{"id":68599,"date":"2025-12-30T11:04:39","date_gmt":"2025-12-30T11:04:39","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=68599"},"modified":"2026-01-03T16:04:00","modified_gmt":"2026-01-03T16:04:00","slug":"in-vitro-evaluation-of-methotrexate-cytotoxicity-and-antiproliferative-effects-of-withania-somnifera-on-human-peripheral-blood-mononuclear-cells-using-mtt-and-comet-assay","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol18no4\/in-vitro-evaluation-of-methotrexate-cytotoxicity-and-antiproliferative-effects-of-withania-somnifera-on-human-peripheral-blood-mononuclear-cells-using-mtt-and-comet-assay\/","title":{"rendered":"In Vitro Evaluation of Methotrexate Cytotoxicity and Antiproliferative Effects of Withania somnifera on Human Peripheral Blood Mononuclear Cells Using MTT and Comet Assay"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Methotrexate (MTX) earlier known as Amethopterin is a metabolic inhibitor commonly used as an anti-cancer or antineoplastic drug. It acts as an immunosuppressant for various autoimmune diseases. MTX mainly consists of Aminopterin as its parent component which was first drug that was used for the successful treatment of leukaemia. It is used as a treatment for many types of cancer \u2013 lung cancer, breast cancer, myeloma, lymphoma etc. In the treatment of cancer Methotrexate acts as the antifolate metabolic inhibitor.<\/p>\n

Withania somnifera <\/em>(Ashwagandha) is<\/em> an evergreen shrub belonging to the family Solanaceae that grows in India, the middle east and parts of Africa. It is a short shrub which grows to a height of 35-75 cm. The leaves are dark green & flowers are small, not brightly coloured and the ripe fruit is small & red. The name Somnifera indicates that it is sleep inducing and in fact calms the mind. It is used in herbal medicine and dietary supplements to treat various diseases and health conditions.1<\/sup> The word \u2018Ashwagandha\u2019 refers to a horse like smell the source of which is the root. The main phytochemical constituents of the plant extract are withanolides which are a group of triterpene lactones. \u00a0Its extracts have been found to have anti-inflammatory, anti-bacterial, and anti-tumor activities also. \u00a0Role of Withania somnifera <\/em>in the management of male infertility was investigated by Pallav Sengupta.2 <\/sup>Withanolidses isolated from the plant also inhibited the growth of cancerous cells, breast and colon cell lines.3<\/sup><\/p>\n

Hence it was thought that the study of the effect of Withania sominifera<\/em> leaf extract on the human mononuclear cells from peripheral blood which were pre-treated with various concentrations of methotrexate at 4 and 18 hrs incubation would yield interesting results.\u00a0<\/strong><\/p>\n

Materials and Methods<\/strong><\/p>\n

The research work involves the use of mononuclear cells isolated from Human peripheral blood of a pre-informed healthy normal blood donor. The present investigation has been approved by the institutional ethical committee of AVIKA biological research foundation lab, Jabalpur, Madhya Pradesh (where research work was carried out). For the research work Methotrexate injection IP 25 mg\/ml (trade name- Folitrax-25), Ipca laboratories ltd., Mumbai was purchased from a local pharmacy shop. This was the drug used to treat the cells. The injection was treated as the stock solution and dilutions were made in sterile distilled water to yield solutions containing 75, 125 & 250 \u00b5g\/ml of the drug respectively.<\/p>\n

Preparation of Withania sominifera Leaf extract<\/strong><\/p>\n

Plant leaves were cleaned, dried and ground to a fine powder. Then 2.9 g of dried leaf powder along with ethanol and water in 1:1 ratio was subjected to extraction process in Soxhlet for 10hrs at 80\u1d3cC. This extract was filtered with Whatman filter paper and the filtrate was evaporated in hot air oven at 55\u1d3cC. Then stock solution of this was prepared @ 25mg\/ml.4<\/sup><\/p>\n

Isolation of Lymphocytes from whole blood<\/strong><\/p>\n

In a sterile heparinised vial, 3.5ml of blood was collected from a healthy male young volunteer donor after informed consent. This was diluted to 7ml with PBS (1X). 3.5ml HisepTM<\/sup> Lymphocyte separation medium (HiMedia, LSM) was transferred aseptically into a centrifuge tube. This was then gently layered with 7ml of the diluted blood. It was centrifuged for 30 minutes at 400g at room temperature (RT). The erythrocytes were sedimented at the bottom and lymphocytes formed a layer above the Hisep layer. The lymphocyte layer along with half of Hisep layer was carefully pipetted out into a separate centrifuge tube after the supernatant was removed. After that, it was washed twice with isotonic PBS by adding 5ml PBS 1X to the WBC thus separated & centrifuging at 2300 RPM for 10 min. One more similar wash was given & 500\u00b5l of the suspension was used to count the cells in Hemocytometer in which 28.5 \u00d7 104<\/sup> cells\/ml or 2.85 \u00d7 105<\/sup> cells\/ml of cells were observed. The cells were appropriately diluted with TC 199 Medium (HiMedia) supplemented with FBS 1% (Fetal Bovine serum ) and PHA (Phytohemaglutinin) 15\u00b5l to achieve the final concentration of 5698 cells\/well if 180 \u00b5l of cells+medium suspension was used to fill each well.<\/p>\n

MTT Assay<\/strong><\/p>\n

Two 96 well plates of ELISA microplate reader (Lisaquant-TS, Tulip Diagnostics) were loaded (one for 4 hrs incubation and the other for 18 hrs of Incubation). First row was kept empty and the loading started from row B which contained only 180 \u00b5l cells + medium in 9 wells (forming 3 sets of triplicates of the control cells). The 3 subsequent rows were similarly loaded with 180 \u00b5l cells + medium + 20 \u00b5l of the 3 drug concentrations taken up for the experiment (D1, D2, D3 representing 75, 125 & 250 \u00b5g\/ml of MTX). The next 3 rows, the cells + medium +10 \u00b5l of the drug conentration + 10\u00b5l of WE was loaded in the manner described above. 3 replicates of 20\u00b5l of each of the 3 concentrations of the drug and WE were loaded in separate cells .5,6<\/sup><\/p>\n

Prior to loading, the reference reading of the plates was taken at 630nm. The OD reading of the drug only wells was taken the momemt they were loaded. The plate was then incubated for 4hrs at 37\u1d3cC. Similar pattern of loading was followed for 18hrs of incubation. After 4 hrs 20\u00b5l of MTT (0.25 mg\/ml) was added to the first plate & put for incubation for further 2hrs. After that 100\u00b5l of DMSO was added to each well and again incubated for 1 hour. OD readings were taken at 550nm. Similar procedure was followed for plate for 18hrs of incubation.<\/p>\n

Calculations were done after making appropriate deductions of the readings of the empty plate at 630 nm, the drug and WE. Viability and inhibition was calculated according to the following formulas<\/p>\n

Viability% = OD of the sample \/ OD of the control \u00d7 100<\/p>\n

Inhibition% = 1- (OD of the sample \/ OD of the control) \u00d7 100<\/p>\n

COMET Assay<\/strong><\/p>\n

Comet Assay is a method used to detect DNA damage at the level of individual cells and it is valuable to assess the damage to the genetic material due to toxic chemicals and pharmaceuticals. This test was optimised for alkaline conditions7 <\/sup>and it helps to assess single or double stranded DNA breaks at alkali labile cells.<\/p>\n

Precoating of slides<\/strong>: Slides were thoroughly clean and dried. They were then uniformly coated with a 1% solution of NMPA (Normal melting point agarose) in PBS, and dried at 37\u1d3cC overnight. The slide should be perfectly dried and stored in a slide box whose lid is sealed with parafilm.8<\/sup><\/p>\n

Solution of 0.5% LMPA (low melting point agarose) in PBS was also prepared and kept at 4\u1d3cC.<\/p>\n

Before performing the experiment, the blood was subjected to 3 concentrations os MTX in eppendorf tubes each containing 100\u00b5l of blood and 50 \u00b5l each of 75,125 & 250 \u00b5g\/ml concentrations of MTX for 1hrs 30mins.<\/p>\n

This treated blood + LMPA (maintained at 37\u1d3cC) was loaded on the NMPA coated slides (30 \u00b5l of blood + 90 \u00b5l of LMPA), out of which 50 \u00b5l was added on slide. Coverslip was placed and the slides were put in the freezer for 5minutes. Coverslip was then removed gently and the slide was then put in a Petri dish in cold lysis (working solution) for 90 minutes.<\/p>\n

They were then put in cold electrophoresis buffer and the slides were made to lie in the buffer for 20 minutes in the electrophoresis tank.<\/p>\n

After this the power was run for 20-25 minutes.<\/p>\n

Neutralisation: <\/strong>The slides were gently lifted out of the tank and flooded with neutralisation buffer for 5 minutes. This was repeated 3 times. The slides were then dried and stored.<\/p>\n

Staining: <\/strong>The slides were hydrated in cold distilled water for 7 minutes & stained in 1\u00b5M Ethidium bromide for 15 minutes.<\/p>\n

They were observed in the florescence microscope in green excitation filter and red emission filter where the cells appeared red on a black background.<\/p>\n

Results<\/strong><\/p>\n

MTT Assay<\/strong><\/p>\n

After 4hrs of incubation with MTX only<\/em><\/p>\n

If the OD (readings after appropriate deductions and calculations) of the control were taken to be 100%, the addition of 20\u00b5l of each of the 3 concentrations of MTX only taken for study (75, 125 & 250 \u00b5g\/ml) resulted in a dose dependent decrease in viability because at 250\u00b5g\/ml it dropped to 82.6% which was statistically significant (p\u02c2 0.05).<\/p>\n

Fig 1 shows that there was dose dependent decrease in viability with MTX only at 4hrs. If 20\u00b5l of Withania somnifera <\/em>(WE) leaf extract is also added to the reaction mixture in the wells, the viability drops till further. In this case the controls showed a viability of 82.38% and there was a drop in viability at every concentration of MTX if tested with WE, reaching the lowest level of 58.13% at 250 \u00b5g\/ml but the decrease was non-significant.\u00a0 These viability values were lower than those found with only MTX. Hence it maybe said that this was due to the antiproliferative effect of WE.<\/p>\n

At 18 hrs incubation with only MTX, more dose dependent reduction in viability was observed at every concentration. The addition of WE in this case resulted in a further decrease in viability values. The drops with 125 and 250\u00b5g\/ml were found to be statistically significant. Thus, the antiproliferative effect of WE was more evident at 18hrs of incubation.<\/p>\n\n\n\n
\"\"<\/td>\nFigure 1: Average Viability % of cells when treated with various concentrations of MTX (with and without WE) at 4 hrs and 18hrs of incubation.<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Antiproliferative\/inhibitory effect of MTX and MTX+WE<\/strong><\/p>\n

At 4 hrs incubation the results showed that 75 \u00b5g\/ml of MTX if used alone gave inhibition of 10.15%, but if used with WE it showed 13.17%. Similarly, at 125 \u00b5g\/ml MTX alone depressed cell viability by 11.28% but the inhibitory effect was found to be 19.24% with WE. At 250 \u00b5g\/ml of MTX alone the inhibition was only 17.38% but with WE it was 24.15%.<\/p>\n

At 18hrs of incubation it was found that MTX only caused more inhibition to cell viability than observed at 4 hrs and with the addition of WE still more inhibition of viability was evident.<\/p>\n

COMET Assay<\/strong><\/p>\n

The comets were scored in more than 150 cells\/ treatment and they were classified into 5 categories9<\/sup> \u2013 0, 1, 2, 3 & 4. According to this system TCS (total comet score ) is calculated as-<\/p>\n

TCS= 0(n)+ 1(n)+ 2(n)+ 3(n)+ 4(n)<\/p>\n

where n indicates the number of cells in each class. 10<\/sup><\/p>\n

The TCS of control cells thus calculated was found to be 69.3% and the TCS values showed a dose dependent increase with the treatment of the cells with 75, 125 & 250 \u00b5g\/ml of MTX. At 75 \u00b5g\/ml the TCS value was found to be 120.66% which is considerably higher than the control value and indicates the DNA damage caused by the drug. At 125 \u00b5g\/ml, the TCS value obtained was 213.66% and 250 \u00b5g\/ml it raised to 259.43%.<\/p>\n

At 125 \u00b5g\/ml comets of the category 2 & 3 showed the maximum occurrence and only 9 cells were in the category 4. At 250 \u00b5g\/ml 89 cells showed category 4 comets which was the maximum score of this class. Comets of class 1 showed the maximum occurrence at 75 \u00b5g\/ml and at 125 \u00b5g\/ml maximum number of comets were of category 2 & 3 as shown in Table 1 and Fig 2.<\/p>\n

Table 1: Mean frequency of each comet class per 150 cells (\u00b1 Standard deviation) & overall total cell count % of lymphocytes exposed to different concentrations of MTX .<\/strong><\/p>\n\n\n\n\n\n\n\n\n
<\/td>\nComet class<\/strong><\/td>\n<\/tr>\n
Concentration of MTX<\/strong><\/td>\n0<\/strong><\/td>\n1<\/strong><\/td>\n2<\/strong><\/td>\n3<\/strong><\/td>\n4<\/strong><\/td>\nTCS (in %)<\/strong><\/td>\n<\/tr>\n
Control<\/td>\n87 \u00b1 5.81<\/td>\n36 \u00b1 4.12<\/td>\n16 \u00b1 1.63<\/td>\n12 \u00b1 2.25<\/td>\n0<\/td>\n69.3<\/td>\n<\/tr>\n
75 \u00b5g\/ml<\/td>\n90 \u00b1 5.88<\/td>\n82 \u00b1 3.70<\/td>\n61 \u00b1 2.39<\/td>\n29 \u00b1 3.10<\/td>\n9 \u00b1 1.25<\/td>\n120.66<\/td>\n<\/tr>\n
125 \u00b5g\/ml<\/td>\n6 \u00b1 1.0<\/td>\n37 \u00b1 6.11<\/td>\n56 \u00b1 6.48<\/td>\n53 \u00b1 3.01<\/td>\n9 \u00b1 0.83<\/td>\n213.66<\/td>\n<\/tr>\n
250 \u00b5g\/ml<\/td>\n2 \u00b1 0<\/td>\n64 \u00b1 2.72<\/td>\n69 \u00b1 3.87<\/td>\n57 \u00b1 2.09<\/td>\n89 \u00b1 3.09<\/td>\n259.43<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 2: Showing comets of different classes ( classes1,2,3 &4).<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion <\/strong><\/p>\n

Barani M.11<\/sup> et al<\/em>, 2021 worked on the effect of MTX on normal and malignant cells and found that it causes dose dependent reduction in viability of cancerous and non-cancerous cells, and the toxic doses were found to be from 0-320 \u00b5g\/ml (in-vivo and in vitro in rats).<\/p>\n

Barbisan F.12<\/sup> et al, <\/em>(2014) worked on the possibility that the MTX related response on human peripheral blood mononuclear cells may be modulated by Ala16 Val-SOD2 <\/sub>gene polymorphism. They found no appreciable decrease in viability at 10 & 100 \u00b5M of MTX treated cells showing a SOD frequency of 27.6%. Some reduction in viability was found at both concentration in AV cells (SOD frequency 48.7%).<\/p>\n

Invitro cytotoxic studies on antineoplastic drugs on human lymphocytes was carried out by Vanshi Krishna et al<\/em> (2009). 13<\/sup> 100\u00b5l of lymphocytes containing approximately 50000 cells were incubated with different concentrations of various anticancer drugs (50\u00b5l) for 2 hrs and the effects were judged by MTT Assay. The lowest viability in MTX was found to be 32.01% with 400\u00b5M of MTX.<\/p>\n

Probably the reason for reduction in cell viability caused by MTX treatment is that it is a competitive inhibitor of dihydrofolate reductase. As tetrahydrofolate is necessary for de novo<\/em> synthesis of DNA, it hinders the proliferation of lymphocytes used in the experiment.<\/p>\n

The comet assay findings also show a dose dependent increase in DNA damage as evident by class 3 & class 4 comets when the cells are treated with MTX.<\/p>\n

Conclusion<\/strong><\/p>\n

In the present investigation it was found that MTX with WE caused a dose dependent decrease in viability of the lymphocytes which was found to be more at 18 hrs of incubation. This confirmed the antiproliferative effect of Withania on normal human lymphocytes. The DNA damage caused by MTX was evidenced by the findings of the Comet assay studies.<\/p>\n

Acknowledgment<\/strong><\/p>\n

The work was done in AVIKA Biological Research Foundation Lab, Jabalpur, (M.P.). The authors are grateful to the AVIKA Society for providing research facilities.<\/p>\n

Funding Source<\/strong><\/p>\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

The author(s) do not have any conflict of interest.<\/p>\n

Data Availability Statement<\/strong><\/p>\n

This statement does not apply to this article.<\/p>\n

Ethics Statement<\/strong><\/p>\n

This research did not involve human participants, animal subjects, or any material that requires ethical approval.<\/p>\n

Informed Consent Statement<\/strong><\/p>\n

This study did not involve human participants, and therefore, informed consent was not required.<\/p>\n

Clinical Trial Registration<\/strong><\/p>\n

This research does not involve any clinical trials<\/p>\n

Permission to reproduce material from other sources\u00a0<\/strong><\/p>\n

Not Applicable<\/p>\n

Author Contributions<\/strong><\/p>\n