INTRODUCTION:

Indian flora has always been beneficial to us in providing herbs of medicinal significance¹. Chemical constituents embedded in the herbs such as carbohydrates, flavonoids, alkaloids, glycosides and lignans have been found to possess medicinal benefits in the mitigation of liver disorders and tumors². The usage of plant based medicines and herbal drug therapy by number of patients is increasing exponentially³. Human consumption of herbal drugs for the treatment of ailiments has a long history since from primitive ages⁴. With the above informative background the present research was planned to extract and to evaluate the cytotoxic potentials of Chloris barbata leaves (family poaceae) by using in vitro models. The leaves of the plant are rich in alkaloids, glycosides andflavonoids^5,6. Chloris barbata leaves are known to possess medicinal properties such as antidiabetic, antimicrobial, antiurolithiac, hepatoprotective and also used in cancer⁷. Cancer is a dangerous and dreadful disease affecting a wide number of populations every year. There is no documented evidence regarding the anticancer activity of the Chloris barbata leaves. Hence the present research is essential and is reasonable.

MATERIALS AND METHODS:

The fresh plant of Chloris barbata belonging to the family Poaceae was collected from Nallajerla, Westgodavari District, Andhra Pradesh on November 2018. The plant was identified and authenticated by Dr. Ramadevi, associated professor of Horticulture, Dr. Y.S.R Horticultural University Horticultural College and Research Institute, Venkatramanagudem. After collecting the leaves, they were washed and left for shade drying on the floor above the newspapers for 10 days. After that the leaves were dried in hot air oven at 40⁰C for an hour just before starting the extraction process to remove the equilibrium moisture content. After drying the leaves were size reduced by grinder. The dried powdered material was weighed and subjected to extraction by the process of Soxhlation using organic solvents such as petroleum ether, chloroform, hexane, ethyl acetate, methanol and water for 3 days with occasional stirring⁸. After the extraction, the extracts were filtered by Whatman filter paper and filtrate was concentrated to obtain a crude extract⁹. The dried extracts were collected and transferred in to sterile bottles and stored at 40⁰C for the phytochemical screening and for the Cytotoxic studies. The obtained extracts were subjected to phytochemical tests for detection of phytoconstituents present in it viz. alkaloids, carbohydrates, glycosides, phytosterols, fixed oils and fats, phenolic compounds and tannins, proteins and free amino acids, gums and mucilage, flavonoids, lignans and saponins¹⁰. The methanolic extract was found to contain a large amount of flavonoids, alkaloids, glycosides, saponins, carbohydrates, triterpenoids and lignans. Hence methanolic extract of Chloris barbata leaves was selected for further in vitro cytotoxic investigations.

IN VITRO CYTOTOXIC ACTIVITY OF CHLORIS BARBATA LEAF EXTRACT¹¹:

MATERIALS:

Chloris barbata plant extract and 5- Fluorouracil (standard drug) procured from Sigma-Aldrich, Bengaluru, India.

CELL LINES:

Human epidermoid larynx carcinoma (Hep 2), African greenmonkey kidney normal (Vero), Dalton’s Ascitic Lymphoma (DAL), cell lines were utilized for the current cytotoxic evaluation. These cells were collected from Amala Research Center for Cancer, Thrissur, Kerala, India.

CULTURES USED:

Minimum Essential Media (MEM) added with heat inactivated fetal calf serum 10% (FCS) including 5% of combination of Gentamicin, Streptomycin (100µg/ml) and Penicillin (100Units/ml) in presence of Co₂ (5%) at 37ºC for 3-4 days. Dulbecco’s eagle medium modified (DMEM) media with 5% Gentamycin, Streptomycin and Penicillin.

REAGENTS USED:

Tryphan blue, MTT, PBS, Trichloroacetic acid (TCA) and Dimethyl sulfoxide (DMSO) (All are obtained from Sisco Research Chemicals Laboratory, Mumbai).5-Fluorouracil, Neutral red and other chemicals are procured from Sigma Aldrich, Bengaluru, India.

Dose of Chloris barbata plant extract selected for in vitro anticancer activity:

The following doses of MECP i.e 10, 25, 50, 100 and 200µg/ml are selected.

Dose of 5-Fluorouracil (5-FU) (standard drug) selected for Cytotoxic assays:

5µg/ml to 100µg/ml with water for injection (sterile).

COUNTING OF CELLS:

The live cells were counted by utilizing haemocytometer by diluting the solution (stock) to 1x10⁶ cells and then transferred cells within the 96 well plates in tryphan blue dye method. Tryphan blue dye evaluation involves addition of 0.5ml Trypan Blue (0.4 %) to 0.5ml cell suspension and the cells have been loaded over a haemocytometer after 5 mins of incubation. The numerous nonviable cells were evaluated and the % of non-viable cells was estimated. Cells which are viable will reject the dye while cells that are nonviable absorb the dye and emerge in blue colour.

TRYPHAN BLUE DYE EXCLUSION TEST:

The cytotoxic activity of Chloris barbata was assessed by in vitro technique on DAL, HEp2 and Vero cells utilizing Tryphan Blue dye procedure. Short term cytotoxicity analyses were done on DAL, HEp2 and Vero cells by tryphan blue dye technique. The Tryphan blue dye technique is employed to find out the number of survival cells in cell suspension. The principle involved in this technique is that viable cells contain intact plasma membrane that excludes certain dyes like tryphan blue, eosin or propidium while nonviable cells take over the stain. About 1 million cells have been taken for cell cytotoxicity studies. Different concentrations of Chloris barbata plant extract (10, 25, 50, 100 and 200µg/ml) are infused within cells and with one ml of demineralized H₂O volume has been fulfilled. DAL, HEp2 and Vero cell suspensions were incubated at 37⁰C in Co₂incubator with continuous flow of CO₂. After incubation, cell suspension (0.2ml) was taken and diluted with phosphate-buffered saline, PBS (0.3ml) and tryphan blue (0.2ml) to 0.5ml, mixed thoroughly and kept aside for 5 mins. The nonviable total cells were enumerated and the nonviable cellular % was estimated.

Nonviable cells (%) = Nonviable cells/total cells x 100.

CELL VIABILITY ASSAY BY MTT:

This colorimetric technique determines the reduction of MTT (yellow) by succinate dehydrogenase (mitochondrial) in viable cells to a purple colored (insoluble) product known as formazan which is quantified spectroscopically.

PROCEDURE:

MTT solution preparation: Add 5mg/ml MTT incorporated in PBS (stock solution)

MTT working solution: 1:10 dilution of stock in PBS

DAL, HEp2 and Vero cell lines were employed for the MTT assay. The cell lines have been cultured in tissue culture bottles possessing minimum essential media (MEM) added with heat inactivated fetal calf serum (FCS) 10% including 5% of mixture of Gentamicin (100Units/ml), Streptomycin (100µg/ml) and Penicillin (100Units/ml) in presence of Co₂(5%) at 37ºC for 3-4 days. Subsequent to 3 days the supernatant media was removed and replaced with DMEM media with 5% Gentamicin, Streptomycin and Penicillin. Incubate overnight. The growth of DAL, HEp2 and Vero cells were observed microscopically and after sufficient surge of cells in the DMEM media, the supernatant media was rejected and the cell lines were collected as individual cell suspension. The cell suspensions were processed with various concentrations of Chloris barbata plant extract diluted with demineralized H₂O and further the cell lines diluted with DMEM medium. Control group contains only DMEM media and cell lines. DAL, HEp2 and Vero cell lines were injected in 96 wells microtitre plate with incubation (24 hrs) at 37⁰C with 5% CO₂,after the medium was substituted with MTT solution (20µl) with 4 hrs incubation. The supernatant cautiously aspirated and the deposited ‘Formazan blue’ crystals were dissolved by adding DMSO (200µl) and OD was evaluated (570nm).

The result was represented as the average of three readings.

Mean OD (test compound)

(%) Surviving cells = ---------------------------------- X 100

Mean OD (control)

SULPHORODAMINE B ASSAY:

SRB, a radiant aminoxanthine pink dye having sulfonic groups (two), under acidic environment attaches to the amino acid residues (basic) in TCA stained cells offers an index to determine cellular protein substance. The cell culture layer (mono) was trypsinized and cell enumeration was adapted (0.5 x 1.0 x 10⁵ cells per ml) by utilizing medium possessing 10% new born sheep serum. Individual wells of 96 well plates were added with 0.1ml of cell suspension (approx. 10,000 cells). The supernatant was wiped off, cleansed and 100µl of various concentrations of Chloris barbata Plant extract were incorporated to the cells in plates. Further plates are incubated (37⁰C, 72hrs, 5%CO₂), microscopic observation was done and observations noted (once in 24 hrs). Following 72 hrs, 2.5µl of TCA (50%) has been added to the wells with gentle care so that a delicate layer is formed above the test compounds with 10% strength. Incubation of plates was done for 1 hr (4⁰C). Flicking off the plates followed by 5 times cleansing with tap water to eliminate medium, sample and serum traces and exposed for drying. The plates (dried) were stained with 100µl SRB, stored for half an hour at room temperature. The dye which is unbound is eliminated with rapid cleansing with acetic acid (1%) for four times. The plates are air dried. To this 100µl tris-base (10mM) incorporated for solubilization of dye. Vigorous agitation of the plates was carried for 5 minutes. Absorbance was recorded at 540nm. % growth suppression was evaluated as follows.

Percent cell suppression= 100-[A_t-A_b/A_c- A_b] X 100

A_t= Absorbance of test

A_b= Absorbance of blank

A_c= Absorbance of control

NEUTRAL RED UPTAKE ASSAY:

Neutral red is a dye which penetrates only in viable cells. After disrupting the colored viable cells, NR is liberated and assessed by recording the absorbance. Cells were located in 96 well plates (10,000cells/200µL/Well). Twenty-four hours afterwards, cells were placed in contact with Chloris barbata plant extract at various concentrations (10, 25, 50, 100, 200µg/ml). The cell lines (DAL, Vero and Hep-2) were exposed for 72 hrs, the medium from individual well was removed and is substituted with 200µl of freshly arranged neutral red solution (50µg/ml). The cells were further reincubated for 4 hr at 37⁰C, rinsed with 200µL of PBS (to remove extracellular Neutral red) and then lysed with acetic acid solution (200µl)/50% ethanol (1:99 v/v). Absorbance was calculated (540nm) and matched with wells of untreated cells. Cytotoxicity of each test agent is expressed as IC₅₀ value.

RESULTS AND DISCUSSION:

Chloris barbata is an Indian grass family plant belonging to poaceae family. It is an annual herb and is used traditionally for various disorders like diabetes, microbial infections, kidney stones and in cancer^12,13. Chloris barbata plant leaves are subjected to shade drying followed by pulverization in to coarse powder and then subjected to soxhlation with polar and nonpolar solvents like petroleum ether, chloroform, hexane, ethyl acetate, methanol and water. Methanolic extract revealed the presence of high amount of carbohydrates, alkaloids, glycosides, flavonoids, triterpinoids and saponins during phytochemical investigations. Hence methanolic extract was selected for further investigations. In tryphan blue dye exclusion test for invitro cytotoxic activity it was found that the methanolic extract of Chloris barbata leaves at dose 200mg/kg exhibited significant anticancer activity by increasing the percentage of dead vero, DAL and Hep-2 cells to 92±0.49, 76±0.47 and 82±0.36 in comparison to control group. Whereas with MTT cell viability assay it was found that Chloris barbata leaves at dose 200mg/kg exhibited significant anticancer activity by decreasing percentage of survival of DAL, Hep-2 and vero cells to 0.941±0.95, 0.714±1.62 and 1.364±1.48. 5-Fluorouracil is used as reference standard. In SRB assay, the percentage of cell lines growth inhibition has been found to be 56.362 (vero cells), 69.128 (DAL cells) and 61.354 (Hep-2 cells) when compared to control group. IC₅₀ values of leaf extract obtained from NRU assay shows a potential cytotoxic activity of plant extract. IC₅₀values of plant extract was found to be 24.11±3.82 (vero), 47.16±0.14 (DAL) and 31.26±2.15 (Hep-2 cells) when compared to control. The results are depicted in the table 1 to 3 and figure 1 to 5.

Table.1 Percentage of dead Vero cells with Chloris barbata (C.B) extract and standard 5-FU by tryphan blue dye exclusion assay

Groups	Drug concentrations in µg/ml	Percentage (%) of dead Vero cells
Control	-	0
C.B extract	10	16 ± 0.17
	25	46 ± 1.28
	50	61 ± 0.91
	100	83 ± 1.73
	200	92 ± 0.49**
Standard 5-FU	5	100 ± 1.49***

Results are expressed in Mean ± SEM. One way ANOVA with *P<0.05, **P<0.01, ***P<0.001 by using Tukey-kramer multiple comparison test

Figure 1. Chromatogram representing Cytotoxic activity of C.B extract by tryphan blue dye exclusion test on Vero cell lines

Table 2. Percentage of dead DAL cells with C.B extract and standard 5-FU by tryphan blue dye exclusion assay

Groups	Drug concentrations in µg/ml	Percentage (%) of dead DAL cells
Control	-	0
C.B extract	10	17 ± 0.32
	25	24 ± 0.94
	50	43 ± 1.21
	100	61 ± 1.63
	200	76 ± 0.47**
Standard 5-FU	5	94± 1.35***

Results are expressed in Mean ± SEM. One way ANOVA with *P<0.05, **P<0.01, ***P<0.001 by using Tukey-kramer multiple comparison test.

Figure 2. Chromatogram representing Cytotoxic activity of C.B extract by tryphan blue dye exclusion test on DAL cell lines

Table 3. Percentage of dead Hep-2 cells with C.B extract and standard 5-FU by tryphan blue dye exclusion assay

Groups	Drug concentrations in µg/ml	Percentage (%) of dead Hep-2 cells
Control	-	0
C.B extract	10	8± 0.15
	25	24 ± 1.36
	50	32 ± 0.11
	100	63 ± 1.82
	200	82± 0.36**
Standard 5-FU	5	99± 0.21***

Results are expressed in Mean ± SEM. One way ANOVA with *P<0.05, **P<0.01, ***P<0.001 by using Tukey-kramer multiple comparison test.

Figure 3. Chromatogram representing Cytotoxic activity of C.B extract by tryphan blue dye exclusion test on Hep-2 cell lines

Figure 4. Percentage viability of Vero cells in Micro-Plate Reader (MTT assay)

Figure 5. Percentage viability of DAL cells in Micro-Plate Reader (MTT assay)

CONCLUSION:

Phytochemical screening of the methanolic extract of leaves of Chloris barbata has confirmed the presence of alkaloids, carbohydrates, flavonoids, triterpinoids, glycosides and saponins. Literature survey reveals that the plant leaves were used traditionally in the treatment of cancer. Widely accepted in vitro cytotoxic models are used for anticancer studies such as Tryphan blue dye assay, MTT assay, NRU and sulphorhodamine assay with DAL, Vero and Hep-2 cell lines. The results indicate that the methanolic extract of Chloris barbata leaves has significant cytotoxic potentials with increase in percentage of dead cell lines, decrease in percentage viability of cells and percentage growth inhibition of cell lines. The results indicate that the leaf extract of Chloris barbata has cytotoxic potentials. Hence it can be hypothesized that a wide variety of active constituents present in the leaf extract may be responsible for in vitro anticancer activity.

CONFLICT OF INTEREST:

This is to inform that the authors declare that they have no conflicts of interest regarding this article.

ACKNOWLEDGEMENT:

The authors are grateful to the management of A.K.R.G. College of Pharmacy Nallajerla, W.G. District and National Cancer Institute, Pune for providing facilities to carry out the present research.

REFERENCES:

1. Sheetal Verma and S.P. Singh. Current and future status of herbal medicines. Veterinary World, vol.1 (11), November 2008: 347-350.

2. Venugopal R, Liu RH (2012). Phytochemicals in diets for breast cancer prevention. The importance of resveratrol and ursolic acid. Food Sci Hum Wellness 1:1-13.

3. Rasika D Bhalke, Mahendra A Giri, Subodh C Pal. Antiulcer Activity of Methanol Extract of Leaves of Bauhinia racemosa Linn (Leguminaceae). Research J. Pharm. and Tech. 4 (1): January 2011; Page 124-127.

4. Mukesh Kumar Singh, A. Prathapan, Kushagra Nagori, S. Ishwarya, K.G Raghu. Cytotoxic and Antimicrobial Activity of Methanolic Extract of Boerhaavia diffusa L. Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1061-1063.

5. Rohan Sharadanand Phatak, Anup Subhash Hendre, Pushpa Prakash Durgawale. Phytochemical Composition of Methanolic Extract of Phyllanthus acidus L (Skeels) Fresh Leaves by GC/MS Analysis. Research J. Pharm. and Tech. 2016; 9(5): 559-561

6. Durga Devi M , Banu N. Anti-Proliferative activity of Chlorophyllin from Phyllanthus emblica L. against MCF-7 and Vero Cell line. Research J. Pharm. and Tech. 2017; 10(2): 516-524

7. A. Tamil Selvan, R. Suthakaran, B. Karun Kumar, R. Naveen Kumar, M. Naveena Mala, D. Jai Ram, M. Praveen. Pharmacognostical and Pharmacological Evaluation of the Polyherbal Extract on Rodents. Res. J. Pharmacognosy and Phytochem. 2014; 6(2): 80-83.

8. Yeshwante SB, Juvekar AR, Pimprikar RB, Kakade RT, Tabrej M, Kale MK, Firke SD. Anti-Diarrheal Activity of Methanolic and Aqueous Extracts of Dillenia indica L. Research J. Pharmacology and Pharmacodynamics. 2009; 1(3):140-142.

9. Sandhya Mittal, Nidhi Rao, Sudhanshu, Ekta Menghani. Phytochemical and antimicrobial activity of Tectona grandis L. Research J. Science and Tech. 2012; 4(5): 188-191

10. Seema V. Pattewar, Deepak N. Patil. Formulation of herbal antibacterial cream by using extract from Kalanchoe pinnata leaves. Research J. Topical and Cosmetic Sci. 5(1):Jan.–June 2014 page 1-4

11. Sarav A. Desai, Prakash S. Sukhramani, Maulik P. Suthar, Vipul P. Patel. Biological Cytotoxicity Evaluation of Sulfonamide Derivatives as Anti-Lung and Anti-Breast Cancer Activity. Asian J. Research Chem. 4(4): April, 2011; Page 671-677.

12. P. Kamleshiya, V.G. Meshram, A.H. Ansari. The in-vitro Bacteriostatic Potential of Some Traditionally used Indian Spices. Asian J. Research Chem. 5(4): April 2012; Page 492-496.

13. Cheyma Bensaci, Zineb Ghiaba, Messaouda Dakmouche, Mokhtar Saidi, Mohamed Hadjadj, Assia Belfar, Mahdi Belguidoum. Studies on Antibacterial Compounds from Methanolic Extracts of Date Palm (Phoenix dactylifera L.) fruits from Algeria. Asian J. Research Chem. 2019; 12(6):338-340.

Received on 09.10.2019 Modified on 11.12.2019

Research J. Pharm. and Tech. 2020; 13(7): 3227-3231.

DOI: 10.5958/0974-360X.2020.00571.5