Identification of VOCs in essential oils extracted using ultrasound- and microwave-assisted methods from sweet cherry flower

The floral fragrance of plants is an important indicator in their evaluation. The aroma of sweet cherry flowers is mainly derived from their essential oil. In this study, based on the results of a single-factor experiment, a Box–Behnken design was adopted for ultrasound- and microwave-assisted extraction of essential oil from sweet cherry flowers of the Brooks cultivar. With the objective of extracting the maximum essential oil yield (w/w), the optimal extraction process conditions were a liquid–solid ratio of 52 mL g−1, an extraction time of 27 min, and a microwave power of 435 W. The essential oil yield was 1.23%, which was close to the theoretical prediction. The volatile organic compounds (VOCs) of the sweet cherry flowers of four cultivars (Brooks, Black Pearl, Tieton and Summit) were identified via headspace solid phase microextraction (SPME) and gas chromatography–mass spectrometry (GC–MS). The results showed that a total of 155 VOCs were identified and classified in the essential oil from sweet cherry flowers of four cultivars, 65 of which were shared among the cultivars. The highest contents of VOCs were aldehydes, alcohols, ketones and esters. Ethanol, linalool, lilac alcohol, acetaldehyde, (E)-2-hexenal, benzaldehyde and dimethyl sulfide were the major volatiles, which were mainly responsible for the characteristic aroma of sweet cherry flowers. It was concluded that the VOCs of sweet cherry flowers were qualitatively similar; however, relative content differences were observed in the four cultivars. This study provides a theoretical basis for the metabolism and regulation of the VOCs of sweet cherry flowers.

40 °C and 230 °C, respectively. The temperature-rise program was followed by an initial temperature of 5 °C for 5 min, 20 °C min −1 with a maximum rate of 250 °C and then held constant for 2.5 min. Mass spectrometry (EI + , 70 eV) was determined by a full-scan method with a range from 200 to 400 (m z −1 ). The classification was referred from NIST 2014 28-30 . Statistical analyses. Statistical analysis was performed using the SPSS Statistics Version 21.0 software (Chicago, IL, USA). Duncan's multiple comparisons test (p < 0.05) was performed to assess the statistically significant differences between the mean values (means) of three replications. Origin 9.1 (Origin Lab, Northampton, MA, USA) was employed to construct the graphs.

Results and discussion
Selection of factors and their levels by single-factor analysis. With an extraction time of 20 min and a microwave power of 400 W, the influence of the liquid-solid ratio on yield was investigated; the results are shown in Fig. 2. As the amount of solvent increases, the yield of sweet cherry flower essential oil increases and then becomes stable. When the liquid-solid ratio is 60 mL g −1 , the yield approaches the maximum, perhaps because as the amount of solvent increases, the contact area between the solvent and the raw material increases, and the essential oil can be fully dissolved. However, when the amount of solvent is too high, the solvent absorbs more microwave energy, and the absorption of microwave energy by the raw material decreases, which causes a decrease in the ability of the microwave to damage raw material cells, and the yield of essential oils only slightly changes 31 . After comprehensive consideration, a liquid-solid ratio of 40 mL g −1 is selected.   www.nature.com/scientificreports/ With a liquid-solid ratio of 40 mL g −1 and microwave power of 400 W, the effect of extraction time on the yield of essential oils was investigated. It can be seen from Fig. 2 that with an extension of extraction time, the yield increases and then gradually becomes stable with minimal change after 20 min. In a certain period, with an extended microwave treatment time, the raw material absorbs more microwave energy; the cell rupture is more sufficient; and the essential oil dissolves more. Thus, the yield continues to rise. However, as the microwave treatment time continues to increase, the essential oil inside the raw material is completely extracted, and further increases in the extraction time have a minimal effect on the yield 32 . Therefore, 20 min is an appropriate extraction time.
With a liquid-solid ratio of 40 mL g −1 and an extraction time of 20 min, the influence of microwave power on yield was investigated. The results are shown in Fig. 2. As the microwave power increases, the yield gradually increases and reaches a maximum at 400 W. At a power greater than 400 W, the yield decreases slightly. A microwave extracts essential oils by desorption. With an increase in microwave power, the heating rate increases; the cell wall quickly ruptures; and a large amount of essential oils dissolves. However, when the microwave power is too high, the heat-sensitive compounds in the essential oil will oxidize and decompose, which causes a decrease in yield. The experimental results of this study are consistent with the viewpoint of Routray and Valérie 33 . Usually, the microwave extraction yield will increase with the temperature, and after a certain optimum temperature is reached, further heating will not increase the yield. This result may be due to the increase in molecular migration and solute dissolution rate during the heating process. However, if the temperature is too high, the energy consumption will increase, and the extraction yield will not increase significantly. Therefore, the optimum microwave power is 400 W.
Model fitting and effect of UMAE factors on yield. Based on the results of a single-factor experiment, three factors were selected as independent variables: the liquid-solid ratio (A), extraction time (B), and microwave power (C). The essential oil yield (Y) was the response value. The factors and levels are shown in Table 3. The Box-Behnken design and results are shown in Table 4. The binary polynomial regression model equation fitted by the software is: A variance analysis was performed using the regression model equation, and a significance analysis was performed using the model coefficients. As shown in the Table 5 model variance analysis results, the Model P-value of 0.0025 implies that the model is significant. The probability of this large "Model F-Value" is due to noise. Values of "Prob > F" less than 0.05 indicate that the model terms are significant. R 2 = 0.9318 and Adj R 2 = 0.8442 are near 1, which means that a 93.18% variation in the yield (w/w) of sweet cherry flower essential oil can be explained   34 . Design Expert 8.0 software was utilized to perform quadratic multiple regression fitting of the data in Table 4. The response surface results of the quadratic regression equation are shown in Fig. 3. As shown in Fig. 3a, as the liquid-solid ratio increased, the yield appears to increase and then decrease in certain microwave power conditions. As shown in Fig. 3e, in certain liquid-solid ratio conditions, with an increase in the microwave power, the yield also appears to increase and then decrease. In this case, A, C, A 2 , and C 2 are significant model terms, and B, AB, AC, BC, and B 2 are nonsignificant model terms. By the response surface analysis of the regression model, with the goal of maximizing the yield of sweet cherry flower essential oil, the optimal extraction conditions for UMAE are a liquid-solid ratio of 52.06 mL g −1 , an extraction time of 26.83 min, and microwave power of 435.94 W. In these extraction conditions, the regression equation model predicts the yield of sweet cherry flower essential oil to be 1.24667%. Considering the feasibility of the actual operating conditions, the extraction condition parameters were revised to a liquid-solid ratio of 52 mL g −1 , an extraction time of 27 min, and microwave power of 435 W. The actual yield of essential oils measured by 3 parallel experiments is 1.23%, which is near the predicted value and indicates that the regression equation model can better simulate and predict the yield of sweet cherry flower essential oils.
Identification of VOCs of sweet cherry flower essential oil. The extraction of VOCs from samples is a key link in the analysis of aromatic components. In the past, solvent extraction was employed to extract aromatic compounds, but due to its defects, this method could not completely extract VOCs from the samples, www.nature.com/scientificreports/ which affects the accuracy of the analysis results [35][36][37] . In this study, the HS-SPME was utilized to enrich the volatile and semivolatile components in the sample, combined with GC-MS technology to analyze, identify, and compare the aroma components of sweet cherry flower essential oil. This method offers high sensitivity and simple operation and is convenient and fast. The total ion chromatogram of the sweet cherry flower essential oils by HS-SPME/GC-MS separation analysis is shown in Fig. 4 The total ion chromatogram in four cultivars is similar, but the peak area with the same retention time is different. In this study, a total of 155 VOCs (Table 6) of 11 different chemical groups were separated and identified in the Brooks, Black Pearl, Tieton and Summit sweet cherry cultivars. Sweet cherry flower essential oils from different cultivars have different numbers and relative contents of VOCs. The VOCs possessed by plant flowers are secondary metabolites, which have a vital role in the pollination process of plants. Lilac alcohol, dimethyl sulfide, acetaldehyde, 3-methyl-1-butanol, 3-methyl butanal, (E)-2-hexenal, benzaldehyde, etc. in the VOCs of sweet cherry flowers can be employed in natural floral flavors. Among them, lilac alcohol has a lilac fragrance. Dimethyl sulfide is one of the key odorants in the production of corn, tomato, potatoes, dairy product, and pineapple 38 . In addition, 3-methylbutanal is mainly employed to formulate various fruit flavors. Ethanol, linalool, 4-methoxy-benzaldehyde and lilac aldehyde have been shown to have certain pharmacological effects 39 . A large amount of ethanol is observed in the flowers of sweet cherry during the full Classification analysis of VOCs. To identify the main aroma compounds in sweet cherry flower essential oil, the differences in the VOCs of the essential oils of the flours of the four sweet cherry cultivars were compared. The VOCs were grouped according to their chemical families as alcohols, aldehydes, esters, ether, furan, alkane, olefin, terpenes, ketones, organic acids, and other VOCs. As shown in Fig. 5, in the Brooks, Black Pearl, Tieton, and Summit cultivars, 97 compounds, 107 compounds, 112 compounds and 111 compounds, respectively, were detected. Among the 155 VOCs, ethanol, linalool, dimethyl sulfide, acetaldehyde, (E)-2-hexenal, and benzaldehyde are the main compounds that comprise the aroma of the sweet cherry flower essential oil from the four cultivars.
It can be seen from Fig. 6 that the aldehyde content is the largest in the VOCs, followed by alcohols. These two substances are the main sources of sweet cherry flower aroma. Benzaldehyde was detected in four sweet cherry cultivar flowers and hyacinth, citronella, cinnamon, iris and rose. Moreover, (E)-2-hexenal has a fresh green leaf fragrance and can be used as a blending fragrance for essential oils and various floral fragrances 42 . Acetaldehyde is also present in the aroma of four sweet cherry cultivar flowers and naturally exists in round pomelo, pear, apple, raspberry, strawberry, pineapple, coffee, and orange juice 43 . After dilution, acetaldehyde has a fruity, coffee, wine, green fragrance. Benzaldehyde and benzeneacetaldehyde are important aldehyde flavor ingredients 44 , and benzaldehyde has the aroma of bitter almond, cherry and nut. Benzaldehyde, which is a common component of plant volatiles 45 , attracts many pest species. A recent study has indicated that benzaldehyde can be recognized by adult A. lucorum and can affect its behavior 46,47 . Benzaldehyde is produced by enzymolysis of amygdalin in flowers, and the importance of this substance has been emphasized in previous studies 48 . In this study, the concentrations of benzaldehyde and its derivative benzyl alcohol in the essential oil of the four sweet cherry cultivar flowers are relatively high, which shows the best fragrance of the flowers. This result confirms the importance of benzaldehyde to the sweet cherry flower aroma. Hexanal is a fatty acid that is produced under the catalytic action of lipoxygenase (LOX) 49 . Hexanal has the fragrance of grass and can increase the perceived intensity of fruit aroma. Linalool and phenylethyl alcohol are important alcohol flavor ingredients, among which linalool is extensively applied in cosmetic flavors and food fruit flavors with antibacterial and antiviral effects. Phenylethyl alcohol is one of the two main aroma components of rose essential oil 50 . The fresh, sweet smell of phenylethyl has calming and soothing effects and anti-inflammatory and antibacterial effects. Lilac alcohol isomers (lilac alcohol B and lilac alcohol C) and lilac aldehyde isomers (lilac aldehyde B and lilac aldehyde C) in sweet cherry flowers are the characteristic aroma components. Most esters can impart plant fruit fragrance. 4-Methoxybenzoic acid ethyl ester is the main ester component of sweet cherry flowers, with aromas similar to fruits and anise. Among the olefins, beta-pinene, myrcene, trans-beta-ocimene, and β-ocimene are the main components, and beta-pinene is only detected in the Summit cultivar 51 . Ocimene has a certain role in the prevention and treatment of cancer. An antidepressant experiment in mice has also shown that ocimene can effectively reduce depression traits in mice 52 . Ethers and ketones are relatively rare in flowers. Ketones are also typical aromatic components, and 4-hydroxy-2-butanone is typical of sweet cherry flowers. These VOCs provide the unique aromatic quality of sweet cherry flowers, which indicates that sweet cherry flowers are an important natural spice raw material, which has important scientific value and excellent development prospects.

Conclusions
Ultrasound-and microwave-assisted extraction is an effective method that is suitable for the extraction of essential oil from sweet cherry flowers. Response surface methodology, as part of the experimental design and optimization, showed that the liquid-solid ratio and microwave power had a notable influence on the extraction yield. HS-SPME/GC-MS is an accurate, fast and effective method for determining the aromatic components of sweet cherry flower essential oil. www.nature.com/scientificreports/ of these compounds to each cultivar were observed. Regardless of the cultivar, the most abundant alcohols and aldehyde compounds were ethanol and benzaldehyde, respectively. The principal volatiles were ethanol, linalool, lilac alcohol, acetaldehyde, (E)-2-hexenal, benzaldehyde and dimethyl sulfide, and their concentrations were highly dependent on each cultivar. These VOCs are the main sources of the aroma of sweet cherry flowers. Ethanol, linalool, 4-methoxy-benzaldehyde and lilac aldehyde have various biological activities. The research results provide a basis for the health benefits of sweet cherry flowers.