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Optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (Origanum majorana L.) using response surface methodology
Ultrasonics Sonochemistry 19 (2012) 582–590
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Optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (Origanum majorana L.) using response surface methodology Mohammad B. Hossain a,b,⇑, Nigel P. Brunton b, Ankit Patras d, Brijesh Tiwari c, C.P. O’Donnell d, Ana B. Martin-Diana e, Catherine Barry-Ryan a a
School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Ireland Teagasc, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland Manchester Metropolitan University, Manchester M14 6HR, UK d School of Biosystems Engineering, UCD, Dublin 4, Ireland e Agricultural Technological Institute of Castilla and Leon, Government of Castilla and Leon, Finca Zamadueñas, Valladolid, Castilla and Leon, Spain b c
a r t i c l e
i n f o
Article history: Received 22 July 2011 Received in revised form 24 October 2011 Accepted 1 November 2011 Available online 23 November 2011 Keywords: Antioxidant Spice Ultrasound assisted extraction Total phenols RSM
a b s t r a c t The present study optimized the ultrasound assisted extraction (UAE) conditions to maximize the antioxidant activity [Ferric ion Reducing Antioxidant Power (FRAP)], total phenol content (TP) and content of individual polyphenols of extracts from marjoram. Optimal conditions with regard to amplitude of sonication (24.4–61.0 lm) and extraction temperature (15–35 °C) and extraction time (5–15 min) were identified using response surface methodology (RSM). The results showed that the combined treatment conditions of 61 lm, 35 °C and 15 min were optimal for maximizing TP, FRAP, rosmarinic acid, luteolin-7O-glucoside, apigenin-7-O-glucoside, caffeic acid, carnosic acid and carnosol values of the extracts. The predicted values from the developed quadratic polynomial equation were in close agreement with the actual experimental values with low average mean deviations (E%) ranging from 0.45% to 1.55%. The extraction yields of the optimal UAE were significantly (p < 0.05) higher than solid/liquid extracts. Predicted models were highly significant (p < 0.05) for all the parameters studied with high regression coefficients (R2) ranging from 0.58 to 0.989. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Marjoram has been traditionally used for the treatment of gastrointestinal disturbances, cough and bronchial diseases. Marjoram is used in mouthwashes for oral hygiene and also applied topically to relieve symptoms of the common cold, such as nasal congestion [1]. Several studies reported that methanolic extracts of marjoram had high antioxidant capacity [2,3] mostly due to the polyphenolic compounds present in them. Recently, interest has increased considerably in naturally occurring antioxidants for use in foods or medicinal materials as replacements for synthetic antioxidants such as BHA and BHT, whose use is restricted due to concerns over safety [4,5]. Natural antioxidants can protect the human body from free radicals and are reported to retard the progress of many chronic diseases as well as lipid oxidative rancidity in foods [6–8]. A host of potentially beneficial physiological effects have been postulated for antioxidants over the past three decades which are supported by extensive animal studies. Among these are ⇑ Corresponding author at: School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Ireland. Fax: +353 (0) 18059550. E-mail address: [email protected] (M.B. Hossain). 1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2011.11.001
beneficial influences on lipid metabolism, efficacy as anti-diabetic, ability to stimulate digestion, antioxidant property, anti-carcinogenic and anti-inflammatory potential [9,10]. Oxidation of polyunsaturated fatty acids not only lowers the nutritional value of food [11], but is also associated with cell membrane damage, aging, heart disease and cancer in living organisms [12]. Therefore the addition of natural antioxidants to food products has become popular as a means of increasing self life and to reduce wastage and nutritional losses by inhibiting and delaying oxidation [13]. However, an efficient extraction technique is required in order to harvest the benefits of natural antioxidants present in marjoram. A number of techniques are available for the extraction of natural antioxidants from plants, including ultrasound-assisted extraction, supercritical fluid extraction, microwave-assisted extraction, and solvent extraction [14,15]. Among these, ultrasound-assisted extraction (UAE] offers an inexpensive, environmentally friendly, less time consuming and efficient alternative to conventional extraction techniques. The enhancement in extraction obtained by using ultrasound is mainly attributed to the effect of acoustic cavitations produced in the solvent by the passage of an ultrasound wave [16,17]. Ultrasound also offers a mechanical effect allowing greater penetration of solvent into the sample matrix, increasing the contact surface area between
Carnosic acid
1.16 ± 0.05 1.19 ± 0.03 1.19 ± 0.06 0.95 ± 0.02 1.31 ± 0.07 1.51 ± 0.09 1.39 ± 0.06 1.28 ± 0.04 1.31 ± 0.03 0.75 ± 0.01 1.31 ± 0.04 1.29 ± 0.03 1.24 ± 0.01 1.19 ± 0.02 1.17 ± 0.03 1.24 ± 0.05 1.24 ± 0.04
Apigenin-7-Oglucoside mg/g DW
42.70 24.40 24.40 42.70 61.00 61.00 61.00 42.70 42.70 24.40 42.70 61.00 42.70 24.40 42.70 42.70 42.70
(0) (1) (1) (0) (+1) (+1) (+1) (0) (0) (1) (0) (+1) (0) (1) (0) (0) (0)
25 25 35 15 25 35 25 15 25 15 35 15 25 25 25 35 25
(0) (0) (+1) (1) (0) (+1) (0) (1) (0) (1) (+1) (1) (0) (0) (0) (+1) (0)
10 (0) 5 (1) 10 (0) 5 (1) 5 (1) 10 (0) 15 (+1) 15 (+1) 10 (0) 10 (0) 15 (+1) 10 (0) 10 (0) 15 (+1) 10 (0) 5 (1) 10 (0)
7.90 ± 0.18 7.49 ± 0.14 8.01 ± 0.12 8.16 ± 0.11 7.78 ± 0.15 9.62 ± 0.10 8.36 ± 0.09 7.92 ± 0.13 7.78 ± 0.12 7.74 ± 0.14 9.10 ± 0.16 8.21 ± 0.15 8.00 ± 0.17 7.95 ± 0.16 7.69 ± 0.08 8.17 ± 0.11 8.00 ± 0.18
14.27 ± 0.21 13.76 ± 0.22 15.07 ± 0.25 14.94 ± 0.21 14.66 ± 0.19 17.08 ± 0.27 16.53 ± 0.17 14.61 ± 0.20 14.36 ± 0.22 13.68 ± 0.16 16.72 ± 0.12 15.29 ± 0.27 14.55 ± 0.24 13.45 ± 0.21 14.45 ± 0.23 14.67 ± 0.29 14.55 ± 0.28
23.01 ± 0.38 21.88 ± 0.25 22.90 ± 0.29 22.01 ± 0.34 23.30 ± 0.41 24.41 ± 0.62 23.74 ± 0.38 23.49 ± 0.31 22.78 ± 0.33 21.92 ± 0.36 24.09 ± 0.37 23.27 ± 0.29 23.07 ± 0.33 22.45 ± 0.30 22.85 ± 0.38 23.53 ± 0.41 23.07 ± 0.39
0.12 ± 0.005 0.12 ± 0.004 0.12 ± 0.008 0.14 ± 0.014 0.12 ± 0.009 0.15 ± 0.010 0.12 ± 0.008 0.13 ± 0.005 0.12 ± 0.007 0.13 ± 0.011 0.13 ± 0.008 0.14 ± 0.010 0.11 ± 0.004 0.12 ± 0.003 0.12 ± 0.006 0.12 ± 0.009 0.11 ± 0.005
4.53 ± 0.08 4.10 ± 0.15 4.11 ± 0.10 4.59 ± 0.11 4.84 ± 0.12 5.25 ± 0.18 4.96 ± 0.14 4.65 ± 0.11 4.63 ± 0.09 4.42 ± 0.14 4.86 ± 0.07 4.73 ± 0.10 4.62 ± 0.13 4.00 ± 0.11 4.58 ± 0.10 4.66 ± 0.16 4.62 ± 0.12
Luteolin-7-Oglucoside Caffeic acid Rosmarinic acid FRAP (g Trolox/100 g DW) TP (g GAE/100 g DW)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Solid/liquid extractions were carried out according to the method of Shan et al. [3] with slight modifications. Dried and ground samples (0.5 g) were homogenized for 1 min at 24,000 rpm using an Ultra-Turrax T-25 Tissue homogenizer (Janke and Kunkel, IKAÒ-Labortechnik, Saufen, Germany) in 25 mL of 80% methanol at room temperature (23 °C). The homogenized sample suspension was shaken at 1500 rpm for 3 h at room temperature (ffi25 °C) using a V400 Multitude Vortexer (Alpha laboratories, North York, Canada). The sample suspension was then centrifuged for 15 min at 2000g (MSE Mistral 3000i, Sanyo Gallenkamp, Leicestershire, UK) and filtered through 0.45 lm polytetrafluoethylene (PTFE) filters. The extracts were stored at 20 °C until subsequent analysis. The experiment was performed in two batches and included three replications of each sample.
Time (min)
2.3. Conventional solid/liquid extraction
Temperature (°C)
A 1500 W ultrasonic processor (VC 1500, Sonics and Materials Inc., Newtown, USA) with a 19 mm diameter probe was used for sonication. Samples were processed at a constant frequency of 20 kHz. The energy input was controlled by setting the amplitude of the sonicator probe. Extrinsic parameters of amplitude (24.4– 61.0 lm), temperature (15–35 °C) and extraction time (5–15 min) were varied with pulse durations of 5 s on and 5 s off. Dried leaf particles of marjoram (1 g) were placed in a 50 mL jacketed vessel through which water was circulated at 15 ± 0.5, 25 ± 0.5 and 35 ± 0.5 °C with a flow rate of 0.5 L/min. Sonication at the desired amplitude level was started once the set temperature was reached. The ultrasound probe was submerged to a depth of 25 mm in the sample. The input range of the selected variables was determined by preliminary experiments. The three independent variables were coded at three levels (1, 0, +1), which resulted in an experimental design of 17 experimental points, including five central points. The actual values of the factors with their coded terms for the experimental designs are given in Table 1. Statistical analyzes were carried out on the basis of actual values. All treatments were carried out in triplicate.
Amplitude (lm)
2.2. Sonication treatment
Table 1 Combinations of amplitude, temperature and time with their coded terms obtained from RSM and the respective values of TP, FRAP and content of different phenolic compounds.
2.1. Samples and reagents
Run
2. Materials and methods
Dried and ground marjoram leaf was provided by Allinall Ingredients Limited, Dublin 12. The country of origin of the spices was Turkey. The plants were grown in sunny and well drained land with annual rainfall of around 38 cm. According to the product specifications the samples were air dried at ambient temperature (23 °C) after heat treatment (steam sterilization at 120 °C for 30 s). Folin–Ciocalteu Reagent, gallic acid, sodium acetate anhydrous, ferric chloride hexahydrate, 2,4,6-Tri(2-pyridyl)-s-triazine, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sodium carbonate, caffeic acid, rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, carnosic acid and carnosol were purchased from Sigma–Aldrich.
9.79 ± 0.17 8.77 ± 0.15 8.58 ± 0.21 9.78 ± 0.20 9.81 ± 0.18 10.29 ± 0.15 9.87 ± 0.17 9.74 ± 0.15 9.67 ± 0.21 8.90 ± 0.18 9.85 ± 0.19 9.79 ± 0.14 9.74 ± 0.12 8.76 ± 0.11 9.67 ± 0.17 9.91 ± 0.20 9.74 ± 0.19
Carnosol
the solid and liquid phase, and as a result, the solute more rapidly diffuses from the solid phase into the solvent [18,19]. In this study, UAE parameters such as extraction temperature, extraction time and amplitude of ultrasound were optimized using response surface methodology (RSM), by employing a Box–Behnken design to maximize extraction of antioxidant polyphenolic compounds from marjoram.
1.42 ± 0.08 1.42 ± 0.07 1.32 ± 0.05 1.32 ± 0.08 1.54 ± 0.09 1.76 ± 0.07 1.66 ± 0.04 1.48 ± 0.06 1.54 ± 0.03 1.28 ± 0.02 1.51 ± 0.06 1.66 ± 0.09 1.43 ± 0.07 1.50 ± 0.06 1.45 ± 0.06 1.58 ± 0.04 1.49 ± 0.07
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A
8.58 8.37
8.98 8.78
8.17
30.00
25.00
7.67
7.76
7.86
FRAP (g Trolox/100 g DW)
35.00
Temperature (°C)
Temperature (°C)
B
TP (g GAE/100 g DW)
35.00
7.97
20.00
15.00
16.33 16.01 15.70 15.38
30.00
14.76 15.07 14.44 25.00
14.13 13.81
20.00
15.00
24.40
33.55
42.70
51.85
61.00
24.40
33.55
Amplitude (µm)
C
D
TP (g GAE/100 g DW)
15.00
42.70
51.85
61.00
Amplitude (µm)
FRAP (g Trolox/100 g DW)
15.00
16.17
8.36
15.84
8.24
12.50
15.51
12.50
Time (min)
Time (min)
15.18 8.11 7.86
7.73
10.00
7.98
13.76
14.84
13.95
10.00
14.18
14.51
7.61 7.50
7.50
7.48
5.00
5.00
24.40
33.55
42.70
51.85
61.00
24.40
Amplitude (µm)
E
42.70
51.85
61.00
Amplitude (µm)
F
TP (g GAE/100 g DW)
15.00
33.55
FRAP (g Trolox/100 g DW)
15.00
16.17 8.78
15.51
8.56
8.33 7.82 7.89 7.98 8.11
7.89 10.00
15.84
12.50
7.85
Time (min)
Time (min)
12.50
14.39
7.78
14.51
10.00
14.84
14.29 14.51
7.50
15.18
14.35
14.22
7.50
7.71 14.15 5.00 15.00
5.00 20.00
25.00
30.00
35.00
Temperature (°C)
15.00
20.00
25.00
30.00
35.00
Temperature (°C)
Fig. 1. Contour plots showing the effect of amplitude, temperature and time on total phenol content (A, C, E) and antioxidant activity as measured by FRAP (B, D, F).
2.4. Determination of total phenol (TP) The total phenolic content was determined using Folin– Ciocalteu Reagent (FCR) as described by Singelton et al. [20]. The
experiment was performed in two batches which included three replications of each sample and standard. Methanolic gallic acid solutions (10–400 mg/L) were used as standards. In each replicate, 100 lL of the appropriately diluted sample extract, 100 lL
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methanol, 100 lL FCR and finally 700 lL Na2CO3 (20%) were added together and vortexed. The mixture was incubated for 20 min in the dark at room temperature. After incubation the mixture was centrifuged at 13,000 rpm for 3 min. The absorbance of the supernatant was measured at 735 nm using a spectrophotometer. The total phenolic content was expressed as gallic acid equivalent (GAE)/100 g dry weight (DW) of the sample. 2.5. Ferric ion reducing antioxidant power (FRAP) assay The FRAP assay was carried out as described by Stratil et al. [21] with slight modifications. The FRAP reagent was prepared by mixing 38 mM sodium acetate anhydrous in distilled water pH 3.6, 20 mM FeCl36H2O in distilled water and 10 mM 2,4,6-Tri (2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl in a proportion of 10:1:1. This reagent was freshly prepared before each experiment. Hundred microlitre of appropriately diluted sample extract and 900 lL of FRAP reagent was added to each sample and the mixture was incubated at 37 °C for 40 min in the dark. In the case of the blank, 100 lL of methanol was added to 900 lL of FRAP reagent. The absorbance of the resulting solution was measured at 593 nm using a spectrophotometer. Trolox (6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) (a synthetic antioxidant) at concentrations from 0.1 to 0.4 mM was used as a reference antioxidant standard. FRAP values were expressed as g Trolox/100 g DW of the sample. 2.6. HPLC analysis of the extracts Reversed phase high performance liquid chromatography (RP-HPLC) of the filtered sample extracts was carried out according to the method of Tsao and Yang [22]. The chromatographic system (Shimadzu-Model No. SPD-M10A VP, Mason Technology, Dublin 8, Ireland) consisted of a pump, a vacuum degasser, a diode-array detector and was controlled through EZ Start 7.3 software (Shimadzu) at 37 °C. An Agilent C18 column (15 cm 4.6 cm, 5 lm, Agilent Technologies, USA) was utilized with a binary mobile phase
of 6% acetic acid in 2 mM sodium acetate (final pH 2.55, v/v, solvent A) and acetonitrile (solvent B). Solvent A was prepared first by mixing 2 mM sodium acetate water solution, with acetic acid at a ratio of 94:6 by volume. All solvents were filtered through a 0.45 lm diameter membrane filter prior to analysis. The flow rate was kept constant at 1.0 mL/min for a total run time of 80 min. The following gradient program was carried out: 0–15% B in 45 min, 15–30% B in 15 min, 30–50% B in 5 min, 50–100% B in 5 min and 100–0% B in 5 min. The injection volume for all the samples was 10 lL. All the standards for quantification purposes were dissolved in methanol. The detection wavelength of 280 nm was used for the detection of carnosol and carnosic acid. Rosmarinic acid, caffeic acid and apigenin-7-O-glucoside were detected at 320 nm while luteilon-7-O-glucoside was detected at 360 nm. Identification of the compounds was achieved by comparing their retention times and UV–Vis spectra with those of authenticated standards by using the inline DAD with a 3D feature. Results are expressed as mean values of three assays for each replicated experiment. 2.7. Experimental design and data analysis Polynomial regression equations were developed to describe the effects of the 3 independent processing parameters; ultrasound amplitude (X1, lm), extraction temperature (X2, °C) and extraction time (X3, min) on total phenol (TP), antioxidant activity as measured by FRAP and different polyphenolic compounds such as rosmarinic acid, caffeic acid, luteolin-7-O-glucoside, apigenin-7-Oglucoside, carnosol and carnosic acid. Independent variables of amplitude level (X1) (24.4, 42.7, and 61 lm), temperature (X2) (15, 25, and 35 °C), and extraction time (X3) (5, 10 and 15 min) were varied to investigate the effects on the dependent variables mentioned above. The general form of the quadratic polynomial model regression equation employed in this study is presented in Eq. (1). By using this equation, linear (X1, X2, X3), quadratic (X 21 , X 22 , X 23 ) and interactive (X1X2, X1X3, X2X3) effects of independent variables, temperature (X1), amplitude level (X2), and time (X3) on dependent variable (Y) were determined:
Table 2 Analysis of the variance of the regression coefficients of the fitted polynomial quadratic equation for TP (g GAE/ 100 g DW), FRAP (g Trolox/100 g DW) and different polyphenols (mg/g DW).
ns
Coefficients
TP
FRAP
Rosmarinic acid Luteolin-7-O-glucoside Apigenin-7-O-glucoside Caffeic acid
b0 (Intercept)
+11.87
+20.10
+19.89
Carnosic acid
Carnosol
+4.96
+0.447
+0.248
+7.64
+1.17
Linear b1 (Amplitude) 1.98 102c 7.65 103d +3.8 102d b2 (Temperature) 3.3 101c 4.3 101d 4.98 102d b3 (Time) 1.01 101b 4.69 101c +1.91 101d
+1.72 102d 1.02 101a –
+8.04 103c +1.23 102b –
– – –
+1.1 101d – –
+7.45 103c – –
Quadratic b11 b22 b33
– +4.83 103c –
– +7.48 103d –
– +3.04 103b –
2.80 104a +1.20 103b –
– – –
– 1.27 103d – +1.50 104b +9.14 104a – – – –
Cross product b12 b13 b23
+1.55 103a – +5.79 103a
– – 4.61 103a
+1.13 103d
+5.95 103c +1.19 102c
– – –
+3.72 105a +1.1 103b – – – –
– – –
R2 Predicted R2 CV p Lack of fit F-value
0.917 0.635 2.39 <0.0001 0.1804 17.89
0.976 0.905 1.36 <0.0001 0.1046 68.32
0.964 0.872 0.71 <0.0001 0.183 30.34
0.966 0.906 1.52 <0.0001 0.147 55.27
0.721 0.450 8.04 0.0007 0.137 11.23
0.750 0.271 4.97 0.0013 0.223 9.02
0.58 0.44 5.70 0.0004 0.108 20.61
Not significant. a Significant at b Significant at c Significant at d Significant at
p 6 0.05. p 6 0.01. p 6 0.001. p 6 0.0001.
0.989 0.951 0.64 <0.0001 0.347 200.12
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A
23.95 23.72
24.17
0.13 0.13 0.13
23.50
30.00
22.82 23.05
23.27
22.60
25.00
22.38 22.15 20.00
30.00
0.11
33.55
42.70
51.85
C
0.12 0.12
20.00
0.14
24.40
61.00
0.13 0.13
0.13 33.55
42.70
51.85
61.00
Amplitude (µm)
Amplitude (µm)
35.00
0.12
25.00
15.00
15.00 24.40
Caffeic acid (mg/g DW )
35.00
Temperature (°C)
Temperature (°C)
B
Rosmarinic acid (mg/g DW)
35.00
D
Luteolin-7-O-glucoside (mg/g DW )
35.00
5.18 5.07
Apigenin-7-O-glucoside (mg/g DW ) 1.41
30.00
4.84
4.18 4.62
4.29
25.00
Temperature (°C)
Temperature (°C)
4.96
4.40
4.73
4.51
20.00
30.00
1.35 1.30 1.22
25.00
1.26
1.16 1.11
20.00
1.06 1.00
15.00 24.40
15.00 33.55
42.70
51.85
24.40
61.00
33.55
E
51.85
61.00
Amplitude (µm)
Amplitude (µm)
F
Carnosic acid (mg/g DW )
35.00
42.70
Carnosol (mg/g DW)
35.00
10.15
30.00
Temperature (°C)
Temperature (°C)
1.64 10.01 9.93 25.00
8.87 9.72 9.84 9.10 9.33 9.56 9.86
9.84
20.00
15.00 24.40
30.00
1.61 1.42
25.00
1.45
1.49
1.53
1.57
1.38 20.00
1.34
15.00 33.55
42.70
51.85
61.00
Amplitude (µm)
24.40
33.55
42.70
51.85
61.00
Amplitude (µm)
Fig. 2. Contour plots showing the effect of amplitude and temperature on the extraction of rosmarinic acid (A), caffeic acid (B), luteolin-7-O-glucoside (C), apigenin-7-Oglucoside (D), carnosic acid (E) and carnosol (F) at treatment time of 10 min.
Y ¼ b0 þ
3 X i¼1
bi X i þ
3 X i¼1
bii X 2i þ
2 3 X X i¼1 j¼iþ1
bij X i X j
ð1Þ
where Y is the predicted response; b0 the constant (intercept); bi the linear coefficient; bii the quadratic coefficient and bij is the cross
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M.B. Hossain et al. / Ultrasonics Sonochemistry 19 (2012) 582–590
product coefficient. Xi and Xj are independent variables. The response surface regression was used to analyze the experimental data using Design Expert Version 7.1.3 software (Stat-Ease Inc.,
A
Minneapolis, MN). Two dimensional contour plots were developed while holding a variable constant in the second order polynomial models. All processing trials were conducted in triplicate.
B
Rosmarinic acid (mg/g DW)
15.00
Caffeic acid (mg/g DW )
15.00
23.74 0.11 23.57 12.50
23.36 10.00
22.75
22.48
Time (min)
Time (min)
12.50
22.98 23.18
0.11 0.11
0.12 0.11 0.11
10.00
22.20
0.12
0.12
0.12
0.11 7.50
7.50
0.11 21.92 5.00 24.40
0.11 5.00 33.55
42.70
51.85
61.00
24.40
33.55
C
51.85
61.00
Amplitude (µm)
Amplitude (µm)
15.00
42.70
D
Luteolin-7-O-glucoside (mg/g DW )
15.00
Apigenin-7-O-glucoside (mg/g DW) 1.38
12.50
12.50
10.00
4.20
4.34
4.48
4.70 4.61
Time (min)
Time (min)
4.88
4.81 4.75
7.50
1.33 1.29
1.06
33.55
42.70
51.85
61.00
24.4
33.55
Amplitude (µm)
E
42.7
51.85
61
Amplitude (µm)
F
Carnosic acid (mg/g DW )
15.00
Carnosol (mg/g DW)
15.00
1.62
12.50
9.93 8.94 9.19 9.41 9.61 9.76 9.87 9.92
9.92
Time (min)
12.50
Time (min)
1.24
5.00
24.40
10.00
1.20
1.10 7.50
5.00
1.15
10.00
1.40
10.00
9.93
1.44
1.47
1.51
1.55
1.59
1.36
7.50
7.50
9.93 5.00 24.40
5.00 33.55
42.70
Amplitude (µm)
51.85
61.00
24.40
33.55
42.70
51.85
61.00
Amplitude (µm)
Fig. 3. Contour plots showing the effect of amplitude and time on the extraction of rosmarinic acid (A), caffeic acid (B), luteolin-7-O-glucoside (C), apigenin-7-O-glucoside (D), carnosic acid (E) and carnosol (F) at treatment temperature of 25 °C.
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2.8. Model validation The predictive performance of the developed models describing the combined effect of amplitude (X1), temperature (X2) and time
A
(X3) on independent variables (FRAP, TP, rosmarinic acid, caffeic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, carnosol and carnosic acid) of marjoram were validated with optimal extraction conditions as predicted by the design.
B
Rosmarinic acid (mg/g DW)
15.00
Caffeic acid (mg/g DW )
15.00
0.11
23.74
12.50
12.50
0.13
23.03 10.00
23.36
Time (min)
Time (min)
23.18 23.57
22.88 22.74
0.12
0.13 0.12
0.12 0.12
10.00
0.12 0.12 0.11
22.59 7.50
7.50
22.41
0.11
22.20 5.00
5.00 15.00
20.00
25.00
30.00
15.00
35.00
20.00
Temperature (°C)
C 15.00
25.00
30.00
35.00
Temperature (°C)
D
Luteolin-7-O-glucoside (mg/g DW )
15.00
Apigenin-7-O-glucoside (mg/g DW )
4.59 1.30323 12.50
4.75 10.00
4.61
4.60 4.59 4.60
4.70 4.61 4.65
4.58
Time (min)
Time (min)
12.50
1.22
1.25733
1.28464
1.16 10.00
1.11 1.06
7.50
7.50
4.56
1.00 1.30323
5.00
5.00
15.00
20.00
25.00
30.00
35.00
15.00
20.00
Temperature (°C)
E 15.00
25.00
30.00
35.00
Temperature (°C)
F
Carnosic acid (mg/g DW )
Carnosol (mg/g DW)
15.00
1.50 1.50 12.50
9.72 10.00
9.74
9.83 9.74 9.76 9.79
9.72
Time (min)
Time (min)
12.50
1.49 1.48 1.45
10.00
1.43
1.50
1.40 7.50
7.50
1.53
1.35
9.72 5.00 15.00
1.38
5.00 20.00
25.00
30.00
Temperature (°C)
35.00
15.00
20.00
25.00
30.00
35.00
Temperature (°C)
Fig. 4. Contour plots showing the effect of time and temperature on the extraction of rosmarinic acid (A), caffeic acid (B), luteolin-7-O-glucoside (C), apigenin-7-O-glucoside (D), carnosic acid (E) and carnosol (F) at treatment amplitude of 42.70 lm.
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E ð%Þ ¼
n V E V P 1 X 100 ne i¼1 V E
ð2Þ
where ne is the number of experimental data, VE is the experimental value and VP is the predicted value. 3. Results and discussion 3.1. Effect of thermosonication on total phenol content Fig. 1A, C, and E present the contour plots showing the effect of UAE parameters of ultrasound amplitude, temperature and time on the total phenol content of marjoram extract. All the three factors had a significant (p < 0.05) positive effect on the total phenol content of marjoram extract. Among the factors, amplitude showed the highest effect followed by temperature and time. Higher amplitude of ultrasound could have damaged more cell walls releasing more antioxidants including phenolic compounds to the solvents. An increase in temperature increases target compound solubility, solvent diffusion rate and mass transfer, while solvent viscosity and surface tension decrease [23]. Reduced viscosity and surface tension facilitate solvent penetration deeper into sample matrix which enhances the extraction efficiency by exposing more surface area of the sample to the solvent used. Increased extraction time allowed the solutes to be in contact with solvent for longer facilitating higher diffusion of the target compounds [24]. Among the treatments generated by RSM, the highest total phenolic content (9.62 g GAE/100 g DW) was observed in the extracts obtained at UAE conditions of 61 lm amplitude, 35 °C and an extraction time of 10 min of treatment. This value was 98.39% higher than that obtained with the reference solid/liquid extraction method. 3.2. Effect of thermosonication on ferric reducing antioxidant power All three factors had a significant effect on enhancing the FRAP values of the marjoram extracts. Among the factors, ultrasonic amplitude showed the highest effect followed by temperature and time (Fig. 1B, D, and F and Table 2). Temperature also had a significant (p < 0.05) effect at the quadratic level. The other two factors did not show significant (p < 0.05) quadratic effects. The effect of temperature and time was in agreement with the finding of Ghafoor et al. [24] who investigated the antioxidant activity of grape extracts obtained using a fixed level of ultrasound. The interactions of the factors, except the interaction between amplitude and temperature, showed a significant effect on the extraction. Among the ultrasonication treatments used, the amplitude level of 61 lm at a temperature of 35 °C for 10 min showed the highest FRAP value (17.08 g Trolox/100 g DW). This treatment increased the antioxidant activity as measured by FRAP by 89.76% compared to conventional solid/liquid extraction (9.00 g Trolox/100 g DW). In fact, all the ultrasonication treated extracts showed significantly (p < 0.05) higher FRAP values than that of solid/liquid extraction. When the ultrasonication amplitude was increased from the lowest level (24.4 lm) to the highest level (61 lm), the FRAP value showed an increase of 26.98%. In spices, antioxidant activity is related to their total phenol content. The high Pearsons correlation coefficients (r = 0.90) reflect the importance of phenols in the antioxidant capacity of marjoram. 3.3. Effect of thermosonication on different polyphenols The principal polyphenol of marjoram extract was rosmarinic acid [23]. Rosmarinic acid content of the extracts were significantly
(p < 0.05) affected by amplitude, temperature and time. All the factors had positive effects on the rosmarinic acid content (Figs. 2–4A and Table 2). This result was in agreement with the finding of Albu et al. [25]. The effect of amplitude was higher than that of other factors. Only temperature had a significant (p < 0.05) quadratic effect. It also had an interaction effect with time. Higher levels of time and temperature would have further increased the extraction of antioxidant polyphenols. But this would have increased the cost of extraction and an environmentally friendly extraction method requires minimal extraction time and temperature [24]. Therefore, in the present study, time and temperature range was kept low. The lowest value of rosmarinic acid content was observed at an amplitude level of 24.4 lm treated for 5 min at 25 °C. With an increase of ultrasound amplitude the rosmarinic acid content of the extracts increased gradually. At the highest amplitude and temperature investigated, the content of rosmarinic acid was 24.41 mg/g DW which was approximately two times higher than that of solid/liquid extraction (12.08 mg/g DW) (Fig. 5). An increase in rosmarinic acid extraction from dried rosemary with higher ultrasound amplitude has also been reported by Paniwnyk et al. [26]. The other hydroxycinnamic acid derivative investigated, caffeic acid, was affected by temperature at a quadratic level showing higher extractions at two ends of the temperature range used (Figs. 2B, and 4B). Temperature also showed significant (p < 0.05) positive effect in interaction with amplitude. The major flavonoids of Lamiaceae spices are luteolin-7-O-glucoside and apigenin-7-O-glucoside. Both these flavonoids showed significant (p < 0.05) increase with the increase of amplitude and temperature, while time did not have any significant effect (Figs. 2–4C, and D). In the case of luteolin-7-O-glucoside, temperature also had quadratic and interaction effects with amplitude. Amplitude played the dominant role in extracting flavonoids. The antioxidant volatile polyphenols carnosic acid and carnosol showed significant (p < 0.05) increases with the increase of amplitude (Figs. 2–4E, and F). Paniwnyk et al. [26] also found an increase of extraction of carnosic acid from rosemary with increased amplitude of ultrasonication. The other factors on linear term did not have significant effect on both the compounds. Temperature at the quadratic level and in interaction with amplitude had significant (p < 0.05) effects on the extraction of carnosic acid. 3.4. Model fitting The analysis of variance showed that the R-squared statistic of all the parameters was in the range of 0.580–0.989, indicating high representation of the variability of the parameters by the models. The quadratic polynomial models generated were highly significant with p-value ranging from 0.0013 to <0.0001. The lack of fit
1000
4
800
mAu
The criterion used to characterize the fitting efficiency of the data to the model was the multiple correlation coefficients (R2) and the average mean deviation (Eq. (2)):
600
2 1
3
400
6 5
A
56
B
4
200 2
1
3
0
0
0
20
40 Minutes
60
80
Fig. 5. HPLC chromatograms of UAE extracts of marjoram obtained at 61 lm, 35 °C and 10 min (A), in comparison to solid/liquid extraction (B) showing the changes in peaks of different polyphenols (1 = caffeic acid, 2 = luteolin-7-O-glucoside, 3 = apigenin-7-O-glucoside, 4 = rosmarinic acid, 5 = carnosol and 6 = carnosic acid).
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M.B. Hossain et al. / Ultrasonics Sonochemistry 19 (2012) 582–590
Table 3 Predicted and experimental values of the parameters tested at optimal UAE conditions in comparison to the conventional solid/liquid extraction values and average mean deviation between predicted and experimental values of optimal UAE. Parameter
Optimum UAE condition for all the parameters combined
Predicted values at optimal UAE
Desirability
Experimental values at optimal UAE
(E%)
Solid/liquid extraction values
TP (g GAE/ 100 g DW) FRAP (g Trolox/100 g DW) Rosmarinic acid (mg/g DW) Luteolin-7-O-glucoside (mg/g DW) Apigenin-7-O-glucoside (mg/g DW) Caffeic acid (mg/g DW) Carnosic acid (mg/g DW) Carnosol (mg/g DW)
61 lm, 35 °C and 15 min
9.90 18.56 24.53 5.38 1.54 0.15 10.25 1.72
0.984
9.51 ± 0.10 18.96 ± 0.19 24.86 ± 0.45 5.27 ± 0.14 1.60 ± 0.13 0.14 ± 0.01 10.63 ± 0.26 1.81 ± 0.16
1.34 0.70 0.45 0.65 1.31 1.55 1.20 1.36
4.85 ± 0.05 9.00 ± 0.17 12.08 ± 0.03 2.69 ± 0.02 0.86 ± 0.01 0.10 ± 0.01 3.56 ± 0.15 0.72 ± 0.02
statistics of all the parameters were not significant (p > 0.05) and high degree of F-value (range 9.02–200.12) further strengthened the reliability of the models (Table 2). The predicted values obtained by the quadratic polynomial equations showed strong correlation with actual experimental values with Pearsons correlation coefficients (r) from 0.88 to 0.98.
caffeic acid, carnosic acid and carnosol values of the extract. The developed prediction models showed good correlation with the experimental data at a 95% confidence level. The absolute extraction yields of the optimal UAE were significantly (p < 0.05) higher than those obtained with reference solid/liquid extraction. UAE appears to have great potential as a method for the extraction of antioxidant materials from foods of plant origin.
3.5. Optimization and model validation Acknowledgments The RSM guided optimization demonstrated that the optimum treatment conditions for maximizing the TP and FRAP values were 60.60 lm (amplitude), 34.84 °C (temperature), 14.80 min (time) and 54.34 lm (amplitude), 34.76 °C (temperature), 14.79 min (time) respectively. The antioxidant polyphenols namely rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, caffeic acid, carnosic acid and carnosol had optimum extraction conditions at the amplitude level from 58 to 61 lm with a range of extraction times from 11 to 15 min at 35 °C. The predicted optimal extraction condition for all the parameters combined was 61 lm, 35 °C and 15 min which predicted the TP, FRAP, rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, caffeic acid, carnosic acid and carnosol values as 9.90 g GAE/100 g DW, 18.56 g Trolox/100 g DW, 24.53 mg/g DW, 5.38 mg/g DW, 1.54 mg/g DW, 0.15 mg/g DW, 10.25 mg/g DW and 1.72 mg/g DW respectively. These values were significantly (p < 0.05) higher than those of solid/liquid extraction (Table 3). The predicted values were in close agreement with experimental values (Table 3) and were found to be not significantly different at p > 0.05 using a paired t-test. In addition, variations between the predicted and experimental values obtained for total antioxidant activity by FRAP assay, TP content and antioxidant polyphenols were within an acceptable error range as depicted by average mean deviation (E%, Table 3); therefore, the predictive performance of the established model may be considered acceptable. 4. Conclusions The ultrasound assisted extraction of phenolic antioxidants from marjoram appeared very effective in comparison to solid liquid extraction. Optimal process conditions with regard to amplitude of sonication (24.4–61.0 lm) and extraction temperature (15–35 °C) and treatment time (5–15 min) were identified using response surface methodology (RSM). The results demonstrated that the combined treatment of 61 lm, 35 °C and 15 min was optimal for maximizing key antioxidant parameters namely TP, FRAP, rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside,
This work was supported by the Irish Department of Agriculture, Fisheries and Food under the Food Institutional Research Measure and by the ABBEST scholarship program of Dublin Institute of Technology, Ireland. The authors would also likely to acknowledge AllinAll Ingredients Ltd. for providing spice samples. References [1] J. Bruneton. Pharmacognosy, Phytochemistry, Medicinal Plants, Technique and Documentation, Paris 1999. [2] M.B. Hossain, N.P. Brunton, C. Barry-Ryan, A.B. Martin-Diana, M. Wilkinson, Rasayan J. Chem. (2008) 751–756. [3] B. Shan, Y.Z. Cai, M. Sun, H. Corke, J. Agric. Food Chem. 53 (2005) 7749–7759. [4] M. Ito, K. Murakami, M. Yoshino, Food Chem. Toxicol. 43 (2005) 461–466. [5] W. Zheng, S.Y. Wang, J. Agric. Food Chem. 49 (2001) 5165–5170. [6] I.C. Arts, P.C. Hollman, Am. J. Clin. Nutr. 81 (2005) 317S–325S. [7] L.S. Lai, S.T. Chou, W.W. Chao, J. Agric. Food Chem. 49 (2001) 963–968. [8] G. Williamson, C. Manach, Am. J. Clin. Nutr. 81 (2005) 243S–255S. [9] K. Srinivasan, Food Res. Int. 38 (2005) 77–86. [10] J.W. Lampe, Am. J. Clin. Nutr. 78 (2003) 579S–583S. [11] V. Kristinová, R. Mozuraityte, I. Storrø, T. Rustad, J. Agric. Food Chem. 57 (2009) 10377–10385. [12] J.P. Cosgrove, D.F. Church, W.A. Pryor, Lipids 22 (1987) 299–304. [13] T. Tsuda, T. Osawa, K. Ohshima, S. Kawakishi, J. Agric. Food Chem. 42 (1994) 248–251. [14] S.B. Glisic, M. Ristic, D.U. Skala, Ultrason. Sonochem. 18 (2010) 318–326. [15] M. Vinatoru, Ultrason. Sonochem. 8 (2001) 303–313. [16] Y. Ma, X. Ye, Y. Hao, G. Xu, D. Liu, Ultrason. Sonochem. 15 (2008) 227–232. [17] D.T. Velickovic, D.M. Milenovic, M.S. Ristic, V.B. Veljkovic, Biochem. Eng. J. 42 (2008) 97–104. [18] M. Toma, M. Vinatoru, L. Paniwnyk, T.J. Mason, Ultrason. Sonochem. 8 (2001) 137–142. [19] T.J. Mason, Ultrason. Sonochem. 10 (2003) 175–179. [20] V.L. Singelton, R. Orthofer, R.R. Lamuela-Raventos, Methods Enzymol. 299 (2009) 152–178. [21] P. Stratil, B. Klejdus, V. Kuban, J. Agric. Food Chem. 54 (2006) 607–616. [22] R. Tsao, R.J. Yang, Chromatography A 1018 (2003) 29–40. [23] M.B. Hossain, C. Barry-Ryan, A.B. Martin-Diana, N.P. Brunton, Food Chem. 126 (2010) 339–346. [24] K. Ghafoor, Y.H. Choi, J.Y. Jeon, I.H. Jo, J. Agric. Food Chem. 57 (2009) 4988– 4994. [25] S. Albu, E. Joyce, L. Paniwnyk, J.P. Lorimer, T.J. Mason, Ultrason. Sonochem. 11 (2004) 261–265. [26] L. Paniwnyk, H. Cai, S. Albu, T.J. Mason, R. Cole, Ultrason. Sonochem. 16 (2009) 287–292.
Contents lists available at SciVerse ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (Origanum majorana L.) using response surface methodology Mohammad B. Hossain a,b,⇑, Nigel P. Brunton b, Ankit Patras d, Brijesh Tiwari c, C.P. O’Donnell d, Ana B. Martin-Diana e, Catherine Barry-Ryan a a
School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Ireland Teagasc, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland Manchester Metropolitan University, Manchester M14 6HR, UK d School of Biosystems Engineering, UCD, Dublin 4, Ireland e Agricultural Technological Institute of Castilla and Leon, Government of Castilla and Leon, Finca Zamadueñas, Valladolid, Castilla and Leon, Spain b c
a r t i c l e
i n f o
Article history: Received 22 July 2011 Received in revised form 24 October 2011 Accepted 1 November 2011 Available online 23 November 2011 Keywords: Antioxidant Spice Ultrasound assisted extraction Total phenols RSM
a b s t r a c t The present study optimized the ultrasound assisted extraction (UAE) conditions to maximize the antioxidant activity [Ferric ion Reducing Antioxidant Power (FRAP)], total phenol content (TP) and content of individual polyphenols of extracts from marjoram. Optimal conditions with regard to amplitude of sonication (24.4–61.0 lm) and extraction temperature (15–35 °C) and extraction time (5–15 min) were identified using response surface methodology (RSM). The results showed that the combined treatment conditions of 61 lm, 35 °C and 15 min were optimal for maximizing TP, FRAP, rosmarinic acid, luteolin-7O-glucoside, apigenin-7-O-glucoside, caffeic acid, carnosic acid and carnosol values of the extracts. The predicted values from the developed quadratic polynomial equation were in close agreement with the actual experimental values with low average mean deviations (E%) ranging from 0.45% to 1.55%. The extraction yields of the optimal UAE were significantly (p < 0.05) higher than solid/liquid extracts. Predicted models were highly significant (p < 0.05) for all the parameters studied with high regression coefficients (R2) ranging from 0.58 to 0.989. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Marjoram has been traditionally used for the treatment of gastrointestinal disturbances, cough and bronchial diseases. Marjoram is used in mouthwashes for oral hygiene and also applied topically to relieve symptoms of the common cold, such as nasal congestion [1]. Several studies reported that methanolic extracts of marjoram had high antioxidant capacity [2,3] mostly due to the polyphenolic compounds present in them. Recently, interest has increased considerably in naturally occurring antioxidants for use in foods or medicinal materials as replacements for synthetic antioxidants such as BHA and BHT, whose use is restricted due to concerns over safety [4,5]. Natural antioxidants can protect the human body from free radicals and are reported to retard the progress of many chronic diseases as well as lipid oxidative rancidity in foods [6–8]. A host of potentially beneficial physiological effects have been postulated for antioxidants over the past three decades which are supported by extensive animal studies. Among these are ⇑ Corresponding author at: School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Ireland. Fax: +353 (0) 18059550. E-mail address: [email protected] (M.B. Hossain). 1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2011.11.001
beneficial influences on lipid metabolism, efficacy as anti-diabetic, ability to stimulate digestion, antioxidant property, anti-carcinogenic and anti-inflammatory potential [9,10]. Oxidation of polyunsaturated fatty acids not only lowers the nutritional value of food [11], but is also associated with cell membrane damage, aging, heart disease and cancer in living organisms [12]. Therefore the addition of natural antioxidants to food products has become popular as a means of increasing self life and to reduce wastage and nutritional losses by inhibiting and delaying oxidation [13]. However, an efficient extraction technique is required in order to harvest the benefits of natural antioxidants present in marjoram. A number of techniques are available for the extraction of natural antioxidants from plants, including ultrasound-assisted extraction, supercritical fluid extraction, microwave-assisted extraction, and solvent extraction [14,15]. Among these, ultrasound-assisted extraction (UAE] offers an inexpensive, environmentally friendly, less time consuming and efficient alternative to conventional extraction techniques. The enhancement in extraction obtained by using ultrasound is mainly attributed to the effect of acoustic cavitations produced in the solvent by the passage of an ultrasound wave [16,17]. Ultrasound also offers a mechanical effect allowing greater penetration of solvent into the sample matrix, increasing the contact surface area between
Carnosic acid
1.16 ± 0.05 1.19 ± 0.03 1.19 ± 0.06 0.95 ± 0.02 1.31 ± 0.07 1.51 ± 0.09 1.39 ± 0.06 1.28 ± 0.04 1.31 ± 0.03 0.75 ± 0.01 1.31 ± 0.04 1.29 ± 0.03 1.24 ± 0.01 1.19 ± 0.02 1.17 ± 0.03 1.24 ± 0.05 1.24 ± 0.04
Apigenin-7-Oglucoside mg/g DW
42.70 24.40 24.40 42.70 61.00 61.00 61.00 42.70 42.70 24.40 42.70 61.00 42.70 24.40 42.70 42.70 42.70
(0) (1) (1) (0) (+1) (+1) (+1) (0) (0) (1) (0) (+1) (0) (1) (0) (0) (0)
25 25 35 15 25 35 25 15 25 15 35 15 25 25 25 35 25
(0) (0) (+1) (1) (0) (+1) (0) (1) (0) (1) (+1) (1) (0) (0) (0) (+1) (0)
10 (0) 5 (1) 10 (0) 5 (1) 5 (1) 10 (0) 15 (+1) 15 (+1) 10 (0) 10 (0) 15 (+1) 10 (0) 10 (0) 15 (+1) 10 (0) 5 (1) 10 (0)
7.90 ± 0.18 7.49 ± 0.14 8.01 ± 0.12 8.16 ± 0.11 7.78 ± 0.15 9.62 ± 0.10 8.36 ± 0.09 7.92 ± 0.13 7.78 ± 0.12 7.74 ± 0.14 9.10 ± 0.16 8.21 ± 0.15 8.00 ± 0.17 7.95 ± 0.16 7.69 ± 0.08 8.17 ± 0.11 8.00 ± 0.18
14.27 ± 0.21 13.76 ± 0.22 15.07 ± 0.25 14.94 ± 0.21 14.66 ± 0.19 17.08 ± 0.27 16.53 ± 0.17 14.61 ± 0.20 14.36 ± 0.22 13.68 ± 0.16 16.72 ± 0.12 15.29 ± 0.27 14.55 ± 0.24 13.45 ± 0.21 14.45 ± 0.23 14.67 ± 0.29 14.55 ± 0.28
23.01 ± 0.38 21.88 ± 0.25 22.90 ± 0.29 22.01 ± 0.34 23.30 ± 0.41 24.41 ± 0.62 23.74 ± 0.38 23.49 ± 0.31 22.78 ± 0.33 21.92 ± 0.36 24.09 ± 0.37 23.27 ± 0.29 23.07 ± 0.33 22.45 ± 0.30 22.85 ± 0.38 23.53 ± 0.41 23.07 ± 0.39
0.12 ± 0.005 0.12 ± 0.004 0.12 ± 0.008 0.14 ± 0.014 0.12 ± 0.009 0.15 ± 0.010 0.12 ± 0.008 0.13 ± 0.005 0.12 ± 0.007 0.13 ± 0.011 0.13 ± 0.008 0.14 ± 0.010 0.11 ± 0.004 0.12 ± 0.003 0.12 ± 0.006 0.12 ± 0.009 0.11 ± 0.005
4.53 ± 0.08 4.10 ± 0.15 4.11 ± 0.10 4.59 ± 0.11 4.84 ± 0.12 5.25 ± 0.18 4.96 ± 0.14 4.65 ± 0.11 4.63 ± 0.09 4.42 ± 0.14 4.86 ± 0.07 4.73 ± 0.10 4.62 ± 0.13 4.00 ± 0.11 4.58 ± 0.10 4.66 ± 0.16 4.62 ± 0.12
Luteolin-7-Oglucoside Caffeic acid Rosmarinic acid FRAP (g Trolox/100 g DW) TP (g GAE/100 g DW)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Solid/liquid extractions were carried out according to the method of Shan et al. [3] with slight modifications. Dried and ground samples (0.5 g) were homogenized for 1 min at 24,000 rpm using an Ultra-Turrax T-25 Tissue homogenizer (Janke and Kunkel, IKAÒ-Labortechnik, Saufen, Germany) in 25 mL of 80% methanol at room temperature (23 °C). The homogenized sample suspension was shaken at 1500 rpm for 3 h at room temperature (ffi25 °C) using a V400 Multitude Vortexer (Alpha laboratories, North York, Canada). The sample suspension was then centrifuged for 15 min at 2000g (MSE Mistral 3000i, Sanyo Gallenkamp, Leicestershire, UK) and filtered through 0.45 lm polytetrafluoethylene (PTFE) filters. The extracts were stored at 20 °C until subsequent analysis. The experiment was performed in two batches and included three replications of each sample.
Time (min)
2.3. Conventional solid/liquid extraction
Temperature (°C)
A 1500 W ultrasonic processor (VC 1500, Sonics and Materials Inc., Newtown, USA) with a 19 mm diameter probe was used for sonication. Samples were processed at a constant frequency of 20 kHz. The energy input was controlled by setting the amplitude of the sonicator probe. Extrinsic parameters of amplitude (24.4– 61.0 lm), temperature (15–35 °C) and extraction time (5–15 min) were varied with pulse durations of 5 s on and 5 s off. Dried leaf particles of marjoram (1 g) were placed in a 50 mL jacketed vessel through which water was circulated at 15 ± 0.5, 25 ± 0.5 and 35 ± 0.5 °C with a flow rate of 0.5 L/min. Sonication at the desired amplitude level was started once the set temperature was reached. The ultrasound probe was submerged to a depth of 25 mm in the sample. The input range of the selected variables was determined by preliminary experiments. The three independent variables were coded at three levels (1, 0, +1), which resulted in an experimental design of 17 experimental points, including five central points. The actual values of the factors with their coded terms for the experimental designs are given in Table 1. Statistical analyzes were carried out on the basis of actual values. All treatments were carried out in triplicate.
Amplitude (lm)
2.2. Sonication treatment
Table 1 Combinations of amplitude, temperature and time with their coded terms obtained from RSM and the respective values of TP, FRAP and content of different phenolic compounds.
2.1. Samples and reagents
Run
2. Materials and methods
Dried and ground marjoram leaf was provided by Allinall Ingredients Limited, Dublin 12. The country of origin of the spices was Turkey. The plants were grown in sunny and well drained land with annual rainfall of around 38 cm. According to the product specifications the samples were air dried at ambient temperature (23 °C) after heat treatment (steam sterilization at 120 °C for 30 s). Folin–Ciocalteu Reagent, gallic acid, sodium acetate anhydrous, ferric chloride hexahydrate, 2,4,6-Tri(2-pyridyl)-s-triazine, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sodium carbonate, caffeic acid, rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, carnosic acid and carnosol were purchased from Sigma–Aldrich.
9.79 ± 0.17 8.77 ± 0.15 8.58 ± 0.21 9.78 ± 0.20 9.81 ± 0.18 10.29 ± 0.15 9.87 ± 0.17 9.74 ± 0.15 9.67 ± 0.21 8.90 ± 0.18 9.85 ± 0.19 9.79 ± 0.14 9.74 ± 0.12 8.76 ± 0.11 9.67 ± 0.17 9.91 ± 0.20 9.74 ± 0.19
Carnosol
the solid and liquid phase, and as a result, the solute more rapidly diffuses from the solid phase into the solvent [18,19]. In this study, UAE parameters such as extraction temperature, extraction time and amplitude of ultrasound were optimized using response surface methodology (RSM), by employing a Box–Behnken design to maximize extraction of antioxidant polyphenolic compounds from marjoram.
1.42 ± 0.08 1.42 ± 0.07 1.32 ± 0.05 1.32 ± 0.08 1.54 ± 0.09 1.76 ± 0.07 1.66 ± 0.04 1.48 ± 0.06 1.54 ± 0.03 1.28 ± 0.02 1.51 ± 0.06 1.66 ± 0.09 1.43 ± 0.07 1.50 ± 0.06 1.45 ± 0.06 1.58 ± 0.04 1.49 ± 0.07
M.B. Hossain et al. / Ultrasonics Sonochemistry 19 (2012) 582–590
583
584
M.B. Hossain et al. / Ultrasonics Sonochemistry 19 (2012) 582–590
A
8.58 8.37
8.98 8.78
8.17
30.00
25.00
7.67
7.76
7.86
FRAP (g Trolox/100 g DW)
35.00
Temperature (°C)
Temperature (°C)
B
TP (g GAE/100 g DW)
35.00
7.97
20.00
15.00
16.33 16.01 15.70 15.38
30.00
14.76 15.07 14.44 25.00
14.13 13.81
20.00
15.00
24.40
33.55
42.70
51.85
61.00
24.40
33.55
Amplitude (µm)
C
D
TP (g GAE/100 g DW)
15.00
42.70
51.85
61.00
Amplitude (µm)
FRAP (g Trolox/100 g DW)
15.00
16.17
8.36
15.84
8.24
12.50
15.51
12.50
Time (min)
Time (min)
15.18 8.11 7.86
7.73
10.00
7.98
13.76
14.84
13.95
10.00
14.18
14.51
7.61 7.50
7.50
7.48
5.00
5.00
24.40
33.55
42.70
51.85
61.00
24.40
Amplitude (µm)
E
42.70
51.85
61.00
Amplitude (µm)
F
TP (g GAE/100 g DW)
15.00
33.55
FRAP (g Trolox/100 g DW)
15.00
16.17 8.78
15.51
8.56
8.33 7.82 7.89 7.98 8.11
7.89 10.00
15.84
12.50
7.85
Time (min)
Time (min)
12.50
14.39
7.78
14.51
10.00
14.84
14.29 14.51
7.50
15.18
14.35
14.22
7.50
7.71 14.15 5.00 15.00
5.00 20.00
25.00
30.00
35.00
Temperature (°C)
15.00
20.00
25.00
30.00
35.00
Temperature (°C)
Fig. 1. Contour plots showing the effect of amplitude, temperature and time on total phenol content (A, C, E) and antioxidant activity as measured by FRAP (B, D, F).
2.4. Determination of total phenol (TP) The total phenolic content was determined using Folin– Ciocalteu Reagent (FCR) as described by Singelton et al. [20]. The
experiment was performed in two batches which included three replications of each sample and standard. Methanolic gallic acid solutions (10–400 mg/L) were used as standards. In each replicate, 100 lL of the appropriately diluted sample extract, 100 lL
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methanol, 100 lL FCR and finally 700 lL Na2CO3 (20%) were added together and vortexed. The mixture was incubated for 20 min in the dark at room temperature. After incubation the mixture was centrifuged at 13,000 rpm for 3 min. The absorbance of the supernatant was measured at 735 nm using a spectrophotometer. The total phenolic content was expressed as gallic acid equivalent (GAE)/100 g dry weight (DW) of the sample. 2.5. Ferric ion reducing antioxidant power (FRAP) assay The FRAP assay was carried out as described by Stratil et al. [21] with slight modifications. The FRAP reagent was prepared by mixing 38 mM sodium acetate anhydrous in distilled water pH 3.6, 20 mM FeCl36H2O in distilled water and 10 mM 2,4,6-Tri (2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl in a proportion of 10:1:1. This reagent was freshly prepared before each experiment. Hundred microlitre of appropriately diluted sample extract and 900 lL of FRAP reagent was added to each sample and the mixture was incubated at 37 °C for 40 min in the dark. In the case of the blank, 100 lL of methanol was added to 900 lL of FRAP reagent. The absorbance of the resulting solution was measured at 593 nm using a spectrophotometer. Trolox (6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) (a synthetic antioxidant) at concentrations from 0.1 to 0.4 mM was used as a reference antioxidant standard. FRAP values were expressed as g Trolox/100 g DW of the sample. 2.6. HPLC analysis of the extracts Reversed phase high performance liquid chromatography (RP-HPLC) of the filtered sample extracts was carried out according to the method of Tsao and Yang [22]. The chromatographic system (Shimadzu-Model No. SPD-M10A VP, Mason Technology, Dublin 8, Ireland) consisted of a pump, a vacuum degasser, a diode-array detector and was controlled through EZ Start 7.3 software (Shimadzu) at 37 °C. An Agilent C18 column (15 cm 4.6 cm, 5 lm, Agilent Technologies, USA) was utilized with a binary mobile phase
of 6% acetic acid in 2 mM sodium acetate (final pH 2.55, v/v, solvent A) and acetonitrile (solvent B). Solvent A was prepared first by mixing 2 mM sodium acetate water solution, with acetic acid at a ratio of 94:6 by volume. All solvents were filtered through a 0.45 lm diameter membrane filter prior to analysis. The flow rate was kept constant at 1.0 mL/min for a total run time of 80 min. The following gradient program was carried out: 0–15% B in 45 min, 15–30% B in 15 min, 30–50% B in 5 min, 50–100% B in 5 min and 100–0% B in 5 min. The injection volume for all the samples was 10 lL. All the standards for quantification purposes were dissolved in methanol. The detection wavelength of 280 nm was used for the detection of carnosol and carnosic acid. Rosmarinic acid, caffeic acid and apigenin-7-O-glucoside were detected at 320 nm while luteilon-7-O-glucoside was detected at 360 nm. Identification of the compounds was achieved by comparing their retention times and UV–Vis spectra with those of authenticated standards by using the inline DAD with a 3D feature. Results are expressed as mean values of three assays for each replicated experiment. 2.7. Experimental design and data analysis Polynomial regression equations were developed to describe the effects of the 3 independent processing parameters; ultrasound amplitude (X1, lm), extraction temperature (X2, °C) and extraction time (X3, min) on total phenol (TP), antioxidant activity as measured by FRAP and different polyphenolic compounds such as rosmarinic acid, caffeic acid, luteolin-7-O-glucoside, apigenin-7-Oglucoside, carnosol and carnosic acid. Independent variables of amplitude level (X1) (24.4, 42.7, and 61 lm), temperature (X2) (15, 25, and 35 °C), and extraction time (X3) (5, 10 and 15 min) were varied to investigate the effects on the dependent variables mentioned above. The general form of the quadratic polynomial model regression equation employed in this study is presented in Eq. (1). By using this equation, linear (X1, X2, X3), quadratic (X 21 , X 22 , X 23 ) and interactive (X1X2, X1X3, X2X3) effects of independent variables, temperature (X1), amplitude level (X2), and time (X3) on dependent variable (Y) were determined:
Table 2 Analysis of the variance of the regression coefficients of the fitted polynomial quadratic equation for TP (g GAE/ 100 g DW), FRAP (g Trolox/100 g DW) and different polyphenols (mg/g DW).
ns
Coefficients
TP
FRAP
Rosmarinic acid Luteolin-7-O-glucoside Apigenin-7-O-glucoside Caffeic acid
b0 (Intercept)
+11.87
+20.10
+19.89
Carnosic acid
Carnosol
+4.96
+0.447
+0.248
+7.64
+1.17
Linear b1 (Amplitude) 1.98 102c 7.65 103d +3.8 102d b2 (Temperature) 3.3 101c 4.3 101d 4.98 102d b3 (Time) 1.01 101b 4.69 101c +1.91 101d
+1.72 102d 1.02 101a –
+8.04 103c +1.23 102b –
– – –
+1.1 101d – –
+7.45 103c – –
Quadratic b11 b22 b33
– +4.83 103c –
– +7.48 103d –
– +3.04 103b –
2.80 104a +1.20 103b –
– – –
– 1.27 103d – +1.50 104b +9.14 104a – – – –
Cross product b12 b13 b23
+1.55 103a – +5.79 103a
– – 4.61 103a
+1.13 103d
+5.95 103c +1.19 102c
– – –
+3.72 105a +1.1 103b – – – –
– – –
R2 Predicted R2 CV p Lack of fit F-value
0.917 0.635 2.39 <0.0001 0.1804 17.89
0.976 0.905 1.36 <0.0001 0.1046 68.32
0.964 0.872 0.71 <0.0001 0.183 30.34
0.966 0.906 1.52 <0.0001 0.147 55.27
0.721 0.450 8.04 0.0007 0.137 11.23
0.750 0.271 4.97 0.0013 0.223 9.02
0.58 0.44 5.70 0.0004 0.108 20.61
Not significant. a Significant at b Significant at c Significant at d Significant at
p 6 0.05. p 6 0.01. p 6 0.001. p 6 0.0001.
0.989 0.951 0.64 <0.0001 0.347 200.12
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A
23.95 23.72
24.17
0.13 0.13 0.13
23.50
30.00
22.82 23.05
23.27
22.60
25.00
22.38 22.15 20.00
30.00
0.11
33.55
42.70
51.85
C
0.12 0.12
20.00
0.14
24.40
61.00
0.13 0.13
0.13 33.55
42.70
51.85
61.00
Amplitude (µm)
Amplitude (µm)
35.00
0.12
25.00
15.00
15.00 24.40
Caffeic acid (mg/g DW )
35.00
Temperature (°C)
Temperature (°C)
B
Rosmarinic acid (mg/g DW)
35.00
D
Luteolin-7-O-glucoside (mg/g DW )
35.00
5.18 5.07
Apigenin-7-O-glucoside (mg/g DW ) 1.41
30.00
4.84
4.18 4.62
4.29
25.00
Temperature (°C)
Temperature (°C)
4.96
4.40
4.73
4.51
20.00
30.00
1.35 1.30 1.22
25.00
1.26
1.16 1.11
20.00
1.06 1.00
15.00 24.40
15.00 33.55
42.70
51.85
24.40
61.00
33.55
E
51.85
61.00
Amplitude (µm)
Amplitude (µm)
F
Carnosic acid (mg/g DW )
35.00
42.70
Carnosol (mg/g DW)
35.00
10.15
30.00
Temperature (°C)
Temperature (°C)
1.64 10.01 9.93 25.00
8.87 9.72 9.84 9.10 9.33 9.56 9.86
9.84
20.00
15.00 24.40
30.00
1.61 1.42
25.00
1.45
1.49
1.53
1.57
1.38 20.00
1.34
15.00 33.55
42.70
51.85
61.00
Amplitude (µm)
24.40
33.55
42.70
51.85
61.00
Amplitude (µm)
Fig. 2. Contour plots showing the effect of amplitude and temperature on the extraction of rosmarinic acid (A), caffeic acid (B), luteolin-7-O-glucoside (C), apigenin-7-Oglucoside (D), carnosic acid (E) and carnosol (F) at treatment time of 10 min.
Y ¼ b0 þ
3 X i¼1
bi X i þ
3 X i¼1
bii X 2i þ
2 3 X X i¼1 j¼iþ1
bij X i X j
ð1Þ
where Y is the predicted response; b0 the constant (intercept); bi the linear coefficient; bii the quadratic coefficient and bij is the cross
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product coefficient. Xi and Xj are independent variables. The response surface regression was used to analyze the experimental data using Design Expert Version 7.1.3 software (Stat-Ease Inc.,
A
Minneapolis, MN). Two dimensional contour plots were developed while holding a variable constant in the second order polynomial models. All processing trials were conducted in triplicate.
B
Rosmarinic acid (mg/g DW)
15.00
Caffeic acid (mg/g DW )
15.00
23.74 0.11 23.57 12.50
23.36 10.00
22.75
22.48
Time (min)
Time (min)
12.50
22.98 23.18
0.11 0.11
0.12 0.11 0.11
10.00
22.20
0.12
0.12
0.12
0.11 7.50
7.50
0.11 21.92 5.00 24.40
0.11 5.00 33.55
42.70
51.85
61.00
24.40
33.55
C
51.85
61.00
Amplitude (µm)
Amplitude (µm)
15.00
42.70
D
Luteolin-7-O-glucoside (mg/g DW )
15.00
Apigenin-7-O-glucoside (mg/g DW) 1.38
12.50
12.50
10.00
4.20
4.34
4.48
4.70 4.61
Time (min)
Time (min)
4.88
4.81 4.75
7.50
1.33 1.29
1.06
33.55
42.70
51.85
61.00
24.4
33.55
Amplitude (µm)
E
42.7
51.85
61
Amplitude (µm)
F
Carnosic acid (mg/g DW )
15.00
Carnosol (mg/g DW)
15.00
1.62
12.50
9.93 8.94 9.19 9.41 9.61 9.76 9.87 9.92
9.92
Time (min)
12.50
Time (min)
1.24
5.00
24.40
10.00
1.20
1.10 7.50
5.00
1.15
10.00
1.40
10.00
9.93
1.44
1.47
1.51
1.55
1.59
1.36
7.50
7.50
9.93 5.00 24.40
5.00 33.55
42.70
Amplitude (µm)
51.85
61.00
24.40
33.55
42.70
51.85
61.00
Amplitude (µm)
Fig. 3. Contour plots showing the effect of amplitude and time on the extraction of rosmarinic acid (A), caffeic acid (B), luteolin-7-O-glucoside (C), apigenin-7-O-glucoside (D), carnosic acid (E) and carnosol (F) at treatment temperature of 25 °C.
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2.8. Model validation The predictive performance of the developed models describing the combined effect of amplitude (X1), temperature (X2) and time
A
(X3) on independent variables (FRAP, TP, rosmarinic acid, caffeic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, carnosol and carnosic acid) of marjoram were validated with optimal extraction conditions as predicted by the design.
B
Rosmarinic acid (mg/g DW)
15.00
Caffeic acid (mg/g DW )
15.00
0.11
23.74
12.50
12.50
0.13
23.03 10.00
23.36
Time (min)
Time (min)
23.18 23.57
22.88 22.74
0.12
0.13 0.12
0.12 0.12
10.00
0.12 0.12 0.11
22.59 7.50
7.50
22.41
0.11
22.20 5.00
5.00 15.00
20.00
25.00
30.00
15.00
35.00
20.00
Temperature (°C)
C 15.00
25.00
30.00
35.00
Temperature (°C)
D
Luteolin-7-O-glucoside (mg/g DW )
15.00
Apigenin-7-O-glucoside (mg/g DW )
4.59 1.30323 12.50
4.75 10.00
4.61
4.60 4.59 4.60
4.70 4.61 4.65
4.58
Time (min)
Time (min)
12.50
1.22
1.25733
1.28464
1.16 10.00
1.11 1.06
7.50
7.50
4.56
1.00 1.30323
5.00
5.00
15.00
20.00
25.00
30.00
35.00
15.00
20.00
Temperature (°C)
E 15.00
25.00
30.00
35.00
Temperature (°C)
F
Carnosic acid (mg/g DW )
Carnosol (mg/g DW)
15.00
1.50 1.50 12.50
9.72 10.00
9.74
9.83 9.74 9.76 9.79
9.72
Time (min)
Time (min)
12.50
1.49 1.48 1.45
10.00
1.43
1.50
1.40 7.50
7.50
1.53
1.35
9.72 5.00 15.00
1.38
5.00 20.00
25.00
30.00
Temperature (°C)
35.00
15.00
20.00
25.00
30.00
35.00
Temperature (°C)
Fig. 4. Contour plots showing the effect of time and temperature on the extraction of rosmarinic acid (A), caffeic acid (B), luteolin-7-O-glucoside (C), apigenin-7-O-glucoside (D), carnosic acid (E) and carnosol (F) at treatment amplitude of 42.70 lm.
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E ð%Þ ¼
n V E V P 1 X 100 ne i¼1 V E
ð2Þ
where ne is the number of experimental data, VE is the experimental value and VP is the predicted value. 3. Results and discussion 3.1. Effect of thermosonication on total phenol content Fig. 1A, C, and E present the contour plots showing the effect of UAE parameters of ultrasound amplitude, temperature and time on the total phenol content of marjoram extract. All the three factors had a significant (p < 0.05) positive effect on the total phenol content of marjoram extract. Among the factors, amplitude showed the highest effect followed by temperature and time. Higher amplitude of ultrasound could have damaged more cell walls releasing more antioxidants including phenolic compounds to the solvents. An increase in temperature increases target compound solubility, solvent diffusion rate and mass transfer, while solvent viscosity and surface tension decrease [23]. Reduced viscosity and surface tension facilitate solvent penetration deeper into sample matrix which enhances the extraction efficiency by exposing more surface area of the sample to the solvent used. Increased extraction time allowed the solutes to be in contact with solvent for longer facilitating higher diffusion of the target compounds [24]. Among the treatments generated by RSM, the highest total phenolic content (9.62 g GAE/100 g DW) was observed in the extracts obtained at UAE conditions of 61 lm amplitude, 35 °C and an extraction time of 10 min of treatment. This value was 98.39% higher than that obtained with the reference solid/liquid extraction method. 3.2. Effect of thermosonication on ferric reducing antioxidant power All three factors had a significant effect on enhancing the FRAP values of the marjoram extracts. Among the factors, ultrasonic amplitude showed the highest effect followed by temperature and time (Fig. 1B, D, and F and Table 2). Temperature also had a significant (p < 0.05) effect at the quadratic level. The other two factors did not show significant (p < 0.05) quadratic effects. The effect of temperature and time was in agreement with the finding of Ghafoor et al. [24] who investigated the antioxidant activity of grape extracts obtained using a fixed level of ultrasound. The interactions of the factors, except the interaction between amplitude and temperature, showed a significant effect on the extraction. Among the ultrasonication treatments used, the amplitude level of 61 lm at a temperature of 35 °C for 10 min showed the highest FRAP value (17.08 g Trolox/100 g DW). This treatment increased the antioxidant activity as measured by FRAP by 89.76% compared to conventional solid/liquid extraction (9.00 g Trolox/100 g DW). In fact, all the ultrasonication treated extracts showed significantly (p < 0.05) higher FRAP values than that of solid/liquid extraction. When the ultrasonication amplitude was increased from the lowest level (24.4 lm) to the highest level (61 lm), the FRAP value showed an increase of 26.98%. In spices, antioxidant activity is related to their total phenol content. The high Pearsons correlation coefficients (r = 0.90) reflect the importance of phenols in the antioxidant capacity of marjoram. 3.3. Effect of thermosonication on different polyphenols The principal polyphenol of marjoram extract was rosmarinic acid [23]. Rosmarinic acid content of the extracts were significantly
(p < 0.05) affected by amplitude, temperature and time. All the factors had positive effects on the rosmarinic acid content (Figs. 2–4A and Table 2). This result was in agreement with the finding of Albu et al. [25]. The effect of amplitude was higher than that of other factors. Only temperature had a significant (p < 0.05) quadratic effect. It also had an interaction effect with time. Higher levels of time and temperature would have further increased the extraction of antioxidant polyphenols. But this would have increased the cost of extraction and an environmentally friendly extraction method requires minimal extraction time and temperature [24]. Therefore, in the present study, time and temperature range was kept low. The lowest value of rosmarinic acid content was observed at an amplitude level of 24.4 lm treated for 5 min at 25 °C. With an increase of ultrasound amplitude the rosmarinic acid content of the extracts increased gradually. At the highest amplitude and temperature investigated, the content of rosmarinic acid was 24.41 mg/g DW which was approximately two times higher than that of solid/liquid extraction (12.08 mg/g DW) (Fig. 5). An increase in rosmarinic acid extraction from dried rosemary with higher ultrasound amplitude has also been reported by Paniwnyk et al. [26]. The other hydroxycinnamic acid derivative investigated, caffeic acid, was affected by temperature at a quadratic level showing higher extractions at two ends of the temperature range used (Figs. 2B, and 4B). Temperature also showed significant (p < 0.05) positive effect in interaction with amplitude. The major flavonoids of Lamiaceae spices are luteolin-7-O-glucoside and apigenin-7-O-glucoside. Both these flavonoids showed significant (p < 0.05) increase with the increase of amplitude and temperature, while time did not have any significant effect (Figs. 2–4C, and D). In the case of luteolin-7-O-glucoside, temperature also had quadratic and interaction effects with amplitude. Amplitude played the dominant role in extracting flavonoids. The antioxidant volatile polyphenols carnosic acid and carnosol showed significant (p < 0.05) increases with the increase of amplitude (Figs. 2–4E, and F). Paniwnyk et al. [26] also found an increase of extraction of carnosic acid from rosemary with increased amplitude of ultrasonication. The other factors on linear term did not have significant effect on both the compounds. Temperature at the quadratic level and in interaction with amplitude had significant (p < 0.05) effects on the extraction of carnosic acid. 3.4. Model fitting The analysis of variance showed that the R-squared statistic of all the parameters was in the range of 0.580–0.989, indicating high representation of the variability of the parameters by the models. The quadratic polynomial models generated were highly significant with p-value ranging from 0.0013 to <0.0001. The lack of fit
1000
4
800
mAu
The criterion used to characterize the fitting efficiency of the data to the model was the multiple correlation coefficients (R2) and the average mean deviation (Eq. (2)):
600
2 1
3
400
6 5
A
56
B
4
200 2
1
3
0
0
0
20
40 Minutes
60
80
Fig. 5. HPLC chromatograms of UAE extracts of marjoram obtained at 61 lm, 35 °C and 10 min (A), in comparison to solid/liquid extraction (B) showing the changes in peaks of different polyphenols (1 = caffeic acid, 2 = luteolin-7-O-glucoside, 3 = apigenin-7-O-glucoside, 4 = rosmarinic acid, 5 = carnosol and 6 = carnosic acid).
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M.B. Hossain et al. / Ultrasonics Sonochemistry 19 (2012) 582–590
Table 3 Predicted and experimental values of the parameters tested at optimal UAE conditions in comparison to the conventional solid/liquid extraction values and average mean deviation between predicted and experimental values of optimal UAE. Parameter
Optimum UAE condition for all the parameters combined
Predicted values at optimal UAE
Desirability
Experimental values at optimal UAE
(E%)
Solid/liquid extraction values
TP (g GAE/ 100 g DW) FRAP (g Trolox/100 g DW) Rosmarinic acid (mg/g DW) Luteolin-7-O-glucoside (mg/g DW) Apigenin-7-O-glucoside (mg/g DW) Caffeic acid (mg/g DW) Carnosic acid (mg/g DW) Carnosol (mg/g DW)
61 lm, 35 °C and 15 min
9.90 18.56 24.53 5.38 1.54 0.15 10.25 1.72
0.984
9.51 ± 0.10 18.96 ± 0.19 24.86 ± 0.45 5.27 ± 0.14 1.60 ± 0.13 0.14 ± 0.01 10.63 ± 0.26 1.81 ± 0.16
1.34 0.70 0.45 0.65 1.31 1.55 1.20 1.36
4.85 ± 0.05 9.00 ± 0.17 12.08 ± 0.03 2.69 ± 0.02 0.86 ± 0.01 0.10 ± 0.01 3.56 ± 0.15 0.72 ± 0.02
statistics of all the parameters were not significant (p > 0.05) and high degree of F-value (range 9.02–200.12) further strengthened the reliability of the models (Table 2). The predicted values obtained by the quadratic polynomial equations showed strong correlation with actual experimental values with Pearsons correlation coefficients (r) from 0.88 to 0.98.
caffeic acid, carnosic acid and carnosol values of the extract. The developed prediction models showed good correlation with the experimental data at a 95% confidence level. The absolute extraction yields of the optimal UAE were significantly (p < 0.05) higher than those obtained with reference solid/liquid extraction. UAE appears to have great potential as a method for the extraction of antioxidant materials from foods of plant origin.
3.5. Optimization and model validation Acknowledgments The RSM guided optimization demonstrated that the optimum treatment conditions for maximizing the TP and FRAP values were 60.60 lm (amplitude), 34.84 °C (temperature), 14.80 min (time) and 54.34 lm (amplitude), 34.76 °C (temperature), 14.79 min (time) respectively. The antioxidant polyphenols namely rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, caffeic acid, carnosic acid and carnosol had optimum extraction conditions at the amplitude level from 58 to 61 lm with a range of extraction times from 11 to 15 min at 35 °C. The predicted optimal extraction condition for all the parameters combined was 61 lm, 35 °C and 15 min which predicted the TP, FRAP, rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, caffeic acid, carnosic acid and carnosol values as 9.90 g GAE/100 g DW, 18.56 g Trolox/100 g DW, 24.53 mg/g DW, 5.38 mg/g DW, 1.54 mg/g DW, 0.15 mg/g DW, 10.25 mg/g DW and 1.72 mg/g DW respectively. These values were significantly (p < 0.05) higher than those of solid/liquid extraction (Table 3). The predicted values were in close agreement with experimental values (Table 3) and were found to be not significantly different at p > 0.05 using a paired t-test. In addition, variations between the predicted and experimental values obtained for total antioxidant activity by FRAP assay, TP content and antioxidant polyphenols were within an acceptable error range as depicted by average mean deviation (E%, Table 3); therefore, the predictive performance of the established model may be considered acceptable. 4. Conclusions The ultrasound assisted extraction of phenolic antioxidants from marjoram appeared very effective in comparison to solid liquid extraction. Optimal process conditions with regard to amplitude of sonication (24.4–61.0 lm) and extraction temperature (15–35 °C) and treatment time (5–15 min) were identified using response surface methodology (RSM). The results demonstrated that the combined treatment of 61 lm, 35 °C and 15 min was optimal for maximizing key antioxidant parameters namely TP, FRAP, rosmarinic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside,
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