Ultrasound pre-treatment combined with microwave-assisted hydrodistillation of essential oils from Perilla frutescens (L.) Britt. leaves and its chemical composition and biological activity

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Ultrasound pre-treatment combined with microwave-assisted hydrodistillation of essential oils from Perilla frutescens (L.) Britt. leaves and its chemical composition and biological activity Fengli Chena,*, Shasha Liua, Ziyue Zhaoa, Wenbin Gaoa, Yibo Maa, Xiaoxia Wanga, Shuangmei Yanb, Duqiang Luoa,* a b

College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Perillafrutescens(L.) Britt. Essential oils Ultrasound pre-treatment Antioxidant activity Antifungal activity Cytotoxic activity

Plant essential oil is an important resource of natural medicinal products, thus development of efficient techniques for the isolation of essential oils is one of the research hotspots. In this work, ultrasound was applied to pre-treat Perilla frutescens (L.) Britt. leaves for the following microwave-assisted hydrodistillation (UP-MAHD) of essential oils. The influence of ultrasound pre-treatment and microwave-assisted hydrodistillation procedure factors on the kinetics and essential oil composition were investigated. Additionally, the kinetic curves under different conditions were fitted using models, which revealed that the first-order kinetic model was more suitable. Compared with reference techniques, the ultrasound pre-treatment procedure could significantly improve the yield of essential oil and the percentage of oxygenated components, which might be attributed to the synergy between ultrasound and microwave irradiation. Gas chromatography-mass spectrometer analyzed that the essential oils were rich in perilla ketone (80.76%), other major components included β-caryophyllene (1.65%), linalool (1.15%), caryophyllene oxide (1.12) and apiol (1.19%). The essential oils from UP-MAHD showed higher antioxidant activity (IC50 = 2.60 μL/mL). Moreover, Colletotrichum gloeosporioides and Fusarium fujikuroi were two of the most sensitive fungal strains to essential oils. As compared with paclitaxel, essential oils presented better cytotoxic activity against human gastric cancer cell (MGC-803) and lung cancer cell (A549). Overall, the developed technique remarkably improves the yield of essential oil and its biological activity, which is a promising alternative and provides a foundation for the efficient utilization of P. frutescens resource and other crops.

1. Introduction Perilla frutescens (L.) Britt., an aromatic annual herb of the family Lamiaceae, is widely distributed in many Asian countries, especially in China, Japan, Korea and Vietnam etc. (Heci, 2001). P. frutescens is commonly known as “Zisu” in China, where it has been cultivated as one of the important economic crops for more than 2000 years (Lee and Kim, 2007). In China, P. frutescens leaves have been widely used as a culinary herb and a representative flavor and food agent due to the aromatic taste (Huang et al., 2011). The characteristic aromatic odor of P. frutescens leaves is attributed to the essential oil components that exists in the glandular trichomes distributed on the abaxial surface of leaves (Ito et al., 2002; Kimura et al., 2000). Furthermore, its leaf

essential oils are listed among generally regarded as safe (GRAS) food flavorings for use in baked goods, beverages, frozen dairy products, puddings, and processed vegetables and soups (Smith et al., 2001). Except for the edible usages, P. frutescens has received widespread attention to be used as folk medicine to deal with cold, cough, headache, poisoning from fish and crab (Ji et al., 2014; Yu et al., 2017). Nowadays, extensive biological and pharmacological researches on the leaf, stem and seed of P. frutescens have been reported, which contain antioxidant (En-Shyh et al., 2014), anti-inflammatory (Chen et al., 2015), antibacterial (Ghimire et al., 2017a, 2017b), antifungal (Tian et al., 2014), anti-allergic (Makino et al., 2003), anticancer (Wang et al., 2013), antidepressant (Lee et al., 2014) and antiproliferative activity (Lin et al., 2010). The pharmacological activities are related to the high

⁎ Corresponding author at: College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China. E-mail addresses: fl[email protected] (F. Chen), [email protected] (D. Luo).

https://doi.org/10.1016/j.indcrop.2019.111908 Received 10 July 2019; Received in revised form 20 September 2019; Accepted 25 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Fengli Chen, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111908

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diphenyltetrazolium bromide (MTT) was purchased from Solarbio. For antioxidant assay, 2,2-diphenyl-1-picrylhydroazyl (DPPH) was bought from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Trypsin was purchased from Sigma–Aldrich Inc. (St. Louis, MO). For essential oil treatment, anhydrous sodium sulfate was bought from Tianjin Fuchen Chemical reagent Co., Ltd. (Tianjin, China), and nHexane was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China).

content of bioactive compounds isolated and identified from P. frutescens, especially including flavonoids, essential oils, unsaturated fatty acids, triterpenes and phenolic compounds (Yu et al., 2017). Essential oil, a complex mixture of secondary metabolites (Koul et al., 2008), has been synthesized and produced by aromatic and spices plants. As reported, monoterpenes and sesquiterpenes are the main components of essential oils, whereas aromatic and phenolic components are also found in essential oils. Due to the existence and diversity of essential oils in aromatic medicinal plants, the ethnic and traditional use including antioxidant, anti-inflammatory, antimicrobial and anticancer activity (Yu et al., 2017), has attracted a growing interest. The researches on the volatile essential oil components of P. frutescens indicate that it is an especially good source of perilla aldehyde, perilla ketone, β-caryophyllene, benzaldehyde and limonene (Ahmed and Tavaszi-Sarosi, 2019; Tabanca et al., 2015). The type and relative content of these components differ substantially based on separation method applied, affecting its performance on the nutritional and medicinal application (Zhang et al., 2009; Tian et al., 2014). Accordingly, selecting a technique for efficient separation of essential oils is extremely important in pharmaceutical and chemical industries. For the isolation of essential oils, steam distillation and hydrodistillation (HD) are the most commonly used techniques (Gawde et al., 2014; Conde-Hernández et al., 2017; Sintim et al., 2015). However, the methods are time- and energy-consuming, in addition, the prolonged high temperature treatment easily causes the variation of essential oil composition. Recently, more concerns have been paid to the reduction of cost and environmental pollution (such as CO2 emission), which encourages researchers to develop green alternatives technique on the basis of cost-effective and sustainable criteria. As respected, microwaveassisted hydrodistillation (MAHD) has been successfully developed and employed for the separation of various plant essential oils, to overcome the aforementioned drawbacks (Mollaei et al., 2019; Megawati et al., 2019). Furthermore, several advanced techniques have been designed for further improving the separation efficiency of essential oils (Gavahian and Chu, 2018; Chen et al., 2018). Ultrasound has been considered as a “green” extraction technology for the destruction of cell wall structure and the release of intracellular components, based on several mechanisms including fragmentation, erosion, cavitation and detexturation (Chemat et al., 2017). As inferred, materials underwent ultrasound pre-treatment before extraction is capable of enhancing the extraction efficiency and shortening the processing time. It is well acknowledged that no researches has been reported on the ultrasound pre-treatment followed by MAHD of active constituents from natural plants. In this work, ultrasound pre-treatment was applied to improve the MAHD of essential oils from the leaves of P. frutescens. The influence on the kinetics and essential oil composition caused by the ultrasound pretreatment and MAHD procedure factors were investigated, followed by model fitting. The advantage of ultrasound pre-treatment combined with MAHD (UP-MAHD) was highlighted through comparison with other reference techniques, including kinetic curves and chemical composition of essential oils. Moreover, the antioxidant, antifungal and cytotoxic activity of P. frutescens essential oils was evaluated to provide the basis for its further application.

2.2. Ultrasound pre-treatment combined with microwave-assisted hydrodistillation (UP-MAHD) apparatus and procedures Before MAHD, the sample solution was subjected to ultrasound pretreatment that was conducted using an ultrasonic bath (KQ-200VDB, Kunshan, China, 300 mm × 150 mm × 150 mm). The device has four centrosymmetric transducers, three working frequencies of 45, 80, and 100 kHz, and adjustable power of 80–200 W. The temperature was controlled through the circulation of propagation medium from the inlet to the outlet, to avoid the variation in temperature caused by the ultrasound irradiation process. After ultrasound pre-treatment, the sample solution was directly processed by MAHD that was consisted of a microwave oven, a Clevenger apparatus and a condenser. The application of P70F23P_G5 (S0) microwave oven (Glanz, Guangdong, China, 197 mm × 340 mm × 338 mm) for irradiation and providing heat is equipped with an irradiation frequency of 2.45 GHz and an adjustable maximum output power of 700 W. The volatile components were released and evaporated with water steam in the boiling state, which was then condensed for the separation from water. The essential oils were collected, dehydrated and stored at 4 °C until analysis. The yield of essential oil (%) is expressed as the percentage of the average mass of essential oils (g) to the samples before distillation (g). 2.3. Kinetic model In order to reasonably interpret the distillation process of essential oils and to clarify the mechanism, first- and second-order kinetic model were chosen and compared to fit the release kinetic curves of essential oils. 2.3.1. First-order kinetic model

dYt = K1 (Ye − Yt ) dt

(1)

where Yt (%) and Ye (%) are the yield of essential oil at any time t (min) or at equilibrium, respectively; t (min) is the time; K1 is the rate constant. As considering the initial and boundary conditions: Yt = 0 to Yt at t = 0 to t, Eq. (1) is integrated and rearranged to a nonlinearized equation, as following:

(

Yt = Ye 1 − e−K1t

)

(2)

2.3.2. Second-order kinetic model

2. Experimental

dYt = K2 (Ye − Yt )2 dt

2.1. Materials and chemicals

where Yt (%) and Ye (%) are the yield of essential oil at any time t (min) or at equilibrium, respectively; t (min) is the time; K2 is the rate constant. After rearranging, the integrated rate equation can be expressed as the nonlinear form in Eq. (4) under the initial and boundary conditions, namely, Yt = 0 and Yt = Yt at t = 0 and t = t, respectively.

The acrial parts of P. frutescens were bought from Anguo medicine market (Hebei, China), and were authenticated by Prof. Hongliang Tang from the Hebei University in China. After natural drying under room temperature, the leaves and stems were separated manually and then crushed. The same batch of samples was used in all the experiments. For cytotoxic activity assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-

Yt = 2

K2Ye2 t 1 + K2 Ye t

(3)

(4)

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30 °C. The essential oils were diluted by 5% polysorbate-80 solution (v/v) to obtain a series of concentrations, which was added to the inoculated Potato Dextrose Broth medium. After incubation for 72 h at 30 °C, the minimal inhibitory concentration (MIC) and minimum fugicide concentration (MFC) were measured as the lowest concentration that no obvious growth or complete kill of fungi strains.

According to the conclusion obtained by Moussout et al. (2018), the nonlinear forms of first- and second-order kinetic model performed more appropriate for describing the separation process than the linear forms. Hence, Eq. (2) and Eq. (4) were used to fit the essential oil isolation processes, which was carried out using the OriginPro 9.0 software (OriginLab Inc., USA). 2.4. Chemical composition analysis

2.8. Cytotoxic activity Gas chromatography–mass spectrometry (GC–MS) instrument (7890B/7000C, Agilent Technologies, Santa Clara, CA) that was consisted of a gas chromatograph equipped with a HP-5MS Ultra Inert column (19091S-433UI, Agilent Technologies, 30 mm × 0.25 mm, 0.25 μm film thickness) and a mass spectrometer was employed to determine and analyze the chemical composition of essential oils. Dilution of essential oil samples from different operational conditions of UP-MAHD and other reference techniques using n-hexane was conducted, and then 1 μL of which was injected with a split ratio of 80:1. The oven temperature programming for the separation of essential oil components were: initial constant temperature was set at 50 °C for 5 min, and a following increase to 200 °C at a rate of 5 °C/min was hold for 5 min. The flow rate of carrier gas (helium gas) was 1.2 mL/ min. The injector and detector temperature was set at 250 °C and 280 °C, respectively. The electron ionization mode (EI, 70 eV) with a scan range of 40–400 amu was applied for the mass spectrometer. The chromatographic peak was identified by the comparison of the retention indices (RI) on HP-5MS Ultra Inert column with literatures, and the mass spectra fragmentation patterns with database (Wiley, Mass Spectral Library, NIST14).

The cytotoxic activity of essential oils against human gastric cancer cell (MGC-803), breast cancer cell (MCF-7), cervical cancer cell (Hela), lung cancer cell (A549), hepatoma cancer cell (huh-7 and HepG2) and colon cancer tumor cell (SW620) were estimated. As the cell fusion degree reaching nearly 80%, trypsin (0.25%) was added to digest cells and realize passage. When stable, 100 μL cell suspension (5 × 103) was added into the 96-well plates for cultivation of 24 h at 37 °C, and then 100 μL different concentrations of essential oils-DMSO dilution was added for further cultivation of 48 h. Subsequently, 20 μL 5 mg/mL MTT solution was added in dark conditions for continual cultivation of 4 h, followed by an addition of 100 μL 0.01 M SDS-HCl solution for overnight cultivation. A microplate reader was used to measure the absorbance at 570 nm and to evaluate the inhibition rate and IC50 value. 2.9. Statistical analysis In this work, all experiments were conducted in triplicate, the result of which was expressed as mean ± standard deviation. The significant difference in the yield of essential oil was determined by one-way analysis of variance and Duncan’s multiple range tests.

2.5. Reference techniques

3. Results and discussion

For the MAHD process, the sample solution (liquid-solid ratio of 8 mL/g) was heated by microwave irradiation for 20 min under 540 W, after soaking of 10 min. The ultrasound pre-treatment combined with HD (UP-HD) was conducted by ultrasound pre-treating the P. frutescens sample solution with a liquid-solid ratio of 8 mL/g under 45 kHz and 160 W for 10 min, and which was then subjected to the jacket heating for 60 min under 550 W. In contrast, the ultrasound pre-treatment of sample solution in UP-HD was replaced by soaking for 10 min at room temperature in HD procedure, while other conditions were consistent with UP-HD. After the aforementioned isolation processes, the essential oils were collected, dehydrated and stored at low temperature.

3.1. Kinetics and essential oil composition of ultrasound pre-treatment procedure During ultrasound pre-treatment process, the key driving force is cavitation phenomenon that can cause the high shear force and the micro-jetting of media, which is highly related to the breakdown of plant matrix and the release of intracellular components (Chemat et al., 2017; Tiwari, 2015). Accordingly, the influencing factors of ultrasound pre-treatment process should be investigated in detail, such as ultrasound frequency, power and time.

2.6. Antioxidant activity 3.1.1. Kinetics and essential oil composition of ultrasound frequency Cavitation effect of ultrasound system can facilitate the reaction rate and the extraction efficiency to some extent, the intensity of cavitation is positively correlated with the ultrasound frequency (Yang et al., 2017). Therefore, it is mandatory to evaluate the influence of ultrasound frequency (45, 80 and 100 kHz) on the yield of essential oil. As shown in Fig. 1a, the kinetics illustrated that the yield of essential oil was enhanced initially and then remained constant with the increase in time. Furthermore, the yield of essential oil reduced significantly as the increasing ultrasound frequency from 45 kHz to 100 kHz. The result can be interpreted by that larger number of cavitation bubbles are generated under high frequency, but the intensity of bubble collapse is reduced (Kaur Bhangu et al., 2016). Nevertheless, a transient type of bubbles is predominantly formed as the frequency decreasing, which can grow to a large size and collapse violently with higher intensity. Additionally, the influence of ultrasound frequency on the percentage of major components of essential oils is listed in Table 1. As shown, perilla ketone was the main component with a very high percentage (above 75%), and its percentage was enhanced with the increase of ultrasound frequency from 45 kHz to 100 kHz. Nevertheless, except linalool, the remaining components showed the opposite trend, namely

The antioxidant activity of essential oils was evaluated by detecting its DPPH free radical scavenging activity. The detailed operational process was to mix1 mL different concentrations of essential oils with 3 mL DPPH-95% ethanol solution (40 mg/L), and the control used 95% ethanol solution to substitute sample. The evenly mixed solution was left in the dark for 30 min, and the absorbance at 517 nm was measured using a microplate reader (with 95% ethanol solution as the blank) to calculate the radical scavenging activity. 2.7. Antifungal activity The antifungal activity of essential oils was investigated against five plant pathogenic fungi strains (Phytophthora capsici, Colletotrichum gloeosporioides, Fusarium fujikuroi, Sclerotinia sclerotiorum and Verticillium dahlia). The fungal suspension of strains with a concentration of 106–107 CFU/mL was prepared. Whereafter, 100 μL fungal suspension was evenly inoculated on the surface of Potato Dextrose Agar (PDA) medium. Additionally, filter-paper discs with a diameter of 5 mm were soaked in 6 μL essential oils and then were placed in the middle of the inoculated PDA for inverse incubation of 72 h under 3

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the ultrasound frequency of 45 kHz provided the highest percentages. 3.1.2. Kinetics and essential oil composition of ultrasound power Ultrasound pre-treatment can accelerate the mass transmission of target analytes from the inside of materials to the outside of solvent, thereby improving the separation efficiency (Wang et al., 2016). Several ultrasound powers (80, 120, 160 and 200 W) that determined the cavitation intensify were compared, as the kinetics presented in Fig. 1b. For different ultrasound powers, an initial improvement and then near equilibrium of the yield of essential oil was achieved as the increasing time. The augmentation of ultrasound power from 80 W to 160 W could remarkably enhance the yield of essential oil, whereas the continuous increase in ultrasound power to 200 W did not cause a great impact on the yield. It is likely that high ultrasound power can cause the violent vibration between materials and solvent, contributing to the drastic cell disruption and realizing the satisfactory yield (Sivakumar and Pandit, 2001). In contrast, inadequate energy provided by lower power is not conducive to the extraction of ingredients. Overall, some representative ultrasound mechanisms, such as cavitation, liquid jets and shock waves, are beneficial to the collapse of cytoderm and the diffusion of components and solvent (Al-Dhabi et al., 2017). As listed in Table 1, the influence caused by ultrasound power on the percentage of major components of essential oils illustrated that higher percentage of perilla ketone was achieved under 160 W ultrasound power, too high or too low ultrasound power caused the negative influence on its percentage. For other components, 160 W ultrasound power was not conductive to the separation. 3.1.3. Kinetics and essential oil composition of ultrasound time Higher extraction efficiency and yield can be realized through ultrasound irradiation, whereas there is the possibility for the degradation of heat-sensitive compounds (Yang et al., 2014). Accordingly, it is necessary to explore the impact of a series of ultrasound times (0, 5, 10, 20, 40 and 60 min) on the yield of essential oil. The kinetics shown in Fig. 1c pointed out that the yield of essential oil was enhanced significantly and then tended to be balanced as the time progressing. After ultrasound pre-treatment, the yield of essential oil was remarkably improved, and even the equilibrium time was shortened. Ultrasound time of 10 min performed distinctly in increasing the yield of essential oil, nevertheless, the excessive ultrasound time longer than 10 min did not result in better result. Hence, pre-treating the plant materials using ultrasound before MAHD mightily contributes to the collapse of cytoderm, the mass transmission and the release of essential oils, causing the positive influence on the result (Both et al., 2015). From the influence of ultrasound time on the percentage of major

Fig. 1. Kinetics for the yield of essential oil with different ultrasound frequencies (a), ultrasound powers (b) and ultrasound times (c).

Table 1 Influence of ultrasound pre-treatment procedure on the percentage of major components of essential oils. Major components

Relative area percentage (%) Ultrasound frequency (kHz)

Linalool Carvone Pulegone Perilla ketone Perilla aldehyde Thymol β-Caryophyllene trans-α-Bergamotene α-Humulene Germacrene D Spathulenol Caryophyllene oxide Humulene-1,2-epoxide Apiol

Ultrasound power (W)

Ultrasound time (min)

45

80

100

80

120

160

200

0

5

10

20

40

60

1.31 0.81 0.48 75.70 0.47 0.16 2.11 0.59 0.44 0.17 0.22 1.47 0.17 2.43

0.83 0.66 0.26 78.97 0.30 0.15 1.70 0.56 0.38 0.11 0.21 1.39 0.15 1.82

1.21 0.63 0.25 81.43 0.33 0.12 1.68 0.43 0.34 0.11 0.14 1.06 0.11 1.25

1.24 0.71 0.31 79.74 0.35 0.13 1.62 0.40 0.33 0.11 0.13 1.04 0.11 1.04

1.23 0.72 0.26 79.75 0.28 0.14 1.60 0.41 0.32 0.11 0.15 1.05 0.11 1.07

1.11 0.68 0.26 80.03 0.28 0.13 1.53 0.38 0.31 0.10 0.14 1.02 0.10 1.04

1.31 0.81 0.48 75.70 0.47 0.16 2.11 0.59 0.44 0.17 0.22 1.47 0.17 2.43

1.22 0.62 0.29 82.11 0.34 0.10 1.71 0.42 0.34 0.10 0.15 1.05 0.10 1.01

1.30 0.62 0.33 81.12 0.32 0.12 1.82 0.45 0.36 0.13 0.16 1.06 0.11 0.97

1.25 0.66 0.40 79.93 0.30 0.13 2.03 0.53 0.41 0.16 0.17 1.16 0.11 1.17

1.31 0.81 0.48 75.70 0.47 0.16 2.11 0.59 0.44 0.17 0.22 1.47 0.17 2.43

1.17 0.68 0.24 80.59 0.37 0.13 1.55 0.38 0.31 0.11 0.15 1.04 0.10 1.74

1.13 0.67 0.23 80.88 0.32 0.13 1.59 0.39 0.31 0.10 0.13 0.98 0.10 1.77

4

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3.2.1. Kinetics and essential oil composition of liquid-solid ratio A suitable liquid-solid ratio can ensure satisfactory yield of target analytes, avoiding the excess and wastage of solvent caused by higher liquid-solid ratio and the inadequate extraction generated from lower liquid-solid ratio (Matin and Khosrowshahi, 2017). In view of the importance of liquid-solid ratio, a range of 6, 8, 10, 12 and 14 mL/g was selected for comparison. As presented in Fig. 2a, a remarkable enhancement on the yield of essential oil was observed within 10 min for all liquid-solid ratios. Afterwards, the yield of essential oil tended to be constant with the further augmentation of time. The increase in liquidsolid ratio from 6 to 8 mg/mL was favorable for improving the yield of essential oil. By contrast, slight difference in the yield of essential oil with liquid-solid ratio between 8 and 10 mg/mL was achieved. Nevertheless, a moderate reduction of the yield of essential oil was found as the liquid-solid ratio further enhancing. The above result is in agreement with the conclusions from Peng et al. (2012) and Mollaei et al. (2019). With higher liquid-solid ratio, the adequate interaction between materials and solvent (here water) realizes the reduction of mass transfer resistance and the enhancement of essential oil release (Zhu and Liu, 2013). However, the diffusivity of components is hindered to provide a relatively low effectiveness of solvent for extraction at lower liquid-solid ratio (Lau et al., 2015). Liquid-solid ratio affects the percentage of major components of essential oils, which is summarized in Table 2. A first decrease and subsequent increase in the percentage of perilla ketone was obtained when liquid-solid ratio enhanced, which achieved the lowest percentage with liquid-solid ratio of 10 mL/g. In contrast, higher content of linalool, β-caryophyllene, trans-α-bergamotene, α-humulene, germacrene D and spathulenol was achieved with liquid-solid ratio of 8 mL/g, and which was 10 mL/g liquid-solid ratio for the rest of components.

Fig. 2. Kinetics for the yield of essential oil with different liquid-solid ratios (a) and microwave irradiation powers (b).

components of essential oils listed in Table 1, perilla ketone presented an initial reduction and following improvement with the extension of ultrasound time. With ultrasound time of 40 min, lower percentage of perilla ketone and inversely higher percentage of other components were achieved.

3.2.2. Kinetics and essential oil composition of microwave irradiation power Microwave irradiation power is a key factor during the separation process, due to its special mechanisms, such as rapid and instantaneous heating, for facilitating the plant cell fragmentation (Wang and Weller, 2006). The kinetics of essential oil under different microwave irradiation powers (230, 385, 540 and 700 W) are shown in Fig. 2b. The yield of essential oil was positively correlated with duration prior to 10 min under 540 W and 700 W, after that an approximate equilibrium was observed. As the microwave irradiation power reducing to 385 W and 230 W, the time required to reach equilibrium was extended to more than 15 min. Higher microwave irradiation power facilitates the heat transfer and conversion, thus rapidly raising the temperature throughout the system and accelerating the distillation of essential oils (Megawati et al., 2019). Additionally, the kinetics pointed out that

3.2. Kinetics and essential oil composition of microwave-assisted hydrodistillation procedure For the MAHD procedure, liquid-solid ratio, microwave irradiation power and time are three crucial parameters. Accordingly, the kinetics of yield of essential oil versus time under various liquid-solid ratios and microwave irradiation powers were plotted for investigation.

Table 2 Influence of microwave-assisted hydrodistillation procedure on the percentage of major components of essential oils. Major components

Relative area percentage (%) Liquid-solid ratio (mL/g)

Linalool Carvone Pulegone Perilla ketone Perilla aldehyde Thymol β-Caryophyllene trans-α-Bergamotene α-Humulene Germacrene D Spathulenol Caryophyllene oxide Humulene-1,2-epoxide Apiol

Microwave irradiation power (W)

6

8

10

12

14

230

385

540

700

1.28 0.62 0.31 81.63 0.28 0.10 2.16 0.63 0.41 0.17 0.11 0.87 0.09 1.09

1.44 0.73 0.29 77.24 0.41 0.15 2.60 0.81 0.54 0.23 0.17 1.30 0.14 1.62

1.31 0.81 0.48 75.70 0.47 0.16 2.11 0.59 0.44 0.17 0.22 1.47 0.17 2.43

1.22 0.73 0.25 80.80 0.28 0.13 1.65 0.38 0.33 0.12 0.10 1.04 0.11 1.24

1.14 0.64 0.23 80.10 0.25 0.11 1.65 0.44 0.33 0.12 0.18 1.15 0.13 2.28

1.36 0.61 0.31 81.56 0.25 0.11 2.10 0.54 0.40 0.13 0.13 1.02 0.10 0.99

1.38 0.77 0.35 75.66 0.34 0.16 2.73 0.86 0.59 0.25 0.24 1.53 0.17 1.81

1.31 0.81 0.48 75.70 0.47 0.16 2.11 0.59 0.44 0.17 0.22 1.47 0.17 2.43

1.39 0.88 0.38 76.07 0.37 0.17 2.09 0.64 0.47 0.21 0.25 1.45 0.16 1.64

5

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Furthermore, the variation tendency of kinetic parameters (Pre-Ye and K1) as the operational condition changing is also presented in Table 3. It was clearly observed that the reduction of ultrasound frequency (from 100 kHz to 45 kHz) ended up with the increase of Pre-Ye and K1. Nevertheless, an initial increment followed by an obvious decrease of Pre-Ye was achieved as the increasing ultrasound power, ultrasound time and microwave irradiation power, with the inflection point of 160 W, 10 min and 540 W, respectively. However, the augmentation of the three factors above was related to the continual enhancement or further balance of K1. As compared and analyzed, different impacts were caused by liquid-solid ratio. The range of liquidsolid ratio from 6 to 14 mL/g caused the first improvement and then reduction of Pre-Ye, and the maximum Pre-Ye was obtained with liquidsolid ratio of 10 mL/g. Conversely, the decreasing K1 to equilibrium with the increment of liquid-solid ratio was found. As listed in Table 3, the variation of Pre-Ye from the first-order kinetic model under different conditions was in agreement with Exp-Ye, indicating a satisfactory fitting of the actual data.

540 W performed better than others in terms of the time consumption and the yield of essential oil at equilibrium. The reduction of yield was achieved as the microwave irradiation power reaching 700 W, which was possibly caused by the component biodegradation. Similar result was concluded by Megawati et al. (2019) who systematically investigated the influence of microwave power on the essential oil extraction from Myristicae arillus, founding that excessive power could reduce the yield. The variation of the percentage of major essential oil components according to microwave irradiation power is shown in Table 2. The lowest percentage of perilla ketone was obtained under microwave irradiation power of 385 W, higher or lower than the value could enhance its percentage. Interesting, pulegone, perilla aldehyde and apiol reached the highest percentage at microwave irradiation power of 540 W, whereas the remaining components achieved a relatively higher content at 385 W. 3.3. Comparison and selection of kinetic models Selection a suitable kinetic model is important to provide a precise explanation of the essential oil separation process, thus contributing to the elucidation of mechanism. As described in section 2.3, the nonlinear forms of the first- and second-order kinetic models were applied to fit the kinetics. The fitting result under different conditions is summarized in Table 3. Coefficient of determination (R2) determined the goodness of fit, which was used to compare the appropriateness of the aforementioned two models. As shown, all the R2 were higher than 0.99 for the first-order kinetic model, whereas most of R2 were lower than 0.99 for the second-order kinetic model. Accordingly, the result illustrated that the first-order kinetic model was more suitable for fitting and describing the actual experimental data. Additionally, more closer predicted Ye (Pre-Ye) values to the experimental Ye (Exp-Ye) values were derived from the first-order kinetic model under various conditions. Taken as a whole, first-order kinetic model performed suitably for fitting the essential oil separation process of UP-MAHD.

3.4. Comparison with reference techniques In order to highlight the advantage of UP-MAHD on the separation of essential oils, various reference techniques were selected for comparison, including MAHD, UP-HD and HD. The kinetic curves of these methods were plotted for visualization, as presented in Fig. 3. After indepth analysis, the microwave irradiation applied in UP-MAHD and MAHD procedures significantly shortened the entire time consumption (from 60 min to 10 min), as compared with the corresponding UP-HD and HD methods. The unique heating properties of microwave irradiation (localized and instantaneous) could facilitate the destruction of cell walls, and the distillation of water and essential oils (Dan et al., 2010). Significant enhancement (P < 0.05) on the yield of essential oil from UP-MAHD (5.79 mg/g) was obtained at 10 min, as comparing with the process without ultrasound pre-treatment (MAHD, 4.34 mg/g). Additionally, significant result (P < 0.05) was also achieved by

Table 3 Fitting result of the first-order kinetic model and second-order kinetic model under different factors. Factors

Experimental Ye a

Ultrasound frequency (kHz) 45 5.92 80 5.70 100 5.35 Ultrasound power (W) 80 5.42 120 5.75 160 5.96 200 5.92 Ultrasound time (min) 0 4.93 5 5.60 10 5.95 20 5.92 40 5.85 60 5.83 Liquid-solid ratio (mL/g) 6 5.53 8 5.93 10 5.92 12 5.37 14 5.28 Microwave irradiation power (W) 230 5.53 385 5.73 540 5.92 700 5.60 a

First-order kinetic model

Second-order kinetic model

Ye

K1

R2

Ye

K2

R2

6.09 5.93 5.61

0.25 0.24 0.21

0.9967 0.9946 0.9947

7.37 7.27 7.05

0.04 0.04 0.03

0.9844 0.9808 0.9827

5.75 6.03 6.14 6.09

0.19 0.21 0.25 0.25

0.9917 0.9934 0.9970 0.9967

7.36 7.57 7.43 7.37

0.03 0.03 0.04 0.04

0.9785 0.9797 0.9846 0.9844

5.33 5.83 6.14 6.09 5.97 5.91

0.16 0.22 0.24 0.25 0.28 0.29

0.9946 0.9954 0.9970 0.9967 0.9982 0.9988

7.07 7.25 7.50 7.37 7.11 6.99

0.02 0.03 0.04 0.04 0.05 0.05

0.9875 0.9821 0.9851 0.9844 0.9865 0.9875

5.61 6.10 6.09 5.54 5.43

0.28 0.26 0.25 0.25 0.25

0.9974 0.9965 0.9967 0.9968 0.9977

6.63 7.34 7.37 6.73 6.61

0.05 0.04 0.04 0.04 0.04

0.9914 0.9858 0.9844 0.9850 0.9858

5.90 5.96 6.09 5.69

0.16 0.21 0.25 0.28

0.9974 0.9979 0.9967 0.9985

7.82 7.50 7.37 6.75

0.02 0.03 0.04 0.05

0.9935 0.9895 0.9844 0.9906

Ye, the yield of essential oil (%) at equilibrium; K1 and K2, rate constant; R2, coefficient of determination. 6

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Table 4 GC–MS result for the chemical composition of essential oils from Perilla frutescens (L.) Britt. leaves using different techniques. No.

Components

RI

Relative area percentage (%)

α-Pinene 1-Octen-3-ol β-Pinene α-Phellandrene 3-Carene Limonene Fenchone Linalool β-Thujone Camphor Isomenthone Borneol Terpinen-4-ol α-Terpineol Estragole Carvone Chrysanthenyl acetate Pulegone Perilla ketone 3,5-Dimethoxytoluene trans-Shisool Borneol Perilla aldehyde Anethole Safrole Thymol Methyl geranate Pulespenone Eugenol α-Cubebene β-Bourbonene Methyleugenol Isocaryophyllene β-Caryophyllene Nerylacetone trans-α-Bergamotene α-Humulene γ-Elemene Germacrene D β-Ionone α-Farnesene Myristicine Cubebol Elemicin Nerolidol Spathulenol Caryophyllene oxide Humulene-1,2-epoxide Isospathulenol Caryophylla-4(12),8(13)dien-5.beta.-ol 51 τ-Cadinol 52 Neointermedeol 53 () 54 (1R,7S,E)-7-Isopropyl-4,10dimethylenecyclodec-5-enol 55 Phytol Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified components

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Fig. 3. Kinetic curves for the yield of essential oil using UP-MAHD, MAHD, UPHD and HD techniques. Points represent actual values, and lines represent the fitting behavior predicted by first- and second-order kinetic models.

comparing UP-HD (5.77 mg/g) with HD (5.10 mg/g) at 60 min. The above comparison results revealed that the ultrasound pre-treatment process played an important role in increasing the yield of essential oil. Obviously, the combination of ultrasound pre-treatment and microwave irradiation in UP-MAHD produced the improvement of both the yield of essential oil and the time requirement, thus increasing the separation efficiency. Additionally, first- and second-order kinetic models were employed to fit the actual experimental data, thus to elucidate the separation procedure. It was clearly observed from Fig. 3 that the first-order kinetic model was suitable for the description and interpretation of essential oil isolation process due to all R2 exceeding 0.99. In contrast, the R2 for UP-MAHD and MAHD using the second-order kinetic model was lower than 0.99, whereas which performed satisfactorily for UP-HD and HD with relatively higher R2 (0.9988 and 0.9986). Taken as a whole, MAHD of essential oils could be described accurately by first-order kinetic model, while second-order kinetic model was more suitable to the conventional HD process. As reported, the crucial ultrasound mechanism for facilitating the release of target components and enhancing the yield is attributed to the cavitation phenomenon (Tiwari, 2015). Under the impact of ultrasound wave, a series of compressions and rarefactions of medium molecules caused by the high-frequency mechanical vibration can generate the formation of multiple microbubbles at various nucleation sites in the extraction solvent (here water), and result in its gradual growth to realize ultimately collapse (Both et al., 2015). At the moment the bubble burst, localized high temperature and equivalent to several thousand atmosphere pressure are produced, the energy of which is enough to generate the disintegration of cellular structure (Leighton, 2007). Additionally, micro-jets with high shear stress is also formed near the surface of materials, which is another factor influencing the cell disruption (Chemat et al., 2017; Both et al., 2015). In this work, the materials pretreated by ultrasound are conductive to the release of target analytes by accelerating the cell breakage and increasing the mass transfer. The result was in agreement with that from Morsy (2015) who separated essential oils from cardamom seeds after ultrasound pretreatment, and Damyeh et al. (2016) who applied ultrasonic pretreatment followed by HD for isolation of essential oils from Prangos ferulacea Lindl. and Satureja macrosiphonia Bornm. leaves. However, inconsistent conclusion was found by Périno-Issartier et al. (2013) for separation of lavandin essential oils, no significant change of time and yield was achieved after ultrasound pre-treatment as compared with HD. The reason was speculated to be attributed to the difference in the

a

UP-HD

MAHD

UP-MAHD

1168 1271 1283 1293 1297 1321 1329 1353 1354 1377 1399 1408 1423 1434 1441 1443 1465 1472 1488 1503 1519 1522 1552 1565 1571 1578 1606 1638 1644

0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.90 0.02 0.01 0.04 0.01 0.00 0.01 0.02 0.61 0.03 0.31 74.00 0.03 0.04 0.01 0.31 0.26 0.26 0.22 0.07 0.02 0.15 0.04 0.04 0.04 0.01 4.09 0.03 1.32 0.84 0.12 0.44 0.12 0.07 0.03 0.08 0.03 0.26 0.19 1.49 0.18 0.03 0.05

0.01 0.01 0.01 0.01 0.00 0.01 0.00 1.15 0.01 0.01 0.07 0.01 0.01 0.01 0.01 0.69 0.01 0.36 79.69 0.02 0.04 0.01 0.48 0.11 0.11 0.14 0.07 0.00 0.09 0.02 0.01 0.04 0.00 1.81 0.02 0.53 0.36 0.05 0.18 0.08 0.03 0.02 0.04 0.02 0.14 0.16 1.08 0.11 0.02 0.03

0.01 0.01 0.00 0.00 0.01 0.01 0.00 1.22 0.01 0.01 0.06 0.01 0.01 0.01 0.02 0.70 0.03 0.34 80.46 0.02 0.03 0.01 0.23 0.23 0.23 0.16 0.06 0.01 0.09 0.02 0.01 0.03 0.00 1.89 0.02 0.51 0.36 0.03 0.18 0.08 0.02 0.02 0.04 0.02 0.12 0.12 0.92 0.10 0.02 0.02

0.02 0.01 0.01 0.00 0.00 0.00 0.00 1.15 0.01 0.01 0.07 0.02 0.01 0.01 0.00 0.72 0.02 0.39 80.76 0.03 0.03 0.01 0.27 0.04 0.04 0.18 0.07 0.00 0.16 0.01 0.01 0.03 0.00 1.65 0.02 0.44 0.34 0.03 0.13 0.06 0.02 0.02 0.04 0.02 0.12 0.16 1.12 0.12 0.03 0.03

1651 1662 1671 1695

0.08 0.03 1.65 0.02

0.05 0.01 1.12 0.02

0.03 0.01 1.08 0.00

0.06 0.01 1.19 0.01

2112

0.03 0.03 76.79 6.96 2.37 2.47 88.62

0.01 0.03 82.78 3.00 1.63 1.61 89.05

0.01 0.03 83.51 3.03 1.36 1.70 89.63

0.01 0.03 83.69 2.64 1.67 1.67 89.70

HD 942 978 980 996 1011 1030 1087 1104 1110 1145 1159 1165 1177 1190 1196 1225 1233 1235 1252 1260

a HD, hydrodistillation; UP-HD, ultrasound pre-treatment combined with hydrodistillation; MAHD, microwave-assisted distillation; UP-MAHD, ultrasound pre-treatment combined with microwave-assisted distillation.

7

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components that were achieved by the synergistic effect between ultrasound and microwave irradiation (Seidi Damyeh et al., 2016). Additionally, the IC50 value that is an essential oil concentration causing 50% radical scavenging activity was calculated. As expected, UP-MAHD essential oils gave the lower IC50 value than others. The ultrasound pretreatment is conductive to the rupture of cell walls, thus further facilitating the release of intracellular components under microwave irradiation (Toma et al., 2001). The result of the present study was similar to that concluded by Hashemi et al. (2018), in which improved antioxidant activity of essential oils was realized after ultrasound treatment of materials. In a word, P. frutescens essential oils can provide excellent antioxidant activity and can be considered as a potential antioxidant for application.

structure of raw materials and the storage site of essential oils. 3.5. Essential oil component analysis Ultrasound pre-treatment of raw materials can cause the component variation of essential oils, as does the synergistic effect between ultrasound and microwave irradiation. Accordingly, the essential oils obtained from UP-MAHD, MAHD, UP-HD and HD techniques were subjected to the GC–MS determination of chemical composition. As listed in Table 4, a total of 55 components were identified for the essential oils from all the four methods. Overall, the chemical composition of P. frutescens essential oils was extremely consistent when using various techniques, but the difference in the relative content was large. Perilla ketone was the crucial component with the highest relative area percentage (RA%), which accounted for more than 74% of total components. Additionally, β-caryophyllene, linalool, caryophyllene oxide, apiol, trans-α-bergamotene, carvone, perilla aldehyde, pulegone were the other more important components. The result of this work was in agreement with that of Ghimire et al. (2017a, 2017b) who investigated the composition of P. frutescens essential oils from various habitats, which revealed that the perilla ketone was the major component. As reported by Ahmed and Tavaszi-Sarosi (2019), perillaldehyde, perilla ketone, β-dehydro-elsholtzia ketone, limonene and trans-shisool were detected as the predominant components. From Table 4, the essential oils from UP-MAHD were analyzed to contain higher percentage of oxygenated monoterpenes and oxygenated sesquiterpenes than MAHD, UP-HD and HD, which was speculated to be attributed to the synergistic effect of ultrasound and microwave irradiation. Additionally, the individual influence of ultrasound or microwave irradiation was reflected in the comparison between MAHD and UP-HD with HD. In conclusion, the raw materials undergoing ultrasound pre-treatment can provide an easy penetration of microwave irradiation, and substantially enhance the essential oil separation.

3.7. Antifungal activity The essential oil components with low molecular weight can disrupt the cytomembrane of microorganisms due to the lipophicity. Several mechanisms for inhibiting microorganisms have been confirmed, including cytomembrane permeability, protein and K+ leakage, etc. processes (Xiang et al., 2018). Therefore, the antifungal activity of P. frutescens essential oils isolated from UP-MAHD, MAHD, UP-HD and HD was estimated. The inhibition zone diameter (IZD), MIC and MFC against P. capsici, C. gloeosporioides, F. fujikuroi, S. sclerotiorum and V. dahlia are summarized in Table 5. The result pointed out that the IZD formed by UP-MAHD essential oils was greater than other techniques, and the MIC and MFC were lower or similar to others. As described by Vilkhu et al. (2008) and Toma et al. (2001), the materials treated by sonication could produce essential oils with higher antibacterial activity than untreated materials, attributing to the high content of main antibacterial components. However, no significant discrepancy in the antibacterial activity of essential oils from sonication treatment or not was found by Seidi Damyeh et al. (2016). After comparison, the two most susceptible fungi to UP-MAHD essential oils was C. gloeosporioides and F. fujikuroi, with IZD of 41.10 mm and 40.65 mm, both MIC of 0.75 μL/mL, MFC of 0.75 μL/mL and 1.00 μL/mL. Nevertheless, V. dahlia presented the lowest susceptibility, with IZD of 16.64 mm, MIC of 1.50 μL/mL, and MFC of 2.50 μL/mL. The difference in the inhibitory activity of essential oils against different fungi was caused by the structure and composition of fungal cytomembrane. In conclusion, P. frutescens essential oils had spectral antifungal activity against the chosen plant pathogenic fungi, and the exploration and interpretation of its antifungal mechanism in the future is of great significance.

3.6. Antioxidant activity For evaluating the antioxidant activity, the essential oils isolated from UP-MAHD, MAHD, UP-HD and HD were subjected to the DPPH free radical-scavenging assay. The result is shown in Fig. 4, which revealed that the radical-scavenging activity was dose-dependent with essential oil concentration. The antioxidant activity of MAHD and UPHD essential oils was distinctly higher than conventional HD. In contrast, the additional enhancement on the activity of UP-MAHD essential oils was probably attributed to the high percentage of oxygenated

3.8. Cytotoxic activity The cytotoxic activity of essential oils can realize the structural destroy of polysaccharides and phospholipid layers of cells, and the further disruption of cells. Therefore, the cytotoxic activity of P. frutescens essential oils obtained using UP-MAHD against MGC-803, MCF7, Hela, A549, huh-7, HepG2 and SW620 cell lines were evaluated, with paclitaxel as the positive control. The IC50 value of essential oils and paclitaxel against various cell lines was calculated. As shown in Fig. 5, essential oils exhibited lower IC50 value of 17.82 ± 5.12 μg/mL against MGC-803, 21.31 ± 0.98 μg/mL against A549, 30.94 ± 0.84 μg/mL against MCF-7, and 34.58 ± 5.27 μg/mL against Hela cell lines. However, the IC50 value for other cell lines were too larger, meaning relatively low cytotoxic activity. The high standard deviation of result was partly attributed to the difference in cell state under culture condition. According to the American National Cancer Institute (de Oleivera et al., 2015), samples that provide a IC50 value lower than 30 μg/mL for inhibiting cancer cell line can be acted as a potential anticancer drug for development. Therefore, P. frutescens essential oils exhibited more active cytotoxic activity to MGC-803 and A549 cell lines. Perilla ketone, a main oxygenated monoterpene accounting for

Fig. 4. DPPH free radical-scavenging activity of essential oils isolated from UPMAHD, MAHD, UP-HD and HD. 8

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Table 5 Antifungal activity of Perilla frutescens (L.) Britt. essential oils. Essential oil

P. capsici

HD UP-HD MAHD UP-MAHD

F. fujikuroi

S. sclerotiorum

V. dahlia

MIC

MFC

IZD

MIC

MFC

IZD

MIC

MFC

IZD

MIC

MFC

IZD

MIC

MFC

24.20 25.45 26.02 27.22

1.25 1.00 1.00 1.00

1.50 1.25 1.25 1.25

37.75 39.04 39.36 41.10

1.00 0.75 0.75 0.75

1.25 1.00 0.75 0.75

36.55 38.14 39.32 40.65

1.00 0.75 0.75 0.75

1.25 1.00 1.00 1.00

15.76 16.96 17.88 19.22

1.25 1.00 1.00 1.00

1.50 1.50 1.50 1.50

14.06 14.72 15.60 16.64

1.50 1.50 1.50 1.50

2.50 2.50 2.50 2.50

IZD a

C. gloeosporioides

b

a HD, hydrodistillation; UP-HD, ultrasound pre-treatment combined with hydrodistillation; MAHD, microwave-assisted distillation; UP-MAHD, ultrasound pretreatment combined with microwave-assisted distillation. b IZD, Inhibition zone diameter (mm); MIC, Minimal inhibitory concentration (μL/mL); MFC, Minimal fugicide concentration (μL/mL).

future efficient separation and application of P. frutescens essential oils. Acknowledgements The authors thank the Advanced Talents Incubation Program of the Hebei University (050001-521000981292), National Natural Science Foundation of China (31672070) and National Key Research and Development Program of China (2017YFD0201400 and 2017YFD0201401) for financial support. References Ahmed, H.M., Tavaszi-Sarosi, S., 2019. Identification and quantification of essential oil content and composition, total polyphenols and antioxidant capacity of Perilla frutescens (L.) Britt. Food Chem. 275, 730–738. Al-Dhabi, N.A., Ponmurugan, K., Jeganathan, P.M., 2017. Development and validation of ultrasound-assisted solid-liquid extraction of phenolic compounds from waste spent coffee grounds. Ultrason. Sonochem. 2017 (34), 206–213. Both, S., Strube, J., Cravatto, G., 2015. Mass transfer enhancement for solid–liquid extractions. In: Chemat, F., Strube, J. (Eds.), Green Extraction of Natural Products. Wiley-VCH, Weinheim, pp. 101–119. Chemat, F., Rombaut, N., Sicaire, A., Meullemiestre, A., Fabiano-Tixier, A., Abert-Vian, M., 2017. Ultrasound assisted extraction of food and natural products. Mechanisms techniques, combinations, protocols and applications. A review. Ultrason. Sonochem. 34, 540–560. Chen, C.Y., Leu, Y.L., Fang, Y., Lin, C.F., Kuo, L.M., Sung, W.C., Tsai, Y.F., Chung, P.J., Lee, M.C., Kuo, Y.T., Yang, H.W., Hwang, T.L., 2015. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and calcium. Sci. Rep. 5, 18204. Chen, F., Jia, J., Zhang, Q., Yang, L., Gu, H.Y., 2018. Isolation of essential oil from the leaves of Polygonum viscosum Buch-ham. using microwave-assisted enzyme pretreatment followed by microwave hydrodistillation concatenated with liquid–liquid extraction. Ind. Crop. Prod. 112, 327–341. Conde-Hernández, L.A., Espinosa-Victoria, J.R., Trejo, A., Guerrero-Beltrán, J.Á., 2017. CO2-supercritical extraction, hydrodistillation and steam distillation of essential oil of rosemary (Rosmarinus officinalis). J. Food Eng. 200, 81–86. Damyeh, M.S., Niakousari, M., Saharkhizc, M.J., 2016. Ultrasound pretreatment impact on Prangos ferulacea Lindl. and Satureja macrosiphonia Bornm. essential oil extraction and comparing their physicochemical and biological properties. Ind. Crop. Prod. 87, 105–115. Dan, Y., Liu, H.Y., Gao, W.W., Chen, S.L., 2010. Activities of essential oils from Asarum heterotropoides var. mandshuricum against five phytopathogens. Crop Prot. 29, 295–299. de Oleivera, P.F., Alves, J.M., Damasceno, J.L., Oliveira, R.A.M., Dias, H.J., Crotti, A.E.M., Tavares, D.C., 2015. Cytotoxicity screening of essential oils in cancer cell lines. Rev. Bras. Farmacogn. 25, 183–188. En-Shyh, L., Chia-Ching, L., Hung-Ju, C., 2014. Evaluation of the antioxidant and antiradical activities of Perilla seed, leaf and stalk extracts. J. Med. Plants Res. 8, 109–115. Gavahian, M., Chu, Y.H., 2018. Ohmic accelerated steam distillation of essential oil from lavender in comparison with conventional steam distillation. Innov. Food Sci. Emerg. 50, 34–41. Gawde, A., Cantrell, C.L., Zheljazkov, V.D., Astatkie, T., Schlegel, V., 2014. Steam distillation extraction kinetics regression models to predict essential oil yield, composition, and bioactivity of chamomile oil. Ind. Crop. Prod. 58, 61–67. Ghimire, B.K., Yoo, J.H., Yu, C.Y., Chung, I.M., 2017a. GC–MS analysis of volatile compounds of Perilla frutescens Britton var. Japonica accessions: Morphological and seasonal variability. Asian Pac. J. Trop. Med. 10, 643–651. Ghimire, B.K., Yu, C.Y., Chung, I.M., 2017b. Assessment of the phenolic profile, antimicrobial activity and oxidative stability of transgenic Perilla frutescens L. overexpressing tocopherol methyltransferase (γ-tmt) gene. Plant Physiol. Biochem. 118, 77–87. Hashemi, S.M.B., Khaneghah, A.M., Koubaa, M., Barba, F.J., Abedi, E., Niakousari, M., Tavakoli, J., 2018. Extraction of essential oil from Aloysia citriodora Palau leaves

Fig. 5. Cytotoxic activity of essential oils isolated from UP-MAHD and paclitaxel.

above 70% of essential oils, has been reported to contain cytotoxic activity (Paborji et al., 1988). Moreover, some other essential oil components with relatively higher content also show stronger cytotoxic activity or can be used as a promotor, such as thymol, trans-α-bergamotene, linalool, β-caryophyllene (Llana-Ruiz-Cabello et al., 2014; Miyashita and Sadzuka, 2013; Monajemi et al., 2005; Wang et al., 2018). Several researches have revealed that essential oils exhibit better cytotoxic activity than individual major components (de Oleivera et al., 2015). After analysis, the cytotoxic activity of P. frutescens essential oils is speculated to be related to the existence of abovementioned components and the synergistic effect. 4. Conclusion This work applied ultrasound pre-treatment for enhancing the MAHD of essential oils from P. frutescens leaves. The kinetics and essential oil composition were subjected to obvious variations under different conditions of ultrasound pre-treatment and MAHD procedures. After fitting, first-order kinetic model performed suitably than the second-order kinetic model. In the UP-MAHD process, the yield of essential oil and the percentage of oxygenated monoterpenes and oxygenated sesquiterpenes were remarkably enhanced, which was speculated to be attributed to the synergistic effect between ultrasound and microwave irradiation. After GC–MS analysis, perilla ketone (80.76%), β-caryophyllene (1.65%), linalool (1.15%), caryophyllene oxide (1.12) and apiol (1.19%) were the major components of essential oils. Additionally, the essential oils isolated from UP-MAHD exerted better DPPH free radical-scavenging activity, antifungal activity against C. gloeosporioides and F. fujikuroi, and cytotoxic activity against MGC-803 and A549. In a word, P. frutescens essential oil is a promising alternative to natural medicinal products, and this work lays a foundation for the 9

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