In vitro Cytotoxic studies of Saraca asoca bark extracts on HT-29 cancer cell Line

 

Jeenathunisa. N1*, Rajan. S2

1Research Scholar, PG and Research Department of Microbiology, M.R. Government Arts College,

Mannargudi-614001, Thanjavur (Dt.), Tamil Nadu, India. Affiliated to Bharathidasan University, Tiruchirappalli-620024.

2Assistant Professor, PG and Research Department of Microbiology, M.R. Government Arts College, Mannargudi-614001, Thanjavur (Dt.), Tamil Nadu, India. Affiliated to Bharathidasan University, Tiruchirappalli-620024.

*Corresponding Author E-mail: jeenathunisa22@gmail.com

 

ABSTRACT:

Asoka-Saraca asoca (Roxb.) de Wilde is one of the traditional plants with well-known medicinal values prescribed widely to treat haematuria, dyspepsia, fever, tumor, burning sensation, visceromegaly, colic, ulcers, menorrhagia, metropathy leucorrhoea, skin infection along with many other pharmacological activities. As globally, we look towards the use of non-toxic plant based products with conventional medicinal usage and the developing countries depends on herbal medicines for their primary health care needs. And so, the aim of this study is to explore the the in vitro cytotoxicity of the bark extracts of Saraca asoca on HT-29 cancer cell line using MTT and SRB assays. The results show a significant inhibition percentage on HT-29 cancer cell line was observed in both assays. In MTT assay the IC50 values was 174.1 and 163.2µg/ml on alcohol and aqueous bark extracts, respectively. And the SRB assay shows the IC50 values 94.76 and 200.5µg/mL on the crude ethanolic and aqueous bark extracts, respectively. Thus, both MTT and SRB assays can be used for cytotoxic screening of Saraca asoca herb towards the development of modern drug.

 

KEYWORDS: MTT assay, SRB assay, HT-29 cancer cell line, IC50 value, cytotoxic activity.

 

 


INTRODUCTION:

Over the past 100 years, chemically synthesized drugs radically changed the health care scenario in most parts of the world. However, almost 70% of population in the developing countries like India depends on traditional herbal medicines to meet out their primary health care needs(­1). The use of traditional medicines is not only restricted to developing countries, but also greatly increased in industrialized countries due to the public interest towards natural therapies for the treatment of various diseases. Medicinal plants constituents have an important role towards growth control of tumour cells(2).

 

Plant and phytoproducts plays a vital role in curing wide range of diseases including cancer. Many plant origin substances reported to have antitumor activity and its possible application in cancer prevention(3). This emphasize the importance of ethnobotanicals in pharmacological research and drug development, though either we use plant constituents directly as therapeutic agents or the starting material for the synthesis of drugs and active pharmacological compounds(4).

 

The GLOBOCAN 2018 data states that colorectal cancer (CRC) is the third and fourth most deadly and commonly diagnosed cancer in the world, respectively. Adoption to “western” way of life, especially, in developing countries shows a constant increasing incidence of CRC. The driving sources for the rise of CRC were obesity, static lifestyle, consumption of redmeat and social drinking. Recent advances in early detection screenings and treatment can reduce CRC mortality rate in developed nations(5).

 

Human cancer cell lines were used commonly for experimental models, as it continue to have the characteristics features of cancer cells that can be propagated and genetically modified to provide consistent results.(6-10). Thus, we believe there is a strong correlation between the uses of appropriate cell line for invitro preclinical studies of cancer drug responses and to the cancer properties.

 

Cytotoxicity assays has different parameters associated with cell death and multiplication(11). which is intensively utilized for the evaluation of toxicology under in-vitro condition(12). Two major techniques are used to assess the cell growth. The first one is the MTT assay(13) used to measure the activity of living cells through mitochondrial dehydrogenases. The key component is 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) is converted to an insoluble purple formazan. The amount formazan formed is directly proportional to the number of active cells(14). Thus, MTT investigation indicates the anticancer potential of the test samples against cancer cell lines(15). The second technique is sulforhodamine B (SRB) assay developed in 1990 investigate the cytotoxicity in cell based studies(16) (17). The amount of bound dye can be used to measure the cell proliferation. Ashoka i.e. Saraca asoca Roxb.de Wilde, syn. S.indica belongs to Caesalpinaceae family is an ancient scared tree seen throughout India(18). According to Ayurvedic system of medicine, Saraca asoca is an excellent source of herb that claims to treat several diseases. Several studies has reported that this legendry evergreen tree is useful in treating gynaecological disorders and also possesses antimicrobial, anti-progestational, anti-ulcer, anti-oxidant and anti-cancer activity(19). As colon cancer cell has a very limited availability of drugs in the market, the current study focus on the bark extracts of Saraca asoca aganist HT-29 colon cancer cell line, which resembles the real colon tissues(20) for the evaluation of invitro anticancer activity(21) to formulate plant based drug source(22).

 

MATERIALS AND METHODS:

Collection of sample:

The bark of Saraca asoca tree was collected from Anaikatti, Coimbatore, Tamil Nadu, India. The stem bark sample was authenticated by Dr. John Britto, Professor, Department of Botany and Director, Rapinat Herbarium, St. Joseph’s College, Tiruchirappalli, Tamil Nadu, India.

 

Processing of plant material:

The collected bark part was manually cleaned to remove course impurities and washed thoroughly with distilled water and shade dried. The dried bark sample was uniformly grinded using mechanical grinder to make a crude powder stored in airtight container for further        use (23)(24).

 

Preparation of extracts:

Extract from the dried stem bark was prepared using ethanol and water by soxhlet extraction method. The residue was left to air dry at room temperature for about 72 hours. The dry residue was stored at 4°C in air-tight bottle.

 

In vitro cytotoxcity:

Invitro cytotoxcity activity was determined by the standard MTT(25) and SRB assay. The colon cancer cell line HT-29 was procured from ATCC, stock cells was cultured in DMEM supplemented with 10% inactivated Fetal Bovine Serum (FBS). All cells were maintained in a humidified atmosphere of 5% CO2 at 37°C until confluent.

 

Cell treatment procedure:

The monolayer cells were trypsinized and the cell count was adjusted to 5.0 × 105 cells/ml with the respective media containing 10% FBS. About 100µl of the diluted cell suspension was seeded to each of the 96 well microtiter plates and incubated for 24 hours to allow cell attachment at 37°C, 5% CO2, 95% air and 100% relative humidity. After incubation the cells were treated with serial two fold dilutions prepared using DMEM plain media for treatment to obtain final concentrations of 10, 20, 40, 80, 160, 320µg/ml. The final volume in each well was maintained as 200µl. The medium with DMSO alone served as control.

 

MTT assay:

MTT is a water soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solution that lacks phenol red. MTT is converted to insoluble purple formazan. The amount of formazan formed indicates the degree of effects caused by the test material. After incubation the test solutions in the wells were discarded and 100µl of MTT (5mg/10ml of MTT in PBS) was added to each well and incubated for 4 hours at 37°C in 5% CO2 atmosphere(26). The supernatant was removed and DMSO of 100µl was added and to solubilise the formed formazan the plates were gently shaken. Then the absorbance was read at 590nm using a microplate reader.

 

SRB assay:

Sulforhodamine B assay does not rely on the metabolic activities of the cell, instead used for the determination of cell density, based on the measurement of cellular protein content(27). After incubation the test solutions in the well were discarded and 25µl cold 50% (wt/vol) TCA was added gently to each well directly to medium supernatant and incubated at 4°C for 1 hr. Then, 50 µl of 0.04% (wt/vol) SRB solution was added to each well to incubate at room temperature for 1 hr. Now the plate was rinsed adequately with 1% (vol/vol) acetic acid to remove unbound dye. Followed by air-dried and 50% of 19mM Tris base solution (pH 10.5) was added to each well and the plate under agitation in orbital shaker for 10 min to solubilise protein-bound dye. Absorbance was measured at 560nm in a plate reader.

 

Calculation of inhibition:

The percentage of growth inhibition can be calculated by the following formula:

                      OD of control – OD of sample

% Inhibition =----------------------------------- X 100

                                 OD of control

 

For cytotoxicity, lower the IC 50 higher the cytotoxicity(28).

 

 

RESULT:

Natural product substances have historically served as the most significant source of new leads for pharmaceutical development(29). HT-29 colon cancer cell line were seeded and exposed to Saraca asoca ethanolic and aqueous extracts at 10, 20, 40, 60, 80, 160, 320 µg/ml of concentration. In MTT assay shows dose-dependent inhibition of cell proliferation in HT-29 cell line by the ethanolic and aqueous bark extracts of Saraca asoca (Figure 1). Table1shows the extracts exposure demonstrated a maximum decrease in cell growth by MTT assay on HT-29 cell line of 62.19% and 65.61% of inhibition at 320µg/ml concentration by ethanolic (S1) and aqueous (S2) bark extract, respectively, compared to DMSO control treatment. The IC50 value for MTT has been determined for ethanolic and aqueous extract as 174.1 and 163.2µg/ml, respectively using Graph Pad Prism 6 software.


Table 1: Percentage of cell inhibition of S.asoca against HT-29 cancer cell line

Compound name

Conc. µg/ml

OD at 590nm

% Inhibition

IC50 µg/ml

Control

0

0.739

0

 

S1

10

0.681

7.88

174.1

20

0.656

11.27

40

0.593

19.76

80

0.498

32.57

160

0.405

45.13

320

0.279

62.19

 

S2

10

0.666

9.90

163.2

20

0.636

13.97

40

0.571

22.77

80

0.474

35.83

160

0.382

48.31

320

0.254

65.61

 

 

Table 2: Percentage of cell inhibition of S.asoca against HT-29 cancer cell line

Compound name

Conc. µg/ml

OD at 560nm

% Inhibition

IC50 µg/ml

Control

0

0.439

0.00

 

S1

10

0.410

6.58

94.76

20

0.376

14.35

40

0.330

24.83

80

0.303

30.98

160

0.199

54.57

320

0.179

59.23

 

 

 

S2

10

0.415

5.47

200.5

20

0.387

11.94

40

0.365

16.86

80

0.302

31.12

160

0.245

44.15

320

0.164

62.55

 


 

Figure 1- Growth inhibition percentage of various S.asoca bark extracts concentrations

 

The SRB assay with the ethanolic and aqueous extracts of Saraca asoca bark sample shows maximum cell inhibition at 59.23% and 62.55%, respectively, at 320 µg/ml concentrations (Figure 2). Table 2 presents the IC50 value of both ethanolic (S1) and aqueous (S2) Saraca asoca bark extracts as 94.76 and 200.5µg/ml, respectively with Graph Pad Prism software

 

Figure 2- Growth inhibition percentage of various S.asoca bark extracts concentrations

 

DISCUSSION:

Humankind have been benefitted by plants, herbs, and ethnobotanicals since early days and still used worldwide for health promotion and to treat diseases. Herbal medicines, due to less toxicity, have great demand across the globe(30). Natural sources and plants are the basis of modern medicine; thereby largely contribute to the preparation commercial drugs(31). At present the most reliable and available invitro screening techniques used to evaluate the anticancer activity of herbal formulations on cancer cell lines are MTT and SRB assay. The MTT and SRB is used for quantitative and for qualitative analysis, respectively(32)(33)(34). The MTT assay is colorimetry method of analysing the color reduction of reagent to estimate cell viability. It determines the cytotoxicity by mitochondrial dehydrogenase activities in living cells. The better linearity of cell number with higher sensitivity is achieved by cell independent staining of SRB assay. In this assay the cell debris does not get stained and so the drug sensitivity data will not get affected (35). It measures whole protein content that is proportional to the cell number(36).

 

Cancer is one of the most dreadful diseases worldwide that increases at a progressive rate. Public awareness towards phytopharmaceu­ticals gains the importance of phytoconstituents as therapeutic agents to provide defensive mechanism(37). Every system of medicine emphasized the importance of most secondary metabolites from herbal plants were used to cure enumerous diseases like diabetics, cancer, arthritis etc. Hence, phytoconstituents can continue to be used as ideal sources for anticancer drug formulations. In this study, the MTT and SRB methods were used to evaluate the cytotoxicity potential of the Saraca asoca bark extracts. Results from those assays revealed that the bark extracts of S.asoca explored a very good percent of cell inhibition with increasing concentration of the bioactive component of the test material.

 

CONCLUSION:

We conclude that the in vitro studies reduce the usage of animals for clinical trials. It helps to assess the larger number of compounds with minimum quantity quickly. The findings suggest that the bark extracts of Saraca asoca have a potential promising novel anticancer activity against HT-29 cancer cell line. Thus, the overall results indicate the potential use of this indigenous tree as a novel chemotherapeutic agent for the treatment of Colon cancer.

 

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Received on 10.04.2020           Modified on 19.05.2020

Accepted on 24.06.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(1):42-46.

DOI: 10.5958/0974-360X.2021.00008.1