INTRODUCTION:

The liver as a vital organ in the body is primarily responsible for the metabolism of endogenous and exogenous agents. It plays in drug elimination and detoxification.

The liver is a reddish-brown, wedge-shaped organ with two lobes of unequal size and shape. A human liver normally weighs approximately 1.5kg (3.3 lb).¹ and has a width of about 15cm (6 in).²It is both the heaviest internal organ and the largest gland in the human body.

There are 2 distinct sources that supply blood to the liver, including the following:

1) Oxygenated blood flows in from the hepatic artery.

2) Nutrient-rich blood flows in from the hepatic portal vein.

Fig:- Anatomy of liver

Structure:

The liver consists of four lobes: the larger right lobe and left lobe, and the smaller caudate lobe and quadrate lobe. The left and right lobe are divided by the falciform ("sickle-shaped" in Latin) ligament, which connects the liver to the abdominal wall. The liver's lobes can be further divided into eight segments, which are made up of thousands of lobules (small lobes). Each of these lobules has a duct flowing toward the common hepatic duet, which drains bile from the liver.³

Microscopic anatomy:

Microscopically, each liver lobe is seen to be made up of hepatic lobules.

The lobules are roughly hexagonal, and consist of plates of hepatocytes, and sinusoids radiating from a central vein towards an imaginary perimeter of interlobular portal triads.

The central vein joins to the hepatic vein to carry blood out from the liver. A distinctive component of a lobule is the portal triad, which can be found running along each of the lobule's corners.

The portal triad consists of the hepatic artery, the portal vein, and the common bile duct.⁴

Causes of liver cancer:

Hepatitis B virus (HBV) infection: HBV can be transmitted in blood, semen, or other body fluids. The infection can be passed from mother to child during childbirth, through sexual contact, or by sharing needles that are used to inject drugs. It can cause inflammation (swelling) of the liver that leads to cancer. Routine HBV vaccination in infancy is reducing the incidence of HBV infection. Chronic HBV infection is the leading cause of liver cancer in Asia and Africa.

Hepatitis C virus (HCV) infection: HCV can be transmitted in the blood. The infection can be spread by sharing needles that are used to inject drugs or, less often, through sexual contact. In the past, it was also spread during blood transfusions or organ transplants. Today, blood banks test all donated blood for HCV, which greatly lowers the risk of getting the virus from blood transfusions. It can cause cirrhosis that may lead to liver cancer. Chronic HCV infection is the leading cause of liver cancer in North America, Europe, and Japan.

Heavy alcohol use: Heavy alcohol use can cause cirrhosis, which is a risk factor for liver cancer. Liver cancer can also occur in heavy alcohol users who do not have cirrhosis. Heavy alcohol users who have cirrhosis are ten times more likely to develop liver cancer, compared with heavy alcohol users who do not have cirrhosis.

Cirrhosis: The risk of developing liver cancer is increased for people who have cirrhosis, a disease in which healthy liver tissue is replaced by scar tissue. The scar tissue blocks the flow of blood through the liver and keeps it from working as it should. Chronic alcoholism and chronic hepatitis infections are common causes of cirrhosis. People with HCV-related cirrhosis have a higher risk of developing liver cancer than people with cirrhosis related to HBV or alcohol use.

Cigarette smoking: Cigarette smoking has been linked to a higher risk of liver cancer. The risk increases with the number of cigarettes smoked per day and the number of years the person has smoked.⁵

CELL LINE AND ITS ROLE IN SCREENING:

Hep2 cell line is the required cell line and it was: Originated from tumors produced in irradiated-cortisonised weanling rats after injecting with epidermoid carcinoma tissue from the larynx of a 56 year old male. This cell line was found to be indistinguishable from HeLa by STR PCR DNA profiling. Therefore, the cell line should be considered as derived from HeLa. HeLa contaminant; adherent; Cells contain human papilloma virus. The cells are positive for keratin by immunoperoxidase staining. The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium (EMEM).⁶

Fig:-Hep G2 Cell line (Normal)

CURCUMIN AS ANTICANCER DRUG:

Curcumin derived from the plant Curcuma longa, is a gold-colored spice commonly used in the Indian subcon-tinent. Curcumin has been shown to exhibit antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anticancer activities and thus has a potential protective role against various malignant diseases, diabetes, allergies, arthritis, Alzheimer’s disease, and other chronic illnesses. It has also been shown to induce cell death in apoptosis resistant cancer cells.⁷

Fig: CURCUMIN

Curcumin helps stop the formation of new blood vessels in tumors by lowering VEGF and HIF-1α levels. It blocks key signaling pathways (like VEGFR2, PI3K-Akt and MAPK), reduces inflammation-related factors, and decreases MMP activity. Because of this, endothelial cells grow, move, and form tubes less effectively, which lowers the number of blood vessels in the tumor.⁸

OBJECTIVES:

· To assess the cytotoxic effect of curcumin on HepG2 liver cancer cells.

· To investigate the morphological changes in HepG2 cells post-curcumin treatment using microscopy.

· To evaluate the apoptotic potential of curcumin in HepG2 cells.

· To extract Curcumin from turmeric rhizomes using the maceration method with ethanol (95%) as a solvent.

· To identify the presence of Curcumin by performing specific chemical tests, including the lead acetate test and the boric acid test.

MATERIALS AND METHODOLOGY FOR EXTRACTION:

Plant material:

The turmeric rhizomes were purchased from local market of Nipani and then were kept in a desiccator at room temperature until used.

Chemicals and reagents:

Use organic solvents for extraction because these are volatile in nature they can be evaporate easily. Example organic solvents like Ethanol, Acetone etc.¹²

Extraction process (Maceration method):

Take fleshy rhizomes of turmeric. The rhizomes of turmeric were dried in oven at 105°C for 3hrs.¹²

Dried rhizome was grinded and obtained uniform powder. The turmeric powder was stored in refrigerator to prevent moisture uptake.¹²

Extraction solvent (ethanol), solid-liquid ratio (1:20 w/v), extraction time (3 days), and extraction temperature (room temperature).⁹

Use a gentle water bath at ~50°C to evaporate the solvent. A thick, orange-yellow residue is obtained — this is crude curcumin extract.

Purification of curcumin:

Dissolve crude extract in a small amount of ethanol. Add distilled water slowly to precipitate pure curcumin. Filter the yellow-orange precipitate. Dry under reduced pressure or in a desiccator.¹⁰

Fig. 1: Maceration method

Fig. 2: Vacumn filtration

Fig. 3: Crude extract

Identification tests for curcumin:-

Lead acetate test:

Two mL of 10% lead acetate filtered, clear solution, and were added to 2mL of the extract. A bulky white precipitate indicates the presence of Tannins and/or Phenolic compounds.¹³

Fig. 4: Rosocyanine

Fig. 5: Identification tests

MATERIALS AND METHODOLOGY:

The materials and methodology used for the screening of the anti-cancer activity of curcumin on Hep G2 cell line as follows:-

Sample: - Curcumin (Solid).

Cell line: - Hep G2 (Human Liver Cancer Cell Line).

Media: -

1. DMEM Medium with high glucose.

2. FBS (Gibco, Invitrogen).

3. Antibiotic- Antimycotic 100X solution (Thermo fisher Scientific).

Equipment’s required:

96 well plate

Biosafety cabinet

ELISA plate reader (BenespheraE21)

Principle of Assay:-

This Colorimetric assay is based on the capacity of Mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow water soluble substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into an insoluble, purple colored formazan product which is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells.

Procedure:-

Hep G2 (Human Liver Cancer Cell line) was procured from National center for cell sciences (NCCS), Pune maintained in DMEM Medium supplemented with 10% fetal bovine serum.

Cells were incubated at a concentration of 1×104 cells/ml in culture medium for 24 h at 37°C and5%CO2.

Cells were seeded at a concentration (70µl) 104 cells/well in 100µl culture medium and 100µl.

i. Samples (10-100µl/ml) into micro plates respectively (tissue culture grade and 96 wells).

Control wells were incubated with DMSO (0.2% in PBS) and cell line. All samples were incubated in triplicate. Controls were maintained to determine the control cell survival and the percentage of live cells after culture.

Cell cultures were incubated for 24 h at 37°C and 5% CO2 in CO2 incubator (Thermo scientific BB150).

After incubation, the medium was completely removed and Added 20μl of MTT reagent (5mg/min PBS).

After addition of MTT, cells incubated for 4hrs at 37oC in CO2 incubator.

i. Observed the wells for formazan crystal formation under microscope. The Yellowish MTT was reduced to dark colored formazan by viable cells only.

After removing the medium completely. Added 200μl of DMSO (keptfor10min) and incubate at 370C (wrapped with aluminum foil).

i. Triplicate samples were analyzed by measuring the absorbance of each sample by an Elisa microplate reader (BenespheraE21) at a wavelength of 570nm.

RESULTS:

Table 1. Effect of 5- Fluorouracil against HepG2 cell line MTT Assay:-

Sr. No	Concentration (µg/ml)	Absorbance (O D)				Cell viability (%)	IC50(µg/ml)
Sr. No	Concentration (µg/ml)	1	2	3	Average	Cell viability (%)	IC50(µg/ml)
1.	Control	2.326	2.325	2.328	2.326	100	____
2.	Standard-5FU 10(µg/ml)	1.253	1.253	1.254	1.253	53.87591	14.22 µg/ml
3.	40(µg/ml)	0.856	0.851	0.854	0.853	36.6958
4.	100(µg/ml)	0.421	0.422	0.423	0.422	18.14013

Fig. 6: Effect of Sample against HepG2 Cell line MTT Assay

Table 1. Effect of Sample against HepG2 Cell line MTT Assay

Sr. No	Concentration (µg/ml)	Absorbance (O D)				Cell viability (%)	IC50(µg/ml)
Sr. No	Concentration (µg/ml)	1	2	3	Average	Cell viability (%)	IC50(µg/ml)
1.	Control	2.326	2.325	2.328	2.326	100	_
2.	Sample-Curcumin 10(µg/ml)	1.523	1.522	1.524	1.523	65.47	37.96 µg/ml
3.	40(µg/ml)	0.953	0.951	0.952	0.852	40.95
4.	100(µg/ml)	0.711	0.712	0.714	0.712	30.62

CONCLUSION:

· The cytotoxic effect of Curcumin on Hep G2 (Human Liver Cancer) cells was evaluated using the MTT assay. The following observations were made:

· The control group showed the highest absorbance (average OD = 2.326), indicating 100% cell viability.

· At a 10µg/ml concentration of Curcumin, the absorbance dropped to 1.523, resulting in 65.47% cell viability, indicating moderate cytotoxicity.

· At 40µg/ml, a significant reduction in viability was observed (40.95%), with an average OD of 0.952.

· At 100µg/ml, cell viability further decreased to 30.62%, showing a strong cytotoxic effect at this concentration.

· The IC₅₀ (concentration at which 50% inhibition of cell viability occurs) was calculated as 37.96µg/ml, suggesting that Curcumin exhibits dose-dependent cytotoxic activity against Hep G2 cells.

LIST OF ABBREVIATIONS USED

Sr. No.	Abbreviations	Full Form
1	HCC	Hepatocellular Carcinoma
2	NF-kB	Nuclear Factor kappa-light-chain-enhancer of activated B cells
3	STAT 3	Signal Transduction And Activation of Transcription 3
4	COX2	Cyclooxygenase-2
5	Bcl2	B-cell lymphoma 2
6	HBV	Hepatitis B Virus
7	HCV	Hepatitis C Virus
8	DMEM	Dulbecco’s Modified Eagle Medium
9	PBS	Phosphate Buffer Saline

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Received on 05.09.2025 Revised on 15.11.2025

Accepted on 26.12.2025 Published on 15.04.2026

Available online from April 18, 2026

Asian J. Pharm. Res. 2026; 16(2):141-146.

DOI: 10.52711/2231-5691.2026.00021

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