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RESEARCH ARTICLE

 

Isolation of Flavonoids and their Anticancer Activity from Sphaeranthus amaranthoides in A549 Cell Line

 

Swarnalatha. Y*

Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Chennai-119.

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

 

ABSTRACT:

Flavonoids are considered as potential explore to treat cancer and they are ubiquitous. The chemical structure and the nature of the polyphenolic flavonoids are responsible for their anticancer actions. Objective: The present investigation concentrates on the cytotoxic effect of flavonoids in A549 adenocarcinoma cell lines. Methodology: the plant extract was  subjected to TLC for confirmation and HPLC confirm the pure form of the flavonoids and the induction of the cytotoxicity was studied using AO/EB, PI and the uptake of the PI was confirmed using the FACS in A549 cells. results: the TLC and HPLC results confirmed the presence of flavonoides. Flow cytometry denotes the percentage of the cells in the G0/G1 and the G2 stage. This indicates the accumulation of the cells in the apoptotic and proapoptotic stages. Conclusion: These findings indicate that the flavonoids from the Sphaeranthus amarathoides may be promising anticancer agents to control the cell growth in A549 cells.  

 

KEYWORDS: Flavonoids; cancer; FACS; A549; Sphaeranthus amaranthoides

 


INTRODUCTION:

Herbal plants are rich in many pharmacologically active compounds like flavonoids, saponins, alkaloids. Flavonoids naturally exist in almost all the plant kingdom. Flavonoids can be found in almost all dietary plants, like fruits and vegetables. Flavonoids were also present in many medicinal plants, and herbal remedies consists flavonoids have been used in folk medicine throughout the world. Flavonoids from Sphaeranthus amaranthoides may be usefull chemopreventive agents for cancer. The polyphenolic compounds from Sphaeranthus amaranthoides shows a remarkable broad spectrum of the action on different biological activities, for example, antianalgesic and anti-inflammatory [1], wound healing [2], antimicrobial activity.

 

 

 

 

 

 

 

 

 

 

Received on 02.01.2015       Modified on 09.01.2015

Accepted on 20.01.2015      © RJPT All right reserved

Research J. Pharm. and Tech. 8(4): April, 2015; Page 462-467

DOI: 10.5958/0974-360X.2015.00077.3

 

The plant Sphaeranthus amaranthoides is a weed in the paddy field of Southern India, particularly in Tanjore, Thirunelveli, Southern Mysore and Travancore area[3],[4]. In folk medicine, it has been proved that the activity of the plant is due to the presence of the flavonoids. In this context the current study concentrates on the cytotoxic activity of flavonoids on the lung cancer cell lines. Lung cancer is one of the major prevalent life-threatening human cancers and increasing in terms of morbidity and mortality.   The current medication is with synthetic drugs leads to drastic side effects. The natural drugs play an essential role to reduce the side effects on the normal cells and increase the cytotoxicity of the tumor cells.

 

Chemicals and Reagents:

Quercitin was used as standard and was obtained from Sigma Aldrich. Methanol and acetonitrile was purchased of analytical grade. TLC plates of silica gel 60 which are not on fluorescent indicator were purchased, and the rest of the chemicals used for the anticancer activity were of kit based.

 

 

METHODOLOGY:

Collection of Leaf material: The leaf material was obtained from Tirunalveli district and it was authenticated by V. Chelladurai, Rtd Botanist. The collected leaf material was shade dried and powdered into coarse and preserved for further use.  Sample preparation for HPLC: 2.0g of Sphaeranthus amaranthoides dried leaf powder was taken and subjected to maceration agitation with 10mL of EtOH/ H2O (1:1 v/v).  The hydroethanolic extracts were filtered, and their final volume was adjusted to 20ml with EtOH/ H2O (1:1 v/v). The hydroethanolic extracts were simply filtered through a Millipore membrane with 0.5µm. For HPTLC and HPLC analysis, the extracts were subjected to liquid-liquid extraction using 5mL of CHCl3; the hydroethanolic layer was filtered through 0.5 µm Millipore membrane for the preparation of the sample for HPLC analysis.

 

HPLC-UV-Pad analysis: the analysis was performed in a Shimadzu Prominence_UFLC XR with Phenomenex Gemini NX C18 column (150×4.6mm, 5µ) injection volume 10 µL wit flow rate 0.5 ml/min. Mobile phase A is 0.01M Ammonium Formate in water and Mobile phase B is Acetonitrile. The column oven was thermostat controlled at 35oC and the detection was monitored at 254 and 350nm.  Thin layer chromatography: The hydro ethanolic extract was used for analyses,  TLC was carried out on silica gel 60 aluminium sheets which are precoated with wit EtOAc/HCOOH/H2O (6:1:1 v/v). The solvent should be dried after developing plots and the flavonoids were visualized under UV at 360 NM [5]. TLC a: TLC was performed according to the Brasseur and Angenot et al 1996. The TLC plates were visualized at 254 NM with the suitable spray reagent.

 

Cell Preparation and Culturing:

A549 lung adenocarcinoma cell line was procured from National Centre for Cell Science (NCCS), Pune, India with the passage number 11. Cells were maintained in Dulbecco’s Minimum Essential Media (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), with 100units/mL penicillin and 100μg/mL streptomycin. Cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C. Cells were grown in 75cm2 culture flask and after a few passages, cells were seeded for experiments. The experiments were done at 70 to 80% confluence. Upon reaching confluence, cells were detached using 0.25% Trypsin-EDTA solution.

 

MTT Assay:

According to Safadi et al [5] the proliferation of A549 cells was assessed by MTT assay. The proliferation test is based on the color reaction of mitochondrial dehydrogenase in living cells by MTT. Cells were plated in 96-well plate at a concentration of 5 × 104 cells/well 24 h after plating. After 24h of cell incubation, the medium was replaced with 100µl medium containing flavonoid fraction at different concentrations (5 – 640µg/ well) and incubated for 24h. Untreated cells served as controls and received only 0.1% DMSO in which the fraction was prepared. At the end of treatment period, media from control and flavonoid-treated cells was discarded and 20μl of MTT (5mg/ml PBS) was added to each well. Cells were then incubated for 4h at 37°C in CO2 incubator. MTT was then discarded and the colored crystals of producing formazan were dissolved in 200μl of DMSO and mixed effectively by pipetting up and down. Spectrophotometrical absorbance of the purple blue formazan dye was measured using an ELISA reader (BIORAD) at 570nm. The optical density of each sample was compared with control optical density and graphs were plotted.

 

Ethidium Bromide/Acridine Orange (Dual Staining):

Ethidium bromide/acridine orange staining was carried out by the method of Gohel et al[6]. A549 cells were plated at a density of 5×104 in 48-well plates. They were allowed to grow at 37°C in a humidified CO2 incubator until they were 70–80% confluent. Then cells were treated with 40µg/ml and 80µg/ml (selected based on the IC50 concentration) of the flavonoid fraction for 24h. The culture medium was aspirated from each well and the cells were gently rinsed twice with PBS at room temperature. Then equal volumes of cells from control and drug treated were mixed with 100μl of dye mixture (1:1) of ethidium bromide and acridine orange) and viewed immediately under Nikon inverted fluorescence microscope (Ti series) at 10x magnification. A minimum of 300 cells was counted in each sample at two different fields. The percentage of apoptotic cells was determined by [% of apoptotic cells = (total number of apoptotic cells/total number of cells counted) ×100].

 

Assessment of Nuclear Morphology after Propidium Iodide Staining:

Propidium iodide staining was performed according to the method of Chandramohan et al[7]. PI was used to quantify the apoptotic cells.  A549 cells were plated at a density of 5 × 104 in 48-well plates. They were allowed to grow at 37°C in a humidified CO2 incubator until they are 70–80% confluent. Then cells were treated with 40µg/ml and 80µg/ml of flavonoid for 24 hrs. The culture medium was aspirated from each well and the cells were gently rinsed twice with PBS at room temperature, before fixing in methanol: acetic acid (3:1 v/v) for 10 min, and stained with 50μg/ml Propidium iodide for 20min before flowcytometry analysis. Nuclear morphology of apoptotic cells with condensed/ fragmented nuclei was examined by fluorescence microscopy and at least 1 ×103cells were counted for assessing apoptotic cell death.

 

 

Flow Cytometry:

The effect of flavonoid fraction on the cell cycle distribution was assessed according to Tu et al [8].  A549 cells (1×105 cells/ml) were treated with 40µg/ml and 80µg/ml cultured for 24h. The treated cells were harvested, washed with phosphate-buffer saline (PBS) and fixed in 75% ethanol at 4C overnight. After washing twice with cold PBS, cells were suspended in PBS containing 40μg/ml propidium iodide (PI) and 0.1mg/ml RNase A followed by shaking at37C for 30min. The stained cells were analyzed with a flow cytometer (Becton-Dickinson, San Jose, CA, USA) and the data were consequently calculated using WinMDI 2.9 software (TSRI, La Jolla, CA, USA).

 

Statistical Analysis:

Data were expressed as mean ± S.E.M and analyzed by Tukey’s test to determine the significance of differences between groups. A p value lower than 0.05, 0.01 or/and 0.001 were considered to be significant.

 

RESULTS AND DISCUSSION:

Sample preparation and optimization of HPLC The flavonoids were isolated using hydro-ethanolic separation and the isolated flavonoids subjected to HPLC (fig.1) then to TLC. The extract appeared in two spots with below Rf values to the quercetin standards in TLC. The orange fluorescent spot indicated the presence of quercetin derivatives [9].

 

The hydro-ethanolic extract is highly efficient in the separation of flavonoids from Sphaeranthus amaranthoides. The compound showed with the 9.67 retention time and with 94% of the area will indicate the purity of the flavonoid (fig 2). Acetonitrile gives better results than using methanol and was therefore selected as the organic solvent in the optimized HPLC conditions for the extracts of the Sphaeranthus amaranthoides [10].

 

Fig 1. TLC of standard and plant extract showing the Rf value 0.7

 

The chromatogram of the Sphaeranthus amaranthoides was showed 8 peaks in that one major peak with 94.78% of flavonoids and retention time lesser than the quercetin at 254-350 NM. These isolated and identified flavonoids were subjected to the anticancer activity in the A549 adenocarcinoma lung cancer cell lines.

 

Anticancer activity of flavonoids:

Inhibition of growth and proliferation of human A549 lung adenocarcinoma cells .The growth inhibitory effect of the flavonoids against A549 lung adenocarcinoma cells was assessed by MTT assay. The treatment of A549 cells with the drug (5 - 640µg) resulted in significant reduction in cell growth ranging from 9.13±0.004 to 71.02±0.002 after 24h. A dose dependent inhibition in the cell growth was observed (Fig 3) with an IC50value of 78.19µg.


 

Fig2. HPLC – chromatogram for flavonoids with retention time

 


Fig 3: Cytotoxic effect of the flavonoid fraction treatment on A549 lung adenocarcinoma cells - % of the inhibition against the concentration.

 

Chemotherapeutic drugs are considered to kill tumor cells (Fig 4) by activating a cascade of events resulting in apoptosis. In agreement with this line of thought, we provided evidence that flavonoids from Sphaeranthus amaranthoides were able to induce apoptosis with the classic feature of apoptosis, including initiator and effector caspase activation and inter-nucleosomal DNA fragmentation.

 

 

Control 0.1% DMSO

20µg/ml

80µg/ml

320µg/ml

Fig  4: Morphological changes induced by different concentrations of Flavonoids

 

Dual Staining Assay:

The type of the cell death can be assessed using dual staining with AO/EB. In this, the morphological changes after double staining with Acridine Orange/Ethidium Bromide (AO/EB) were investigated. AO/EB staining uses a combination of two dyes to visualize cells with aberrant chromatin organization. AO penetrates into living cells, emitting green fluorescence after intercalation into DNA, but it cannot distinguish viable from non-viable cells. To achieve this, a mixture of Acridine Orange and Ethidium Bromide was used. EB can uptake by non living cells so the differential uptake of these two dyes allows the identification of viable and non-viable cells, where the second day, EB emits red fluorescence in the cells with an altered cell membrane (Fig 5).

 

Control

 

40µg/ml

 

80µg/ml

Fig 5: shows induction of apoptosis by the drug in A549

 

Cells:

Control group the green colored cells are live cells, in 40µg/ml showed an intense green colored cells which are early apoptotic cells and late apoptotic cell number are less. In 80µg/ml the cells are more in red and orange color which is in late apoptotic cells but the early apoptotic cells are less.

 

Table 1. Dual Staining assay of flavonoid fraction:

Treatment groups

% No of apoptotic cells

Control

9.83±2.42

40µg/ml

32.15±2.17

80µg/ml

62.83±1.74

 

Fig 6: Bar graph showing the percentage number of apoptotic cells

 

Viable cells with intact DNA and nucleus gave green fluorescence. Early apoptotic cells had fragmented DNA, which exhibited intense green colored nuclei. Late apoptotic and necrotic cell’s DNA was fragmented and stained orange and red. Besides, some cells exhibited typical characteristics of apoptotic cells like plasma membrane blebbing. From the data it was clear that with increasing concentration of drug, the number of viable cells decreased tremendously. The percentage of apoptotic cells after treatment with 40µg and 80µg/ml of the drug was drastically increased (p< 0.001) to 32% and 62% respectively (Fig 6).

 

Propidium Iodide – nuclear fragmentation assay:

Apoptosis was further confirmed by analyzing the nuclear morphology of flavonoids treated A549 cells. Nuclear morphology was evaluated with membrane-permeable PI stain (Fig 7). The treated cells contained a number of apoptotic cells when compared to untreated control cells. There was characteristic nuclear fragmentation of nuclei in treating A549 cells, whereas the untreated control cells did not show any nuclear fragmentation. The apoptotic cells displayed characteristic features of reduced size, intense fluorescence of condensed nuclear chromatin and formation of membrane blebs. The percentage of apoptotic nuclei after treatment with 40μg/ml and 80μg/ml of the drug increased enormously (p<0.001) to 41% and 62%, respectively (Fig 7).

 

Fig 7 Showing the percentage of apoptotic nuclei

 

Control

40µg/ml

80µg/ml

Fig 8: shows the nuclear localization of A549 cells by Propidium iodide staining the cells showed a characteristic reduction of size with the nuclear fragmentation.

Quantification of the cell death was estimated using PI staining [11]. The A549 cells have been selected for the distribution of the cells into three major phases of the cell cycle and make it possible to detect the apoptotic cells with DNA traces when treated with the flavonoids from Sphaernathus amaranthoides are being processed through the FACS for the viability of the cells when tagged through the Propidium iodide stain with the extract of concentration of about 40 µg/ml and 80 µg/ml. Tremendously, there was a gradual increase in the PI uptake with an increase in the concentration of the flavonoids. This explains that, with respective to the concentration of the flavonoids, the number of cells accumulated G0/G1 was increased. The S phase showed rapid accumulation of cells at the 40 µg/ml and at this concentration in G0/G1 phase, there is a linear decrease in the accumulation of the cells. However, the maximum accumulation of the cells at 80 µg/ml was observed in G1 phase. Hence, the above result proves that the increasing in the concentration of the flavonoids increases the anticancer activity this was calculated through the FACS in the analysis of the cytometer (Fig 8).

 

CONCLUSION:

Result in this study concludes that Flavonoids from Sphaeranthus amarnathoides possess anticancer activities and may contribute in the therapeutic effect of this medicinal herb. However, there is a need of detailed scientific study on traditional medical practices to ensure that valuable therapeutic knowledge of the plant is preserved and also to provide scientific evidence for its efficacies.

 

ACKNOWLEDGEMENT:

The authors thank Sathyabama University for the kind support and infrastructure.

 

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