing free radicals, the ethanol and water-based extracts of the stem and leaves of Cissuswoodrowii (Stapf Ex T. Cooke) Santapau demonstrated strong antioxidant activity.

Our goal in conducting this study was to look at the in-vitro cytotoxicity tests that were done on HepG2 liver cancer cells and brine shrimp using an ethanol and water extract of the stem and leaves of Cissuswoodrowii. Aside from that, there have been no comprehensive in-vitro investigations on the anticancer characteristics of those plants. This is why the present research is focusing on the Santapau plant species potential anticancer and cytotoxic effects.

MATERIAL AND METHODS:

The location of the fresh leaves and stem collection of Cissuswoodrowii (Stapf Ex T. Cooke) Santapau is Kolhapur, Maharashtra, India. After being shade dried, the stem and leaves were ground into powder. Then, 2.5 liters of 95% v/v ethanol were used as solvents to subject it to continuous hot percolation for 8 hours after packing 500 grams of it in a Soxhlet device. After being dried in a desiccator, the extract was concentrated under vacuum. By employing the maceration process, an aqueous extract of the stem and leaves was produced ^9,10.

Cell lines and culture medium:

Acquired from the National Centre for Cell Sciences (NCCS) in Pune, we cultivated the HepG2 liver cancer cell line using minimal essential medium (MEM) that contained fetal bovine serum (FBS). Everything was maintained at 370 °C, with 6.5 percent CO2, 95% air, and 100 percent relative humidity in each cell. Every week, maintenance cultures were passed on and the culture medium was changed twice a week.

1. Brine shrimp lethality assay:

All steps of the bioassay experiment were carried out as directed^10-13. A glass capillary was used to extract water and nauplii. A total of ten shrimp were transferred from the capillary stem to each sample vial, which had 4.5ml of brine solution (specific volume brine and yeast suspension), after counting them against a lit background. A mixture of 4.5ml of brine solution with the concentrations given and 0.5ml of Sample 01 and 02 solutions was prepared for each experiment. The control vial was filled with 0.2% DMSO in addition to 0.5ml of synthetic seawater, for a total volume of 4.5ml. Three pairs of magnifying glasses or a well-lit background were used to count survivors after 24hours, and a formula was used to determine the percentage of fatalities.

Percent Mortality= (Totalnaupii–Alivenaupii)/ Totalnaupii x 100

2. Trypan Blue Exclusion Method:

Prepare cell suspension and add 100µg/ml (1 x 10⁴) in Eppendorf tube, at adifferent concentration 10 to 2000 µg/ml of standard and test compound treated with cell suspension and incubate for the 3hrs in Co2 incubator. Take the clean heamocytometer slide and fix the coverslip Add the100µl of cell suspension and 100µl Trypan blue in avial. Take about 20µl in to the tip of a micropipette and transfer immediately to the edge of the coverslip. Leave for1-2min (no longer than 2 min) Placeon microscopeunder 10 X and countthecells.^14,15

Percentage of Viability = Total Cells– Dead cell/Total Cells×100

3. MTT assay:

For three hours at 37°C with 5% CO2, cells were pre-incubated in culture media at a concentration of 1 × 10¹ cells/ml. Microplates with 96 wells and a flat bottom were used. The cells were seeded in 100μl of culture medium with a concentration of 5 × 10¹ cells/well. Plant extract and standard drug flurouracil were added at different concentrations (10, 40, 100μg/ml) in a 2% DMSO solution. Plates were then incubated at 37°C with 5.0% CO₂. A blue formazan promotes cell growth. The test indicates which cells are able to survive exposure to toxins. Afterwards, 10 microliters of the MTT mixture was added and left to incubate at 37 degrees Celsius with 5% CO2 for four hours. We performed each experiment three times. Next, each well was filled with 100 μl of solubilization solution and left to incubate overnight. A microplate (ELISA) reader was used to get spectrophotometric absorbance of the samples. Based on the filters that were accessible for the ELISA reader, absorbance of formazan product was measured at a wavelength ranging from 550nm. It is recommended that the reference wavelength exceed 650 nm ^16-19.

RESULTS AND DISCUSSION:

Table 1: In vitro cytotoxic activity of Ethanol and aqueous extract of leaves and stem of CissusWoodrowii (Stapf Ex T. Cooke) Santapau (stem aqueous (SA), Stem Ethanol (SE), leaves Aqueous (LA), and leaves ethanol (LE)on Percentage inhibition Assay by Trypan Blue Exclusion Method

Sr. No.	Compounds	Conc. µg/ml	No. of LiveCells	Percentage inhibition	IC50 µg/ml
1	Control		280	-
2	Standard 5-flurouracil	10	144	48.58	56.40
		40	131	49.65
		100	120	57.15
3	Sample-SE	10	230	17.86	37.35
		40	195	30.36
		100	170	39.29
4	Sample-SA	10	239	14.65	33.82
		40	176	37.15
		100	153	45.36
5	Sample- LE	10	245	12.50	34.66
		40	210	25.00
		100	195	30.36
6	Sample- LA	10	256	08.58	30.59
		40	195	30.36
		100	186	34.65

According to trypan blue assay, the At the concentration 10µg/ml to 100µg/ml, given sample extract SE 37.35%, SA 33.82%, LE 34.66% and LA 30.59% showed good inhibition of hepG2 cell swhen compared to the standard 56.40%.

From this table, it is evident that maximum cytotoxicity was shown by the stem and leaves of ethanol extract of Cissuswoodrowii (Stapf Ex T. Cooke) Santapau. At all tested concentration the ethanol extract was showing more than 50% cell death.

Sr. No.	Control	STD (5-Fluorouracil )	Stem aqueous
1
	Stem ethanol	Leaves stem	Leaves ethanol
2

Fig. 1. Cell viability Assay by Trypan Blue Exclusion Method on HepG2 Liver cancer cell line

Table 2: In vitro cytotoxic activity of Ethanol and aqueous extract of leaves and stem of Cissuswoodrowii (Stapf Ex T. Cooke) Santapau (SA, SE, LA, and LE) on Brine shrimp lethality assay

S. No	Concentration (µg/ml)	Percentage mortality (%)
S. No	Concentration (µg/ml)	SA	SE	LA	LE
1	10	16.66	20.00	20.00	26.66
2	100	30.00	36.66	26.66	36.66
	1000	46.66	43.33	36.66	43.33
^LC50 (µg/ml)	-	107.73	72.70	114.46	87.31

The brine shrimp were used to conduct the BSL assay. The results show that the ethanol and water-based stem and leaf extracts had a strong effect on brine shrimp nauplii, and the LC50 values were extremely high for all of the extracts.

Fig. 2. Brine shirmp lethality assay in the 24 well plate.

When compared to the standard medicine 5FU, the aqueous and ethanol extracts demonstrated good action against liver cancer cell lines at concentrations ranged from 10 to 100µg/ml. According to IC₅₀ value, less concentration required for the killing of 50 percent cancer cell. So it indicates that it showed promising anticancer activity.^20,21

DISCUSSION:

Finding the percentage A quick way to determine if your cell culture conditions are ideal is to use the Trypan Blue exclusion test. It labels dead cells, whereas it is excluded from living cells. To evaluate cell death^22-24 and to find out whether cultured cells have survived damage or disruption²⁵, trypan blue exclusion testing is helpful.

Aqueous and Ethanolic extract of Cissuswoodrowii (Stapf Ex T. Cooke)Santapau belonging to Vitaceae families were selected for the cytotoxic screening using HepG2 cell lines by 3hr culture using Trypan blue dye exclusion method.

Several plant-based anticancer medicines that are presently used to treat cancers work by regulating the cell cycle, specifically the accumulation of G2/M cells, which inhibits the proliferation of cancer cells^26,27.

This could be an indication of the test materials' potential cytotoxic effects. The opportunity for additional inquiry was identified in crude extracts that produced LC50 values below 250µg/ml, as they were deemed significantly active²⁸. It is evident that maximum lethality on brine shrimp was shown by ethanol and aqueous extract of Cissuswoodrowii (Stapf Ex T. Cooke)Santapau. At all tested concentration the ethanolic and aqueous extract was showing more than 50% of cell death.

A cell viability assay called MTT was employed. Metabolic conversion of MTT to formazan, resulting in a purple color, was utilized in the MTT experiment to assess cytotoxicity^29,30.

In MTT assay, IC50 values were found promisingly cytotoxic after treatment with Cissuswoodrowii (Stapf Ex T. Cooke)Santapau. in Hep G2 (liver carcinoma)cell line

CONCLUSION:

Uncontrolled cell growth and division is a hallmark of cancer and many other diseases. This absence of dysregulation is caused by mutations in genes that code for proteins that regulate the cell cycle. The 50% ethanol-aqueous stem and leaves extract of Cissuswoodrowii (Stapf Ex T. Cooke) Santapu showed a clear cytotoxic and brine shrimp lethality inductive impact on the human hepatoma (HepG2) cell line, suggesting an anticancer activity. The next step is to extract the anticancer properties of each chemical component fromCissuswoodrowii(Stapf Ex T. Cooke) Santapau.

ACKNOWLEDGMENTS:

This study was part of the PhD thesis for this work supporthavegiven by Dr. Shitalkumar S. Patil, Principal, Dr. J.J. Magdum Pharmacy College, Jaysingpur, Kolhapur, MS, India.

CONFLICT OF INTEREST STATEMENT:

The authors declared no conflict of interest in this study.

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Received on 10.06.2024 Revised on 18.10.2024

Accepted on 05.01.2025 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2164-2168.

DOI: 10.52711/0974-360X.2025.00310

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sr. No	Conc. (µg/ml)	% Inhibition on HepG2 Liver cancer cell line
Sr. No	Conc. (µg/ml)	SA	SE	LA	LE	STD (5-Fluorouracil )
1	10	35.28	47.04	32.43	47.65	77.78
2	40	38.97	50.42	39.12	52.19	80.86
3	100	42.58	53.65	41.04	54.65	84.47
^IC50 (µg/ml)	-	39.91	39.75	32.47	36.05	41.20