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Secondary metabolites constituents and antioxidant, anticancer and antibacterial activities of Etlingera elatior (Jack) R.M.Sm grown in different locations of Malaysia
© Ghasemzadeh et al. 2015
Received: 2 June 2015
Accepted: 28 August 2015
Published: 23 September 2015
Etlingera elatior is a well-known herb in Malaysia with various pharmaceutical properties.
E. elatior flowers grown in three different locations of Malaysia (Kelantan, Pahang and Johor), were investigated for differences in their content of secondary metabolites (total phenolics [TPC], total flavonoids [TFC], and total tannin content [TTC]) as well as for their antioxidant, anticancer, and antibacterial properties. Phenolic acids and flavonoids were isolated and identified using ultra-high performance liquid chromatography (UHPLC). Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used to evaluate the antioxidant activities. The anticancer activity of extracts was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
When extracted with various solvents (aqueous and ethanolic), samples from the different locations yielded significantly different results for TPC, TFC, and TTC as well as antioxidant activity. Aqueous extracts of E. elatior flowers collected from Kelantan exhibited the highest values: TPC (618.9 mg/100 g DM), TFC (354.2 mg/100 g DM), TTC (129.5 mg/100 g DM), DPPH (76.4 %), and FRAP (6.88 mM of Fe (II)/g) activity with a half-maximal inhibitory concentration (IC50) of 34.5 μg/mL compared with extracts of flowers collected from the other two locations. The most important phenolic compounds isolated in this study, based on concentration, were: gallic acid > caffeic acid > tannic acid > chlorogenic acid; and the most important flavonoids were: quercetin > apigenin > kaempferol > luteolin > myricetin. Extracts of flowers from Kelantan exhibited potent anticancer activity with a IC50of 173.1 and 196.2 μg/mL against the tumor cell lines MCF-7 and MDA-MB-231 respectively, compared with extracts from Pahang (IC50 = 204.5 and 246.2 μg/mL) and Johor samples (IC50 = 277.1 and 296.7 μg/mL). Extracts of E. elatior flowers also showed antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa with minimal inhibitory concentrations (MIC) ranging from 30 to >100 μg/mL.
In general, therefore, based on the potent antioxidant and anticancer activity of flower extracts, it appears that E. elatior grown in the North-east of Malaysia (Kelantan) is a potential source of therapeutic compounds with anti-cancer activity.
Samples of E. elatior flowers were collected from three different areas of Malaysia: Kelantan (North-east), Pahang (Central), and Johor (South-east). The samples were identified by Mr. Thiyagu Devarajan from the Malaysian Agriculture Research and Development Institute (MARDI) as E. elatior . Flowers were harvested, washed with distilled water and shade dried. After drying process, samples were stored at −20 °C for future analysis.
Dried flowers (50 g) were ground into powder followed by extraction with distilled water and ethanol (1 L). Solutions were refluxed for 2 h at 65 °C, then cooled, and filtered through Whatman filter paper (No. 1) in a filter funnel. This was followed by evaporation under reduced pressure in an Eyela rotary evaporator to remove excess solvent. The residue was freeze-dried and dried extracts were kept at −20 °C for future analysis.
Total phenolic content
Extracts of flowers (200 μL) were diluted in 20 mL of distilled water. Folin-Ciocalteu reagent (10-fold diluted; 1 mL) was added and the mixture was incubated in total darkness for 10 min at room temperature. Sodium carbonate 7.5 % (1 mL) was then added and incubated for 30 min, then the absorbance of the solution was read at 765 nm using a spectrophotometer (UV2550, Shimadzu, Japan) . Different concentrations of gallic acid were used to prepare a calibration curve.
Total flavonoid content
Flower extracts (1 mL) were mixed with NaNO2 in a methanolic solution (4 mL, 1:5, w/v) and incubated at room temperature for 6 min. Then, 0.3 mL of AlCl3 solution (1:10, w/v) was added, the reagents were mixed well, and the reaction was allowed to stand for another 6 min. Immediately after that, 1 M NaOH solution (2.0 mL) was added to each extract and incubated for 10 min at room temperature. The absorbance of the solutions was read at 510 nm using a spectrophotometer (UV2550, Shimadzu, Japan) . Different concentrations of quercetin standard were used to prepare a calibration curve.
Total tannin content
Total tannins content were determined according to the method of Morrison et al.  with some modification. 0.5 mL of extract was diluted with methanol to made up to 5 mL. Extract was mixed with 25 mL of vanillin reagent (1 g vanillin in 100 mL methanol) and 25 mL of 4 % HCl in methanol. The mixture was kept for 15 min at room temperature in dark place, and absorption was measured at 500 nm using a spectrophotometer (UV2550, Shimadzu, Japan). Methanol was used as a blank. All samples were analyzed in triplicate.
Separation and analysis of flavonoids and phenolic acids
Ultra-high performance liquid chromatography (UHPLC, 1290 Infinity Quaternary LC System, Agilent, Santa Clara, CA, USA) was used to separate and identify the phenolic acids. The chromatographic system conditions were set as follows: mobile phase, 0.03 M orthophosphoric acid (A) and methanol HPLC grade (B); detector, UV 280 nm; column, C18 column (5.0 μm, 4.6 mm inner diameter [ID] × 250 mm); column oven temperature, 35 °C; and flow rate, 1.0 mL/min. Gradient elution was performed as follows: 0 min 40 % B, 10 min 100 % B, 15 min 100 % B, and 20 min 40 % B. Linear regression equations were calculated using Y = aX ± b, where X is the concentration of the related compound and Y the peak area of the compound obtained from UHPLC. The linearity was established by the coefficient of determination (R2).
vitro evaluation of antioxidant activity
1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay
Ferric Reducing Antioxidant Potential (FRAP) Assay
The stock solutions consisted of 10 volumes of 300 mM acetate buffer (pH = 3.6), 1 volume of 10 mM TPTZ (2,4,6-tripyridyl-S-triazine) solution in 40 mM HCl, and 1 volume of 20 mM FeCl3 solution. Acetate buffer (25 mL) and TPTZ (2.5 mL) were mixed (FRAP solution), and 2.5 mL FeCl3 was added. Flower extracts (100 μL) and deionized water (300 μL) were added to 3 mL of the FRAP solution. Solution was mixed well and incubated for 30 min in a water bath (at 37 °C). The absorbance of the resultant solution was measured at 593 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan) with acetate buffer used as the blank. A standard curve was prepared using various concentrations of FeSO4 × 7H2O. The value for the blank absorbance was subtracted from that of the sample and used to calculate the FRAP value .
Determination of Anticancer Activity
Cell Culture and Treatment
Human breast carcinoma cell lines (MCF-7 and MDA-MB-231) and normal human mammary epithelial cells (MCF-10A) were cultured in RPMI 1640 media (Roswell Park Memorial Institute) containing 10 % fetal bovine serum (FBS). Cell lines were incubated overnight at 37 °C in 5 % CO2 to allow the cells to attach to the culture plates.
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay
Bacterial cultures and growth conditions
Multi drug resistant (MDR) clinical isolates of Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa) with their antibiotic resistance profiles were obtained from the laboratory of microbial culture collection unit (UNiCC), Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia. All the test strains were maintained on nutrient agar slants at 4 °C and sub-cultured in nutrient broth for 24 h prior to testing. These bacteria served as test pathogens for the antibacterial activity assay.
Antibacterial activity assay
Molten Mueller-Hinton agar, (15 mL at 45 °C, Oxoid, Basingstoke, UK) was poured into sterile Petri dishes (90 mm). Bacterial cell suspensions were prepared and 100 μL was spread evenly onto the surface of the agar plates. Once the plates had been aseptically dried, 6 mm wells were punched into the agar with a sterile Pasteur pipette. The test extracts (10 mg/mL) were dissolved in dimethylsulfoxide (DMSO)/water (1/9) and 80 μL were applied to the wells and incubated at 37 °C for 24 h. Gentamicin (25 μL/well at concentration of 4 μg/mL) and ciprofloxacin (5 μg/mL) were used as positive antibacterial controls. The antibacterial activity was evaluated by measuring the diameter of the circular inhibition zones around the well. Tests were performed in triplicate and values are the average of three replicates. Data are expressed as mean ± standard deviation.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of plant extracts was analyzed by the agar-well diffusion method with a protocol similar to that described in the previous section. A bacterial suspension (105–106 CFU/mL) of each tested microorganism was spread on the nutrient agar plate. Wells (6 mm diameter) were cut out of the agar, and 60 μL of each test extract at different concentrations (10, 20, 25, 50, 75 and 100 μg/mL) dissolved in DMSO, were applied to them. The plates were incubated at 37 °C for 24 h under aerobic conditions, followed by measurement of the diameter of the inhibition zone expressed in millimeter. The MIC was taken as the lowest concentration of test material where there was visually no growth after 24 h. All samples were tested in triplicate.
Results and discussion
Total phenolics, total flavonoids, and total tannin contents
Total phenolic, total flavonoid and total tannins content of E. elatior flowers, extracted with different solvent and collected from three different locations
618.9 ± 16.40a
354.2 ± 11.24a
122.5 ± 5.38a
520.4 ± 15.26c
312.5 ± 10.44c
114.5 ± 5.72b
544.7 ± 15.33b
330.8 ± 12.19b
106.4 ± 4.59c
500.6 ± 14.54d
310.4 ± 11.72c
104.2 ± 4.68c
516.4 ± 15.41c
246.1 ± 12.80d
88.7 ± 3.50d
461.5 ± 20.55e
211.0 ± 11.55e
86.3 ± 4.68d
Antioxidant activity of E. elatior flowers, extracted with different solvent and collected from three different locations
FRAP (mM Fe(II)/g)
76.4 ± 5.89c
34.5 ± 1.42f
6.88 ± 0.62b
63.2 ± 4.31e
41.2 ± 2.16e
5.66 ± 0.75c
70.5 ± 5.22d
44.6 ± 2.41d
6.11 ± 0.68c
58.6 ± 3.81f
59.5 ± 3.17b
5.20 ± 0.70c
62.1 ± 4.79e
52.9 ± 2.88c
5.57 ± 0.63c
40.0 ± 3.33g
139.8 ± 4.52a
4.06 ± 0.51d
92.0 ± 4.91b
19.7 ± 0.87g
6.05 ± 0.55c
98.6 ± 4.57a
12.6 ± 0.81h
7.78 ± 0.89a
The amount of TFC extracted was between 211.0–354.2 mg/100 g DM and was significantly influenced by the different locations and solvents. The TFC (354.2 mg/100 g DM) was the highest in Kelantan extracts followed by Pahang (330.8 mg/g DM) and Johor (246.1 mg/g DM) samples. As for the TPC, aqueous extraction enhanced the level of TFC by about 11.7 % (Kelantan), 6.1 % (Pahang), and 14.2 % (Johor) compared to ethanol extraction. It is apparent from Table 1 that the solubility of polyphenolic compounds is higher in aqueous solvents than in ethanol. The TFC of extracts of E. elatior flowers from Kelantan was higher than that previously reported for herbs including Cymbopogon citratus (3.05 mg/g DM), Mentha piperita (3.01 mg/g DM), Citrus bergamia (2.11 mg/g DM), and Jasminum officinale (3.05 mg/g DM) . Herbs may contain tannins, which are important phytochemicals with a wide range of medicinal properties, including anticancer, anti-inflammatory, antioxidant, and antibacterial activities [21–23]. Variable tannin content was identified in different herbs and plants including Caesalpinia pyramidalis Tul. (817 mg/100 g DM), Anadenanthera colubrina (Vell.) (4.41 mg/100 g DM), and Jatropha mollissima (2.35 mg/100 g DM) .
In the current study, E. elatior flowers from all locations had a high TTC. Aqueous extracts of E. elatior flowers from Kelantan had the highest TTC (122.5 mg/100 g DM) followed by Pahang (106.4 mg/100 g DM), and Johor samples (88.7 mg/100 g DM). The solvent did not appear to affect the TTC and no significant difference between the TTC of aqueous and ethanol extracts from Pahang and Johor was observed. Mailoa et al.  reported that the TTC in extracts of guava leaves decreased from 3.228 mg/g in ethanol 30 % to 2.33 mg/g in 70 % ethanol. A recent study showed that water was a more effective solvent than ethanol-water mixtures for the extraction of condensed tannins from grape skin .
Identified phenolic acids and flavonoids from E. elatior extracts collected from three different locations
129.14 ± 7.54a
102.40 ± 9.07b
87.72 ± 6.74c
82.66 ± 10.6a
66.19 ± 10.56b
53.70 ± 4.62c
75.79 ± 9.61a
70.45 ± 8.46a
88.46 ± 7.20a
58.25 ± 4.56c
69.11 ± 4.02b
89.50 ± 6.55a
77.20 ± 7.80b
64.17 ± 5.66c
71.88 ± 7.19a
60.18 ± 5.06b
40.23 ± 5.21c
62.19 ± 6.58b
70.28 ± 6.22a
55.70 ± 4.83c
46.69 ± 5.19a
35.75 ± 7.21a
20.58 ± 5.17b
5.66 ± 4.29c
The FRAP value was in the range of 4.06–7.78 mM of Fe (II)/g with the highest and lowest FRAP activity observed in the aqueous extracts from Kelantan flowers and ethanol extracts from Johor flowers, respectively. The FRAP activity increased by about 17.7 % (Kelantan), 14.8 % (Pahang), and 37.1 % (Johor) when extraction was with aqueous solvent rather than ethanol. Chan et al.  reported that Etlingera species with high leaf TPC also have high antioxidant capacity and FRAP activity and several studies reported a significant correlation between the antioxidant activity of herbs and the phytochemical content [19, 28, 29]. In the current study, aqueous extracts of E. elatior flowers collected from Kelantan had the highest content of total flavonoids, total phenolics, and total tannins in addition to high antioxidant properties. Earlier, it has been opined that with the change of solvent polarity, viscosity, and vapor pressure, the type of antioxidant compound being dissolved in the solvent also varies. Solvents with low viscosity have low density and high diffusivity that allows them to easily diffuse into the pores of the plant materials to leach out the bioactive constituents .
Separation and identification of phenolic acid and flavonoid compounds
Quercetin has been reported to be a potent antioxidant with anticancer activity [31, 32]. E. elatior flowers had higher levels of quercetin than other herbs such as Silybum marianum (23 mg/100 g DM), Archangelica officinalis (48 mg/100 g DM), and Hypericum perforatum (49 mg/100 g DM), but a lower quercetin content than Chelidonium majus (759 mg/100 g DM), Epilobium hirsutum (214 mg/100 g DM), Juglans regia (460 mg/100 g DM), and Syzygium aromaticum (155 mg/100 g DM) . In addition, luteolin which has been reported to have potent anti- and pro-oxidative activity [31, 33, 34] was detected only in extracts of E. elatior from Kelantan where it was found in quantities higher than those reported for a number of other herbs such as Salvia officinalis (33.4 mg/100 g DM), Poliomintha longiflora (25.1 mg/100 g DM), and Thymus vulgaris (39.5 mg/100 g DM) . The most important flavonoids isolated in this study, based on concentration were quercetin > apigenin > kaempferol > luteolin > myricetin. Comparing the three different sampling locations from the North-east (Kelantan) to South-east (Johor), the concentration of polyphenols decreased in the following order: Kelantan > Pahang > Johor. This variation in the content of phenolic acids and flavonoids in E. elatior flowers could be related to the differences in the weather conditions or soil nutrition and type, which have been reported previously [36–38]. This finding is in agreement with previous studies of current authors which found production and accumulation of secondary metabolites in herbs were influenced by growing area in Malaysia [39, 40].
The anticancer properties of herbs and spices are directly related to their phytochemical content . In the current study, the E. elatior extracts with the highest content of secondary metabolites exhibited the most potent antioxidant and anticancer activity. In general, therefore, it appears that the potent antioxidant and anticancer activity of E. elatior grown in the North-east of Malaysia may be attributed to the high phytochemical content.
Antibacterial activity of E. elatior flower extracts collected from different locations of Malaysia
Inhibition zone (mm)
8.4 ± 0.264b
4.0 ± 0.142e
9.2 ± 0.316a
6.5 ± 0.277d
7.3 ± 0.276c
6.5 ± 0.216a
4.2 ± 0.207e
6.2 ± 0.168b
5.6 ± 0.264c
4.8 ± 0.229d
2.5 ± 0.183b
2.0 ± 0.273c
4.0 ± 0.177a
4.2 ± 0.119a
4.6 ± 0.166b
2.6 ± 0.219c
5.4 ± 0.318a
5.5 ± 0.337a
6.2 ± 0.250c
2.5 ± 0.266e
5.4 ± 0.348d
7.2 ± 0.372a
6.8 ± 0.352b
8.0 ± 0.233a
6.1 ± 0.318c
6.5 ± 0.374b
6.7 ± 0.357b
Minimal inhibitory concentration (MIC)of E.elatiorflower extracts collected from different locations of Malaysia
This study demonstrated that aqueous solvents rather than ethanol are recommended for extraction of phenolic acids, flavonoids, and tannins from E. elatior flowers. The levels of secondary metabolites and the pharmaceutical quality of E. elatior flowers decreased from the South-east to North-east of Malaysia. In general, if the three different sampling locations from North-east (Kelantan) to South-east (Johor) are compared, the concentration of polyphenols, as well as the antioxidant, anticancer, and antibacterial activities decreased in the following order: Kelantan > Pahang > Johor. One of the most significant findings in this study is that the extracts of E. elatior flowers exhibited promising anticancer activity against the MCF-7 and MDA-MB-231 cancer cell lines. The extracts contained substantial amounts of effective phenolic and flavonoid compounds such as gallic acid, caffeic acid, quercetin, luteolin, and myricetin, which can inhibit the growth of breast cancer cell lines. In conclusion, these findings indicate that E. elatior flowers grown in the North-east of Malaysia (Kelantan) are a potential source of therapeutic compounds with antimicrobial activity and suggest areas for further investigation.
The authors are grateful to the Research Management Centre of Universiti Putra Malaysia for financing this work. The authors would like to acknowledge all the staff of the Laboratory, of Nutrition, Department of Nutrition and Dietetics, Faculty of Medicine, and Health Sciences, Universiti Putra Malaysia (UPM) for all the help and guidance provided in order to accomplish this work.
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