The liver is an essential metabolic organ that is unique to vertebrates and is involved in many biological functions. It produces proteins, detoxifies the body, and synthesizes a variety of biochemicals necessary for development and digestion[1]. Over two million deaths worldwide are attributed to liver disease each year, making up 4% of all fatalities (1 in 25). Of them, females account for one in three liver-related fatalities. The number of fatalities from liver cancer alone ranges from 600,000 to 900,000[2].

The hepatoprotective properties of Moringa oleifera (commonly known as the drumstick tree) have been widely researched due to its bioactive compounds. This plant is a rich source of antioxidants, vitamins, minerals, and phytochemicals, all of which play a role in its liver-protective effects[3].

The liver is highly susceptible to damage from various factors, including infections, excessive alcohol consumption, drug toxicity, and metabolic disorders, leading to conditions collectively known as liver diseases. These disorders, ranging from mild hepatic dysfunction to severe conditions like cirrhosis, hepatocellular carcinoma, and liver failure, hepatitis pose significant global health concerns[4, 5].

The ability of a substance (drug, herbal extract, or molecule) to either prevent liver damage or aid in the liver's recovery from injury is known as hepatoprotective action. Alcohol, drugs, illnesses, and pollutants can all have an impact on the liver, which is essential for digestion, metabolism, and detoxification[6].

Name – Moringa OleiferaKingdom – PlantaeSub kingdom – TracheobiontaSuper Division – SpermatophytaDivision – MagnoliophytaClass – MagnoliopsidaSub class – DilleniidaeOrder – CapparalesFamily – MoringceaeGenus – MoringaSpecies – oleifera

Aqueous and alcoholic extracts (methanolic & ethanolic) of leaves and roots of Moringa oleifera exhibit strong in-vitro anti-oxidant and radical scavenging activity[7]. Aqueous extract of Moringa oleifera leaves shows anti-diabetic activity and controls diabetes and thus exhibit glycemic control[8]. Ethanolic extract of Moringa oleifera leaves showed prominent anti-hypertensive or hypotensive activity[9]. Ethanolic extracts of leaves and seeds of Moringa oleifera shows potent anti–tumor activity[10]. The in-vitro anti-urolithiatic activity was performed in aqueous and alcoholic extract of bark of Moringa oleifera. It showed reduction in weight of stone produced using ethylene glycol induced urothiasis. It also possesses both preventive and curative property[11]. Leaves, roots, bark and seeds of Moringa oleifera show anti-microbial activity against bacteria and fungi[12]. Methanolic and aqueous extract of root and bark, methanolic extract of leaves and flowers and ethanolic extract of seeds of Moringa oleifera possess anti-inflammatory activity[13]. Moringa oleifera leaves extract restores mono amine levels of brain, which may be useful in Alzheimer’s disease[14].

Aim: Effectiveness of crude drug of Moringa oliefera in Hepato carcinoma.

Objectives:

It is a population of cells that can be cultured and maintained in lab for an extended period often indefinitely under controlled conditions. Cell lines are derived from various sources including normal tissues, tumors, stem cell and they are commonly used in biological and medical research[15].

Hep G2 is an immortal cell line that was derived in 1975 from the liver tissue of a 15–year–old Caucasian male from Argentina with a well-differentiated hepatocellular carcinoma. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic in nude mice[16]. HepG2 cells, a human hepatoma cell line, are used in research as in vitro model for studying liver-related functions, including drug metabolism, hepatotoxicity, and liver cancer, as well as viral infections and tissue engineering[17].

All Test Samples were filter sterilized using 0.22µ filters and diluted by double dilution method in MEM with FBS[18].

HepG2 cells were cultured in D-Modified Eagle Medium (DMEM) with NEAA media supplemented with 10% (v/v) fetal bovine serum. Cells were cultured at 370C and 5% CO2; the complete medium was changed every 2 to 3 days[19].

All Test Samples were filter sterilized using 0.22µ filters and diluted by double dilution method in MEM with FBS.

The MTT assay is a colorimetric assay for assessing cell metabolic activity. NADPH-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT, which is chemically 3 (4,5dimethylthiazol2yl) 2,5diphenyltetrazolium bromide, to its insoluble formazan, which has a purple colour. The result of MTT Assay is given below[22].

[Table. 2] shows the morphological changes in HepG2 cells treated with different concentrations of Moringa oleifera powder ranging from 5 µg/ml to 250 µg/ml over a 24-hour period.

The table includes images of the HepG2 cells at 0 hours and 24 hours for each concentration. The images help visualize the shape, size, and colour of the cells, which are indicators of cell viability and health.

Control: Shows healthy, viable cells with no treatment.

Treated Cells: As the concentration of Moringa oleifera increases, the number of viable cells decreases, and cell morphology changes (e.g., cells may appear shrunken or fragmented), indicating cytotoxicity.

[Table. 3] provides quantitative data from the MTT assay, including absorbance values, average % cell survival, and the IC50 value.

Concentration (µg/ml): The concentrations tested range from 5 µg/ml to 250 µg/ml.

Absorbance: The absorbance values at 570 nm are recorded for each concentration, reflecting the amount of formazan produced by viable cells.

% Cell Survival: The percentage of cell survival is calculated by comparing the absorbance of treated cells to the control (untreated cells).

IC50 Value: The IC50 value (464 µg/ml) is the concentration at which 50% of the cells are killed. This value indicates the potency of Moringa oleifera in inhibiting HepG2 cell growth.

[Fig. 1] is a column chart that further illustrates the % cell viability at different concentrations of Moringa oleifera.

The X-axis represents the concentrations (Control to 250 µg/ml), and the Y-axis represents the % cell viability.

The chart shows a clear trend of decreasing cell viability with increasing concentrations of Moringa oleifera, supporting the cytotoxic effect of the extract on HepG2 cells. 

Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular directions to specific locations. Cells often migrate in response to specific external signals, including chemical signals and mechanical signals. The result of Cell Migration Assay is given below[23].

[Table. 4] shows the wound healing process (cell migration) in HepG2 cells treated with Moringa oleifera powder at concentrations ranging from 10 µg/ml to 50 µg/ml.

The table includes images of the HepG2 cells at 0 hours, 8 hours, and 24 hours for each concentration.

Control: Shows the natural wound healing process over time.

Treated Cells: The images show how Moringa oleifera affects the migration of HepG2 cells into the scratch area. As the concentration increases, the rate of cell migration decreases, indicating that Moringa oleifera inhibits cell migration, which is a critical factor in cancer metastasis.

[Table. 5] provides quantitative data from the cell migration assay, including the area covered by migrating cells, mean values and minimum/maximum areas.

Concentration (µg/ml): The concentrations tested range from 10 µg/ml to 50 µg/ml.

Time: The observations are recorded at 0 hours, 8 hours, and 24 hours.

Area: The area covered by migrating cells is measured at each time point.

Mean, Min, Max: These values provide a statistical summary of the cell migration data, showing the variability in cell migration at different concentrations and time points.

The data shows that as the concentration of Moringa oleifera increases, the area covered by migrating cells decreases, indicating that Moringa oleifera inhibits cell migration. 

[Fig. 2] is a graphical representation of the cell migration data. It shows the mean area covered by migrating cells at different concentrations of Moringa oleifera over time.

The X-axis represents the concentrations (Control to 50 µg/ml), and the Y-axis represents the mean area covered by migrating cells.

The graph shows a clear trend of decreasing cell migration with increasing concentrations of Moringa oleifera, supporting the anti-migratory effect of the extract on HepG2 cells.

The results demonstrate that Moringa oleifera has a cytotoxic effect on HepG2 cells, with an IC50 value of 464 µg/ml. The morphological images and graphs show a dose-dependent decrease in cell viability, indicating that Moringa oleifera can inhibit the growth of liver cancer cells. Ic50 value of pure extract of Moringa oleifera drug is 10 ± 1μg/ml but as we have used crude form of Moringa oleifera the IC50 value was found to be 464μg/ml.

The results show that Moringa oleifera inhibits the migration of HepG2 cells, which is a critical factor in cancer metastasis. The concentration of 30μg/ml showing the constant decrease in Area and Mean both. The images and graphs demonstrate that higher concentrations of Moringa oleifera significantly reduce cell migration, suggesting its potential as an anti-metastatic agent.

These findings suggest that Moringa oleifera has potential therapeutic effects against liver cancer by inhibiting cell proliferation and migration, making it a promising candidate for further research.