In Vitro Cytotoxic Activity of Quercetin and Silymarin and Their Synergistic Effect on Hep G2 Cell Lines

doi:10.21203/rs.3.rs-2974314/v1

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In Vitro Cytotoxic Activity of Quercetin and Silymarin and Their Synergistic Effect on Hep G2 Cell Lines

https://doi.org/10.21203/rs.3.rs-2974314/v1

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Purpose

One of the biggest causes of mortality among people is cancer. Plants may contain potential bioactive substances that might be used to provide "new leads" in the fight against cancer and other disorders. The main objective of this present study was to evaluate the cytotoxic activities of Quercetin and Silymarin and their Synergistic effect on Hep G2.

Method

The cytotoxic activity of this individual drug and in combination was confirmed by the consideration of IC₅₀ by MTT assay.

Results

The results show that IC₅₀ of Quercetin 50 µg, Silymarin 40 µg, and combination 30 µg respectively. The combination of these drugs shows a significant increase in cytotoxic effect and anti-proliferative effect compared with individual drugs. Thus, the results indicate that this combination has anti -hyper carcinogenic effect.

HepG2

Hepatocellular carcinoma

viability assays

Cytotoxicity

Silymarin

Quercetin.

Hepatocellular carcinoma is one the malignant tumor that affects the liver cells and has the highest rate of morbidity, and mortality in the world[1, 2]. The hepatocellular has various characteristics that show its uniqueness among other cancer like fast progression, recurrence, and a highly malignant property with a high rate of metastasis [1, 3]. Hepatocellular carcinoma is a complex type of disease with the comparison to the other type of cancer [4]. Due to various factors, there is a genetic alteration in the body that genetic alteration leads to hepatocarcinogenesis [2, 5]. Polyphenolic chemicals called flavonoids serve as the primary nutritious components of plant-based foods. Quercetin is a common dietary flavonoid that is found in many plants, including citrus fruits, apples, tea, onions, and red wine. It has a wide variety of pharmacological properties.[4, 6, 7] For many centuries, Silybum marianum (thistle plant) has been regarded as a "hepatic elixir." The Milk Thistle plant (Silybum marianum), exert effects on the liver to inhibit oxidative stress. Silymarin offers hepato-protective effects by reducing ROS free radicals and lipid peroxidation and maintaining redox status. Because it inhibits oxidative stress and proliferation, the bioactive component silibin found in S. marianum has been described as an antioxidant, hepatoprotective, and antitumorigenic effects both in vitro and in vivo.[8, 9]

Hep G2, which are manageable and preserve many of the physical traits of liver parenchymal cells as well as a number of the enzymes needed to activate a variety of xenobiotics.

The MTT test assesses the viability, proliferation, and activation of the cells in a sensitive, quantitative, and reliable colorimetric assay. The test works by converting a yellow, water-soluble substrate called 3-(4, 5-dimethylthiazole-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product that has to the ability of mitochondrial dehydrogenase enzymes in live cells, is insoluble in water.[10, 11]

Viable cells can transform yellow MTT under tetrazolium ring cleavage into a water-insoluble purple blue complexation that precipitates in the cellular cytosol and can be dissolved after cell lysis, whereas cells killed by toxic damage are unable to transform MTT. The amount of this formation is inversely proportional to the amount of cytotoxicity and is proportionate to the number of viable cells. Dehydrogenase, an enzyme connected to the endoplasmic reticulum and the mitochondria, controls the reaction.[10, 12]

Drugs and Reagents:

Quercetin, Silymarin and MTT Was procured for New Nita chemicals Pvt Ltd(Chinchwad). All other chemicals are purchased from Analab fine chemical Mumbai.

Cell Culture:

Hep G2 cell line was purchased from National Centre for Cell Science (NCSS), Pune. Human hepatocellular carcinoma (HepG2) cell lines were maintained at laboratories of School of Bioscience and Bioengineering, Dr. D. Y. Patil International University, Akurdi, Pune. All reagents were of analytical grade.

The Niosomes of this, individual drugs and Combination of drugs were prepared using Ether injection method.

Methods:

Preparation of cell culture and stock drug:[10]

The cell lines used for the study were Hep G2 was maintained in 96 wells micro titter plate containing Minimal essential medium (MEM), supplemented with 10% heat inactivated fetal calf serum (FCS) containing 5% of mixture of gentamicin (10 µg/ml), Penicillin (100 units/ml) and streptomycin (100 µg/ml) in presence of 5% CO2 at 370C for 48–72 hours. In brief, 10 mg of Quercetin, 5 mg Silymarin and 15 mg combination (2:1) was prepared in 1 ml Dimethyl sulfoxide (DMSO) as a stock, from this stock 10, 20, 30,40 50 µl were transferred into the wells containing 100 µl of medium.

MTT Assay:

The 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyl tetrazolium bromide (MTT) test was used to measure the cytotoxicity and the anticancer activity of the sample on Hep G2 cells. [12] In vitro growth inhibition effect of test compound was assessed by colorimetric determination of conversion of MTT into formazan blue by living cells. Cells (1 x 105 /wells) were plated in 0.2 ml of medium/well in 96 well plates. For MTT assay supernatant was removed and fresh MEM solution was added and wells were treated with different concentration of samples (10, 20, 30, 40, 50 µl/ml) from the stock (10 mg/ml) were added to respective wells containing 100 µl of MEM medium, so that the final concentrations were 10, 20, 30, 40 & 50 µg/ml. incubated the plates for 48 hours at 37 ⁰ C in a humidified atmosphere of 5% CO2 .[10]

After incubation, the MTT stock was added to each well (20 µl, 5mg/ml in sterile PBS). Then the plates were further incubated 4 hours, for cytotoxicity. Later the supernatant carefully aspirated and the precipitated crystals of Formazan blue were solubilized by adding 1 ml DMSO and left for 45 sec. presence of viable cells was visualized by the development of purple color due to formation of formazan crystals. Concentration of Formazan was determined on a microplate reader (ELISA plus) at 570 nm. The Cell viability was calculated using DMSO treated cells as the 100% viable control. The results represent the mean of five readings.

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =	𝑀𝑒𝑎𝑛 𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡	× 100
	𝑀𝑒𝑎𝑛 𝑂𝐷 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙

The MTT assay was used to determine the cytotoxic activity of quercetin and silymarin against Hep G2 as well as their combined effect.

Figure 1 – IC ₅₀ Values of Quercetin and Silymarin and their combination on Hep G2 Cell lines

The results of the Hep G2 cytotoxic assay are shown in Table 1. The comparison between quercetin, silymarin, and a combination of the two was shown in the table above. When compared to individual samples of Quercetin and Silymarin, it was suggested that the combined effect of the two compounds had a significant additive effect.

Numerous articles published recently demonstrated the effectiveness of numerous herbal plants in the treatment of a variety of diseases. There are already a number of herbal formulations made from naturally occurring herbs accessible for cancer patients. Since the majority of chemotherapy drugs were harmful to healthy cells and led to medication resistance.[13, 14]

In this present research study individual Quercetin and Silymarin showed a potent cytotoxic effect on Hep G2 cell line. Then, on combination of Quercetin and Silymarin shows increased cytotoxic effect even when it is used in minimal quantity (30µg/ml) as per our study. Through this we can determine that silymarin having synergistic effect with Quercetin, that help in anti-cancer activity. Though this we can also conclude that this formulation could be useful in humans for the anticancer activity, as the results shows positive effects. the increase int number of cells or decrease in the number of viable cells determine the effectiveness of the drugs.

Finally, pure phytoconstituents of quercetin and silymarin showed potent synergistic activity in combinational treatment of quercetin and silymarin at very low concentrations ( 30 µg/ml), at which 50% of cancer cell death occurred, and significantly inhibited cell viability and induced apoptosis in Hep G2 cells.

(IC50) : Inhibitory Concentration 50

ROS : Reactive Oxygen Species

Conflict of interest:

Authors declared no conflict of interest

Financial support:

None

Ethics statement:

None

Acknowledgment:

The authors are grateful to the Dr. D. Y. Patil International University, Akurdi, Pune for providing required facilities for carry out the research study.

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No competing interests reported.

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Version 1

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In Vitro Cytotoxic Activity of Quercetin and Silymarin and Their Synergistic Effect on Hep G2 Cell Lines

Status:

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Abstract

Purpose

Method

Results

Figures

Introduction

Materials & Methods

MTT Assay:

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1