INTRODUCTION:

Cancer can be described as a diseases characterized by groups of aberrant cells that undergo uncontrolled proliferation in the absence of cell cycle regulation. The metastasize cancer cells spread to various locations in the body where their uncontrolled growth invades the normal tissue. Cancer comprises more than hundred different diseases, including malignant tumors of different sites such as breast, cervix, stomach, intestines, colon, lung, mouth etc. There are numerous factors responsible for the cause, like genetic, tobacco, chemicals, environmental factors, hormones etc.

Cancer detection is described by their pathological grade and clinical stage^1,2. The cancer treatment includes surgery, radiation, hormone therapy and chemotherapy³. Medicines from plant sources have gained importance in therapeutic efficacy with minimum adverse effects. Hence there is need for continuous investigation and isolation of secondary plant metabolites for efficient therapeutic system. Lonicera ligustrina shrub belongs to family caprifoliaceae grows in Bhutan, Nepal, India and China. The leaves claimed to have Antioxidant, anti-inflammatory, laxative, Antidiabetic, Anticancer and urinary disorders based on practices^4,5. However, this plant has not been studied for anticancer activity on breast and colorectal carcinoma. Thus we have made an attempt to use this herbal extract to check the efficacy against Breast and Colorectal carcinoma cells^6,7. The present study aimed to evaluate preliminary phytochemical screening and invitro anticancer potential activities against MCF-7(breast) and HCT-116 (colorectal) cancer cell lines.

METHODOLOGY:

Collection and Authentication of Plant:

Lonicera ligustrina leaves was collected from Chittoor, Andhra Pradesh, India. The plant was identified and authenticated by Dr. K. Madhavachetty, Department of Botany, S.V University, Tirupathi. The specimen number 1321.

Preparation of crude drug for extraction:

The fresh leaves was collected and shade dried for a week and powdered by a mechanical grinder. Coarsely powdered leaf material (100g) was extracted with 95% ethanol in soxhlet apparatus. The extract was collected and filtered, and concentrated using rotary evaporator under reduced pressure at 40⁰c. The concentrated extract was stored in a desiccator for further drying. The percentage yield was calculated using following formula and the yield was found to be 5.25%w/w.

Amount of extract obtained

Percentage of Yield = ––––––––––––––––––––––––– ×100

Amount of powder taken

Phytochemical Investigation

The Qualitative test for different phytochemical constituents carried out such as alkaloids, glycosides, tannins, flavonoids, saponins, lignans, Phenols, Anthroquinones and reducing sugars by the standard procedure⁸.

Table 1: Phytoconstituents analysis

S. No	Phytoconstituents	Ethanol Extract
1	Alkaloids	-
2	Glycosides	++
3	Tannins	+
4	Flavonoids	+
5	Saponins	+
6	Phenols	+
7	Lignans	-
8	Anthroquinones	++
9	Reducing sugars	++
(+Present) (- Absent)

In vitro cytotoxic activity in cultured cells by MTT assay:

In our study, all the test compounds was screened for anticancer activity using MTT assay on two cell lines namely MCF-7 and HCT-116 at Skanda Life Sciences Pvt. Ltd, DSIR recognized R&D center, Bangalore. The ability of cells to survive a toxic insult has been is the basic principle of cytotoxic assays^[9,10].

Requirements:

1. MTT (prepared in hank^,s balanced salt solution (HBBS) without phenol red, 2 mg/ml)

2. DMEM (Dulbecco’s modified eagle’s medium) media supplemented with10%foetal bovine serum (FBS)

3. Tissue culture flasks, 96 well micro culture plates from cellstar greiner bio-one.

4. Isopropanol

5. Cell lines used : MCF-7 and HCT-116

Preparation of test solutions:

For cytotoxicity studies, 32mg/ml stocks were prepared using DMSO. Serial two fold dilutions was prepared from 320µg/ml to 10µg/ml using DMEM plain media for treatment.

Maintenance of cell lines:

MCF-7 and HCT-116 cells was grown in 25cm² tissue culture flasks containing DMEM media supplemented with FBS (10%), L-glutamine (1%) and gentamycin sulphate (50µg /ml) at 37⁰C in CO₂incubator in an atmosphere of humidified 5% CO₂ and 95% air. The cells was maintained at routine sub culturing in 25cm²tissue culture flasks¹¹.

Sub culturing process of cell lines:

The culture media from the flasks containing monolayer was aspirated and washed with sterile phosphate buffered saline (PBS). To the flasks 2ml of 0.1% trypsin-EDTA solution was added and after few seconds it was aspirated and the flask kept at 37^oc in CO₂incubator 2-3minutes for detachment. Then the flasks was removed from the incubator and gently tapped to detach all the adhering cells. The cell detachment was confirmed by observing under an inverted microscope. Once the cells was completely detached from the flasks, 2-3ml of media containing 10% FBS was added and mixed well. Cell viability was checked with a small sample of the suspension by tryphan blue dye exclusion test. From the stock cell suspension, 1×10⁴ viable cells /ml suspended in media was seeded in 25cm² tissue culture flask containing about 4ml of fresh media and incubated until the flasks attained 60-70 % confluence¹².

Preservation of the tumor cells:

Tumor cells from the first and second passage of transplantation was stored in liquid nitrogen in cryovials containing respective medium supplemented with 10% FBS and 10% DMSO as preservative at a concentration of 1×10⁶cells/ml. This constituted the tumor bank. After every 10 passages, that tumor cell line was discarded and new passage was started using the original tumor cells from the tumor bank. To obtain a single cell suspension from a monolayer culture, cells was dislodged from the culture flasks by trpsinization.

Procedure:

Exponentially growing cells were harvested from 25ml flask and a stock cell suspension was prepared. A 96-well flat bottom tissue culture plate was seeded with 5×10 ⁴cells/ml in medium and supplemented with 10% FBS and incubated at 37 ⁰c for 24hr in 5% CO₂atmosphere. The partial monolayer was formed after 24hr and supernatant was flicked off, to this 100 µl of different drug concentrations diluted in the medium was added to the different wells containing cells in the micro titer plate. The cells in the control group received no treatment and the plates were incubated at 37 ⁰c for 2 days in 5 % CO₂, After the treatment for 48hr, drug containing media was removed and the plates were washed twice with 100 µl PBS. Then to each well of the 96 well plate, 100µl of MTT reagent (stock: 2 mg/ml) was added and incubated for 4hr at 37 ⁰c. Plates was centrifuged at 2000 rpm for 10 min and inverted tissue paper to remove the media. To solubilize formazan crystals in the wells, 100µl of isopropanol was added to each well and incubated at 37 ⁰c for 30 minutes. The optical density (OD) was measured by an ELISA plate reader at 570 nm. The percentage growth inhibition was calculated using the formula and the IC₅₀value determined as the concentration of sample required to inhibit the growth of 50 % of viable cell population.

RESULTS AND DISCUSSION:

Samples shown significant activity at its higher concentration and the IC50 value of breast cell lines 24.45µM (MCF-7) and colorectal cell lines 9.210µM (HCT-116) inhibition. Standard Doxorubicin tested against MCF-7 and HCT-116 cell lines showed IC50 value of 18.76µM and 16.89µM inhibition. Originally, the MTT assay was not intended for the measurement of IC₅₀values but rather to assess cell viability and metabolic changes in response to exogenous treatment. No linearity was established between MTT ODs and cell viability as external agents was added. In contrast, the innovators suggested that with the administration of certain agents, the per-cell MTT ODs may vary up to seven- or eight-fold. Therefore, this assay was inappropriately adopted for IC₅₀ measurements. Among the three deficiencies we identified, the systematic IC₅₀errors caused by uneven proliferation or growth was the most significant in terms of their misleading effects. The purpose of IC₅₀ measurements was to identify the dose required for a chemotherapeutic agent to kill 50% of a cell population with a definite initial number of cells. However, in MTT analogue assays, this idea was interpreted as the dose required to inhibit 50% of a cell population with an uncertain or changing initial cell number. Therefore, IC₅₀errors are unavoidable. Only the limiting dilution assay, as a direct viability test, ensures that the obtained IC₅₀ data closely approximate actual cancer cell survival. This technique may be more broadly applied, whereas the MTT and its analogue assays need be restricted to the comparison of cancer cell chemo responsiveness under identical drug-concentrations, seeding densities and culture conditions.

Table2: Percentage inhibition by extract and Standard (Doxorubicin) against MCF-7(Breast cancer) cells

S. No	Sample Conc. ppm	Sample % inhibition	Std. Conc. µM	Std. % inhibition
1	0.1	13.90	3.13	26.74
2	1	21.00	6.25	33.10
3	10	30.35	12.5	46.30
4	100	37.53	25	59.61
5	500	46.55	50	67.13
6	1000	53.11	100	79.63
IC50µM				18.76

Table3: Percentage inhibition by extract and Standard (Doxorubicin) against HCT-116 (Colorectal carcinoma) cells

S. No	Sample Conc. ppm	Sample % inhibition	Std. Conc. µM	Std. % inhibition
1	0.1	15.5	3.13	19.91
2	1	29.58	6.25	26.62
3	10	37.22	12.5	41.32
4	100	43.69	25	55.44
5	500	51.76	50	64.70
6	1000	59.11	100	75.58
IC50µM				16.89

Figure 1: Graph of % cell growth inhibition versus logarithmic concentration

Figure 3: Morphological assessment of HCT-116 cells using MTT assay

CONCLUSION:

The preliminary phytochemical screening had revealed the presence of glycosides, tannins, flavonoids, anthroquinones and reducing sugars. The invitro anticancer study offers a paradigm for analyzing density-dependent variations in IC₅₀ values in cancer cells and reveals the underlying mechanisms and practical implications of these phenomena. Based on our findings, we hold the same opinion and propose that inconsistencies in IC₅₀values are a natural property of cancer cells rather than a remediable artifact. Therefore, a dynamic view of IC₅₀ values and the methods used to detect the density-dependent IC₅₀ spectrum of a cancer cell line (primary or passaged) established. This view will benefit patients and the cancer research community as a whole.

ACKNOWLEDGEMENT:

We immensely thankful to the Skanda Life Sciences Pvt. Ltd, Bangalore for providing the laboratory facilities, I would also like to thank Dr. K. Madhavashetty, Department of Botany, S.V University, Tirupathi for providing and authentication of plant extract and support for the study.

CONFLICT OF INTEREST:

None.

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Received on 18.08.2020 Modified on 20.02.2021

Research J. Pharm. and Tech. 2022; 15(8):3485-3489.

DOI: 10.52711/0974-360X.2022.00584