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Optimization of ultrasound-assisted extraction conditions for euphol from the medicinal plant, Euphorbia tirucalli, using response surface methodology
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Optimization of ultrasound-assisted extraction conditions for euphol from the medicinal plant, Euphorbia tirucalli, using response surface methodology Quan V. Vuong a,b,∗ , Van Tang Nguyen a,b,c , Dang Trung Thanh a,b,c , Deep Jyoti Bhuyan a,b , Chloe D. Goldsmith a,b , Elham Sadeqzadeh a,b , Christopher J. Scarlett a,b , Michael C. Bowyer a,b a
Pancreatic Cancer Research, Nutrition Food & Health Research Group, University of Newcastle, NSW, Australia School of Environmental and Life Sciences, University of Newcastle, NSW, Australia c Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Viet Nam b
a r t i c l e
i n f o
Article history: Received 22 June 2014 Received in revised form 25 August 2014 Accepted 28 September 2014 Available online xxx Keywords: Euphol Euphorbia tirucalli Ultrasonic-assisted extraction Optimization Response surface methodology
a b s t r a c t Euphol identified in Euphorbia tirucalli (E. tirucalli) has been linked with various health benefits. This study aimed to optimize ultrasonic extraction conditions for euphol from E. tirucalli leaf. Different solvents were tested to determine the most effective solvent for extraction of euphol. Then, response surface methodology (RSM) was employed to optimize ultrasound-assisted extraction conditions including temperature, time and power for maximal extraction of euphol. Our results showed that ethyl acetate:ethanol (4:1, v/v) was the most effective solvent for the extraction of euphol. Ultrasonic temperature and time had a positive impact, whereas, ultrasonic power had a negative effect on the extraction efficiency of euphol. The optimum ultrasonic extraction conditions for euphol were identified as: solvent-to-fresh sample ratio of 100:32 mL/g; ultrasonic temperature of 60 ◦ C; ultrasonic time of 75 min and ultrasonic power of 60% (150 W). Under these optimum conditions, approximately 4.06 mg of euphol could be obtained from one gram of fresh E. tirucalli leaf. This extract also contained phenolic compounds (2.5 mg GAE/g FW) and possessed potent antioxidant capacity. These optimal conditions are applicable for a larger scale to extract and isolate euphol for potential utilization in the pharmaceutical industry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Euphorbia tirucalli (E. tirucalli), also known as the pencil tree, sticks-on-fire or milk bush, is a small tree native to Madagascar and Africa; however, it has been widely distributed across the globe because of its tolerance to a wide range of climatic conditions (Mwine and Damme, 2011). E. tirucalli has been used as a traditional medicine in the Middle East, India, Africa and South America for the treatment of a range of ailments including syphilis, asthma,
Abbreviations: RSM, response surface methodology; UAE, ultrasound-assisted extraction. ∗ Corresponding author at: Pancreatic Cancer Research Nutrition Food & Health Research Group, School of Environmental and Life Sciences, University of Newcastle, Brush Rd, Ourimbah, NSW 2258, Australia. Tel.: +61 2 4348 4045; fax: +61 2 4348 4145. E-mail address: [email protected] (Q.V. Vuong).
cancer, colic, intestinal parasites and leprosy (Cataluna and Rates, 1997; Gupta et al., 2013). The traditional use of E. tirucalli for purported health benefits has prompted scientific interest in the exploration of its bioactive constituents for pharmacological utilization. A range of di- and triterpene compounds has been identified in E. tirucalli. Of those identified, euphol (Fig. 1) is the most prominent and has been found to exhibit anti-cancer activity against human gastric cancer and breast cancer in vitro (Sadeghi-Aliabadi et al., 2009; Zhang et al., 2012). Consequently, optimization of conditions for maximal extraction of euphol and other bioactives from E. tirucalli is of interest and worthy of further investigation. To date however, no formal studies in this area have been undertaken. Response Surface Methodology (RSM) is a statistical technique that aims to develop a functional relationship between a response of interest and a number of input variables (Khuri and Mukhopadhyay, 2010). In comparison with single variable optimization methods, RSM is a time and cost effective means of simultaneously evaluating the key experimental parameters
http://dx.doi.org/10.1016/j.indcrop.2014.09.057 0926-6690/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Vuong, Q.V., et al., Optimization of ultrasound-assisted extraction conditions for euphol from the medicinal plant, Euphorbia tirucalli, using response surface methodology. Ind. Crops Prod. (2014), http://dx.doi.org/10.1016/j.indcrop.2014.09.057
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2.2. Extraction of euphol using ultrasound-assisted extraction (UAE) To test the impact of solvents on extraction efficiency of euphol, ground fresh leaf was extracted in different solvents including ethanol, acetonitrile:ethanol (4:1, v/v), acetone:ethanol (4:1, v/v), ethyl acetate:ethanol (4:1, v/v), or hexane:ethanol (4:1, v/v) at a solvent-to-sample ratio of 100:32 mL/g. The extraction was conducted using a tunable ultrasonic bath (Soniclean, 220 V, 50 Hz and 250W, Soniclean Pty Ltd, Australia) under ultrasonic conditions of room temperature, 60 min and power of 150 W. To optimize UAE conditions, the most effective solvent (ethyl acetate:ethanol (4:1, v/v)) was used for extraction of fresh leaf at a solvent-to-sample ratio of 100:32 mL/g. UAE was conducted using a tunable ultrasonic bath (Soniclean, 220 V, 50 Hz and 250 W, Soniclean Pty Ltd, Australia) to be set at different conditions as designated by RSM design. An external digital thermometer was also used to measure the temperature of the ultrasonic bath, while tap water was used to cool the water to the required temperature if the ultrasonic bath exceeded the designated temperature due to ultrasonic energy. 2.3. Response surface methodology design
Fig. 1. HPLC chromatogram of euphol standard and euphol structure (A) and the extract E. tirucalli leaf (B).
(Wang et al., 2011), thus contributes to the optimization of euphol extraction from E. tirucalli. Ultrasonic extraction has gained increasing popularity as method to be used both in conjunction with, or in place of, traditional extraction techniques because of its efficient energetics and reduced extraction times (Mierzwa et al., 1997). Ultrasound assisted extraction (UAE) is now well established as a technique for the extraction of low molecular weight compounds from plant sources (Hromadkova et al., 2002), and has been found to be more effective in extracting phytochemicals from plants and is easily adapted to the industrial scale (Vinatoru, 2001). This study aimed to determine the most effective extraction solvents and then utilize RSM to develop optimal conditions for the extraction euphol from E. tirucalli by ultrasonic-assisted extraction as a useful engineering tool for commercial applications to prepare euphol from E. tirucalli for utilization in pharmaceuticals.
Response surface methodology (RSM) with a Box–Behnken design was employed to design the experiment and investigate the influence of the three independent ultrasonic parameters: power (60%, 80%, 100% or 150, 200, 250 W), temperature (30, 45, 60 ◦ C), and time (30, 60, 90 min). After extraction, the extracts were then immediately cooled to room temperature (ice bath), then filtered through a 5 mL syringe fitted with a 0.45 m cellulose syringe filter (Phenomenex Australia Pty. Ltd, NSW, Australia), then transferred into brown glass HPLC vials for HPLC analysis. The independent variables and their code variable levels are shown in Table 1. To express the level of euphol as a function of the independent variables, a second-order polynomial equation (Eq. (1)) was used as follows (Vuong et al., 2011): Y = ˇo +
k i=1
ˇi Xi +
k k−1
i=1
ˇij Xi Xj +
j=2
k
ˇii Xi2
(1)
i=1
i
where various Xi values are independent variables affecting the responses Y; ˇ0 , ˇi , ˇii , and ˇij are the regression coefficients for intercept, linear, quadratic, and interaction terms, respectively; and k is the number of variables. In this study, the three independent ultrasonic parameters: X1 (ultrasonic temperature, ◦ C), X2 (ultrasonic time, min) and X3 (ultrasonic power, %) were applied (Table 2), with euphol extraction efficiency expressed as the following polynomial (Eq. (2)): YEuphol = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ3 X3 + ˇ12 X1 X2 + ˇ13 X1 X3 + ˇ23 X2 X3 + ˇ11 X12 + ˇ22 X22 + ˇ33 X32
(2)
2.4. Determination of euphol 2. Materials and methods 2.1. Plant materials The phylloclades of a E. tirucalli tree were collected on July 16 from a property located in Saratoga, NSW, Australia (33.47◦ S, 151.35◦ E) and immediately transferred to the laboratory and stored at −20 ◦ C. Plylloclade samples were then freeze dried in liquid nitrogen, ground to consistent particle size using a commercial blender, then stored at −20 ◦ C until required.
Euphol standard was prepared from the latex of E. tirucalli in our laboratory using a Shimadzu HPLC system fitted with a semiprep Luna C18 reversed-phase column (Phenomenex, Australia) maintained at 35 ◦ C and coupled to auto fraction collector (Shimadzu Australia, Rydalmere, NSW, Australia). The sample was detected using UV detector set at 210 nm as per the work of Vuong et al. (2012). A representative chromatogram of euphol standard is shown in Fig. 1A. Quantitative analysis of euphol content in different filtered sample extracts was then undertaken using
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Table 1 Box–Behnken design and observed responses of euphol extracted from Euphorbia tirucalli phylloclades using UAE. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a b c
UAE temperatures (◦ C)
Patterna
+−0 0++ 000 0−+ +0+ 000 0−− 000 −0− 0+− −−0 +0− −0+ −+0 000
UAE power (%)b
UAE time (min)
60 45 45 45 60 60 45 45 30 45 30 60 30 30 45
30 90 60 30 60 90 30 60 60 90 30 60 60 90 60
Euphol (mg/g)
80 100 80 100 100 80 60 80 60 60 80 60 100 80 80
Experimental valuesc
Predicted values
3.74 3.62 3.57 2.85 3.29 3.92 2.43 3.57 2.03 3.81 1.97 4.14 2.69 2.09 3.57
3.40 3.33 3.57 3.03 3.45 4.04 2.71 3.57 1.87 3.63 1.85 4.20 2.63 2.43 3.57
Pattern −, 0, + are the minimum values, center points, and maximum values, respectively, of UAE temperature, time and power in the tested ranges. Corresponding values of power were 250 W for 100%; 200 W for 80%; and 150 W for 60%. Values were obtained from triplicated experiments and expressed as mg/g FW.
a reversed-phase analytical column (250 mm × 4.6 mm Synergi 4 mm Fusion-RP 80A column, Phenomenex Australia) maintained at 35 ◦ C with peak detection set at 210 nm. The mobile phase (flow rate = 1 mL/min) consisted of two feed solvents; methanol (A) and acetonitrile (B). A gradient elution schedule was used as follows: 100% A for the first 5 min.; followed by a linear gradient changed to 50% A and 50% B (5–25 min.); then finally, a linear gradient changed back to 100% A (25–30 min.). An auto-injector was used to inject 20 L of all sample extracts onto the HPLC column. A representative chromatogram of euphol in the E. tirucalli leaf is shown in Fig. 1B. The content of euphol was calculated based on the standard curve and expressed as mg of euphol per gram of fresh weight (FW). 2.5. Determination of phenolic content and antioxidant capacity Total phenolic content (TPC) of the extract prepared under optimal conditions was determined using a method described by Vuong et al. (2013). Gallic acid was used as the standard for the construction of a calibration curve, with the results expressed as mg of gallic acid equivalents per gram of fresh weight (FW) (mg GAE/g FW). Three different antioxidant assays described in previous studies were employed to test antioxidant properties of the extract under optimal conditions, including ABTS (2,2 -azino-bis3-ethylbenzothiazoline-6-sulphonic acid) (Thaipong et al., 2006),
DPPH (1,1-diphenyl-2-picrylhydrazyl) assay (Vuong et al., 2013) and FRAP (ferric reducing antioxidant power) assay (Thaipong et al., 2006). Values of ABTS and DPPH were expressed as percentage of inhibition and value of FRAP was expressed as Trolox equivalents per gram of fresh weight (TE/g FW). 2.6. Statistical analysis RSM experimental design and analysis were conducted using JMP software (Version 11). The software was also used to establish the model equation, to graph the 3-D plot, 2-D contour of the response and to predict the optimum values for the three response variables. The student’s T-test was conducted using the SPSS statistical software (Version 20) for comparison of sample means. Differences between the mean euphol concentrations in the different samples were taken to be statistically significant at p < 0.05. 3. Results and discussion 3.1. Impact of solvents on extraction efficiency of euphol Previous studies indicated that different solvents could lead to different extraction efficiencies of bioactive compounds (Vuong et al., 2013; Zhang et al., 2013). Therefore, this study determined
Table 2 Estimated regression coefficients for the quadratic polynomial model and the analysis of variance for the experimental results. Parametera Intercept ˇ0 ˇ1 ˇ2 ˇ3 ˇ12 ˇ13 ˇ23 ˇ11 ˇ22 ˇ33 Lack of fit Pure error R2 PRESS a b *
Predicted coefficients 3.568 0.789 0.306 0.004 0.014 −0.375 −0.154 −0.388 −0.248 −0.144
0.929 8.746
Standard error 0.19 0.12 0.12 0.12 0.17 0.17 0.17 0.17 0.17 0.17
DFb 1 1 1 1 1 1 1 1 1 1 3 Adjusted R2 RMSE
Sum of squares
F value
Prob > F
Model
7.283
0.0208*
4.986 0.750 0.000 0.001 0.565 0.094 0.557 0.228 0.076 . . 0.801 0.33
45.606 6.862 0.001 0.007 5.165 0.864 5.091 2.086 0.698 .
0.001* 0.047* 0.972 0.935 0.072 0.395 0.074 0.208 0.442 .
Coefficients refer to the general model. Degree of freedom. Significant different at p < 0.05.
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Fig. 2. Impact of extraction solvents on extraction efficiency of euphol from E. tirucalli phylloclades. Values (mean ± SD, n = 3) not sharing a letter (on top of the columns) are significantly different at p < 0.05.
the impact of five different extraction solvents with various polarity indexes to identify the most effective solvent for further optimization using response surface methodology. The results (Fig. 2) showed that different extraction solvents significantly affected the extraction efficiency of euphol. Extraction efficiency of euphol was low when using ethanol alone; however, extraction efficiency was significantly improved when it was used in combination with other solvents of lower polarity at the ratio of 1:4 (v/v). A solvent mixture of ethyl acetate:ethanol (4:1, v/v) was found to be the most effective solvent for extraction of euphol from E. tirucalli leaf. The current findings were in agreement with a previous study (Zhang et al., 2013), which reported that a mixture of ethyl acetate:ethanol (4:1 v/v) had higher extraction efficiency than methanol when extracting euphol from root of Euphorbia pekinensis Rupr; and these differences could be explained by the variation of solvent polarities (Vuong et al., 2013). Therefore, solvent of ethyl acetate:ethanol (4:1, v/v) was used for further optimization process. 3.2. Fitting the model for the prediction of euphol Response surface methodology (RSM) is an effective statistical procedure using a minimum set of experiments for determination of the coefficients of a mathematical model and optimization of the conditions (Yemis¸ and Mazza, 2012). However, it is important to test the appropriateness of the RSM mathematical model for predicting the optimal variances and adequately representing the real relationship between the selected parameters. The analysis of variances for the experimental results of the Box–Behnken design (Fig. 3) showed that the coefficient of determination (R2 ) of the model was 0.93, suggesting that 93% of the actual levels can be matched with the model-predicted levels of euphol (Li et al., 2011). The analyzed results (Table 2) also showed that root mean square error (RMSE), which is used to estimate the standard deviation of the random error, was 0.33, further confirming a linear correlation exists between the predicted levels and actual levels of euphol present in the samples analyzed. Results of analysis of the actual experiments (Table 2) revealed that the Predicted Residual Sum of Squares (PRESS) for the model – a measure of how a chosen model fits each point in the design (Zhang et al., 2009) was 8.746, indicating a good measure. In addition, the p and F-values of the model were 0.02 and 7.283, respectively, indicating that the model predictions were significant (Zhang et al., 2009). Moreover, the Lack of Fit report was not generated as a consequence of the analysis of the experimental data, implying that the model was well fitted as it was impossible to assess lack of fit because there were as many estimated parameters as there were observations (SAS-Institute, 2014). Therefore, the results revealed
Fig. 3. Correlation between the predicted and actual levels of euphol.
that the mathematical model was adequate for the prediction of euphol and was fitted to the following second-order polynomial formula (Eq. (3)): YEuphol = 3.568 + 0.789X1 + 0.306X2 + 0.004X3 + 0.014X1 X2 − 0.038X1 X3 − 0.154X2 X3 − 0.388X12 − 0.248X22 − 0.144X32
(3)
3.3. Effect of extraction parameters on the UAE performance The impacts of three UAE parameters on extraction efficiency of euphol were investigated. Results (Fig. 4A) showed that UAE temperature was a significant parameter, with rising UAE temperature correlating to a higher extraction efficiency. At fixed conditions of 75 min and UAE power at 60%, euphol extraction increased from 1.7 mg/g to 4.2 mg/g across a temperatures ranging between 30 and 60 ◦ C. Table 2 shows a p value of 0.001 for UAE temperature, further confirming the significance of temperature on extraction efficiency. The findings are in agreement with previously studies assessing the effectiveness of UAE. Teh and Birch (2014) found that UAE temperature significantly affected the yield of phenolic compounds from the seed cake extracts, while Xu and Pan (2013) reported a similar influence in the extraction of all-trans-lycopene from red grapefruit. The general application of higher UAE temperatures in these studies resulted in higher yields of bioactive compounds and these observations may be explained by the higher system energies could increase the solubility of target compounds, and consequentely improve their liberation from the sample matrix by destroying the integrity of connective and structural tissues (Teh and Birch, 2014). Table 2 also shows that UAE time significantly affected the extraction efficiency of euphol (p = 0.047). Under the fixed conditions of UEA temperature (60 ◦ C) and power (60%), yields of euphol were shown to increase from 3.2 mg/g to 4.2 mg/g for UEA times between 30 and75 min. Beyond 75 min, plateauing of the yield was observed (Fig. 4A), indicating that prolonged sonication did not result in further improvements in extraction efficiency. Similar findings were reported in previous studies relating to the extraction of total phenolic compounds from olive leaf (S¸ahin and S¸amlı, 2013) and procyanidins from Larix gmelinii bark (Sun et al., 2013). The rising profile of the extraction curves for temperature and time may be considered to be the result of two distinct process; namely a fast extraction of euphol from cells close to the surface of
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Fig. 4. Impact of ultrasonic temperature, time and power on the extraction efficiency of euphol (A); and the 3D response surface profilers and 2D contour plots of euphol as affected by ultrasonic temperature, time and power (B).
the plant material, which is quickly solubilized, (a process known as “washing”), and a second, slower, diffusion and osmosis-based processes (known as “slow extraction”) involving the liberation of more deeply embedded euphol (S¸ahin and S¸amlı, 2013). Table 2 showed that extraction efficiency was not significantly affected by UAE power either alone or in combination with changes in temperature or solvent ratio change. Somewhat unexpectedly, Fig. 4A showed that under fixed conditions of temperature (60 ◦ C) and time (75 min), euphol decreased from 4.2 mg/g to 3.4 mg/g as UAE power was increased from 60% to 100% (150–250 W). Our findings contrast with results presented in previous studies, which found a positive correlation between power rise and extraction efficiency for the liberation of both procyanidins from L. gmelinii bark (Sun et al., 2013) and anthocyanins and polyphenols from Nephelium lappaceum L. fruit peel (Prakash Maran et al., 2013). We tentatively apportion our findings to destruction of the steroidal skeleton as a result of the prolonged exposure to ultrasonic waves. A recent report probing the application of ultrasound in the destruction of contaminant steroids in ground- and wastewater identified significant degradation (60–98%) of steroids such as estrone, estriol, equilin, 17-dihydroequilin, and norgestrel following sonication at powers ranging between 0.6 and 4 kW for prolonged time periods (40–60 min) (Suri et al., 2007). While our experiments were at a lower power range, time periods were similar, suggesting the potential for a similar outcome. Attempts to examine the HPLC traces of the high power extracts failed to clearly identify the presence of degradation products due to the complexity of the extract profile. 3.4. Optimization of ultrasonic conditions for extraction of euphol, and determination of phenolic content and antioxidant capacity of the extract Based on the regression coefficients, the scale of impact of the ultrasonic parameters on the euphol extraction efficiency (from the largest to the smallest) could be listed as follows: (i) ultrasonic temperature, (ii) ultrasonic time and (iii) ultrasonic power.
Table 3 Bioactive compounds and antioxidant capacity of E. tirucalli leaf extract prepared under optimal UAE conditions. Bioactive compounds/antioxidant assays
Content/capacity
Euphol (mg/g FW) Phenolic content (mgGAE/g FW) ABTS (%) DPPH (%) FRAP (mM TE/g FW)
4.1 ± 0.1 2.5 ± 0.8 61.7 ± 18.2 40.5 ± 12.8 47.0 ± 17.0
The values are the mean ± standard deviations (n = 4).
Specific interrelationships between the respective parameters were visually illustrated in 3D profiles shown in Fig. 4B. The R2 of 0.93 (Table 2) indicated that a total of 93% of the variation in the extraction efficiency of euphol could be explained while only 7% of the variation could not be explained by the prediction profilers. Therefore, the optimal ultrasonic conditions for maximising the extraction efficiency of euphol were deduced as: UAE temperature of 60 ◦ C, UAE time of 75 min and UAE power of 150 W (60% power). Under optimal ultrasonic conditions, the modeling predicted a euphol yield of 4.37 ± 0.79 mg/g of fresh euphorbia leaf. To validate this prediction, euphorbia leaf was extracted under optimal conditions (n = 4). The results (Table 3) revealed an average experimental yield of 4.1 ± 0.1 mg/g, which was found to be within experimental tolerances of the predicted value (p > 0.05), thereby validating the model. Additionally, phenolic content and antioxidant capacity of the E. tirucalli leaf extract under optimal conditions were also investigated. The results (Table 3) showed that this extract also contained 2.5 mg/g FW of phenolic content. In comparison with other materials, this extract contained higher phenolic content than that of Piper betel (2.12 mg/g FW) and Polyalthia longifolia (2.44 mg/g FW) (Kaur and Mondal, 2014) and loquat (Eriobotrya japonica (Thunb.) (0.129–0.578 mg/g FW) (Polat et al., 2010). Of note, these conditions were not optimal for maximum level of phenolic content; therefore, it is worthy to optimize extraction of phenolic content in
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future study for further isolation of phenolic compounds. In addition, Results (Table 3) also showed that percentage of inhibition from ABTS and DPPH assays were 62% and 40%, respectively. Ferric reducing antioxidant power was found to be 47 mM TE/g FW. The DPPH value was comparable to those of Citrus aurantifolia and P. betel (40 and 35%, respectively) (Kaur and Mondal, 2014) and the FRAP value was higher than that of fresh date (Phoenix dactylifera L.) (11.7–20.6 mM TE/g FW) (Al-Farsi et al., 2005). These data indicated that this E. tirucalli leaf extract exhibited potent antioxidant capacity, and future studies are required to identify the major contributors of the antioxidant capacity. 4. Conclusions The solvents and ultrasonic parameters including temperature, time and power were found to significantly affect the extraction efficiency of euphol from E. tirucalli leaf. The solvent ratio of ethyl acetate:ethanol (4:1, v/v) was the most effective for the extraction of euphol. Utrasonic temperature and time had a positive impact on euphol yield; whereas, ultrasonic power had a negative effect. The optimal ultrasonic conditions for extraction of euphol were as follows: temperature of 60 ◦ C, time of 75 min and power of 150 W. Under these optimal conditions, the extract also contained high levels of phenolic compounds and possessed potent antioxidant capacity. These ultrasonic extraction conditions can be scaled up to pilot and then industrial scale for further isolation and potential utilization of euphol in the pharmaceutical industry. Conflict of interest The authors declare no conflicts of interest. Acknowledgements The authors would acknowledge the following funding support: Ramaciotti Foundation (ES2012/0104); Cancer Australia and Cure Cancer Australia Foundation (1033781). The authors also kindly thank the Vietnamese Government through the Vietnam International Education Development - Ministry of Education and Training (Project 911) and the University of Newcastle for awarding a VIEDTUIT scholarship to VTN and DTT. References Al-Farsi, M., Alasalvar, C., Morris, A., Baron, M., Shahidi, F., 2005. Comparison of antioxidant activity, anthocyanins, carotenoids, and phenolics of three native fresh and sun-dried date (Phoenix dactylifera L.) varieties grown in Oman. J. Agric. Food Chem. 53, 7592–7599. Cataluna, P., Rates, S., 1997. The traditional use of the latex from Euphorbia tirucalli Linnaeus (Euphorbiaceae) in the treatment of cancer in South Brazil. Acta Hort. 501, 289–296. Gupta, N., Vishnoi, G., Wal, A., Wal, P., 2013. Medicinal value of Euphorbia tirucalli: a review. Res. Rev.: J. Pharmacognosy Phytochem. 1, 16–25. Hromadkova, Z., Ebringerova, A., Valachovic, P., 2002. Ultrasound- assisted extraction of water-soluble polysaccharides from the roots of valerian (Valeriana officinalis L.). Ultrason. Sonochem. 9, 37–42.
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Optimization of ultrasound-assisted extraction conditions for euphol from the medicinal plant, Euphorbia tirucalli, using response surface methodology Quan V. Vuong a,b,∗ , Van Tang Nguyen a,b,c , Dang Trung Thanh a,b,c , Deep Jyoti Bhuyan a,b , Chloe D. Goldsmith a,b , Elham Sadeqzadeh a,b , Christopher J. Scarlett a,b , Michael C. Bowyer a,b a
Pancreatic Cancer Research, Nutrition Food & Health Research Group, University of Newcastle, NSW, Australia School of Environmental and Life Sciences, University of Newcastle, NSW, Australia c Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Viet Nam b
a r t i c l e
i n f o
Article history: Received 22 June 2014 Received in revised form 25 August 2014 Accepted 28 September 2014 Available online xxx Keywords: Euphol Euphorbia tirucalli Ultrasonic-assisted extraction Optimization Response surface methodology
a b s t r a c t Euphol identified in Euphorbia tirucalli (E. tirucalli) has been linked with various health benefits. This study aimed to optimize ultrasonic extraction conditions for euphol from E. tirucalli leaf. Different solvents were tested to determine the most effective solvent for extraction of euphol. Then, response surface methodology (RSM) was employed to optimize ultrasound-assisted extraction conditions including temperature, time and power for maximal extraction of euphol. Our results showed that ethyl acetate:ethanol (4:1, v/v) was the most effective solvent for the extraction of euphol. Ultrasonic temperature and time had a positive impact, whereas, ultrasonic power had a negative effect on the extraction efficiency of euphol. The optimum ultrasonic extraction conditions for euphol were identified as: solvent-to-fresh sample ratio of 100:32 mL/g; ultrasonic temperature of 60 ◦ C; ultrasonic time of 75 min and ultrasonic power of 60% (150 W). Under these optimum conditions, approximately 4.06 mg of euphol could be obtained from one gram of fresh E. tirucalli leaf. This extract also contained phenolic compounds (2.5 mg GAE/g FW) and possessed potent antioxidant capacity. These optimal conditions are applicable for a larger scale to extract and isolate euphol for potential utilization in the pharmaceutical industry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Euphorbia tirucalli (E. tirucalli), also known as the pencil tree, sticks-on-fire or milk bush, is a small tree native to Madagascar and Africa; however, it has been widely distributed across the globe because of its tolerance to a wide range of climatic conditions (Mwine and Damme, 2011). E. tirucalli has been used as a traditional medicine in the Middle East, India, Africa and South America for the treatment of a range of ailments including syphilis, asthma,
Abbreviations: RSM, response surface methodology; UAE, ultrasound-assisted extraction. ∗ Corresponding author at: Pancreatic Cancer Research Nutrition Food & Health Research Group, School of Environmental and Life Sciences, University of Newcastle, Brush Rd, Ourimbah, NSW 2258, Australia. Tel.: +61 2 4348 4045; fax: +61 2 4348 4145. E-mail address: [email protected] (Q.V. Vuong).
cancer, colic, intestinal parasites and leprosy (Cataluna and Rates, 1997; Gupta et al., 2013). The traditional use of E. tirucalli for purported health benefits has prompted scientific interest in the exploration of its bioactive constituents for pharmacological utilization. A range of di- and triterpene compounds has been identified in E. tirucalli. Of those identified, euphol (Fig. 1) is the most prominent and has been found to exhibit anti-cancer activity against human gastric cancer and breast cancer in vitro (Sadeghi-Aliabadi et al., 2009; Zhang et al., 2012). Consequently, optimization of conditions for maximal extraction of euphol and other bioactives from E. tirucalli is of interest and worthy of further investigation. To date however, no formal studies in this area have been undertaken. Response Surface Methodology (RSM) is a statistical technique that aims to develop a functional relationship between a response of interest and a number of input variables (Khuri and Mukhopadhyay, 2010). In comparison with single variable optimization methods, RSM is a time and cost effective means of simultaneously evaluating the key experimental parameters
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2.2. Extraction of euphol using ultrasound-assisted extraction (UAE) To test the impact of solvents on extraction efficiency of euphol, ground fresh leaf was extracted in different solvents including ethanol, acetonitrile:ethanol (4:1, v/v), acetone:ethanol (4:1, v/v), ethyl acetate:ethanol (4:1, v/v), or hexane:ethanol (4:1, v/v) at a solvent-to-sample ratio of 100:32 mL/g. The extraction was conducted using a tunable ultrasonic bath (Soniclean, 220 V, 50 Hz and 250W, Soniclean Pty Ltd, Australia) under ultrasonic conditions of room temperature, 60 min and power of 150 W. To optimize UAE conditions, the most effective solvent (ethyl acetate:ethanol (4:1, v/v)) was used for extraction of fresh leaf at a solvent-to-sample ratio of 100:32 mL/g. UAE was conducted using a tunable ultrasonic bath (Soniclean, 220 V, 50 Hz and 250 W, Soniclean Pty Ltd, Australia) to be set at different conditions as designated by RSM design. An external digital thermometer was also used to measure the temperature of the ultrasonic bath, while tap water was used to cool the water to the required temperature if the ultrasonic bath exceeded the designated temperature due to ultrasonic energy. 2.3. Response surface methodology design
Fig. 1. HPLC chromatogram of euphol standard and euphol structure (A) and the extract E. tirucalli leaf (B).
(Wang et al., 2011), thus contributes to the optimization of euphol extraction from E. tirucalli. Ultrasonic extraction has gained increasing popularity as method to be used both in conjunction with, or in place of, traditional extraction techniques because of its efficient energetics and reduced extraction times (Mierzwa et al., 1997). Ultrasound assisted extraction (UAE) is now well established as a technique for the extraction of low molecular weight compounds from plant sources (Hromadkova et al., 2002), and has been found to be more effective in extracting phytochemicals from plants and is easily adapted to the industrial scale (Vinatoru, 2001). This study aimed to determine the most effective extraction solvents and then utilize RSM to develop optimal conditions for the extraction euphol from E. tirucalli by ultrasonic-assisted extraction as a useful engineering tool for commercial applications to prepare euphol from E. tirucalli for utilization in pharmaceuticals.
Response surface methodology (RSM) with a Box–Behnken design was employed to design the experiment and investigate the influence of the three independent ultrasonic parameters: power (60%, 80%, 100% or 150, 200, 250 W), temperature (30, 45, 60 ◦ C), and time (30, 60, 90 min). After extraction, the extracts were then immediately cooled to room temperature (ice bath), then filtered through a 5 mL syringe fitted with a 0.45 m cellulose syringe filter (Phenomenex Australia Pty. Ltd, NSW, Australia), then transferred into brown glass HPLC vials for HPLC analysis. The independent variables and their code variable levels are shown in Table 1. To express the level of euphol as a function of the independent variables, a second-order polynomial equation (Eq. (1)) was used as follows (Vuong et al., 2011): Y = ˇo +
k i=1
ˇi Xi +
k k−1
i=1
ˇij Xi Xj +
j=2
k
ˇii Xi2
(1)
i=1
i
where various Xi values are independent variables affecting the responses Y; ˇ0 , ˇi , ˇii , and ˇij are the regression coefficients for intercept, linear, quadratic, and interaction terms, respectively; and k is the number of variables. In this study, the three independent ultrasonic parameters: X1 (ultrasonic temperature, ◦ C), X2 (ultrasonic time, min) and X3 (ultrasonic power, %) were applied (Table 2), with euphol extraction efficiency expressed as the following polynomial (Eq. (2)): YEuphol = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ3 X3 + ˇ12 X1 X2 + ˇ13 X1 X3 + ˇ23 X2 X3 + ˇ11 X12 + ˇ22 X22 + ˇ33 X32
(2)
2.4. Determination of euphol 2. Materials and methods 2.1. Plant materials The phylloclades of a E. tirucalli tree were collected on July 16 from a property located in Saratoga, NSW, Australia (33.47◦ S, 151.35◦ E) and immediately transferred to the laboratory and stored at −20 ◦ C. Plylloclade samples were then freeze dried in liquid nitrogen, ground to consistent particle size using a commercial blender, then stored at −20 ◦ C until required.
Euphol standard was prepared from the latex of E. tirucalli in our laboratory using a Shimadzu HPLC system fitted with a semiprep Luna C18 reversed-phase column (Phenomenex, Australia) maintained at 35 ◦ C and coupled to auto fraction collector (Shimadzu Australia, Rydalmere, NSW, Australia). The sample was detected using UV detector set at 210 nm as per the work of Vuong et al. (2012). A representative chromatogram of euphol standard is shown in Fig. 1A. Quantitative analysis of euphol content in different filtered sample extracts was then undertaken using
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Table 1 Box–Behnken design and observed responses of euphol extracted from Euphorbia tirucalli phylloclades using UAE. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a b c
UAE temperatures (◦ C)
Patterna
+−0 0++ 000 0−+ +0+ 000 0−− 000 −0− 0+− −−0 +0− −0+ −+0 000
UAE power (%)b
UAE time (min)
60 45 45 45 60 60 45 45 30 45 30 60 30 30 45
30 90 60 30 60 90 30 60 60 90 30 60 60 90 60
Euphol (mg/g)
80 100 80 100 100 80 60 80 60 60 80 60 100 80 80
Experimental valuesc
Predicted values
3.74 3.62 3.57 2.85 3.29 3.92 2.43 3.57 2.03 3.81 1.97 4.14 2.69 2.09 3.57
3.40 3.33 3.57 3.03 3.45 4.04 2.71 3.57 1.87 3.63 1.85 4.20 2.63 2.43 3.57
Pattern −, 0, + are the minimum values, center points, and maximum values, respectively, of UAE temperature, time and power in the tested ranges. Corresponding values of power were 250 W for 100%; 200 W for 80%; and 150 W for 60%. Values were obtained from triplicated experiments and expressed as mg/g FW.
a reversed-phase analytical column (250 mm × 4.6 mm Synergi 4 mm Fusion-RP 80A column, Phenomenex Australia) maintained at 35 ◦ C with peak detection set at 210 nm. The mobile phase (flow rate = 1 mL/min) consisted of two feed solvents; methanol (A) and acetonitrile (B). A gradient elution schedule was used as follows: 100% A for the first 5 min.; followed by a linear gradient changed to 50% A and 50% B (5–25 min.); then finally, a linear gradient changed back to 100% A (25–30 min.). An auto-injector was used to inject 20 L of all sample extracts onto the HPLC column. A representative chromatogram of euphol in the E. tirucalli leaf is shown in Fig. 1B. The content of euphol was calculated based on the standard curve and expressed as mg of euphol per gram of fresh weight (FW). 2.5. Determination of phenolic content and antioxidant capacity Total phenolic content (TPC) of the extract prepared under optimal conditions was determined using a method described by Vuong et al. (2013). Gallic acid was used as the standard for the construction of a calibration curve, with the results expressed as mg of gallic acid equivalents per gram of fresh weight (FW) (mg GAE/g FW). Three different antioxidant assays described in previous studies were employed to test antioxidant properties of the extract under optimal conditions, including ABTS (2,2 -azino-bis3-ethylbenzothiazoline-6-sulphonic acid) (Thaipong et al., 2006),
DPPH (1,1-diphenyl-2-picrylhydrazyl) assay (Vuong et al., 2013) and FRAP (ferric reducing antioxidant power) assay (Thaipong et al., 2006). Values of ABTS and DPPH were expressed as percentage of inhibition and value of FRAP was expressed as Trolox equivalents per gram of fresh weight (TE/g FW). 2.6. Statistical analysis RSM experimental design and analysis were conducted using JMP software (Version 11). The software was also used to establish the model equation, to graph the 3-D plot, 2-D contour of the response and to predict the optimum values for the three response variables. The student’s T-test was conducted using the SPSS statistical software (Version 20) for comparison of sample means. Differences between the mean euphol concentrations in the different samples were taken to be statistically significant at p < 0.05. 3. Results and discussion 3.1. Impact of solvents on extraction efficiency of euphol Previous studies indicated that different solvents could lead to different extraction efficiencies of bioactive compounds (Vuong et al., 2013; Zhang et al., 2013). Therefore, this study determined
Table 2 Estimated regression coefficients for the quadratic polynomial model and the analysis of variance for the experimental results. Parametera Intercept ˇ0 ˇ1 ˇ2 ˇ3 ˇ12 ˇ13 ˇ23 ˇ11 ˇ22 ˇ33 Lack of fit Pure error R2 PRESS a b *
Predicted coefficients 3.568 0.789 0.306 0.004 0.014 −0.375 −0.154 −0.388 −0.248 −0.144
0.929 8.746
Standard error 0.19 0.12 0.12 0.12 0.17 0.17 0.17 0.17 0.17 0.17
DFb 1 1 1 1 1 1 1 1 1 1 3 Adjusted R2 RMSE
Sum of squares
F value
Prob > F
Model
7.283
0.0208*
4.986 0.750 0.000 0.001 0.565 0.094 0.557 0.228 0.076 . . 0.801 0.33
45.606 6.862 0.001 0.007 5.165 0.864 5.091 2.086 0.698 .
0.001* 0.047* 0.972 0.935 0.072 0.395 0.074 0.208 0.442 .
Coefficients refer to the general model. Degree of freedom. Significant different at p < 0.05.
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Fig. 2. Impact of extraction solvents on extraction efficiency of euphol from E. tirucalli phylloclades. Values (mean ± SD, n = 3) not sharing a letter (on top of the columns) are significantly different at p < 0.05.
the impact of five different extraction solvents with various polarity indexes to identify the most effective solvent for further optimization using response surface methodology. The results (Fig. 2) showed that different extraction solvents significantly affected the extraction efficiency of euphol. Extraction efficiency of euphol was low when using ethanol alone; however, extraction efficiency was significantly improved when it was used in combination with other solvents of lower polarity at the ratio of 1:4 (v/v). A solvent mixture of ethyl acetate:ethanol (4:1, v/v) was found to be the most effective solvent for extraction of euphol from E. tirucalli leaf. The current findings were in agreement with a previous study (Zhang et al., 2013), which reported that a mixture of ethyl acetate:ethanol (4:1 v/v) had higher extraction efficiency than methanol when extracting euphol from root of Euphorbia pekinensis Rupr; and these differences could be explained by the variation of solvent polarities (Vuong et al., 2013). Therefore, solvent of ethyl acetate:ethanol (4:1, v/v) was used for further optimization process. 3.2. Fitting the model for the prediction of euphol Response surface methodology (RSM) is an effective statistical procedure using a minimum set of experiments for determination of the coefficients of a mathematical model and optimization of the conditions (Yemis¸ and Mazza, 2012). However, it is important to test the appropriateness of the RSM mathematical model for predicting the optimal variances and adequately representing the real relationship between the selected parameters. The analysis of variances for the experimental results of the Box–Behnken design (Fig. 3) showed that the coefficient of determination (R2 ) of the model was 0.93, suggesting that 93% of the actual levels can be matched with the model-predicted levels of euphol (Li et al., 2011). The analyzed results (Table 2) also showed that root mean square error (RMSE), which is used to estimate the standard deviation of the random error, was 0.33, further confirming a linear correlation exists between the predicted levels and actual levels of euphol present in the samples analyzed. Results of analysis of the actual experiments (Table 2) revealed that the Predicted Residual Sum of Squares (PRESS) for the model – a measure of how a chosen model fits each point in the design (Zhang et al., 2009) was 8.746, indicating a good measure. In addition, the p and F-values of the model were 0.02 and 7.283, respectively, indicating that the model predictions were significant (Zhang et al., 2009). Moreover, the Lack of Fit report was not generated as a consequence of the analysis of the experimental data, implying that the model was well fitted as it was impossible to assess lack of fit because there were as many estimated parameters as there were observations (SAS-Institute, 2014). Therefore, the results revealed
Fig. 3. Correlation between the predicted and actual levels of euphol.
that the mathematical model was adequate for the prediction of euphol and was fitted to the following second-order polynomial formula (Eq. (3)): YEuphol = 3.568 + 0.789X1 + 0.306X2 + 0.004X3 + 0.014X1 X2 − 0.038X1 X3 − 0.154X2 X3 − 0.388X12 − 0.248X22 − 0.144X32
(3)
3.3. Effect of extraction parameters on the UAE performance The impacts of three UAE parameters on extraction efficiency of euphol were investigated. Results (Fig. 4A) showed that UAE temperature was a significant parameter, with rising UAE temperature correlating to a higher extraction efficiency. At fixed conditions of 75 min and UAE power at 60%, euphol extraction increased from 1.7 mg/g to 4.2 mg/g across a temperatures ranging between 30 and 60 ◦ C. Table 2 shows a p value of 0.001 for UAE temperature, further confirming the significance of temperature on extraction efficiency. The findings are in agreement with previously studies assessing the effectiveness of UAE. Teh and Birch (2014) found that UAE temperature significantly affected the yield of phenolic compounds from the seed cake extracts, while Xu and Pan (2013) reported a similar influence in the extraction of all-trans-lycopene from red grapefruit. The general application of higher UAE temperatures in these studies resulted in higher yields of bioactive compounds and these observations may be explained by the higher system energies could increase the solubility of target compounds, and consequentely improve their liberation from the sample matrix by destroying the integrity of connective and structural tissues (Teh and Birch, 2014). Table 2 also shows that UAE time significantly affected the extraction efficiency of euphol (p = 0.047). Under the fixed conditions of UEA temperature (60 ◦ C) and power (60%), yields of euphol were shown to increase from 3.2 mg/g to 4.2 mg/g for UEA times between 30 and75 min. Beyond 75 min, plateauing of the yield was observed (Fig. 4A), indicating that prolonged sonication did not result in further improvements in extraction efficiency. Similar findings were reported in previous studies relating to the extraction of total phenolic compounds from olive leaf (S¸ahin and S¸amlı, 2013) and procyanidins from Larix gmelinii bark (Sun et al., 2013). The rising profile of the extraction curves for temperature and time may be considered to be the result of two distinct process; namely a fast extraction of euphol from cells close to the surface of
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Fig. 4. Impact of ultrasonic temperature, time and power on the extraction efficiency of euphol (A); and the 3D response surface profilers and 2D contour plots of euphol as affected by ultrasonic temperature, time and power (B).
the plant material, which is quickly solubilized, (a process known as “washing”), and a second, slower, diffusion and osmosis-based processes (known as “slow extraction”) involving the liberation of more deeply embedded euphol (S¸ahin and S¸amlı, 2013). Table 2 showed that extraction efficiency was not significantly affected by UAE power either alone or in combination with changes in temperature or solvent ratio change. Somewhat unexpectedly, Fig. 4A showed that under fixed conditions of temperature (60 ◦ C) and time (75 min), euphol decreased from 4.2 mg/g to 3.4 mg/g as UAE power was increased from 60% to 100% (150–250 W). Our findings contrast with results presented in previous studies, which found a positive correlation between power rise and extraction efficiency for the liberation of both procyanidins from L. gmelinii bark (Sun et al., 2013) and anthocyanins and polyphenols from Nephelium lappaceum L. fruit peel (Prakash Maran et al., 2013). We tentatively apportion our findings to destruction of the steroidal skeleton as a result of the prolonged exposure to ultrasonic waves. A recent report probing the application of ultrasound in the destruction of contaminant steroids in ground- and wastewater identified significant degradation (60–98%) of steroids such as estrone, estriol, equilin, 17-dihydroequilin, and norgestrel following sonication at powers ranging between 0.6 and 4 kW for prolonged time periods (40–60 min) (Suri et al., 2007). While our experiments were at a lower power range, time periods were similar, suggesting the potential for a similar outcome. Attempts to examine the HPLC traces of the high power extracts failed to clearly identify the presence of degradation products due to the complexity of the extract profile. 3.4. Optimization of ultrasonic conditions for extraction of euphol, and determination of phenolic content and antioxidant capacity of the extract Based on the regression coefficients, the scale of impact of the ultrasonic parameters on the euphol extraction efficiency (from the largest to the smallest) could be listed as follows: (i) ultrasonic temperature, (ii) ultrasonic time and (iii) ultrasonic power.
Table 3 Bioactive compounds and antioxidant capacity of E. tirucalli leaf extract prepared under optimal UAE conditions. Bioactive compounds/antioxidant assays
Content/capacity
Euphol (mg/g FW) Phenolic content (mgGAE/g FW) ABTS (%) DPPH (%) FRAP (mM TE/g FW)
4.1 ± 0.1 2.5 ± 0.8 61.7 ± 18.2 40.5 ± 12.8 47.0 ± 17.0
The values are the mean ± standard deviations (n = 4).
Specific interrelationships between the respective parameters were visually illustrated in 3D profiles shown in Fig. 4B. The R2 of 0.93 (Table 2) indicated that a total of 93% of the variation in the extraction efficiency of euphol could be explained while only 7% of the variation could not be explained by the prediction profilers. Therefore, the optimal ultrasonic conditions for maximising the extraction efficiency of euphol were deduced as: UAE temperature of 60 ◦ C, UAE time of 75 min and UAE power of 150 W (60% power). Under optimal ultrasonic conditions, the modeling predicted a euphol yield of 4.37 ± 0.79 mg/g of fresh euphorbia leaf. To validate this prediction, euphorbia leaf was extracted under optimal conditions (n = 4). The results (Table 3) revealed an average experimental yield of 4.1 ± 0.1 mg/g, which was found to be within experimental tolerances of the predicted value (p > 0.05), thereby validating the model. Additionally, phenolic content and antioxidant capacity of the E. tirucalli leaf extract under optimal conditions were also investigated. The results (Table 3) showed that this extract also contained 2.5 mg/g FW of phenolic content. In comparison with other materials, this extract contained higher phenolic content than that of Piper betel (2.12 mg/g FW) and Polyalthia longifolia (2.44 mg/g FW) (Kaur and Mondal, 2014) and loquat (Eriobotrya japonica (Thunb.) (0.129–0.578 mg/g FW) (Polat et al., 2010). Of note, these conditions were not optimal for maximum level of phenolic content; therefore, it is worthy to optimize extraction of phenolic content in
Please cite this article in press as: Vuong, Q.V., et al., Optimization of ultrasound-assisted extraction conditions for euphol from the medicinal plant, Euphorbia tirucalli, using response surface methodology. Ind. Crops Prod. (2014), http://dx.doi.org/10.1016/j.indcrop.2014.09.057
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future study for further isolation of phenolic compounds. In addition, Results (Table 3) also showed that percentage of inhibition from ABTS and DPPH assays were 62% and 40%, respectively. Ferric reducing antioxidant power was found to be 47 mM TE/g FW. The DPPH value was comparable to those of Citrus aurantifolia and P. betel (40 and 35%, respectively) (Kaur and Mondal, 2014) and the FRAP value was higher than that of fresh date (Phoenix dactylifera L.) (11.7–20.6 mM TE/g FW) (Al-Farsi et al., 2005). These data indicated that this E. tirucalli leaf extract exhibited potent antioxidant capacity, and future studies are required to identify the major contributors of the antioxidant capacity. 4. Conclusions The solvents and ultrasonic parameters including temperature, time and power were found to significantly affect the extraction efficiency of euphol from E. tirucalli leaf. The solvent ratio of ethyl acetate:ethanol (4:1, v/v) was the most effective for the extraction of euphol. Utrasonic temperature and time had a positive impact on euphol yield; whereas, ultrasonic power had a negative effect. The optimal ultrasonic conditions for extraction of euphol were as follows: temperature of 60 ◦ C, time of 75 min and power of 150 W. Under these optimal conditions, the extract also contained high levels of phenolic compounds and possessed potent antioxidant capacity. These ultrasonic extraction conditions can be scaled up to pilot and then industrial scale for further isolation and potential utilization of euphol in the pharmaceutical industry. Conflict of interest The authors declare no conflicts of interest. Acknowledgements The authors would acknowledge the following funding support: Ramaciotti Foundation (ES2012/0104); Cancer Australia and Cure Cancer Australia Foundation (1033781). The authors also kindly thank the Vietnamese Government through the Vietnam International Education Development - Ministry of Education and Training (Project 911) and the University of Newcastle for awarding a VIEDTUIT scholarship to VTN and DTT. References Al-Farsi, M., Alasalvar, C., Morris, A., Baron, M., Shahidi, F., 2005. Comparison of antioxidant activity, anthocyanins, carotenoids, and phenolics of three native fresh and sun-dried date (Phoenix dactylifera L.) varieties grown in Oman. J. Agric. Food Chem. 53, 7592–7599. Cataluna, P., Rates, S., 1997. The traditional use of the latex from Euphorbia tirucalli Linnaeus (Euphorbiaceae) in the treatment of cancer in South Brazil. Acta Hort. 501, 289–296. Gupta, N., Vishnoi, G., Wal, A., Wal, P., 2013. Medicinal value of Euphorbia tirucalli: a review. Res. Rev.: J. Pharmacognosy Phytochem. 1, 16–25. Hromadkova, Z., Ebringerova, A., Valachovic, P., 2002. Ultrasound- assisted extraction of water-soluble polysaccharides from the roots of valerian (Valeriana officinalis L.). Ultrason. Sonochem. 9, 37–42.
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Please cite this article in press as: Vuong, Q.V., et al., Optimization of ultrasound-assisted extraction conditions for euphol from the medicinal plant, Euphorbia tirucalli, using response surface methodology. Ind. Crops Prod. (2014), http://dx.doi.org/10.1016/j.indcrop.2014.09.057