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Optimization of ultrasound-assisted extraction of flavonoid compounds and their pharmaceutical activity from curry leaf (Murraya koenigii L.) using response surface methodology
© Ghasemzadeh et al.; licensee BioMed Central Ltd. 2014
- Received: 2 June 2014
- Accepted: 21 August 2014
- Published: 28 August 2014
Extraction prior to component analysis is the primary step in the recovery and isolation of bioactive phytochemicals from plant materials.
Response surface methodology was applied to optimize ultrasound-assisted extraction conditions followed by ultra high performance liquid chromatography (UHPLC) to achieve high catechin, myricetin, and quercetin contents, and high antioxidant and anticancer activities in the curry leaf extracts. The antioxidant and anticancer activities of the leaf extracts were determined by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. The central composite experimental design (3-level, 3-factorial) was employed to consider the effects of ultrasonic power (80–150 W), temperature (40–80°C), and methanol dilution (40–80%) on the properties of the curry leaf extracts.
It was found that ultrasonic power of 145.49 W at 55.9°C with 80% methanol was the most appropriate set of conditions for the extraction of catechin, myricetin, and quercetin from curry leaves with the consequent high antioxidant activity. Using the optimum extraction conditions, the extraction yields of catechin, myricetin, and quercetin were 0.482, 0.517, and 0.394 mg/g DW, respectively, and the antioxidant activity was enhanced to 83%. The optimized extract showed more distinct anticancer activity against HeLa cancer cells in a concentration of 67.2 μg/mL (P < 0.01) without toxicity to normal cells.
The results indicated that the pharmaceutical quality of curry leaves could be improved significantly by optimizing the extraction process using response surface methodology.
- Response surface methodology
- Ultra high performance liquid chromatography
- Curry leaf
- HeLa cancer
Medicinal plants are the richest source of bioactive compounds used in traditional and modern medicine . Flavonoids and phenolics are essential groups of plant phytochemicals with superoxide radical scavenging activity, thereby providing anticancer activity [2, 3]. In the herbal medicine industry, the extraction process is the important step for the isolation of phytochemicals from herbs and spices . Extraction of herbs using an ultrasound-assisted process was recommended previously as a one of the most inexpensive and simplest existing extraction systems, and could be suitably operated rapidly for large-scale preparations . The application of ultrasound helps develop interesting and novel methodologies in food processing; these methodologies are often complementary to classical methods . Ultrasound-assisted extraction can accelerate heat and mass transfer, has been successively used in the extraction field, and is well known to have a significant effect on the rate of various processes in the food industry . Ultrasound waves interact with the plant material and alter its physical and chemical properties; furthermore, their cavitational effect facilitates the release of extractable compounds and enhances the mass transport by disrupting the plant cell walls . Previous studies have demonstrated that the extraction yield of flavonoid compounds depends strongly on the extraction technique, solvent polarity, and temperature [8, 9]. A developed model is required for optimizing the independent variables in order to get superior extraction yields from herbs. Response surface methodology (RSM) is a collection of statistical and mathematical techniques that are used to optimize the range of variables in various experimental processes to reduce the number of experimental runs, cost, and time, compared to other methods [10, 11]. Murraya koenigii (L.), generally known as the curry leaf, or Pokok kari (Daun kari) in Malaysia, is one of the traditional folk remedies that contains several interesting bioactive compounds  with anti-tumor , antioxidant [14, 15], anti-inflammatory , anti-hyperglycemic , and hypoglycemic effects . Due to the high beneficial value of this crop, research is required to optimize the extraction process to ensure high nutritional and pharmaceutical quality. However, far too little attention has been paid to the optimization of curry leaf extraction in folk medicine. To the best of our knowledge, there have been no studies to optimize the flavonoid extraction from the curry leaf and following that, improvement of the anticancer and antioxidant activities. The current study is designed in order to optimize the ultrasound-assisted extraction conditions of the Malaysian curry leaf (M. koenigii) to achieve high flavonoid contents and high antioxidant and anticancer activity by using response surface methodology with a central composite design.
Fresh curry leaf samples were obtained from Bachok, Kelantan province, Malaysia. The Malaysian Agriculture Research and Development Institute (MARDI) identified the samples with voucher specimens of MTM0018/1. The leaves were shade-dried (moisture content: 6.2%), powdered (80 mesh), and kept at -20°C.
Experimental design and observed experimental data
Methanol concentration %
Ultrasunic power (W)
0.265 ± 0.011
0.309 ± 0.025
0.177 ± 0.010
67.0 ± 2.20
0.426 ± 0.035
0.470 ± 0.018
0.338 ± 0.018
80.0 ± 7.05
0.274 ± 0.014
0.318 ± 0.021
0.186 ± 0.008
66.2 ± 4.72
0.191 ± 0.002
0.235 ± 0.017
0.103 ± 0.011
52.0 ± 7.76
0.396 ± 0.005
0.440 ± 0.016
0.308 ± 0.017
79.5 ± 2.30
0.436 ± 0.017
0.480 ± 0.007
0.348 ± 0.019
77.0 ± 3.82
0.234 ± 0.008
0.278 ± 0.014
0.146 ± 0.009
55.4 ± 1.81
0.222 ± 0.012
0.266 ± 0.011
0.134 ± 0.014
55.0 ± 2.30
0.347 ± 0.011
0.391 ± 0.014
0.259 ± 0.012
71.0 ± 3.01
0.295 ± 0.007
0.339 ± 0.017
0.207 ± 0.011
69.0 ± 5.56
0.204 ± 0.019
0.244 ± 0.006
0.116 ± 0.007
54.7 ± 4.12
0.474 ± 0.010
0.518 ± 0.026
0.386 ± 0.016
78.0 ± 2.69
0.188 ± 0.015
0.232 ± 0.019
0.100 ± 0.011
51.0 ± 1.88
0.466 ± 0.008
0.510 ± 0.013
0.378 ± 0.017
80.4 ± 3.27
0.480 ± 0.017
0.524 ± 0.019
0.392 ± 0.020
83.0 ± 4.61
0.536 ± 0.014
0.580 ± 0.018
0.448 ± 0.015
87.0 ± 3.55
0.456 ± 0.018
0.500 ± 0.015
0.368 ± 0.013
75.0 ± 2.70
0.403 ± 0.018
0.447 ± 0.008
0.315 ± 0.010
73.0 ± 3.29
0.244 ± 0.014
0.288 ± 0.017
0.156 ± 0.016
58.0 ± 1.77
0.460 ± 0.010
0.504 ± 0.017
0.372 ± 0.019
79.5 ± 2.11
Identification of flavonoids by Ultra High Performance Liquid Chromatography (UHPLC)
The UHPLC system (Agilent, Model 1200) with a C18 (4.6 × 250 mm, 5 μm) column was used for flavonoid separation and identification. In this system, two mobile phases, 0.03 M orthophosphoric acid (A) and HPLC-grade methanol (B), were used. The column temperature, flow rate, and injection volume were adjusted at 35°C, 20 μL, and 1 mL/min, respectively. The range of the detecting wavelength was between 260 and 360 nm. Gradient elution was performed as follows: 0–10 min for 40–100% B; 10–15 min for 100% B; 15–20 min for 100–40% B, and finally, washing of the column. To prepare the standard solutions, catechin (0.0625, 0.125, 0.250, 0.500, and 1 mg/mL), myricetin (0.031, 0.062, 0.124, 0.248, and 0.496 mg/mL), and quercetin (0.09, 0.18, 0.36, 0.72 and 1.44 mg/mL) were dissolved in the HPLC-grade methanol. The linear regression equation was calculated with Y = aX ± b, where X is the concentration of flavonoid and Y is the peak area of flavonoids obtained from UHPLC.
1,1-diphenyl-2-picrylhydrazyl (DPPH) assay
Determination of anticancer activity
The frozen cells were retrieved from a liquid nitrogen cell storage tank and thawed rapidly in cryovials. The contents of the cryovial were carefully transferred to a centrifuge tube and a prewarmed media (10 mL) was gradually added to the cell suspension. The centrifuge tube was spun down at 1000 rpm for 10 min and the resulting pellet gently resuspended in fresh media (10 mL) in a culture flask. Subsequent incubation was carried out in a 37°C humidified incubator supplemented with 5% CO2. After 24 h, the old medium was discarded one day after seeding and 2–3 mL PBS was added to cover the entire surface and discard. Then, 1.5-2 mL trypsinizing solution was added to cover the flask surface, which was then left at room temperature for 3 min until most of the cells detached. Subsequently, 10 mL of the complete medium was added. For the MTT assay, the cell medium (100 μL) containing various concentrations of the extract (20, 40, 60, 80, 100, and 120 μg/mL) was added into all the wells and incubated in a 37°C, 5% CO2 incubator for 72 h. A stock solution of MTT in PBS (5 mg/mL) was prepared and the MTT reagent (20 μL) was added to the cell monolayer. DMSO (dimethyl sulfoxide) (100 μL) was added to each well and mixed thoroughly by pipetting 10–20 times to dissolve the blue formazan crystals. The absorbance of samples was read at 570 nm using an ELISA reader .
Experimental design and statistic analysis
Where Y is the predicted dependent variable; b0 is a constant that fixes the response at the central point of the experiment; b 1 , b 2 and b 3 are the regression coefficients for the linear effect terms; b 1 b 2 , b 1 b 3 and b 2 b 3 are the interaction effect terms and b 1 2 , b 2 2 and b 3 2 are the quadratic effect terms; respectively.
Analysis of variance (ANOVA) and response surface analysis were used to determine the statistical significance of the model. The adequacy of the model was predicted through the ANOVA (P < 0.05) and regression analysis (R2). The relationship between the response and independent variables was demonstrated using a response surface plot.
Statistical significance analysis, response surface and model fitting of catechin extraction
ANOVA for response surface models of all independent variables
Prob > F
Prob > F
Prob > F
Prob > F
Statistical parameters calculated after implementation of 2nd full factorial central composite experimental design
F-value of model
Regression ( P- value)
Lack of fit (F-value)
Lack of fit ( P-value)
Statistical significance analysis, response surface and model fitting of myricetin extraction
Analysis of variance for predicted models implied that the model was highly significant (F-value 19.77, P < 0.0001) with a good coefficient of determination (R2 = 0.97). In addition, lack-of-fit (F-value 1.85, P > 0.05) was not significant.
Statistical significance analysis, response surface and model fitting of quercetin extraction
Degradation of the flavonoid and phenolic compounds has been observed with the use of high temperatures . In addition, the Maillard reaction may occur at high temperatures, forming undesired compounds [23, 24]. Decreasing the flavonoid concentration in the extract at high temperatures by ultrasound-assisted extraction could be related to the degradation of these compounds at higher temperatures (>56°C). Pingret et al. reported that the formation of degradation products increased owing to the treatment performed with a titanium horn in the ultrasound process. Chemat et al. demonstrated that the flavor and composition of some edible oils such as hexanal and limonene are deteriorated by the ultrasound treatment (150 W).
Statistical significance analysis, response surface and model fitting of antioxidant activity
Analysis of variance for predicted models implied that the model was highly significant (F-value 17.21, P < 0.0001) with a good coefficient of determination (R2 = 0.98). Moreover, lack-of-fit (F-value 1.61, P > 0.05) was not significant (Table 3).
Optimization of responses
Verification of the ultrasound-assisted extraction model
An experiment was conducted to verify the adequacy of the developed extraction model with the predicted optimum treatment conditions (55.9°C, 80% methanol, and 145.49 W). Under these conditions, the obtained concentration of catechin, narengine, quercetin, and the antioxidant activity were 0.482, 0.517, 0.394 mg/g DW, and 83%, respectively. The results of response surface analysis for catechin, myricetin, quercetin, and the antioxidant activity were verified by comparing the predicted values with the experimental values. The obtained results from verification experiment were in consent with the predicted values, because an insignificant (P > 0.05) difference was observed between the verification experimental and the predicted values.
Evaluation of anticancer activity of optimized and unoptimized curry leaf extract
This study confirmed that anticancer activity of curry leaf extracts is associated with amount of potent flavonoid compounds because the highest anticancer activity was observed from the optimized extract, which contained higher amounts of flavonoids than the unoptimized extracts. Increased activation in the DPPH assay from the optimized extracts corroborates these earlier findings. The anticancer activity of curry leaf extracts against MDA-MB-231 and MCF-7 cell lines (breast cancer) has been reported previously [15, 29, 30]. The anticancer activity of curry leaves against HeLa cancer cells and the effective dose of this extract have been scarcely reported. Therefore, the findings of this current study could be useful for future studies.
The reduced cost of extraction is clearly advantageous for the proposed ultrasound-assisted extraction method in terms of time, energy, and the enhanced final yield . Conventional procedures such as reflux and maceration are often time and energy consuming, and are generally not always interesting from the industrial point of view. Among the others, many advantages can be pointed out when taking into account the ultrasound-assisted extraction of flavonoids from curry leaves, including fast procedure, reduction of experimental cost (time needed, energy required, equipment size), and no need for any additional treatment or chemicals to complete the experiments.
In this study, ultrasound-assisted extraction of catechin, narengine, and quercetin, and the antioxidant activity of curry leaf extracts were successfully optimized using RSM. The results indicate that the ultrasonic power, methanol dilution, and extraction temperature significantly affect the extraction yields of flavonoids with consequent enhancement of the antioxidant and anticancer activities of the extracts. The results can be easily explained by considering that both the temperature and methanol content have a positive effect on the solubility of flavonoids in the extraction solution. The ANOVA results revealed that the extraction temperature is the most significant factor influencing the response variables investigated. It was found that an ultrasonic power of 145.52 W at 55.9°C with 80% methanol was the appropriate condition for the extraction of catechin, narengine, and quercetin from curry leaves, with the consequent high anticancer and antioxidant activities. The optimized extract showed a more distinct scavenging activity against DPPH and significant anticancer activities against HeLa cancer cell lines at a concentration of 67.2 μg/mL, without toxicity to normal cells. Thus, it could be concluded that flavonoids of the curry leaves contributed to the anticancer activity of the extracts.
The authors would like to thank the Ministry of Higher Education Malaysia and the Research Management Centre, University Putra Malaysia (UPM) for sponsoring this work. This paper was part of post doctoral project (Development of Malaysian Herbal Monograph).
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