Determination of in vitro free radical scavenging and antiproliferative effect of Pennisetum alopecuroides on cultured A549 human lung cancer cells.

CONTEXT: Pennisetum alopecuroides (Poaceae) is a grass predominantly distributed in tropics and sub tropics. It is used as a cattle feed in many regions. AIM: The objective of the present study was to investigate the in vitro free radical scavenging and antiproliferative activity of ethanol extract of P. alopecuroides (EEPA) on cultured A549 human lung cancer cell lines. SETTINGS AND
DESIGN: The anti-oxidant activity of ethanol extract was evaluated at dose level 12.5, 25, 50, 100, and 200 μg/ml. The in vitro antiproliferative activity was measured at doses of 10, 50, and 100 μg/ml.
MATERIALS AND METHODS: The free radical scavenging activity of the EEPA was determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method and in vitro antiproliferative activity on A549 human lung cancer cells was conducted by using MTT assay method.
RESULTS: The phytochemical screening revealed that the P. alopecuroides contained alkaloids, tannins, saponins, and flavonoids as the major secondary metabolites. The IC50 value of DPPH scavenging activity was found to be 44.41 μg/ml and 31.02 μg/ml  for a mixture of EEPA and standard ascorbic acid, respectively. In vitro MTT assay showed that EEPA had anti-proliferation effects on A549 cells in a dose dependent manner.
CONCLUSIONS: This is the 1(st) time a pharmacological exploration of P. alopecuroides grasses has been conducted. We have shown that P. alopecuroides exhibits good free radical scavenging and strong in vitro cytotoxic activities against human lung cancer cell lines.

INTRODUCTION

Cancer is a disease of cells characterized by the loss of normal cellular growth maturation and multiplication which in many cases eventually causes death of the host. Chemotherapy is an effective treatment against various types of cancer either singly or in combination with surgery or radiotherapy. Recently, it has become possible to exploit  the basic information of chemotherapy to develop mechanism based strategies for cancer prevention and treatment.[1] As for cancer protection, it has been estimated that diets rich in phytochemicals can significantly reduce cancer risk by as much as 20%.[2] The chemopreventive properties of plant secondary metabolites are either due to anti-lipid peroxidative action, modulating carcinogen detoxification or by improving the anti-oxidant defense system.[3] Plant extracts generally have a capability to scavenge free radicals thus making them extremely effective anti-oxidants. This anti-oxidant activity is most often due to flavonoids, phenolic acids, phenolic diterpenes, and volatile oils.[4] It is well known that utilization of synthetic anti-oxidants may be associated with a number of side effects. Therefore, the need to find natural and safe sources of anti-oxidants has increased.[5]

The free radicals generated in the cells, upon accumulation, react with macromolecules like lipids, proteins and nucleic acids, and damage cellular functions. Moreover, oxidative damage to the cellular components results in alteration of the membrane properties such as fluidity, ion transport, enzyme activities, and protein cross-linking, eventually resulting in cell death.[6] Phytochemicals containing anti-oxidant properties showed capacity to inhibit carcinogenesis.[78] Pennisetum alopecuroides (Poaceae) is a grass predominantly distributed in tropics and sub tropics and used extensively in cattle feed. It is also known as an ornamental grass often used for its radiating, fine-textured effect in the landscape. They usually remain attractive well into the winter, the leaves turning bright golden-yellow in the fall. This genus is predominantly distributed in tropics and sub tropics and used extensively in cattle feed. It has been noted that it's another species, Pennisetum spicatum has been traditionally used in treating heart diseases, as aphrodisiac in women, tonic, and as an appetizer.[9] Previously our research group synthesized a series of novel 5-[(2E)-1- (1H-benzimidazol-2-yl)-3-substituted phenylprop-2-en-1-ylidene] pyrimidine-2, 4, 6 (1H, 3H, 5H)-triones and evaluated their antitumor activity against Dalton's ascitic lymphoma in mice.[10] A careful survey of the literature revealed that no study of chemopreventive activity has so far been conducted on this plant. The present paper deals with the evaluation of in vitro anti-oxidant and antiproliferative activity of ethanol extract of P. alopecuroides (EEPA).

MATERIALS AND METHODS

Collection and authentication of plant

The whole plant P. alopecuroides were collected in June 2013 from Palakkad district, Kerala, and authenticated by Dr. K. R. Leena, the botanist from Department of Botany, Government Victoria College, Palakkad, Kerala, India.

Preparation of ethanol extract and determination of secondary metabolites

The plant material was shade dried, powdered, and extracted with ethanol in a Soxhlet extractor for 70–72 h. The extract was concentrated to dryness in a rotary evaporator under reduced pressure and controlled temperature. Qualitative phytochemical analysis of the EEPA was conducted as described by Farnsworth.[11]

IN VITRO ANTI-OXIDANT ASSAY

Different volumes (1.25–10 μl) of EEPA were made up to 40 μl with dimethyl sulfoxide (DMSO) and 2.96 ml 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (0.1 mM) solution was added. The reaction mixture was incubated in dark condition at room temperature for 20 min. After 20 min, the absorbance of the mixture was read at 517 nm. 3 ml of DPPH was taken as control. The decrease in absorbance was monitored at 0 min, 1 min, 2 min, and every 15 min subsequently until the reaction reached a plateau. The time taken to reach the steady state was determined by one-way analysis of variance. The DPPH free radical scavenging activity, expressed as percentage of radical scavenging activity and measured as follows:[121314]

Maintenance of cell line

A549 lung cancer cell lines were purchased from National Centre for Cell Science Pune was maintained in Dulbecco's Modified Eagles Media (HiMedia) supplemented with 10% fetal bovine serum (invitrogen) and grown to confluency at 37° in 5% CO2 (NBS, Eppendorf, Germany) in a humidified atmosphere in a CO2 incubator. The cells were trypsinized with a composition of 100 μl of 0.025% trypsin in phosphate buffer saline (PBS)/0.5 mM EDTA solution (HiMedia) for 2 min and passaged to 24 well plates in aseptic conditions. Extracts were added to grown cells at a concentration of 10 μg, 50 μg and 100 μg from a stock of 10 mg/ml and incubated for 24 h. The percentage difference in viability was determined by standard MTT assay after 24 h of incubation.

In vitro antiproliferative activity

Antiproliferative activity of EEPA (dissolved in DMSO) was tested in cultured A549 human lung cancer cell lines, using the MTT assay as described earlier.[15] The cell culture suspension was washed with 1X PBS and then 30 μl of MTT solution was added to the culture (MTT - 5 mg/ml dissolved in PBS). It was then incubated at 37°C for 3 h. MTT was removed by washing with 1X PBS and 200 μl of DMSO was added to the culture. Incubation was done at room temperature for 30 min until the cells got lysed and color was obtained. The solution was transferred to centrifuge tubes and centrifuged at 13,000 rpm for 2 min to precipitate cell debris. Optical density (OD) was read at 540 nm using DMSO as blank.[16]

% viability = (OD of test/OD of control) × 100

RESULTS AND DISCUSSION

The phytochemical screening test showed that EEPA contained several potentially active secondary metabolites [Table 1]. The presence of flavonoidal content in the EEPA aimed to evaluate the anti-oxidant and cytotoxic properties of the plant. Majority of the phenolic class of flavonoids exhibited their anticancer property by free radical scavenging ability. The anti-oxidant activity of the EEPA was determined by DPPH method. Anti-oxidants react with DPPH and reduce it to DPPH-H and as consequence the absorbance decreases. The degree of discoloration indicates the scavenging potential of the anti-oxidant compounds or extracts in terms of hydrogen donating ability. The anti-oxidant efficiency of EEPA is shown in Table 2. The IC50 value of DPPH scavenging was found to be 44.41 μg/ml and 31.02 μg/ml of EEPA and standard ascorbic acid, respectively. In vitro antiproliferative activity on A549 human lung cancer cell was undertaken using MTT assay method which showed that EEPA had obvious anti-proliferation effects on A549 cells in a dose dependent manner [Table 3]. The sample showed considerable toxicity only at higher concentration and IC50 values will be approximately 100 μg/ml. Morphological analysis showed slight variation from untreated control. Morphological changes in A549 human lung cancer cells were observed under an inverted microscope. Cells were treated with various concentrations of EEPA (10, 50, and 100 μg/ml) and examined as compared to control group. The untreated cells served as the negative control. The morphological changes of the cells were observed under the normal inverted microscope 48 h after-treatment.[17] Treated group showed significant decrease in adherent cells which accompanied an increase in floating cells in culture medium. This study also showed that the EEPA treated A549 cells acquired a round and shrunken shape significantly in contrast to normal polyclonal structure of normal cells [Figure 1].

Table 1

Phytochemicals of Pennisetu alopecuroides

Table 2

Free radical scavenging effect of EEPA compared with the standard ascorbic acid

Table 3

Cytotoxic activity of EEPA

Figure 1

Effects of ethanol extract of Pennisetum alopecuroides on morphological characteristics of A549 cells

Lung cancer is one of the leading cause of cancer deaths throughout the world. Approximately, 80–85% of all lung cancers are classified as nonsmall cell lung cancer. Current therapies includes active surveillance, surgery, radiation therapy, chemotherapy, and immunotherapy. Chemotherapy still plays an important role in this setting, however, these drugs are highly toxic with a low survival profile.[18] Herbal medicines have been proven to be a major source of novel agents with various pharmaceutical applications. Natural products have provided a rich source of secondary metabolites to treat many diseases including cancer.[1920]

In the present investigation, EEPA recorded a consider amount of flavonoid and alkaloidal content. DPPH is a stable free radical that possesses a characteristic absorption maximum at 517 nm, which is diminished in the presence of an anti-oxidant compound capable of reducing it to its hydrazine form by hydrogen/electron donation.[21] The strong scavenging capacity of EEPA on DPPH might possibly be due to the phenolic nature of flavonoid content which could act as a hydrogen donor.[22] The cytotoxicity of EEPA can be related to the anti-oxidant activity and synergistic effect of multiple components in the extract.

CONCLUSION

This is the first attempt of the pharmacological exploration of P. alopecuroides. The crude EEPA was found to exert a strong in vitro cytotoxic activity against human lung cancer cell lines and certainly merits further study to isolate the potentially promising bioactive components. Further studies are needed to identify the unknown anti-oxidant and cytotoxic secondary metabolites present in the plant material to establish their pharmacological properties.