Antioxidant and cytotoxicity to liver cancer HepG2 cells in vitro of Korarima (Aframomumcorrorima (Braun) P.C.M. Jansen) seed extracts

ABSTRACT This study examined the total phenolic contents, in vitroantioxidant activity and anti-cancer effect of petroleum ether and aqueous: methanol (20:80, v/v) seed extracts of Aframomum corrorima (Ethiopian cardamom). The aqueous methanol extract contained higher total phenolic content (25 ± 5 mg gallic acid equivalent/100 g of dried extract and total flavonoid content (19 ± 0.4 mg quercetin equivalent/100 g of dried extract. The extract showed stronger antioxidant activity than the petroleum ether extract, EC50 = 97 ± 4 μg/mL in DPPH radical scavenging assay, and EC50 = 258 ± 15 μg/mL in ferric reducing antioxidant power assay. In the antiproliferative assay, the petroleum ether extract showed stronger (EC50 = 105 ± 7 μg/mL) effect than the aqueous:methanol (20:80, v/v) extract (EC50 = 282 ± 43 μg/mL), on hepatocellular carcinoma (HepG2) cells when incubated for 24 h. This is the initial report to demonstrate antioxidant activity and anti-cancer in vitro effect of seed extracts of A. corrorima.


Introduction
Afromomum corrorima, locally known as Korarima or Ethiopia cardamom, is a monocotyledonous flowering plant belonging to the family Zingiberaceae. [1] It is widely cultivated in the south and southwestern part of Ethiopia. The spice obtained from the plant's seeds (usually dried), is extensively used in Ethiopian cuisine and as herbal medicine to flavor food and beverages. The seeds (usually dried, sometimes fresh) ground with other spices are used to flavor all kinds of traditional sauces in the country. They have been used in the preparation of spiced hot red pepper, [2] paste of chili pepper [3,4] and to flavor butter, coffee, tea, and bread. [1] Different parts of A. corrorima plant (leaf, seed, and pod) have been used in traditional medicine by the people of southern Ethiopia. The leaf is used to treat animal body swelling and skin wounds. [5] A decoction of the rhizomes has been used against parasitic nematodes in ruminant animals. [6] The seed is effective against tonic convulsions, carminative, purgative, headache, stomachache, skin wounds, and sore throat when taken orally (Abegaz, 1994). [7] Seed extract and steam distilled essential oil of theseed of this plant have shown antimicrobial activities. [8] The seeds of A. corrorima contain different types of essential oil components with a typical odor. According to Baser and Kürkcüoglu. [9] and Hymete et al. [10] the main constituents found in the seed oil were 1, 8-Cineole and sabinene, whereas sesquiterpenestructures such as (E)-nerolidol, βcaryophyllene and caryophyllene oxide, dominated the husk oil of the sample collected from the local market. According to Eyob et al. [10] studies conducted on fresh materials, the major constituents of the oil were found to be γ-terpinene in pods and 1, 8-Cineole in seeds. Also Eyob et al. [11] reported the essential oil component of leaf and rhizome. The volatile oil of the leaf was dominated by β-caryophyllene and γ-terpinene and β-pinene was the major monoterpene in rhizome. Recently, Hailu et al. [12] reported the existence of noticeable variability with regards to composition and characteristics ofthe essential oil of fruit and seed samples of A. corrorima collected from different parts of Ethiopia.
Although A. corrorima is widely used as a food flavoring and traditional medicine, in Ethiopia, scarce information is available on total phenolic content and antioxidant activity. [11] To our knowledge, there is no report on anti-cancer effects of the seed extracts. Therefore, the objective of the present study was to estimate the total phenolic and flavonoid contents, in vitro antioxidant activity (using various assays) and cytotoxicity effect on hepatocellular carcinoma (HepG2) cell of petroleum ether and aqueous methanol (20:80), v/v) extracts of the dried seed.

Sample preparation and extraction
Fresh fruit of A. corrorima was collected from farm, 6 km North-East of Yirgalem Town, Sidama Regional State, Southern Ethiopia. The fruits were air-dried for 10 days and then the seeds were separated from the husk and ground to fine powder using an electric grinder (FM100 model, China). The petroleum ether and aqueous: methanol (20:80, v/v) extracts were prepared by dissolving 3 g of the seeds fine powder separately in 30 mL of each solvent. The mixture was then subjected to sonication (model 750D, VWR Intl. Ltd., Montreal, QC, Canada) for 15 min x 3 times, with 10 min intervals in between sonication cycles to keep the temperature below 30℃ during the extraction. The crude extract was centrifuged (model Durafuge 300, Precision Scientific, Richmond, VA, USA) at 3000 g for 15 min. Then the supernatant was filtered using Whatman number 1 filter paper. The aqueous: methanol (20:80, v/v) and petroleum ether extracts were evaporated to dryness under N 2 and the remaining water was freeze-dried for 8 h in a freeze dryer (model 2085C0000, Kinetics Thermal Systems, Stone Ridge, NY, USA). Samples of each treatment were extracted and analyzed in triplicate and immediately stored in amber vials at −20°C until used for the analysis.

Determination of total phenolic contents (TPC)
Total phenolic content was estimated by Folin-Ciocalteu method as described in Jundi et al. [13] To 0.1 mL of the extract (1 mg/mL), 1 mL Folin-Ciocalteu reagent (diluted ten times) was added and the mixture was left for 5 min and then 1 mL (75 g/L) of sodium carbonate was added. The absorbance of the resulting blue color was measured at 765 nm with a double beam UV-visible spectrophotometer (JENWAY, 96500, UK) after incubation for 90 min at room temperature. The total phenolic content was estimated from gallic acid (1-100 µg/mL) calibration curve (y = 0.015x + 0.023, R 2 = 0.99) and results were expressed as (mgGAE/100 g of dried extract).

Determination of total flavonoids (TFC)
The TFC was determined as described in Engeda et al. [14] with minor modifications. The analysis was based on the formation of the yellow color of flavonoid-aluminum complex. Aluminum chloride (2 mL, 2%) was mixed with the same volume of the seed extract (1 mg/ mL). Then absorbance at 415 nm was taken after 1 h of incubation at room temperature against a blank sample. The TFC was determined using a standard curve of quercetin at (1-40 μg/mL) and values were calculated as (mgQE/100 g of dried extract) using the equation, y = 0.024x + 0.112, R 2 = 0.99.

DPPH Radical Scavenging Method
The DPPH radical scavenging activity of the extracts from A. corrorima seed was determined as described by Engeda et al. [15] Different concentrations (50 to 100 µg/mL) of the extracts were taken in different test tubes. Freshly prepared DPPH solution (2 mL, 0.006%, w/v) in methanol was added in each of the test tubes containing 1 mL of the extract. The reaction mixture and the reference standards (ascorbic acid and BHT) were vortex mixed and left to stand at room temperature in the dark for 30 min. The absorbance of the resulting solution was then taken at 717 nm. Methanol was used as blank. The ability to scavenge the DPPH radical was calculated as DPPH scavenging (%) = [(Ac-As)/ Ac] × 100, where Ac is the absorbance of the control and As is the absorbance in presence of the sample extracts. The antioxidant activity of the extract was expressed as EC 50 . The EC 50 value was defined as the concentration (in μg/mL) of extracts that scavenges the DPPH radical by 50%.

Ferric reducing antioxidant power
The ferric reducing power was determined according to the method of Yishaket al. [16] Plant extract (1 mL) solution (final concentration 50-1000 µg/mL) was mixed with 2.5 mL potassium phosphate buffer (0.1 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. Then at 50°C the mixture was incubated for 20 min. Trichloroacetic acid (2.5 mL, 10%) was added to the mixture, which was then centrifuged at 3000 rpm (Centurion, 1000 series, UK) for 5 min. Finally, 2.5 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 0.5 mL FeCl 3 (0.1%) and absorbance was measured at 700 nm. All tests were performed in triplicate. An increase in absorbance of the reaction indicated a higher reducing power. EC 50 values (µg/mL) were calculated and indicate the effective concentration at which the absorbance was 0.5 for reducing power.

Cell lines and culture conditions
Human hepatocellular carcinoma cells (HepG2) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as recommended by the ATCC. HepG2 cells were grown in Eagle's modified minimum essential media (EMEM) supplemented with 10% FBS (FBS; ATCC, Rockville, MD, USA) and 1% penicillin-streptomycin as described before. Briefly cells were maintained at 37 ∘ C in an incubator under 5% CO 2 /95% air atmosphere at above 85% relative humidity constantly. The cells were cultured (about 80% of confluence) once a week in T-75 flasks. Cells were counted using a hemocytometer (Bright-Line Hemacytometer, Sigma-Aldrich, Mississauga, ON, Canada) and were plated in 96-well format for 24 h prior to the addition of test compounds. All the test samples were solubilized in sterile-filtered DMSO (< 0.5% in the culture medium) prior to addition to the culture media. Control cells were also run in parallel and subjected to the same changes in media with <0.5% DMSO.

Cytotoxicity assay
Cytotoxicity to HepG2 cells Cytotoxicity to HepG2 was determined using the Cell Titer 96 TM Aqueous one solution cell proliferation (MTS) assay kit (Promega Madison, WI, USA) according to the manufacturer's instructions. [17] HepG2 cells (1 × 10 4 cells/100 μL/well) were seeded in a sterile flat bottom 96-well plate (BD Biosciences, Mississauga, ON, Canada) and incubated at 37°C, 5% CO 2 for overnight. The aqueous: methanol (20:80, v/v) and petroleum ether extracts of A. corrorimaseeds or sorafenib were prepared in media and 100 μL of each treatment was added to each well, each treatment in three replications. Thereby, cells were exposed to various concentrations (10, 50, 100, 200, 300, and 500 μg/ mL) of each treatment. Controls consist of cells with media containing DMSO (< 0.5%) and blank wells contained media with no cells. After 24 and 48 h of incubation, 20 μL of the MTS reagent in combination with phenazinemethosulfate (the electron coupling agent) was added to the wells, and cells were incubated in a humidified CO 2 incubator for 3 h. Absorbance at 490 nm (OD490) was monitored with a plate reader (FLUO star Optima, BMG Labtech, Durham, NC, USA) to obtain the number of viable cells relative to the control population. Percentage of viability in the test compound treated cells was expressed as a percentage compared to control (<0.5% DMSO), using the following formula: % cell viability = (OD490 of treated cells/OD490 of control cells) × 100. Data were expressed as mean values ±SD and obtained from three different experiments against each cell line (n = 3 per plate per time point).

Morphological observation under inverted phase-contrast microscope
HepG2 cells were equally seeded in 96-well flat-bottom tissue culture-treated plates (BD Biosciences) and then treated with various concentrations of the extracts, sorafenib, or DMSO (< 0.5%) control. After 24 h of treatment, the morphology of HepG2 cells was observed under an inverted phasecontrast microscope (Nikon Eclipse E 100, Nikon, ON, Canada) and images were captured at 100x magnification using Infinity digital microscopy camera (Lumenera corporation, ON, Canada).

Statistical analysis
Statistical analysis was performed using Statistical Analysis System (SPSS, Version 20). The data were subjected to analysis of variance (ANOVA). Duncan's multiple range test was used for mean comparisons at p < .05. Linear regression analysis was used to calculate EC 50 values.

Total phenolic and flavonoid contents
The total phenolic and flavonoid contents of aqueous: methanol (20:80, v/v) extract were higher (p < 0.05) than that of petroleum ether extract (Table 1). According to the study conducted by Eyob et al. [11] the total phenolic content in seeds (3.98 mg/g dried weight) was significantly higher than in pods (1.32 mg/g dried weight). These values were lower than that of the present study.

DPPH radical scavenging activity
The DPPH radical scavenging effects of A. corrorimaseed extracts were shown in Figure 1. DPPH radical is scavenged by antioxidants through the donation of hydrogen forming the reduced DPPH. The color changes from purple 2, 2-diphenyl-1-picrylhydrazyl radical to reduced yellow diamagnetic 2, 2-diphenyl-1-picrylhydrazine molecule, which can be quantified by its absorbance reduction at wavelength 520 nm. [18] At the concentration of 1 mg/mL, the DPPH radical scavenging effects were decreased in the order of L-ascorbic acid (98 ± 4%) > BHT (95 ± 11%) > aqueous: methanol (20:80, v/ v) extract (91 ± 10%) > petroleum ether extract (57 ± 7%). As the concentration of the sample increased, the percent inhibition of DPPH radical also increased. [4] This suggested that dried seed extracts of A. corrorima contain compounds that can donate electron/hydrogen easily and stabilizes free radicals. The IC 50 values were calculated from plotted graph of percentage scavenging activity against the concentration of the extracts ( Table 2). The lower the EC 50 value, the higher is the scavenging potential. Stronger scavenging activity (lower EC 50 values) was recorded for aqueous: methanol (20:80, v/v) extract which appeared more than seven times stronger than the scavenging activity of petroleum ether. This might be in accordance with the total phenolic or flavonoid contents of A. corrorima. [19] The DPPH scavenging activities of petroleum ether and aqueous: methanol (20:80, v/v) extracts were found to be significantly weaker (p < 0.05) than that of BHT and L-ascorbic acid.

Ferric reducing power
The antioxidant potential of the A. corrorima was estimated for its ability to reduce Fe (III) to Fe (II). Similar to DPPH scavenging as the concentration of the extracts increased, the ferric reducing power increased ( Figure 2). In this study, the aqueous methanol extract showed stronger ferric reducing power. Significant differences (p < 0.05) were found between the two extracts (Table 2), the aqueous methanol extract showed three times stronger (smaller EC 50 ) than the ferric reducing power of petroleum ether extract, however this extract showed weaker reducing power than that of butylated hydroxytoluene (BHT).

Microscopic evaluation of morphological changes in HepG2 cells
To examine the effect of the petroleum ether and aqueous: methanol (20:80, v/v) extracts on cell morphology, HepG2 cells were treated with 10, 50, 100, 200, 300, and 500 μg/mL of the extracts, sorafenib (positive control) and DMSO (< 0.5%) for 24 h and morphological changes were observed by phase-contrast microscopy. The images (Figure 3) showed that the petroleum ether extract induced severe morphological changes of cell death, including rounding and shrinkage of cells in a dosedependent manner. The pattern of cell death was similar to liver cancer drug sorafenib at a concentration of 300 µg/mL and above. The control cells were well adhered to, displaying the normal morphology of HepG2 cells. In contrast, the majority of HepG2 cells treated with petroleum ether extract at the concentration of 300 μg/mL or more became round and shrunken and could not be affixed to the walls and floating in the medium (×100 magnification).

Inhibition of HepG2 cell proliferation
HepG2 cells were treated with increasing concentrations (10,50,100,200, 300, and 500 µg/mL) of aqueous methanol (20:80, v/v) extract, petroleum ether extract or sorafenib and cell viability were assayed at 24 and 48 h after treatment (Figure 4). Cell viability decreased in a dose-dependent manner and decreased with the increasing concentration of the extract. A strong negative correlation between the percentage of cell viability and concentration of the extract was observed (Figure 4). Petroleum ether extract inhibited the proliferation of HepG2 cells more than aqueous methanol (20:80, v/v) extract, with increasing inhibitory activity as the concentrations of extracts increased. After 24 and 48 h, petroleum ether extract showed 11 ± 2% and 0.5 ± 0.0% cell viability of HepG2 cells, respectively, at a concentration of 300 μg/mL, while at the same concentration, aqueous: methanol (80:20, v/v) extract showed 40 ± 11% and 34 ± 15% cell viability, respectively. There is a significant difference of EC 50 value between petroleum ether (EC 50 = 105 ± 7 μg/mL) and aqueous methanol (20:80, v/v) (EC 50 = 282 ± 43 μg/mL) extracts treated for 24 h. Also, the EC 50 values of petroleum ether and aqueous methanol (20:80, v/v) extracts were significantly higher (p < 0.05) than the EC 50 value of sorafenib (positive control) at any time ( Table 3). The relatively stronger cytotoxicity activity of the petroleum ether extract, containing low phenolic composition, suggests that the nature of compounds is determinant for these activities rather than their amounts. Studies showed that essential oils [20] are playing a dominant role in anti-cancer activity rather than the polar phenolics..Under this study, the stronger cytotoxicity activity of petroleum ether extract may be because of bioactive monoterpene, 1, 8-Cineole is the major component of the essential oil (44.3%) of seed of A. corrorima. [11] A number of scientific reports have shown the antiproliferative activity of 1, 8-Cineole against different cell lines. According to Rodenaket al. [21] 1, 8-Cineole, the main compound present in the N. binaloudensis oil has been reported to inhibit the growth of liver-derived (HepG2) cell lines. The recent study [22] on 1, 8-Cineole-containing n-hexane extract of Callistemon citrinus, also showed cytotoxicity effect against the cancer cells. Therefore, the antiproliferative efficacy of petroleum ether extract may be related to the presence of 1, 8-Cineole and other phytochemical components in seed extract of A. corrorima.

Conclusion
The results obtained showed the in-vitro studies performed using the hepatocellular carcinoma (HepG2) cell reveals that the petroleum ether seed extract of A. corrorima showed a moderate anticancer activity; however, the aqueous methanol (20:80, v/v) extract exhibited weaker activity. Even though there was an increase in the cell growth inhibition when the concentration of the sample was increased, the EC 50 values were more than 100 μg/mL for the cell lines studied as shown by the MTS assay method. In this study, however, the findings did not show any relationship between antiproliferative activity and phenolic content, and antioxidant activity. Petroleum ether extract, which contained a lower phenolic composition and weaker antioxidant activity, exhibited a stronger antiproliferative activity. The overall results suggested that seed petroleum ether extract of A. corrorima containing the largest amount of 1, 8-Cineole has shown potential antiproliferative activity in a dosedependent manner. Based on these preliminary results, A. corrorima could represent an important source of substances with antiproliferative activity. Therefore supplementing a balanced diet with A. corrorima seeds may have a beneficial effect in treating liver cancer.