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Antioxidant Activity and Anticancer Study on Phytochemicals Extract from Tubers of Gloriosa superba against Human Cancer Cell (Hep-G2)

Simon SE1* and Jayakumar FA2

1Department of Biotechnology, Bharathidasan University, India

2Department of Biotechnology, Malaysia University of Science and Technology, Malaysia

*Corresponding Author:
Samson Eugin Simon
Department of Biotechnology
Bharathidasan University, India
E-mail: samson191847@gmail.com

Received date: 20/06/2016; Accepted date: 27/09/2016 Published date: 07/10/2016

Visit for more related articles at Research & Reviews: Journal of Pharmacognosy and Phytochemistry

Abstract

Gloriosa superba is an alkaloid plant containing a large amount of alkaloid components like colchicine and gloriosine. This study involves anti-cancer examination on Hep-G2 cells (human liver cancer cells) using phytochemical extract obtained from G. superba tuber. G. superba tubers were identified and collected. The collected sample is shade dried and subjected to pulverisation. The powdered sample is followed for the solvent extraction using soxhlet apparatus for 23 cycles for 48 h in the corresponding temperature of the solvent used. The solvents which are used for the extractions are methanol, water and petroleum ether. Phytochemical qualitative analysis was done. The quantitative analysis was followed to the specific phytochemicals such as saponins and alkaloids which have the anti-cancer properties mentioned from earlier literature studies. The analysed solvents are subjected to anti-oxidant activity by DPPH assay. From each solvent the methanolic extract hold the higher value of anti-oxidant property. The solvent which has the highest antioxidant property were preceded to the anti-cancer test against Hep-G2 cells (human liver cancer cells) by MTT assay. The cell death percentage of Hep-G2 cells were calculated from cell viability obtained by MTT assay. Triplicate values were obtained. From the triplicate value the mean value was determined to obtain the average value for each concentration 5 μg, 10 μg, 25 μg, 50 μg, 100 μg. The higher concentration of 100 μg has the lower viable rate and 50% inhibition of viability (IC50) was determined graphically. Death rate of cells indicated the cancer inhibition rate which is due to Hep-G2 cells failed to retain the viability. Inhibition rate of 100 μg has the higher inhibition range of 54.3%. Death rate of Hep-G2 cells may be due to various reasons like protein binding, DNA replication interaction, receptor binding inhibitors, etc. this current study shows the G. superba tubers has the potential to inhibit the growth of Hep-G2 cell.

Keywords

Antioxidant, Alkaloid, Cytotoxicity, Cancer, Phytochemicals

Introduction

Plants are natural sources of bioactive compounds to treat life threatening diseases such as heart diseases and cancer [1]. Gloriosa superba belongs to Liliaceae and is a well-known species of perennial herb. It is also called as “glory lily”. This plant G. superba Linn has various phytochemicals, mostly alkaloids like colchicine, gloriosine, colchine, etc. which means that it can be used for treating cancer [2,3]. Seventy-five percentages of the raw materials can be used in this plant; so far the parts of the plants such as tubers, leaves, seeds and flowers are used. These parts of the plants are used in Ayurveda and Yunani as a reputed medicine [4]. G. superba plant has various medicinal properties and each part of the plant is used to treat diseases [5]. Plants have anti-oxidant molecules in form of phytochemicals that protect our cells from damage caused by free radicals [6]. G. superba contains numerous phytochemicals like alkaloids, glycosides, flavonoids and saponins, which can serve as anti-oxidant and it may reduce the risk of cancer and improve heart health [7]. Cancer is a group of diseases involving abnormal cell growth with the potential of spreading to other parts of the body by invading cells [8]. Hep-G2 is an immortal cell line which is consists of human liver carcinoma cells. They can secret many plasminogen proteins; stimulated by human growth hormone [9]. Cancer is a deadly disease; more than 100 types of cancer affects humans. Treating cancer is so painful process as they involve chemotherapy and radio therapies. Phytochemicals does many works in our body. Based upon their work it can also be anti-cancerous agent for various types of cancer. Plant source for treating cancer may reduce painful outcomes unlike from chemotherapy [1]. The Phytochemicals from G. superba can act as anti-oxidant and anticancer by hormonal action, enzymes stimulators, physical action (contact within the cells) and interference with DNA replication.

Methodology

Collection of the Plant Material

G. superba tubers were collected from cultivation field around Ariyalur and Perambalur district of Tamil Nadu. About 5 kg of tubers were collected and authenticated by Head of the Department, Meenakshi Ramasamy Arts and Science College, Tamil Nadu, India. Then tubers were pulverized obtaining 1.4 kg of sample powder, the extraction was done using soxhlet apparatus in Greens Med Lab, Chennai.

Preparation of Plant Extract

The air dried plant material was cut into small pieces and dried in shade for two days till it completely dried. Then it was pulverised into fine powder. The 500 g of powdered materials were packed in soxhlet apparatus and successive extraction was performed using petroleum ether, methanol and water solvents. The solution of the extract was filtered through Whattman filter paper no. 1 and concentrated using rotary flash evaporator and dried under vacuum.

Phytochemical Analysis

Phytochemical screening was performed to assess the qualitative chemical composition of different samples of crude extract using commonly employed precipitation and coloration reactions to identify the major secondary metabolites like alkaloids, flavonoids, glycosides, Proteins, phenolic compounds, saponins, starch, steroids, tannins and terpenoids.

The phytochemical analyses were carried out using standard procedures. The extracts of G. superba were screened for the presence of secondary metabolites using the procedures. The observations were recorded for total starch, soluble protein, steroids using steroid test, flavonoids and tannins using shinoda test, alkaloids by Dragendroff’s test, proteins and glycosides by Limbermann’s tests, saponins using forth test, total phenol by ferric chloride test and reducing sugar using Benedict’s test.

Quantitative Analysis

Quantification of the alkaloids and saponins can help to understand the test for antioxidant and anti-cancer. Quantitative analysis was done for alkaloids and saponins in an applied standard procedure.

Anti-Scavenging Activity

The free radical scavenging activity of the solvent extracts was determined by 1,1-diphenyl-2-picryl-hydrazil (DPPH). Antioxidant activity was measured. To a fresh tube add 3 ml of methanol which act as a blank. To the second tube add 1 ml of methanol and 3 ml of 0.1 M DPPH. This serves as the control. To the other tubes add respective volume of the sample and make up to 1 ml with Methanol and finally add 3 ml of 0.1 mM DPPH then vortex. Seal the tube with aluminium foil and incubate all the tubes in dark for 30 min at room temperature. Read the absorbance at 517 nm. The capability to scavenge the DPPH radical was calculated using the following equation:

DPPH scavenging effect (%)=[(ABS control–ABS sample)/(ABS control)] × 100

where ABS control is absorbance of negative control and ABS sample is the absorbance of the reaction mixture containing the sample extract.

In vitro Assay for Cytotoxicity Activity (MTT Assay)

The Cytotoxicity of samples on Hep-G2 cells were determined by MTT assay. Cells (1 × 105/well) were plated in 1 ml of medium/well in 24-well plates (Costar Corning, Rochester, NY). After 48 h incubation, the cell reaches the confluence. Then, the cells were incubated in the presence of various concentrations of the methanol extract in 0.1% DMSO for 48 h at 37°C. After removal of the sample solution and washing with phosphate buffered saline (pH 7.4), 200 μl/well (5 mg/ml) of 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl--tetrazolium bromide cells (MTT) phosphate- buffered saline solution was added. After 4 h of incubation, 0.04 M HCl/isopropanol were added. Viable cells were determined by the absorbance at 570 nm. Measurements were performed and the concentration required for 50% inhibition of viability (IC50 was determined graphically. The absorbance at 570 nm was measured with a UV-Spectrophotometer using wells without sample containing cells as blanks.

The effect of the samples on the proliferation of Hep-G2 was expressed as the % cell viability, using the formula:

% Cell viability=A570 of treated cells/A570 of control cells × 100Result and Discussion

The main objective of this study is to analyse the potential of tuber extract of G. superba to prevent Hep-G2 cancer cells by MTT assay [10]. G. superba is an alkaloid plant containing a large amount of alkaloid components like colchicine and gloriosine. Other than alkaloids various phytochemicals compounds like terpenoids, glycoside, tannin, phenol etc., are also present in lower concentration [4]. These phytochemicals were extracted in solvents such as methanol, water and petroleum ether using soxhlet extraction method [11]. The phytochemicals were analysed for qualitative (Table 1) and quantification of alkaloid and saponin. Quantity of alkaloid (1.1311 g/100 g) and saponin (1.7285 g/100 g) were obtained in Table 2.

Phytochemical Chemical test Solvents
Methanol Petroleum Ether Water
Alkaloids Dragendroff’s test ++ + ++
Flavanoids Shinoda test + ++ +
Glycosides Liebermann tests + ++ ++
Saponins Froth’s test ++ - ++
Steroids Steroids test ++ - -
Phenols Ferric chloride test ++ + -

Table 1: Qualitative analysis of phytochemicals.

Test for Phytochemicals Quantitative Values (g/100g) Test Method
Alkaloids 1.1311 g/100g Whattman Filter Paper
Saponins 1.7285 g/100g Soxhlet Apparatus

Table 2: Quantitative analysis for alkaloids and saponins.

Phytochemical were qualitatively analysed in three different solvents extracts (methanol, petroleum ether, water). Standard procedure was followed and the presence of various phytochemicals was detected by colouration and precipitation. i.e. (‘++’ presence; ‘+’ faint; ‘-’ negative).

The solvent extracts are followed with DPPH assay to obtain the anti-scavenging of free radicals [12]. The values of water (100 μl: 41.48%) & (200 μl: 65.42%), petroleum ether (100 μl: 46.45%) & (200 μl: 58.95%) and methanol (100 μl: 91.04%) & (200 μl: 91.75%) were obtained (Table 3). Anti-scavenging of different extract was determined from calculating the OD value obtained from absorbance in 517 nm and calculation.

Extract Test (µl) Absorbance Value (517nm) Percentage of anti-scavenging
Methanol Test 1 (100µl) 0.101 91.04%
Test 2 (200µl) 0.093 91.75%
Petroleum Ether Test 1 (100µl) 0.604 46.45%
Test 2 (200µl) 0.463 58.95%
Water Test 1 (100µl) 0.66 41.48%
Test 2 (200µl) 0.39 65.42%

Table 3: Anti-scavenging activity by DPPH assay.

The methanolic extract has the higher value (100 μl: 91.04%) & (200 μl: 91.75%). As the anti-scavenging is the important factor to prevent cancer and heart disease [5]. MTT test were followed using the extract with higher anti-oxidant value [8]. Hep-G2 cells are used in MTT assay with different concentration of the methanolic extract 5 μg, 10 μg, 25 μg, 50 μg and 100 μg. A triplicate value was obtained using UV-Spectrophotometry analysis of formazan formation [13]. Cell viability percentage was obtained from calculation of absorbent using a formula (Figure 1).

pharmacognosy-phytochemistry-anti-scavenging

Figure 1: Bar diagram for anti-scavenging activity by DPPH assay.

DPPH assay was performed to analyse the antiscavenging property of solvent extract from G. superba tubers. Each solvent was tested with two concentration 100 μl/ml as test 1, 200 μl/ml as test 2 and bar diagram was plotted from values mentioned in Table 3.

Hep-G2 Cytotoxicity Analysis

MTT (3-(4, 5–dimethylthiazol–2–yl)–2, 5-diphenyltetrazolium bromide) assay, it is a homogenous cell viability assay works on the principle of formazan formation. Formazan are measured using UV-spectrophotometer [14]. The viable cells convert MTT into formazan due to its active metabolism, producing colouration which acts as a marker indicator of viability of cells. Formazan crystals are solubilized for absorbance [14].

Hep-G2 is a human liver cancer cell line, used in in vitro studies to analysis cytotoxicity of compounds. In this study Hep-G2 cells are used to analyse the cancer inhibition potential of G. superba tuber extract. Plant extract with higher antioxidant property are used to analyse the cytotoxicity assay on Hep-G2 cells, antioxidant are good cancer preventers. Methanolic extract of G. superba holds the higher value of antioxidant activity hence methanolic extract were used to analyse the MTT assay with different concentration [10].

Concentration of 5, 10, 25, 50, 100 μg/ml methanolic sample was tested for cytotoxicity activity on Hep-G2 cancer cell line (Figure 2).

pharmacognosy-phytochemistry-Microscopic-view

Figure 2: Microscopic view of MTT assay on Hep-G2 cells.

G. superba tuber’s methanolic extract was tested on Hep-G2 cells with different concentration of 5, 10, 25, 50 and 100 μg/ ml. The colouration of the cell is due to the formation of formazan crystals. The microscopic view, show the cell death in different concentration. The Figure 2A: control, shows Hep-G2 cell line and Figure 2F: 100 μg/ml shows the higher inhibition rate of Hep-G2 cells.

The variant concentration shows triplicate value from absorbance using UV–Spectrophotometry at 570 nm. The absorbance of each concentration shows the viable cell rate with indicating colour of formazan crystals and 5 μg (0.854, 0.856, 0.85), 10 μg (0.739, 0.741, 0.735), 25 μg (0.642, 0.645, 0.638), 50 μg (0.522, 0.524, 0.52), 100 μg (0.436, 0.438, 0.432) were obtained.

The absorbent value was then calculated for total viability of Hep-G2 cells form various concentration. Using standard formula, total cell viability % is obtained in triplicate value (Figure 3).

pharmacognosy-phytochemistry-Bar-diagram-view

Figure 3: Bar diagram for cell viability & inhibition of Hep-G2 cell by MTT assay and IC50 graph for inhibition rate %.

The bar diagram was plotted from the triplicate value of cell viability which mentioned in Table 4. Measurements were made and IC50 graph was plotted with linear regression curve for 50% of inhibition of Hep-G2 cells, IC50=19 and 5 μg (89.51782, 89.72746, 89.09853), 10 μg (77.46331, 77.67296, 77.04403), 25 μg (67.2956, 67.61006, 66.87631), 50 μg (54.71698, 54.92662, 54.50734), 100 μg (45.70231, 45.91195, 45.28302) were obtained (Table 4).

Tested Concentrations (µg/ml) Percentage of cell variability (triplicate value)
5 89.518 89.727 89.099
10 77.463 77.673 77.044
25 67.296 67.61 66.876
50 54.717 54.9266 54.5073
100 45.7023 45.912 45.283
Control 100 - -

Table 4: Cell viability in triplicate value.

The variant concentration shows triplicate value from absorbance using UV–Spectrophotometery at 570 nm. Percentage of triplicate viability value was obtained from calculating the standard formula.

Cell viability was obtained higher from lower the concentration applied, 5 μg has higher viable cells and the higher concentration of 100 μg has the lower viable rate. Death rate of cells indicated the cancer inhibition rate which is due to Hep-G2 cells failed to retain the viability [15].

Mean values (M ± SD) for the triplicate values of 5 μg (89.4479 ± 0.32022), 10 μg (77.3934 ± 0.32023), 25 μg (67.2606 ± 0.36812), 50 μg (54.7169 ± 0.20964), 100 μg (45.6324 ± 0.32024) measurements were performed and the concentration required for 50% inhibition of viability (IC50=19.5145 ± 388.9) (19.93%) was determined graphically. The inhibition rate was calculated from subtracting the viable cell to total control 100% from each concentration (Mean-control). Inhibition rate of 5 μg (10.5520), 10 μg (22.6065), 25 μg (32.7393), 50 μg (45.2830), 100 μg (54.3675) were obtained (Table 5), where 100 μg has the higher inhibition range of 54.3%. From evaluation of IC50 value (19.93%) cell death indicates the cancer inhibition.

Concentration (µg/ml) Average value of viability % (M ± SD) Inhibition rate % (mean-control)
5 89.44972 ± 0.32022 10.552
10 77.39343 ± 0.32023 22.607
25 67.26066 ± 0.36812 32.78
50 54.71698 ± 0.20964 45.283
100 45.63243 ± 0.32024 54.3676

Table 5: Average value of cell viability and inhibition rate.

Mean average value of viability % from triplicate value; n=3, M ± SD. Inhibition rate % was determined from (mean–control).

This study concludes the information on G. superba tubers has the potential to inhibit Hep-G2 (human liver cancer cells) cell line.

Conclusion

The plants are natural source of bio-active compounds which can treat various diseases and life threatening cancer [1]. The G. superba methanolic extracts reveals the presence of different types of phyto constituents which has the capacity of anti-oxidant and cytotoxicity effect on Hep-G2 cells. Thus G. superba has the potentiality to inhibit the human carcinoma cell line growth.

Acknowledgement

It is a pleasant aspect that I now have the opportunity to express my gratitude for all who accompanied and supported during my dissertation work.

Conflict of Interest

The authors declare no financial or commercial conflict of interest.

References