Extraction of phenolic compounds from Mentha aquatica: the effects of sonication time, temperature and drying method

  • Pedram Safaiee
  • Amirhossein Taghipour
  • Fatemeh Vahdatkhoram
  • Kamyar MovagharnejadEmail author
Original Paper


The aim of the current research is to study the effect of parameters including: extraction time (sonication time), extraction temperature, and drying method on the extraction yield of phenolics from water mint (Mentha aquatica) under ultrasonic irradiation. Therefore, three drying methods (microwave-drying, oven-drying, and freeze-drying) were performed. The dried samples were then ground and extracted at three different temperatures (20, 40, and 60 °C) in an ultrasonic bath. The maximum total phenolic compounds (TPCs) of 0.2451 mg/g were attained by freeze-drying method regarding the following process conditions: the extraction time of 5 min, and the extraction temperature of 60 °C. The results demonstrated that the drying method and temperature, respectively, are the most effective parameters in the extraction of phenolic compounds from Mentha aquatica. Besides, observations indicated that sonication (under 42 kHz and 160 W sonication condition) would not bring any sensible effect on the extraction yield. Additionally, a cost evaluation of presented experiments has been made to investigate the economical evaluation. The calculations showed that the consumed electrical power in freeze-drying method is very high which would not be cost effective.


Mentha aquatica Drying method Extraction yield Total phenolic content Sonication time 


Mentha aquatica or briefly M. aquatica is a perennial flowering plant in the family Lamiaceae which can be found in the temperate and humid areas near the channels of water streams and shallow margins of swamps, wet meadows and so forth (Dorman et al. 2003). Mint family plants such as spearmint, holy basil, and hyssop have been used widely in the pharmaceutical, food and hygiene industries (Kamkar et al. 2009). In northern regions of Iran, water mint is used as flavoring in some kinds of foods. Additionally, the dried water mint is used as a medicinal plant and a common vegetable in home remedies for many types of diseases. Moreover, the effect of polyphenols on cancer risk reduction has been also reported in numerous studies (Lin et al. 1999; Mukhtar and Ahmad 2000; Thangapazham et al. 2007). Therefore, the studies on determination of phenolic compounds in different parts of plants, extraction techniques, and effective parameters have been investigated in some studies (Hussein et al. 2018; Lopes et al. 2018). Additionally, the effect of hydrothermal conditions on extraction yield was studied and the results indicated that the extracted phenolics increase by increasing the operating pressure and temperature. In another study, the effect of extraction time, temperature, solvent:solid ratio and solvent composition on the extraction yield was studied by Bilek (2010). Considering the selected parameters, the observations indicated that there is an optimum condition for extraction of the highest TPCs and unexpectedly, increasing parameters such as extraction temperature and/or extraction time will not always increase the extraction efficiency (Barba et al. 2016).

The conventional extraction techniques consume a higher amount of solvent, energy and time (Lopes et al. 2018; Patra et al. 2018). In the past few years, different methods such as supercritical fluid extraction (SFE) (Santos et al. 2012; Barba et al. 2017), microwave-assisted extraction (MAE) (Nguyen et al. 2015; Oussaid et al. 2018), and ultrasonic-assisted extraction (UAE) (Pietrzak et al. 2014) have been employed alone or in conjunction with conventional methods for extraction of analytes from different matrices. Propagation of ultrasonic waves in liquids results in turbulent fluid movement and a great microscale velocity gradient around the cavitational bubbles (Leighton 1994; Roy 1999; Chen et al. 2006). Ultrasonically movement of fluid enhances the physical mass transfer processes between the solid-bulk and gas-bulk interfaces. As a consequence, the associated sonophysical effects can facilitate mixing, desorption, extraction, cleaning processes, etc.

In the presented study, we aim to investigate the effect of three drying methods [microwave-drying, hot-air oven-drying, and freeze-drying (lyophilization)] on the extraction yield of total phenolic compounds from Mentha aquatica. The extraction process was assisted with ultrasonic waves in a water bath at different temperatures. Afterward, the effect of operating parameters including sonication time and temperature was also studied. The experimental steps of the current study are summarized in Fig. 1. Additionally, a simple economic evaluation of experiments was carried out to aid decision-making process in scale-up production.
Fig. 1

Experimental steps for ultrasonic-assisted extraction of phenolic compounds from Mentha aquatica


Sodium carbonate purchased was from Merck Co. (Darmstadt, Germany), Gallic acid and Folin–Ciocalteu reagent were purchased from Sigma chemicals Co. Cadyson CD-4820 ultrasonic bath (160 W, 42 kHz) was used for sonicating the extraction vessel.


Water mint (Mentha aquatica) was collected from Bishe Mahalle, Karipey Rural District, Lalehabad District, Babol Country, Mazandaran Province, Iran (latitude of 36.54°, longitude of 52.35°) in June 2018. The harvested plants were then immediately transported to the laboratory at Babol Noshirvani University of Technology. Then, the leaves were separated gently from stems, washed and dehumidified to remove dust and dirt and stored at − 20 °C before the drying process.


The effect of three different drying methods including freeze-drying, hot-air oven-drying, and microwave-drying on extraction yield were studied. The drying conditions of each technique are summarized in Table 1. After drying, the moisture of some dried leaves (as a sample of total dried leaves) was measured using AND MX50 moisture analyzer on wet basis. The dried leaves were then ground and sieved to a uniform size (US mesh 40). They were stored in − 10 °C before being used for the experiments (Fig. 2).
Table 1

Details of the drying conditions

Drying method


Hot-air oven-drying


Temp (°C)

− 45


Pressure (Pa)




Power (W)




Duration (min)




Fig. 2

The dried, ground leaves


The phenolic compounds of 0.1 ± 0.001 g of each sample were extracted in 100 mL of solvent (water) under ultrasonic irradiation. The extraction temperature was preciously controlled using a water bath. The extraction experiments were conducted at different temperatures of 30, 40, and 60 °C and different sonication times of 5, 10, and 20 min. Then, the solution was filtered into a volumetric flask and stored at − 10 °C in dark for further analysis.

TPC analysis

The total phenolic content was measured according to Folin–Ciocalteu method (Singleton and Rossi 1965). Afterwards, 10 mL of distilled water and 0.5 mL of herbal extract were mixed with 0.25 mL Folin–Ciocalteu reagent in a 15-mL volumetric flask. After 5 min, 2 mL of 6% w/w sodium carbonate solution was then added to the volumetric flask. The solution was shacked and kept in a dark place for 1 h at room temperature. Sample absorbance was measured at 750 nm in a UV–Vis spectrophotometer (Chrom-Tech CT-2200, Taiwan). The blank solution was prepared using distillated water instead of the herbal extract. The standard diagram was plotted using gallic acid and total phenolics expressed in milligrams of gallic acid equivalent per g of dry matter (d.m.).

Experimental design

The major defects of old strategies such as One-factor-at-a-time (OFAT) approach for statistical evaluation of different factors in experimentations led to appearance and development of more advanced experimental design procedures. Design of experiments or simply DOE is a method employed to determine the effect of different parameters (independent variables) on one or more responses (dependent variables). In the current study, DOE steps (setting objects, selecting process variables, choosing an experimental design, and significance of independent variables) were performed using Design Expert® software version 7. Considering the small size of the extracted data, statistical evaluations such as interaction analysis and repeatability of the experiments were not presented here. Response surface method (RSM) was also applied to evaluate the effects of sonication time (min), temperature of the extraction vessel (°C), and drying method on total phenolic contents of extracts (mg/g dried plant). According to the effective factors, a three level user-defined design consists of two numeric factors (sonication time and temperature) and one categorical factor (drying method) were employed (which needs 27 runs). Equation mental design is shown in Table 2. Optimal conditions based on the independent variables were obtained using the predictive equation of RSM.
Table 2

Independent variables, corresponding coded values and range used for optimization

Independent variable



Coded alpha level

− 1


+ 1

Sonication time












Drying method





Results and discussion

Although the results can provide a good understanding of the effectiveness of parameters, but they does not provide any specific value to specify the significance of each parameter. Therefore, analysis of variance (ANOVA) technique was used to estimate the source of variation and finding the most important factor(s) (Table 3). In this table, the factors with probability values (P value) less than 0.05 are considered as significant parameters and those with P values greater than 0.10 are nonsignificant (Roosta et al. 2014). Additionally, the calculated F value is another criterion to estimate the significance of a factor, i.e., the more the F value of the design factor is, the more its influence on the response (Shadmehr et al. 2018).
Table 3

Independent variables and corresponding coded values and range used for optimization


Sum of squares


Mean square

F value

P value




4.31E − 03


< 0.0001


6.31E − 06


6.31E − 06




2.82E − 03


2.82E − 03


< 0.0001

C-drying method





< 0.0001


1.02E − 04


1.02E − 04




1.48E − 05


7.41E − 06




9.19E − 04


4.59E − 04




9.52E − 04


5.60E − 05


Cor total




R2-squared = 0.9761, adjusted R2 = 0.9634, predicted R2 = 0.9414

Considering the presented data in Table 3, it can be concluded that the independent variables including drying method and temperature bring the most impacts on the extraction yield, respectively. In contrast, extraction time (sonication time) seems to be an ineffective factor because of its high P value (0.7413). The ineffectiveness of sonication time is in contrast with the findings of Ghafoor et al. (2009) and Revilla et al. (1998) who obtained higher yield of phenolic compounds in longer time. On the contrary, the poor effect of extraction time on the extracted TPC can also be found in the literature (Liyana-Pathirana and Shahidi 2005; Bilek 2010). The ineffectiveness of sonication time might be happened because of the early complete extraction of phenolics before the shortest time period (5 min). Therefore, a new set of experiments were designed to investigate the influence of the sonication in shorter time periods. Extracted data for freeze-dried samples are shown in Table 4. The extracted data showed that using ultrasonic bath (42 kHz and 160 W) even in smaller time periods cannot effectively intensify the extraction process.
Table 4

Extracted phenolics (mg/g) from freeze-dried samples at different temperatures and sonication time range of 30–180 s

Time (s)

Temperature (°C)

TPC (mg/g)




























The effect of drying method

The effect of drying technique on the quality and quantity of products have been investigated in different researches (OrphAnides et al. 2013; Aydin and Gocmen 2015; Nunes et al. 2016). Moreover, the selection and use of an optimal drying method plays a crucial role in retaining bioactive compounds (Nguyen et al. 2015). In the presented study, Mentha aquatica leaves were dried using different drying methods including: microwave-drying, oven-drying, and freeze-drying. According to the ANOVA results (Table 3), drying method plays a statistically significant role (P < 0.0001) in extraction of phenolic compounds from Mentha aquatica leaves. Additionally, the high amount of F value (312.04) clearly indicates the major effect of drying method on the extraction yield. Freeze-drying possessed the highest TPC yield among the three different methods used in this study. The same results were reported by Vu et al. (2017), so, freeze-drying technique seems to be an excellent drying method for preserving bioactive materials of plants. A comparison between the extracted TPC versus drying method at 30 °C, 40 °C, and 60 °C is shown in Figs. 3 and 4. The oven-dried samples seem to miss a considerable amount of their bioactive compounds during the drying process. The loss of bioactive compounds could be the result of the extreme drying conditions (high temperature and radiative heat transfer from heating element). Our observations showed that the extracted bioactive compounds from microwave-dried samples are significantly more than those for oven-dried. One possible explanation for this difference could be due to the lower drying temperature and drying time in microwave driers. Furthermore, in microwave driers, the high-frequency radio waves (300 GHz–300 MHz) simply penetrate inside the samples, so they can dried uniformly.
Fig. 3

Extraction yield of phenolics from samples dried by different methods [oven-dried (square), microwave-dried (diamond), and freeze-dried (filled triangle)] at different extraction temperatures after 5 min

Fig. 4

Extraction yield of phenolics from samples dried by different methods [oven-dried (square), microwave-dried (diamond), and freeze-dried (filled triangle)] at different extraction temperatures after 10 min

The effect of temperature

Since the mobilization of phenolic compounds from substrate may occur up to a certain level and the possibility of decomposition at high temperature, the experiments were performed over a moderate range of temperature (20–60 °C). Our observations indicated that the temperature is a significant parameter. As shown in Figs. 3 and 4, the TPC values increased by increasing the temperature. Furthermore, the freeze-dried samples were highly affected by temperature variations, while the extracted phenolics from oven-dried samples were not changed significantly by temperature change. It can be interpreted that the phenolics can be preserved better in the substrate in freeze-dried samples and release gradually by increasing the extraction temperature. On the other hand, during the oven-drying process a considerable amount of phenolics decomposed or released from the substrate because of the cell destruction under extreme thermal condition. As a consequence, increasing the temperature did not change the extraction yield remarkably.

Process economics

As shown by several studies, among the drying methods, freeze-drying can effectively preserve the bioactive compounds of plants. Furthermore, the extracted experimental data of the presented research indicated that increasing the extraction temperature leads to a significant growth of extraction yield. However, the objective of increasing the extracted phenolics is ultimately related to the minimization of process costs; if the cost of the drying technique is too high and the extraction process proceeds in high temperature, this will defeat the main purpose of its application. Therefore, the cost of consumed electricity is calculated as the sum of consumed power of the dryer and the heater (for increasing the extraction temperature). Accordingly, the overall costs of each experiment are calculated and summarized in Table 5. (The average price people in Iran pay for electricity is about 7209 Iranian rial (0.22 US dollar) per kW-h).
Table 5

The total cost of experiments according to the consumed electrical power at 40 °C (kW-h); 0.22 × (A + B)


5 min

10 min

20 min


0.22 × (0.0833 + 32.0000) = 7.0583

0.22 × (0.1666 + 32.0000) = 7.0766

0.22 × (0.3333 + 32.0000) = 7.1133


0.22 × (0.0833 + 0.1160) = 0.0438

0.22 × (0.1666 + 0.1160) = 0.0621

0.22 × (0.3333 + 0.1160) = 0.0988


0.22 × (0.0833 + 0.6666) = 0.1649

0.22 × (0.1666 + 0.6666) = 0.1833

0.22 × (0.3333 + 0.6666) = 0.2199

Consumed electricity by the heater. Consumed electricity by the dryer

Although using the freeze-drying technique, the extraction yield was maximized between 12 and 90%, the consumed energy considerably increased about 65–160 times. Against the microwave- and oven-drying methods, freeze-drying was a time-consuming process, carried out in partial vacuum (0.1 Pa) and low temperature (− 40 to − 58 °C) which significantly increases the required electrical power.


The effects of three different parameters including drying method, extraction temperature, and extraction time (under ultrasonic irradiation) were studied in the presented paper. We used Design Expert software to design the experiments and randomize the runs. The RSM method and corresponding ANOVA results were then utilized to prioritize the parameters affecting the extraction yield. Additionally, the cost of each experiment was taken into account to give an economic comparison between the three methods. Having investigated the extraction experiments and corresponding costs, the following results were obtained:
  1. 1.

    Drying method is the most significant factor in the extraction of phenolic compounds from Mentha aquatica and the freeze-drying technique is the best drying method when the extraction yield is taken into consideration.

  2. 2.

    The extraction yield increases by increasing the extraction temperature from 30 to 60 °C.

  3. 3.

    Sonication with an ultrasonic water bath (42 kHz and 160 W) is an insignificant parameter in the extraction of phenolics from Mentha aquatica.

  4. 4.

    Economic investigation of experiments indicated that the utilized freeze-drying method consumes a remarkable amount of energy which is not cost effective. Additionally, also the extraction yield increased by increasing the extraction temperature, but the additional required power should be considered in the scale-up process for industrial production.




The authors acknowledge the funding support of Babol Noshiravani University of Technology through Grant Program no. BNUT/370,675/97.

Compliance with ethical standards

Conflict of interest

On behalf of all the authors, the corresponding author states that there is no conflict of interest.


  1. Aydin E, Gocmen D (2015) The influences of drying method and metabisulfite pre-treatment on the color, functional properties and phenolic acids contents and bioaccessibility of pumpkin flour. LWT Food Sci Technol 60:385–392CrossRefGoogle Scholar
  2. Barba FJ, Zhu Z, Koubaa M, Sant’Ana AS, Orlien V (2016) Green alternative methods for the extraction of antioxidant bioactive compounds from winery wastes and by-products: a review. Trends Food Sci Technol 49:96–109. CrossRefGoogle Scholar
  3. Barba FJ, Putnik P, Bursać Kovačević D, Poojary MM, Roohinejad S, Lorenzo JM, Koubaa M (2017) Impact of conventional and non-conventional processing on prickly pear (Opuntia spp.) and their derived products: from preservation of beverages to valorization of by-products. Trends Food Sci Technol 67:260–270. CrossRefGoogle Scholar
  4. Bilek SE (2010) The effects of time, temperature, solvent: solid ratio and solvent composition on extraction of total phenolic compound from dried olive (Olea europaea L.) leaves. GIDA J Food 35:411–416Google Scholar
  5. Chen D, Weavers LK, Walker HW (2006) Ultrasonic control of ceramic membrane fouling by particles: effect of ultrasonic factors. Ultrason Sonochem 13:379–387CrossRefGoogle Scholar
  6. Dorman HJ, Kosar M, Kahlos K, Holm Y, Hiltunen R (2003) Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars. J Agric Food Chem 51:4563–4569. CrossRefGoogle Scholar
  7. Ghafoor K, Choi YH, Jeon JY, Jo IH (2009) Optimization of ultrasound-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from grape (Vitis vinifera) seeds. J Agric Food Chem 57:4988–4994CrossRefGoogle Scholar
  8. Hussein SR, Abdel Latif RR, Marzouk MM, Elkhateeb A, Mohammed RS, Soliman AAF, Abdel-Hameed E-SS (2018) Spectrometric analysis, phenolics isolation and cytotoxic activity of Stipagrostis plumosa (Family Poaceae). Chem Papers 72:29–37. CrossRefGoogle Scholar
  9. Kamkar A, JebeliJavan A, Jamshidi R (2009) Antioxidant capacity of essential oil and extract of Iranian Mentha spicat. J Vet Lab Res 1:69–77. Google Scholar
  10. Leighton T (1994) 5—Effects and mechanisms. In: Leighton T (ed) The acoustic bubble, Academic Press, pp 439–590.
  11. Lin J-K, Liang Y-C, Lin-Shiau S-Y (1999) Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. Biochem Pharmacol 58:911–915CrossRefGoogle Scholar
  12. Liyana-Pathirana C, Shahidi F (2005) Optimization of extraction of phenolic compounds from wheat using response surface methodology. Food Chem 93:47–56CrossRefGoogle Scholar
  13. Lopes AP, Petenuci ME, Galuch MB, Schneider VVA, Canesin EA, Visentainer JV (2018) Evaluation of effect of different solvent mixtures on the phenolic compound extraction and antioxidant capacity of bitter melon (Momordica charantia). Chem Pap 72:2945–2953CrossRefGoogle Scholar
  14. Mukhtar H, Ahmad N (2000) Tea polyphenols: prevention of cancer and optimizing health. Am J Clin Nutr 71:1698S–1702SCrossRefGoogle Scholar
  15. Nguyen VT, Van Vuong Q, Bowyer MC, Van Altena IA, Scarlett CJ (2015) Effects of different drying methods on bioactive compound yield and antioxidant capacity of Phyllanthus amarus. Drying Technol 33:1006–1017. CrossRefGoogle Scholar
  16. Nunes JC, Lago MG, Castelo-Branco VN, Oliveira FR, Torres AG, Perrone D, Monteiro M (2016) Effect of drying method on volatile compounds, phenolic profile and antioxidant capacity of guava powders. Food Chem 197:881–890CrossRefGoogle Scholar
  17. OrphAnides A, GOulAs V, GekAs V (2013) Effect of drying method on the phenolic content and antioxidant capacity of spearmint. Czech J Food Sci 31:509–513CrossRefGoogle Scholar
  18. Oussaid S, Madani K, Houali K, Rendueles M, Diaz M (2018) Optimized microwave-assisted extraction of phenolic compounds from Scirpus holoschoenus and its antipseudomonal efficacy, alone or in combination with Thymus fontanesii essential oil and lactic acid. Food Bioprod Process 110:85–95CrossRefGoogle Scholar
  19. Patra JK, Das G, Lee S, Kang S-S, Shin H-S (2018) Selected commercial plants: a review of extraction and isolation of bioactive compounds and their pharmacological market value. Trends Food Sci Technol 82:89–109. CrossRefGoogle Scholar
  20. Pietrzak W, Nowak R, Olech M (2014) Effect of extraction method on phenolic content and antioxidant activity of mistletoe extracts from Viscum album subsp. abietis. Chemical Pap 68:976–982. CrossRefGoogle Scholar
  21. Revilla E, Ryan J-M, Martín-Ortega G (1998) Comparison of several procedures used for the extraction of anthocyanins from red grapes. J Agric Food Chem 46:4592–4597CrossRefGoogle Scholar
  22. Roosta M, Ghaedi M, Daneshfar A, Sahraei R, Asghari A (2014) Optimization of the ultrasonic assisted removal of methylene blue by gold nanoparticles loaded on activated carbon using experimental design methodology. Ultrason Sonochem 21:242–252CrossRefGoogle Scholar
  23. Roy RA (1999) Cavitation sonophysics. In: Crum LA, Mason TJ, Reisse JL, Suslick KS (eds) Sonochemistry and sonoluminescence. Springer, Dordrecht, pp 25–38. CrossRefGoogle Scholar
  24. Santos SA, Villaverde JJ, Silva CM, Neto CP, Silvestre AJ (2012) Supercritical fluid extraction of phenolic compounds from Eucalyptus globulus Labill bark. J Supercrit Fluids 71:71–79CrossRefGoogle Scholar
  25. Shadmehr J, Mirsoleimani-azizi SM, Zeinali S, Setoodeh P (2018) Electrocoagulation process for propiconazole elimination from wastewater: experimental design for correlative modeling and optimization. Int J Environ Sci Technol. Google Scholar
  26. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
  27. Thangapazham RL, Singh AK, Sharma A, Warren J, Gaddipati JP, Maheshwari RK (2007) Green tea polyphenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo. Cancer Lett 245:232–241CrossRefGoogle Scholar
  28. Vu HT, Scarlett CJ, Vuong QV (2017) Effects of drying conditions on physicochemical and antioxidant properties of banana (Musa cavendish) peels. Dry Technol 35:1141–1151CrossRefGoogle Scholar

Copyright information

© Institute of Chemistry, Slovak Academy of Sciences 2019

Authors and Affiliations

  1. 1.Faculty of Chemical EngineeringBabol Noshirvani University of TechnologyBabolIran

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