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Essential oils from three kinds of fingered citrons and their antibacterial activities
Industrial Crops & Products 147 (2020) 112172
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Essential oils from three kinds of fingered citrons and their antibacterial activities
T
Feiyan Wanga, Huaqian Youa, Yanhong Guoa, Yukun Weib, Pengguo Xiaa, Zongqi Yanga, Min Renc, Hui Guoc, Ruilian Hana, Dongfeng Yanga,* a
College of Life Sciences and Medicine, Key Laboratory of Plant Secondary Metabolism and Regulation in Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China b Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602, China c School of Life Science and Technology, Xinxiang University, Xinxiang 453000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fingered citron Essential oil Extraction process Component analysis Antibacterial activity
Fingered citron (Citrus medica L. var. sarcodactylis Swingle, Foshou in Chinese) is well known as an excellent and popular Chinese herbal medicine and functional vegetable. Fingered citron is rich of essential oil, which exhibits antioxidant, antiradical, and antimicrobial properties. In order to improve the extraction rate of essential oil from three kinds of fingered citrons and understand their activities, the optimal auxiliary extraction conditions of essential oils and their antibacterial activities were analyzed in this work. Fingered citron essential oil (FEO) extracted by the reflux method was analyzed by Gas Chromatography Mass Spectrometry (GC–MS), and their antimicrobial activities were detected by the disc diffusion method. The results showed that the best auxiliary condition for FEO extraction was ultrasonic combined with 9 % NaCl. The extraction rate of 9 % NaCl combined with ultrasonic was the highest (1.80 %) compared with the control (1.15 %). The compositions of FEOs extracted from three kinds of fingered citrons were mainly composed of alkenes, such as D-limonene and γ-terpinene. However, contents of the compositions were significantly different in three kinds of fingered citrons. The Gold fingered citron essential oil possessed the highest content of D-limonene which close to 50 %, and that in the Cantonese fingered citron essential oil was only 27.60 %. A certain amount of ethanol contained in the Sichuan fingered citron essential oil and aldehyde contained in the Cantonese fingered citron essential oil. Moreover, the three FEOs had different antibacterial effects. In three kinds of fingered citron, the antibacterial effect of the Gold fingered citron essential oil on Staphylococcus aureus was the strongest. The Cantonese citron essential oil had the strongest antibacterial effect on Enterococcus faecalis, and the Sichuan citron essential oil had the strongest antibacterial activity against Escherichia coli. These findings could help us to develop citron essential oils as natural antibacterial agents, new drugs, food additives and other products.
1. Introduction
processed into food, drinks and spices. Fingered citron-based activated carbon, a new type of activated carbon, plays an important role in material chemistry analysis (Dai et al., 2014). The highest content of essential oil in fresh fingered citron could reach nearly 1.6 %. Despite its unstable properties (Penbunditkul et al., 2012) and the difficulties during its production, storage and packaging (Rafiee et al., 2007), FEO has become one of the hot spots in the field of essential oil research in recent years due to its mood soothing (Watanabe et al., 2015), antidepressant, antibacterial, anticancer (Bayala et al., 2014), and antiproliferative effects (Navarra et al., 2015a,Navarra et al., 2015b). At present, the reflux method is the most widely used method for essential oil extraction (Belsito et al., 2007), although there are still some
Fingered citron (Citrus medica L. var. sarcodactylis Swingle), also called nine-claw wood, five fingered orange, citrus chirocarpus and so on, which was known as an excellent Chinese herbal medicine. The plant is a shrub or small tree. The stem has short spines and its leaves are alternate. During the fruit development, the fingered citron is divided into finger shaped meat strips. Fingered citron is mainly planted in Zhejiang, Guangxi, Sichuan and other places in China. There are three kinds of citron according to its geographical divisions, including the Gold fingered citron, the Cantonese fingered citron and the Sichuan fingered citron. Fingered citron could be used as medicine and
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Corresponding author. E-mail address: [email protected] (D. Yang).
https://doi.org/10.1016/j.indcrop.2020.112172 Received 28 October 2019; Received in revised form 31 December 2019; Accepted 25 January 2020 0926-6690/ © 2020 Published by Elsevier B.V.
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drawbacks remain. For example, it takes a long time, the quality of essential oil decreases or even degrades at high temperatures, and the extraction rate of essential oil is not high. In recent years many researchers have optimized the extraction conditions to improve extract quality of essential oil, such as ultrasonic (Yang et al., 2014) and microwave extraction (Yi et al., 2016), and explored the optimal parameters of objective conditions. As far as we know a combination of both assisted extraction methods has not yet been reported. We combined these two assisted extraction methods for the first time to maximize the extraction rate of the three kinds of essential oils. This optimum auxiliary condition has many advantages. For example, it improved the fingered citron essential oil extraction rate significantly and is easy to operate. This condition is applicable not only in the small-scale laboratory but also for large-scale industrial production. The purposes of this research were to find the optimal extraction method of fingered citron essential oil and improve the oil extraction rate to lay the foundation for industrial production for FEO and provide a feasible reference scheme. Fingered citron essential oil was recognized for their biological activities, which are generally closely related to its compositions. Only with a full understanding of the main chemical constituents could we provide the corresponding theoretical basis for the development and application of FEO-related products. Therefore, another purpose of this study was to identify the compositions of three kinds of citron essential oils. Secondary metabolites of plants can have a killing or inhibiting effect on microorganisms. When plants are induced by pathogenic microorganisms, such as fungi and bacteria, they will develop a defensive response. Finally, we wanted to investigate the antibacterial activity of three kinds of fingered citron essential oils. The disc diffusion method was used to test the antibacterial activity of three different fingered citron essential oils on Escherichia coli, Streptococcus pneumoniae, Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis.
Fig. 1. Effect of three kinds of auxiliary conditions on the oil extraction rate, Means ± standard deviation (S.D.) (n = 3) are shown.
2.3. Box-Behnken experiment to optimize the extraction conditions The material-liquid ratio, the reflux time and the content of NaCl were considered as significant factors influencing the fingered citron essential oil extraction rate. They were divided into three levels (low, intermediate and high, labelled as -1, 0 and 1, respectively) to carry out Box-Behnken experiments to improve the extract rate. The extraction conditions were shown in Supplement Table 1. First, a total of 100 g of Gold fingered citron pieces was put into 500 mL, 200 mL and 125 mL of extraction solution (9 % NaCl) and ground into pulp. Then, it was extracted by the ultrasonic method for 30 min at 200 W and refluxed for 2 h. Second, the Gold fingered citron pieces were put into 200 mL of solution (9 % NaCl) and ground into pulp. Then, they were extracted by the ultrasonic method for 30 min at 200 W and refluxed for 1 h, 2 h and 3 h. Next, the Gold fingered citron pieces were put into 200 mL of extraction solution (6 %, 9 % and 12 % NaCl) and ground into pulp. Then, they were extracted by the ultrasonic method for 30 min at 200 W and refluxed for 2 h. The response surface analysis was carried out by Expert Design software.
2. Materials and methods 2.1. Samples and chemicals The Gold fingered citron mature fruits were collected from Jinhua in Zhejiang Province on October 2015; the Cantonese fingered citrons were collected from Liuzhou in Guangxi Province on October 2015; and the Sichuan fingered citrons were collected from the Nantianmen Fingered Citron Cultivation Base in Sichuan Province on October 2015. The species were identified by professor Zongsuo Liang of Zhejiang Scitech University. Five bacterial strains including Bacillus subtilis ATCC 6051, Streptococcus pneumoniae ATCC 49619, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8099 and Staphylococcus aureus ATCC 6538 were obtained from the Zhejiang Center for Disease Control and Prevention and stored at -20 ℃ with glycerol. Cells were inoculated into 5 test tubes with 5 mL of liquid medium. After activation on a shaking table at 37 °C for 48 h, bacterial suspensions were made for testing.
2.4. Composition analysis by the GC–MS method The compositions of the FEOs extracted from three kinds of fingered citrons (Jinhua, Sichuan, Guangxi) were analyzed by GC–MS. The conditions were as follows: chromatographic column: Rtx-5Sil MS 30 m × 0.25 mm × 0.25 μm; inlet temperature: 280 ℃; injection mode: split injection; split ratio 100:1; column temperature programme: 40 ℃ (5 min), 5/min, 300 ℃ (5 min); constant line speed: 36.1 cm/sec; sample size: 1 μL; ionization mode: EI; ion source temperature: 230 ℃; interface temperature: 250 ℃; acquisition mode: scan; mass number range: m/z, 35∼650, amu. To exclude the influence of added NaCl, the components of the Gold fingered citron essential oil obtained without adding NaCl were analyzed.
2.2. Optimization of the FEO extraction process Gold fingered citron (100 g) was extracted by ultrasonic with 9 % NaCl extraction solution (Vilkhu et al., 2008). A total of 100 g of fingered citron pulp was added to 200 mL of distilled water as a control and refluxed for 2 h, and the extraction rate was calculated. The experiment was performed three times. In group 1, the essential oil in 100 g citron fruit pulp was extracted by the reflux method with 200 mL of 9 % NaCl extraction solution. In group 2, the fingered citron pulp was pretreated by ultrasonic at 200 W for 30 min and then extracted by the reflux method. In group 3, the fingered citron pulp was pretreated by ultrasonic with 200 mL of 9 % NaCl solution for 30 min and then extracted by the reflux method.
2.5. Antibacterial activity experiment A preliminary screen about the antibacterial activity of the essential 2
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Fig. 2. The response maps and contour maps of material liquid ratio (A), the content of NaCl (C), reflux time (B) and extraction rate. The closer the contour plots were to the ellipse, the more obvious the interaction between the two factors, if the contour plots were closer to the circle, the less significant the interaction between the two factors.
3
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Table 1 GC–MS qualitative analysis results of the Gold fingered citron essential oil (with NaCl). Number
Retention time (min)
Component name
CAS#
Similarity (%)
Area percentage (%)
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
10.792 11.088 12.590 12.768 13.298 13.849 14.258 14.532 15.230 15.490 16.113 16.817 17.255 18.949 19.903 20.374 20.672 21.268 21.328 21.670 22.054 22.561 23.284 23.735 24.909 25.163 25.708 26.604 27.021 27.329 27.779 27.973 28.654 29.036 29.293
alpha-Thujene alpha-Pinene 4(10)-Thujene beta-Pinene beta-Myrcene 3-Thujene (+)-4-Carene p-Cymene D-Limonene beta-cis-Ocimene gamma-Terpinene Isoterpinolene Linalool (R)- (+)-Citronellal Terpinen-4-ol alpha-Terpineol Decanal cis-Geraniol Citronellol beta-Citral Geraniol alpha-Citral 2-Undecanone Undecanal Citronellol acetate Nerol acetate Geranyl acetate Dodecanal Caryophyllene trans-alpha-Fingered citronene (E)-beta-Famesene Humulene Germacrene D Germacrene beta-Bisabolene
2867-05-2 80-56-8 3387-41-5 127-91-3 123-35-3 2867-05-2 29050-33-7 99-87-6 5989-27-5 3338-55-4 99-85-4 586-63-0 78-70-6 2385-77-5 562-74-3 98-55-5 112-31-2 106-25-2 106-22-9 106-26-3 106-24-1 141-27-5 112-12-9 112-44-7 150-84-5 141-12-8 105-87-3 112-54-9 87-44-5 13474-59-4 18794-84-8 6753-98-6 23986-74-5 15423-57-1 495-61-4
97 97 97 97 97 96 96 95 95 99 97 97 97 97 96 97 96 93 94 97 96 98 95 97 94 96 97 97 96 96 95 97 94 92 96
2.10 4.40 0.49 4.03 4.48 0.18 1.08 0.71 48.0 3.25 21.1 2.40 0.23 0.17 0.49 0.65 0.26 0.21 0.14 0.32 0.28 0.42 0.10 0.25 0.10 0.14 0.12 0.20 0.73 0.79 0.07 0.09 0.61 0.27 1.16
oil was conducted. A Sterile 6 mm filter paper discs was put into the essential oil for 2 h. The cell suspensions of the tested strains were gradually diluted to 10−1, 10−2, 10−3, 10−4 and 10−5 with sterile water. Then, the plates were coated with 0.2 mL of cell suspensions. The assay was carried out three times for each strain. Each bacterial strain had three repetitions. Then, the filter paper was attached to it and cultured at 37 ℃ for 37 h (with three repetitions). The diameter of the inhibition zones was measured by the cross method. The essential oil was diluted with petroleum ether to a concentration of 0, 3, 6, 12, 24, 48, 96 mg mL−1 (0 mg mL−1 pure petroleum ether solution served as a negative control), and then filter paper discs were added to different concentrations of essential oil for 2 h. The MIC value was recorded.
the target variable (Supplement Table 3). The results were shown in Supplement Table 4, Supplement Table 5 and Fig. 2. The contour plots can reflect the intensity of the interaction of the two factors. The ellipse indicates that the two factors interact significantly, while the circle is the opposite. We could conclude that Except for the material-liquid ratio and the content of NaCl, the interaction between independent and dependent variables was significant was significant. While the interaction between the material-liquid ratio and reflux time was more significant than the interaction between the reflux time and the content of NaCl. The results showed that when the extraction rate reached the maximum, the value of each factor was A = 0.59, B = 2.33, and C = 8.89, and the experimental conditions were as follows: material liquid ratio 59/100, distillation time 2.33 h, and 8.89 % NaCl auxiliary added. Then, these parameters were substituted into the regression equation: R1=+0.88833-0.041667 * A + 0.12167 * B + 0.063611 * C + 0.16667 * A * B + 0.02778 * A * C + 0.025 * B *C -0.55 * A2-0.095 * B2-0.00070 * C2; the maximum extraction rate was approximately 1.40 %, which had a slight deviation from the measured value. The measured value did not reach the maximum value because of the limited experimental conditions.
3. Results and discussion 3.1. The optimum extraction process of FEO 3.1.1. Auxiliary condition screening Three different pretreatment methods were used to extract the Gold fingered citron essential oil. The average extraction rate of the control was approximately 1.15 % (Supplement Table 2 and Fig. 1). Compared with the control, the three pretreatment methods can improve the oil extraction rate in different degrees. The extraction rate after refluxing with a pretreatment of 9 % NaCl auxiliary and ultrasonic was the highest and reached 1.80 %.
3.2. GC–MS components analysis The Gold fingered citron essential oil extracted with (Table 1) and without NaCl auxiliary (Table 2) were analyzed to study the effect of NaCl auxiliary on the essential oil components. The qualitative analysis results of the fingered citron essential oil from Cantonese and Sichuan were shown in Tables 3 and 4. The essential oil of the Gold fingered citron was mainly composed of alkenes. The content of D-limonene was the most (close to 50 %),
3.1.2. Box-Behnken experimental results Three factors (reflux time, material-liquid ratio and the content of NaCl) and three levels of the Box-Behnken design were conducted by using Design-Expert software, and the extraction rate was selected as 4
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compositions of different FEOs helped provide a relevant theoretical basis for new product development and new drug research.
Table 2 GC–MS qualitative analysis results of the Gold fingered citron essential oil (without NaCl). Retention time (min)
1 2 3 4 5 6 7 8 9 10
10.794 11.092 12.592 12.774 13.299 13.847 14.262 14.531 15.238 15.490
11
16.138
12 13 14
16.827 17.261 18.954
15 16 17 18 19 20 21 22 23 24 25
19.898 20.374 20.671 21.270 21.337 21.682 22.053 22.576 23.284 23.734 24.909
26 27 28 29 30
25.163 25.709 26.604 27.024 27.330
31
27.778
32 33 34 35
27.973 28.652 29.037 29.295
Component name
alpha-Thujene alpha-Pinene 4(10)-Thujene beta-Pinene beta-Myrcene 3-Thujene (+)-4-Carene p-Cymene D-Limonene beta-cisOcimene gammaTerpinene Isoterpinolene Linalool (R)(+)-Citronellal Terpinen-4-ol alpha-Terpineol Decanal cis-Geraniol Citronellol beta-Citral Geraniol alpha-Citral 2-Undecanone Undecanal Citronellol acetate Nerol acetate Geranyl acetate Dodecanal Caryophyllene trans-alphaFingered citronene (E)-betaFamesene Humulene Germacrene D Germacrene beta-Bisabolene
CAS#
Similarity (%)
3.3. Antibacterial activity analysis of FEO
Area percentage (%)
2867-05-2 80-56-8 3387-41-5 127-91-3 123-35-3 2867-05-2 29050-33-7 99-87-6 5989-27-5 3338-55-4
97 97 97 97 97 96 96 94 95 99
2.28 4.44 0.76 4.35 4.44 0.18 1.06 0.79 46.0 3.20
99-85-4
97
21.4
586-63-0 78-70-6 2385-77-5
97 97 98
2.42 0.30 0.31
562-74-3 98-55-5 112-31-2 106-25-2 106-22-9 106-26-3 106-24-1 141-27-5 112-12-9 112-44-7 150-84-5
96 97 96 94 95 98 96 98 95 97 95
0.29 0.65 0.26 0.21 0.14 0.32 0.28 0.42 0.10 0.25 0.10
141-12-8 105-87-3 112-54-9 87-44-5 13474-59-4
96 97 97 96 96
0.14 0.12 0.20 0.73 0.79
18794-84-8
96
0.07
6753-98-6 23986-74-5 15423-57-1 495-61-4
96 94 92 96
0.09 0.60 0.31 1.25
The antibacterial activities of the three kinds of different essential oils were assessed by inhibition zone diameter using agar disc diffusion method. And the inhibition zone could reflect the antibacterial activity of the essential oils. It can be seen from the data shown in Supplement Table 6 that the essential oils all had antibacterial activity on the tested strains, but the antibacterial activities of the essential oils varied greatly. The Gold fingered citron essential oil had better antibacterial activity on Staphylococcus aureus and Streptococcus pneumoniae and the inhibition zone diameters were all larger than 10 mm. The Cantonese fingered citron essential oil had a significant inhibitory effect on Enterococcus faecalis, Escherichia coli and Streptococcus pneumoniae, and the inhibition zones all reached approximately 11 mm. The essential oil of Sichuan fingered citron effectively inhibited Enterococcus faecalis and Escherichia coli, especially the growth of E. coli, and the average diameter of the inhibition zone reached 12.79 mm. Further MIC assays were performed on the bacteria that were inhibited. Essential oils at different concentrations were prepared with petroleum ether. The essential oils began to exhibit antibacterial activity when the inhibition zone was larger than 6 mm, and the minimum inhibitory concentration (MIC) was recorded. The greater the MIC value was, the weaker the antibacterial activity was, the smaller the MIC value was, the stronger the antibacterial activity was. The results showed that the MIC of the Gold fingered citron essential oil against Streptococcus pneumoniae was 24 mg mL−1, and the MIC against Staphylococcus aureus was 12 mg mL−1 (Table 5), illustrating that the antibacterial activity against Staphylococcus aureus was better than that against Streptococcus pneumoniae. The Cantonese fingered citron essential oil had similar antibacterial activity against Escherichia coli and Streptococcus pneumoniae, both of which began to be inhibited at the concentration of 48 mg mL−1. In contrast, the Cantonese fingered citron essential oil had the best antibacterial effect against Enterococcus faecalis, with the MIC of 12 mg mL−1 reflects the highest susceptibility of this strain when compared with the Escherichia coli and Streptococcus pneumoniae (Table 5). According to the data (Table 5), the Sichuan fingered citron essential oil had the strongest antibacterial activity against Escherichia coli, and the MIC was 12 mg mL−1, which was significantly better than that against Enterococcus faecalis, with the MIC of 48 mg mL−1. Cantonese fingered citron essential oil had obvious inhibitory effects on the three tested strains to a certain extent, indicating that it could be used as a raw material for the development of natural antibacterial drugs. The compositions and the contents of compounds and antibacterial activities of the three fingered citron essential oils were different, indicating that their antibacterial activity had a certain correlation with the composition and content. The antibacterial activity of the essential oils was influenced by the chemical composition, the content of the components, and the interactions between the compounds (Marino et al., 2016). D-limonene was the main component of the Gold fingered citron essential oil, which could significantly inhibit the growth of Staphylococcus aureus. A previous study also showed that monoterpenes could inhibit the growth of Staphylococcus aureus. The intensity of inhibition was related to the content of monoterpene. (Rahman et al., 2011). D-limonene could destroy the integrity of the microbial plasma membrane and inhibit the activity of respiratory enzymes, thereby inhibiting the growth of bacteria (Rhayour et al., 2003). According to the data with Escherichia coli, the activity of Escherichia coli inhibition by Sichuan and Cantonese fingered citron essential oils may be positively correlated with the content of oxygenated monoterpenes in essential oil components (Rashed et al., 2017). The content
followed by gamma terpinene with a content of over 20 % (Table 1). The results also indicated that the addition of NaCl adjuvant does not significantly affect the composition of citron essential oil. The main component of Cantonese fingered citron essential oil was also alkenes (Table 3), the content of D-limonene was the highest, reaching 40 %, but was significantly lower than that in the Gold fingered citron essential oil. The γ-terpinene content was approximately 22.8 %. In addition, the Cantonese fingered citron essential oil also contained a certain amount of aldehydes, particularly a high citral content. Similarly, it was found that the essential oil extracted from the Sichuan fingered citron was still mainly composed of D-limonene (27.6 %). It also contained a certain amount of ethanol and esters (Table 4). In summary, GC–MS analysis results showed that the main component of the essential oils extracted from the three kinds of fingered citrons was D-limonene; the high percentage of the D-limonene contained in the three kinds of fingered citron essential oil is consistent with the results obtained in other studies (Sun, 2018; Wang, 2014). However, the content was somewhat different among the three types of essential oils. The strongest fragrance of Gold fingered citron essential oil may be related to the alkenes (Hayouni et al., 2008). The Cantonese fingered citron essential oil contained more aldehydes, and the Sichuan fingered citron oils contained more esters and ethanol, which may be related to its unique pharmacological activity. Therefore, analyzing the 5
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Table 3 GC–MS qualitative analysis results of the Cantonese fingered citron essential oil (with NaCl). Number
Retention time (min)
Component name
CAS#
Similarity (%)
Area percentage (%)
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
10.799 11.105 12.598 12.787 13.300 13.847 14.258 14.588 15.136 15.467 16.156 16.836 17.263 18.952 19.908 20.383 21.298 21.375 21.747 22.082 22.660 25.171 25.714 27.026 27.328 28.658 29.035 29.290
alpha-Thujene alpha-Pinene 4(10)-Thujene beta-Pinene beta-Myrcene 3-Thujene (+)-4-Carene p-Cymene D-Limonene beta-cis-Ocimene gamma-Terpinene Isoterpinolene Linalool (R)-(+)-Citronellal Terpinen-4-ol alpha-Terpineol cis-Geraniol Citronellol beta-Citral Geraniol alpha-Citral Nerol acetate Geranyl acetate Caryophyllene trans-alpha-Fingered citronene Germacrene D Bicyclogermacrene beta-Bisabolene
2867-05-2 80-56-8 3387-41-5 127-91-3 123-35-3 2867-05-2 29050-33-7 527-84-4 5989-27-5 3338-55-4 99-85-4 586-63-0 78-70-6 2385-77-5 562-74-3 98-55-5 106-25-2 106-22-9 106-26-3 106-24-1 141-27-5 141-12-8 105-87-3 87-44-5 13474-59-4 23986-74-5 24703-35-3 495-61-4
97 97 97 96 97 95 95 95 95 99 97 97 98 97 96 97 95 94 98 96 98 96 97 96 96 94 92 96
2.50 4.67 0.66 4.56 3.89 0.19 1.27 1.34 39.0 2.59 22.8 2.75 0.35 0.26 0.58 0.78 0.51 0.15 2.83 0.25 3.73 0.44 0.27 0.91 0.66 0.80 0.23 1.01
Table 4 GC–MS qualitative analysis results of the Sichuan fingered citron essential oil (with NaCl). Number
Retention time (min)
Component name
CAS#
Similarity %
Area percentage (%)
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
10.785 11.081 12.792 13.288 14.260 15.080 15.423 16.053 16.786 17.318 18.952 20.003 20.538 21.468 21.770 22.229 22.630 23.630 24.938 25.267 25.804 27.035 27.346 28.673 29.313 31.134 32.361 33.724 39.953 42.522
2867-05-2 80-56-8 127-91-3 123-35-3 29050-33-7 5989-27-5 3338-55-4 99-85-4 586-63-0 78-70-6 2385-77-5 20126-76-5 98-55-5 106-22-9 106-26-3 106-24-1 141-27-5 499-75-2 150-84-5 141-12-8 105-87-3 87-44-5 13474-59-4 23986-74-5 495-61-4 6750-60-3 88395-46-4 515-69-5 57-10-3 61447-89-0
97 97 96 97 96 96 99 97 97 98 92 97 98 93 98 96 98 94 94 97 97 96 96 94 96 94 95 95 96 90
0.95 2.81 3.57 1.48 0.81 27.6 0.45 14.0 1.52 2.43 0.47 4.36 6.55 5.31 2.31 4.33 1.82 0.63 0.59 4.23 3.40 0.60 0.87 0.99 1.62 0.62 0.64 0.76 2.74 0.62
31
43.198
alpha-Thujene alpha-Pinene beta-Pinene beta-Myrcene (+)-4-Carene D-Limonene beta-cis-Ocimene gamma-Terpinene Isoterpinolene Linalool (R)-(+)-Citronellal (-)-4-Terpinenol alpha-Terpineol Citronellol beta-Citral Geraniol alpha-Citral Carvacrol Citronellol acetate Nerol acetate Geranyl acetate Caryophyllene trans-alpha-Fingered citronene Germacrene D beta-Bisabolene (+)-Spathulenol Isospathulenol alpha-Bisabolol n-Hexadecanoic acid 6-Methyl-4,6-bis(4-methyl-pent-3-en-1-yl) cyclohexa −1,3-dienecarbaldehyde cis, cis-Linoleic acid
60-33-3
91
0.89
of linalool in the Sichuan fingered citron essential oil was higher than that in the Cantonese fingered citron, and the inhibitory activity of the Sichuan fingered citron essential oil on Escherichia coli was stronger than that of the Cantonese fingered citron essential oil. Previous studies have shown that the linalool in the essential oil of Ocimum basilicum was
high (11.2–43.8 %) and had good antibacterial activity against Escherichia coli (Sharopov et al., 2016). In addition, it has been reported that monoterpene ethanol has a hydrophilic property, which disrupts the cell membrane of gram-negative bacteria (Hayouni et al., 2008). The inhibitory effect of the Cantonese fingered citron essential oil 6
Industrial Crops & Products 147 (2020) 112172 8.93 ± 0.10 10.83 ± 0.24 7.77 ± 0.22 9.14 ± 0.22
8.21 ± 0.17 6.00 ± 0.00
6.00 ± 0.00 7.14 ± 0.24 6.00 ± 0.00 6.00 ± 0.00
6.00 ± 0.00 6.00 ± 0.00
6.00 ± 0.00 6.00 ± 0.00
on Enterococcus faecalis was stronger than the inhibitory effect on Escherichia coli and Streptococcus pneumoniae. It was probably caused by aldehydes such as citral. Previous research showed that perillaldehyde had a stronger inhibitory effect on gram-positive bacteria than gramnegative bacteria (Dahmane et al., 2017). The similar antibacterial activity on Escherichia coli and Streptococcus pneumoniae may be caused by the activity of the test strain. Recently, related literature found that fingered citron flavonoids not only had a great effect on human hyperlipidaemia and atherosclerosis (Cappello et al., 2016), but also had significant anti-inflammatory and antioxidant effects (Daniela et al., 2016). The essential oil composition was close to the corresponding pharmacological activities, and the differences among the three essential oil components reflected their distinct biological activities, which further reflected the diverse value in practical application. In addition, other pharmacological activities of fingered citron essential oil, such as antioxidation effects (Cosentino et al., 2014) and anti-depression effects (Saiyudthong and Mekseepralard, 2015) are also worth further exploration. However, the antinociceptive and antiallodynic effects are very important in complementary medicine (Rombolà et al., 2016). Another significant discovery was that fingered citron essential oil has lipid-lowering activity, so it can be used to replace statins with side effects (Giglio et al., 2016). Based on previous comprehensive reports and the results of the antibacterial activity test in this experiment, the differences in the compositions of the three essential oils and their different biological activities indicated that the compositions were closely related to the pharmacological activities, reflecting the diverse value of fingered citron essential oil in practical applications. Fingered citron essentiol oil exhibited resistances on many bacterial strains (Cirmi et al., 2016). We only tested its resistances on five bacteria strains. There are still many representative strains that deserve further research. These results may help us develop antibacterial and anti-inflammatory drugs and bring hope for the solutions of drug resistance.
7.29 ± 0.37 9.02 ± 0.14 7.56 ± 0.24 8.77 ± 0.24 8.21 ± 0.17 8.93 ± 0.10 48 96
6.00 ± 0.00 7.14 ± 0.24 24
Note： “-” indicate that the essential oil have no inhibitory activity on the tested strain at this concentration.
– – – – __ 9.99 ± 0.14 10.82 ± 0.20
– –
8.99 ± 0.68 9.81 ± 0.20
6.00 ± 0.00 –_ –_ 8.65 ± 0.17
–_
8.01 ± 0.28
6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.66 ± 0.24 – –_ – –_ – –_ 6.00 ± 0.00 6.00 ± 0.00 6 12
6.00 ± 0.00 6.00 ± 0.00
6.00 ± 0.00 3
6.00 ± 0.00
– – _ – _ – –
6.00 ± 0.00 7.93 ± 0.10
–_ –_ 6.00 ± 0.00
–_
6.00 ± 0.00
6.00 ± 0.00
– _ – _ – – _ – _ – – _ 6.00 ± 0.00 –_ –_ 6.00 ± 0.00 0
6.00 ± 0.00
– _ –
6.00 ± 0.00
–_
6.00 ± 0.00
Cantonese FEO Gold FEO Cantonese FEO Gold FEO Sichuan FEO Cantonese FEO Gold FEO Gold FEO
Cantonese FEO
Sichuan FEO
Staphylococcus aureus Streptococcus pneumoniae Concentr-ation (mg/mL)
Table 5 The MIC values of three kinds of fingered citron essential oils on the tested strains.
Enterococcus faecalis
Sichuan FEO
Escherichia coli
Sichuan FEO BEO
F. Wang, et al.
3.4. Statistical analysis Design-Expert software was used to study the effects of the material liquid ratio, reflux time and content of NaCl on the exaction rate. The data were analyzed by GraphPad Prism7. All data were reported as a source of variance squared and the degree of freedom, mean square, F value and P value.
4. Conclusion In this study, we found that ultrasonic pretreatment with 9 % NaCl solution was the best auxiliary method to extract fingered citron essential oil. The most abundant component of the essential oil from three areas of China was D-limonene. However, contents of the compositions were significantly different in three kinds of fingered citron. In addition, the strong fragrance of Gold fingered citron may be related to the high content of olefins in essential oil. The Cantonese fingered citron essential oil contained more aldehydes, and the Sichuan fingered citron essential oil contained more esters and ethanol, which may be related to its unique pharmacological activity. Three kinds of essential oils had different antimicrobial strengths on diverse strains. The MIC revealed that Streptococcus pneumoniae, and Enterococcus faecalis were less susceptible than Staphylococcus aureus to the Gold fingered citron essential oil. The Enterococcus faecalis showed the highest susceptibility to the Cantonese citron essential oil compared to Streptococcus pneumoniae and Escherichia coli. The Sichuan citron essential oil had the strongest antibacterial activity against Escherichia coli. These findings could help us to develop citron essential oils as natural antibacterial agents, new drugs, food additives and other products.
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Giglio, R.V., Patti, A.M., Nikolic, D., 2016. The effect of fingered citron on dyslipidemia. J. Phytomed. 23, 1175–1181. Hayouni, E.A., Bouix, M., Abedrabba, M., Leveau, J.Y., Hamd, M., 2008. Mechanism of action of melaleuca armillaris (Sol. Ex Gaertu) Sm. Essential oil on six LAB strains as assessed by multiparametric flow cytometry and automated microtiter-based assay. J. Food. Chem. 111, 707–718. Marino, M., Bersani, C., Comi, G., 2016. Impedance measurements to study the antimicrobial activity of essential oil from Lamiaceae and Compositae. Int. J. Food Microbiol. 200167, 187–195. Navarra, M., Ferlazzo, N., Cirmi, S., Trapasso, E., Bramanti, P., Lombardo, G.E., Minciullo, P.L., Calapai, G., Gangemi, S., 2015a. Effects of fingered citron essential oil and its extractive fractions on SH ‐ SY5Y human neuroblastoma cell growth. J. Pharm. Pharmacol. 67, 1042–1053. Navarra, M., Mannucci, C., Delbò, M., Calapai, G., 2015b. Citrus bergamia essential oil: from basic research to clinical application. J. Front Pharmacol. 6. https://doi.org/10. 3389/fphar.2015.00036. Penbunditkul, P., Yoshii, H., Ruktanonchai, U., Charinpanitkul, T., Assabumrungrat, S., Soottitantawat, A., 2012. The loss of OSA ‐modified starch emulsifier property during the high‐pressure homogeniser and encapsulation of multi‐flavour fingered citron oil by spray drying. Int. J. Food Sci. Technol. 47, 2325–2333. Rafiee, S., Jahromi, M.K., Jafari, A., Sharifi, M., Mirasheh, R., Mobli, H., 2007. Determining some physical properties of fingered citron (Citrus medica). J. Int. Agrophys. 21, 293–297. Rahman, A., Shanta, Z.S., Rashid, M.A., Parvin, T., Afrin, S., Khatun, M.K., Sattar, M.A., 2011. In vitro antibacterial properties of essential oil and organic extracts of Premnaintegrifolia Linn. Arab. J. Chem. 9, 475–479. Rashed, M.M.A., Tong, Q., Nagi, A., 2017. Isolation of essential oil from Lavandula angustifolia by using ultrasonic-microwave assisted method preceded by enzymolysis treatment, and assessment of its biological activities. J. Ind. Crops Prod. 100, 236–245. Rhayour, K., Bouchikhi, T., Elaraki, A.T., Sendide, K., Remmal, A., 2003. The mechanism of bactericidal action of Oregano and Clove essential oil and of their phenolic major components on escherichia coli and bacillus subtilis. J. Essent. Oil Res. 15, 286–292. Rombolà, L., Amantea, D., Russo, R., Adornetto, A., Berliocchi, L., Tridico, L., Corasaniti, M., Sakurada, S., Sakurada, T., Bagetta, G., Morrone, L.A., 2016. Rational basis for the use of fingered citron essential oil in complementary medicine to treat chronic Pain. J. Mini-Rev. Med. Chem. 16, 721–728. Saiyudthong, S., Mekseepralard, C., 2015. Effect of inhaling fingered citron oil on depression-related behaviors in chronic stressed rats. J. Med. Assoc. Thai. 9, 152–159. Sharopov, F.S., Satyal, P., Ali, N.A., 2016. The essential oil compositions of Ocimum basilicum from three different regions: nepal, tajikistan, and Yemen. J. Chem. Biodivers. 13, 241–248. Sun, X.H., 2018. Extraction of Jinhua Bergamot Essential Oil, Preparation of Nanoemulsion and Activity. Thesis for Master’ Degree. Jiangnan University. Vilkhu, K., Mawson, R., Simons, L., Bates, D., 2008. Applications and opportunities for ultrasound assisted extraction in the food industry — a review. J. Innov. Food Sci. Emerg. 9, 161–169. Wang, Q., 2014. Extraction and Application of Bergamot Aroma Components and Their Essential Oils. Thesis for Master’ Degree. Fujian A&F University. Watanabe, E., Kuchta, K., Kimura, M., Rauwald, H.W., Kamei, T., Imanishi, J., 2015. Effects of fingered citron (Citrus bergamia (Risso) Wright & Arn.) essential oil aromatherapy on mood states, parasympathetic nervous system activity and salivary cortisol levels in 41 healthy females. J. Forsch. Komplementärmed. 22, 43–49. Yang, X.S., Wang, L.J., Dung, C., Lui, E.M.K., Ren, G.X., 2014. Optimization of ultrasonicassisted extraction process of polysaccharides from Americould Ginseng and evaluation of its immunostimulating activity. J. Integr. Agric. 12, 2807–2815. Yi, Z.H., Cao, J.P., Wei, J.J., Ma, W.J., 2016. Microwave-assisted hydrodistillation of essential oil from Thyme (Thymus Vulgaris). J. Med. Plant. 7, 5–8.
Credit author statement I solemnly declare the manuscript is the result of independent research. Except for the cited results, this manuscript does not include manuscripts published or written by others or groups. The legal consequences of this statement shall be borne by myself. Declaration of Competing Interest Authors have no conflict of interest to declare. Acknowledgments We were appreciated the financially supported by National Natural Science Foundation of China (81673536, 81973415, 31570370, 31871694), and Open Fund of Shanghai Key Laboratory of Plant Functional Genomics and Resources (PFGR201901). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112172. References Bayala, B., Bassole, I.H., Scifo, R., Gnoula, C., Morel, L., Lobaccaro, J.M., Simpore, J., 2014. Anticancer activity of essential oil and their chemical components - a review. Am. J. Cancer Res. 4, 591–607. Belsito, E.L., Carbone, C., Di, G.M., Leggio, A., Liguori, A., Perri, F., Siciliano, C., Viscomi, M.C., 2007. Comparison of the volatile constituents in cold-pressed fingered citron oil and a volatile oil isolated by vacuum distillation. J. Agric. Food Chem. 55, 7847–7851. Cappello, A.R., Dolce, V., Iacopetta, D., Martello, M., Fiorillo, M., Curcio, R., Muto, L., Dhanyalayam, D., 2016. Fingered citron (Citrus bergamia Risso) flavonoids and their potential benefits in human hyperlipidemia and atherosclerosis: an overview. J. MiniRev. Med. Chem. 16, 619–629. Cirmi, S., Bisignano, C., Mandalari, G., Navarra, M., 2016. Anti-infective potential of Citrus bergamia Risso et Poiteau (fingered citron) derivatives: a systematic review. J. Phytother. Res. 30, 1404–1411. Cosentino, M., Luini, A., Bombelli, R., Corasaniti, M.T., Bagetta, G., Marino, F., 2014. The essential oil of fingered citron stimulates reactive oxygen species production in human polymorphonuclear leukocytes. J. Phytother. Res. 28, 1232–1239. Dahmane, D., Dob, T., Krimat, S., 2017. Chemical composition, antioxidant and antibacterial activities of the essential oil of medicinal plant Ammodaucus leucotrichus from Algeria. J. Essent. Oil Res. 29, 48–55. Dai, W., Gong, R., Hu, J., Zhou, L.M., 2014. Thiophene capture by an oxidation-modified activated carbon derived from fingered citron. J. Sep. Sci. Technol. 49, 367–375. Daniela, I., Marika, C., Michela, C., Enrico, G., Emanuela, E., Filippo, B., Salvatore, C., Michele, N., 2016. Anti-inflammatory and antioxidant effects of Flavonoid-Rich Fraction of fingered citron Juice (BJe) in a Mouse Model of Intestinal Ischemia/ Reperfusion Injury. J. Front. Pharm. 7. https://doi.org/10.3389/fphar.2016.00203.
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Essential oils from three kinds of fingered citrons and their antibacterial activities
T
Feiyan Wanga, Huaqian Youa, Yanhong Guoa, Yukun Weib, Pengguo Xiaa, Zongqi Yanga, Min Renc, Hui Guoc, Ruilian Hana, Dongfeng Yanga,* a
College of Life Sciences and Medicine, Key Laboratory of Plant Secondary Metabolism and Regulation in Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China b Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602, China c School of Life Science and Technology, Xinxiang University, Xinxiang 453000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fingered citron Essential oil Extraction process Component analysis Antibacterial activity
Fingered citron (Citrus medica L. var. sarcodactylis Swingle, Foshou in Chinese) is well known as an excellent and popular Chinese herbal medicine and functional vegetable. Fingered citron is rich of essential oil, which exhibits antioxidant, antiradical, and antimicrobial properties. In order to improve the extraction rate of essential oil from three kinds of fingered citrons and understand their activities, the optimal auxiliary extraction conditions of essential oils and their antibacterial activities were analyzed in this work. Fingered citron essential oil (FEO) extracted by the reflux method was analyzed by Gas Chromatography Mass Spectrometry (GC–MS), and their antimicrobial activities were detected by the disc diffusion method. The results showed that the best auxiliary condition for FEO extraction was ultrasonic combined with 9 % NaCl. The extraction rate of 9 % NaCl combined with ultrasonic was the highest (1.80 %) compared with the control (1.15 %). The compositions of FEOs extracted from three kinds of fingered citrons were mainly composed of alkenes, such as D-limonene and γ-terpinene. However, contents of the compositions were significantly different in three kinds of fingered citrons. The Gold fingered citron essential oil possessed the highest content of D-limonene which close to 50 %, and that in the Cantonese fingered citron essential oil was only 27.60 %. A certain amount of ethanol contained in the Sichuan fingered citron essential oil and aldehyde contained in the Cantonese fingered citron essential oil. Moreover, the three FEOs had different antibacterial effects. In three kinds of fingered citron, the antibacterial effect of the Gold fingered citron essential oil on Staphylococcus aureus was the strongest. The Cantonese citron essential oil had the strongest antibacterial effect on Enterococcus faecalis, and the Sichuan citron essential oil had the strongest antibacterial activity against Escherichia coli. These findings could help us to develop citron essential oils as natural antibacterial agents, new drugs, food additives and other products.
1. Introduction
processed into food, drinks and spices. Fingered citron-based activated carbon, a new type of activated carbon, plays an important role in material chemistry analysis (Dai et al., 2014). The highest content of essential oil in fresh fingered citron could reach nearly 1.6 %. Despite its unstable properties (Penbunditkul et al., 2012) and the difficulties during its production, storage and packaging (Rafiee et al., 2007), FEO has become one of the hot spots in the field of essential oil research in recent years due to its mood soothing (Watanabe et al., 2015), antidepressant, antibacterial, anticancer (Bayala et al., 2014), and antiproliferative effects (Navarra et al., 2015a,Navarra et al., 2015b). At present, the reflux method is the most widely used method for essential oil extraction (Belsito et al., 2007), although there are still some
Fingered citron (Citrus medica L. var. sarcodactylis Swingle), also called nine-claw wood, five fingered orange, citrus chirocarpus and so on, which was known as an excellent Chinese herbal medicine. The plant is a shrub or small tree. The stem has short spines and its leaves are alternate. During the fruit development, the fingered citron is divided into finger shaped meat strips. Fingered citron is mainly planted in Zhejiang, Guangxi, Sichuan and other places in China. There are three kinds of citron according to its geographical divisions, including the Gold fingered citron, the Cantonese fingered citron and the Sichuan fingered citron. Fingered citron could be used as medicine and
⁎
Corresponding author. E-mail address: [email protected] (D. Yang).
https://doi.org/10.1016/j.indcrop.2020.112172 Received 28 October 2019; Received in revised form 31 December 2019; Accepted 25 January 2020 0926-6690/ © 2020 Published by Elsevier B.V.
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drawbacks remain. For example, it takes a long time, the quality of essential oil decreases or even degrades at high temperatures, and the extraction rate of essential oil is not high. In recent years many researchers have optimized the extraction conditions to improve extract quality of essential oil, such as ultrasonic (Yang et al., 2014) and microwave extraction (Yi et al., 2016), and explored the optimal parameters of objective conditions. As far as we know a combination of both assisted extraction methods has not yet been reported. We combined these two assisted extraction methods for the first time to maximize the extraction rate of the three kinds of essential oils. This optimum auxiliary condition has many advantages. For example, it improved the fingered citron essential oil extraction rate significantly and is easy to operate. This condition is applicable not only in the small-scale laboratory but also for large-scale industrial production. The purposes of this research were to find the optimal extraction method of fingered citron essential oil and improve the oil extraction rate to lay the foundation for industrial production for FEO and provide a feasible reference scheme. Fingered citron essential oil was recognized for their biological activities, which are generally closely related to its compositions. Only with a full understanding of the main chemical constituents could we provide the corresponding theoretical basis for the development and application of FEO-related products. Therefore, another purpose of this study was to identify the compositions of three kinds of citron essential oils. Secondary metabolites of plants can have a killing or inhibiting effect on microorganisms. When plants are induced by pathogenic microorganisms, such as fungi and bacteria, they will develop a defensive response. Finally, we wanted to investigate the antibacterial activity of three kinds of fingered citron essential oils. The disc diffusion method was used to test the antibacterial activity of three different fingered citron essential oils on Escherichia coli, Streptococcus pneumoniae, Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis.
Fig. 1. Effect of three kinds of auxiliary conditions on the oil extraction rate, Means ± standard deviation (S.D.) (n = 3) are shown.
2.3. Box-Behnken experiment to optimize the extraction conditions The material-liquid ratio, the reflux time and the content of NaCl were considered as significant factors influencing the fingered citron essential oil extraction rate. They were divided into three levels (low, intermediate and high, labelled as -1, 0 and 1, respectively) to carry out Box-Behnken experiments to improve the extract rate. The extraction conditions were shown in Supplement Table 1. First, a total of 100 g of Gold fingered citron pieces was put into 500 mL, 200 mL and 125 mL of extraction solution (9 % NaCl) and ground into pulp. Then, it was extracted by the ultrasonic method for 30 min at 200 W and refluxed for 2 h. Second, the Gold fingered citron pieces were put into 200 mL of solution (9 % NaCl) and ground into pulp. Then, they were extracted by the ultrasonic method for 30 min at 200 W and refluxed for 1 h, 2 h and 3 h. Next, the Gold fingered citron pieces were put into 200 mL of extraction solution (6 %, 9 % and 12 % NaCl) and ground into pulp. Then, they were extracted by the ultrasonic method for 30 min at 200 W and refluxed for 2 h. The response surface analysis was carried out by Expert Design software.
2. Materials and methods 2.1. Samples and chemicals The Gold fingered citron mature fruits were collected from Jinhua in Zhejiang Province on October 2015; the Cantonese fingered citrons were collected from Liuzhou in Guangxi Province on October 2015; and the Sichuan fingered citrons were collected from the Nantianmen Fingered Citron Cultivation Base in Sichuan Province on October 2015. The species were identified by professor Zongsuo Liang of Zhejiang Scitech University. Five bacterial strains including Bacillus subtilis ATCC 6051, Streptococcus pneumoniae ATCC 49619, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8099 and Staphylococcus aureus ATCC 6538 were obtained from the Zhejiang Center for Disease Control and Prevention and stored at -20 ℃ with glycerol. Cells were inoculated into 5 test tubes with 5 mL of liquid medium. After activation on a shaking table at 37 °C for 48 h, bacterial suspensions were made for testing.
2.4. Composition analysis by the GC–MS method The compositions of the FEOs extracted from three kinds of fingered citrons (Jinhua, Sichuan, Guangxi) were analyzed by GC–MS. The conditions were as follows: chromatographic column: Rtx-5Sil MS 30 m × 0.25 mm × 0.25 μm; inlet temperature: 280 ℃; injection mode: split injection; split ratio 100:1; column temperature programme: 40 ℃ (5 min), 5/min, 300 ℃ (5 min); constant line speed: 36.1 cm/sec; sample size: 1 μL; ionization mode: EI; ion source temperature: 230 ℃; interface temperature: 250 ℃; acquisition mode: scan; mass number range: m/z, 35∼650, amu. To exclude the influence of added NaCl, the components of the Gold fingered citron essential oil obtained without adding NaCl were analyzed.
2.2. Optimization of the FEO extraction process Gold fingered citron (100 g) was extracted by ultrasonic with 9 % NaCl extraction solution (Vilkhu et al., 2008). A total of 100 g of fingered citron pulp was added to 200 mL of distilled water as a control and refluxed for 2 h, and the extraction rate was calculated. The experiment was performed three times. In group 1, the essential oil in 100 g citron fruit pulp was extracted by the reflux method with 200 mL of 9 % NaCl extraction solution. In group 2, the fingered citron pulp was pretreated by ultrasonic at 200 W for 30 min and then extracted by the reflux method. In group 3, the fingered citron pulp was pretreated by ultrasonic with 200 mL of 9 % NaCl solution for 30 min and then extracted by the reflux method.
2.5. Antibacterial activity experiment A preliminary screen about the antibacterial activity of the essential 2
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Fig. 2. The response maps and contour maps of material liquid ratio (A), the content of NaCl (C), reflux time (B) and extraction rate. The closer the contour plots were to the ellipse, the more obvious the interaction between the two factors, if the contour plots were closer to the circle, the less significant the interaction between the two factors.
3
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Table 1 GC–MS qualitative analysis results of the Gold fingered citron essential oil (with NaCl). Number
Retention time (min)
Component name
CAS#
Similarity (%)
Area percentage (%)
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
10.792 11.088 12.590 12.768 13.298 13.849 14.258 14.532 15.230 15.490 16.113 16.817 17.255 18.949 19.903 20.374 20.672 21.268 21.328 21.670 22.054 22.561 23.284 23.735 24.909 25.163 25.708 26.604 27.021 27.329 27.779 27.973 28.654 29.036 29.293
alpha-Thujene alpha-Pinene 4(10)-Thujene beta-Pinene beta-Myrcene 3-Thujene (+)-4-Carene p-Cymene D-Limonene beta-cis-Ocimene gamma-Terpinene Isoterpinolene Linalool (R)- (+)-Citronellal Terpinen-4-ol alpha-Terpineol Decanal cis-Geraniol Citronellol beta-Citral Geraniol alpha-Citral 2-Undecanone Undecanal Citronellol acetate Nerol acetate Geranyl acetate Dodecanal Caryophyllene trans-alpha-Fingered citronene (E)-beta-Famesene Humulene Germacrene D Germacrene beta-Bisabolene
2867-05-2 80-56-8 3387-41-5 127-91-3 123-35-3 2867-05-2 29050-33-7 99-87-6 5989-27-5 3338-55-4 99-85-4 586-63-0 78-70-6 2385-77-5 562-74-3 98-55-5 112-31-2 106-25-2 106-22-9 106-26-3 106-24-1 141-27-5 112-12-9 112-44-7 150-84-5 141-12-8 105-87-3 112-54-9 87-44-5 13474-59-4 18794-84-8 6753-98-6 23986-74-5 15423-57-1 495-61-4
97 97 97 97 97 96 96 95 95 99 97 97 97 97 96 97 96 93 94 97 96 98 95 97 94 96 97 97 96 96 95 97 94 92 96
2.10 4.40 0.49 4.03 4.48 0.18 1.08 0.71 48.0 3.25 21.1 2.40 0.23 0.17 0.49 0.65 0.26 0.21 0.14 0.32 0.28 0.42 0.10 0.25 0.10 0.14 0.12 0.20 0.73 0.79 0.07 0.09 0.61 0.27 1.16
oil was conducted. A Sterile 6 mm filter paper discs was put into the essential oil for 2 h. The cell suspensions of the tested strains were gradually diluted to 10−1, 10−2, 10−3, 10−4 and 10−5 with sterile water. Then, the plates were coated with 0.2 mL of cell suspensions. The assay was carried out three times for each strain. Each bacterial strain had three repetitions. Then, the filter paper was attached to it and cultured at 37 ℃ for 37 h (with three repetitions). The diameter of the inhibition zones was measured by the cross method. The essential oil was diluted with petroleum ether to a concentration of 0, 3, 6, 12, 24, 48, 96 mg mL−1 (0 mg mL−1 pure petroleum ether solution served as a negative control), and then filter paper discs were added to different concentrations of essential oil for 2 h. The MIC value was recorded.
the target variable (Supplement Table 3). The results were shown in Supplement Table 4, Supplement Table 5 and Fig. 2. The contour plots can reflect the intensity of the interaction of the two factors. The ellipse indicates that the two factors interact significantly, while the circle is the opposite. We could conclude that Except for the material-liquid ratio and the content of NaCl, the interaction between independent and dependent variables was significant was significant. While the interaction between the material-liquid ratio and reflux time was more significant than the interaction between the reflux time and the content of NaCl. The results showed that when the extraction rate reached the maximum, the value of each factor was A = 0.59, B = 2.33, and C = 8.89, and the experimental conditions were as follows: material liquid ratio 59/100, distillation time 2.33 h, and 8.89 % NaCl auxiliary added. Then, these parameters were substituted into the regression equation: R1=+0.88833-0.041667 * A + 0.12167 * B + 0.063611 * C + 0.16667 * A * B + 0.02778 * A * C + 0.025 * B *C -0.55 * A2-0.095 * B2-0.00070 * C2; the maximum extraction rate was approximately 1.40 %, which had a slight deviation from the measured value. The measured value did not reach the maximum value because of the limited experimental conditions.
3. Results and discussion 3.1. The optimum extraction process of FEO 3.1.1. Auxiliary condition screening Three different pretreatment methods were used to extract the Gold fingered citron essential oil. The average extraction rate of the control was approximately 1.15 % (Supplement Table 2 and Fig. 1). Compared with the control, the three pretreatment methods can improve the oil extraction rate in different degrees. The extraction rate after refluxing with a pretreatment of 9 % NaCl auxiliary and ultrasonic was the highest and reached 1.80 %.
3.2. GC–MS components analysis The Gold fingered citron essential oil extracted with (Table 1) and without NaCl auxiliary (Table 2) were analyzed to study the effect of NaCl auxiliary on the essential oil components. The qualitative analysis results of the fingered citron essential oil from Cantonese and Sichuan were shown in Tables 3 and 4. The essential oil of the Gold fingered citron was mainly composed of alkenes. The content of D-limonene was the most (close to 50 %),
3.1.2. Box-Behnken experimental results Three factors (reflux time, material-liquid ratio and the content of NaCl) and three levels of the Box-Behnken design were conducted by using Design-Expert software, and the extraction rate was selected as 4
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compositions of different FEOs helped provide a relevant theoretical basis for new product development and new drug research.
Table 2 GC–MS qualitative analysis results of the Gold fingered citron essential oil (without NaCl). Retention time (min)
1 2 3 4 5 6 7 8 9 10
10.794 11.092 12.592 12.774 13.299 13.847 14.262 14.531 15.238 15.490
11
16.138
12 13 14
16.827 17.261 18.954
15 16 17 18 19 20 21 22 23 24 25
19.898 20.374 20.671 21.270 21.337 21.682 22.053 22.576 23.284 23.734 24.909
26 27 28 29 30
25.163 25.709 26.604 27.024 27.330
31
27.778
32 33 34 35
27.973 28.652 29.037 29.295
Component name
alpha-Thujene alpha-Pinene 4(10)-Thujene beta-Pinene beta-Myrcene 3-Thujene (+)-4-Carene p-Cymene D-Limonene beta-cisOcimene gammaTerpinene Isoterpinolene Linalool (R)(+)-Citronellal Terpinen-4-ol alpha-Terpineol Decanal cis-Geraniol Citronellol beta-Citral Geraniol alpha-Citral 2-Undecanone Undecanal Citronellol acetate Nerol acetate Geranyl acetate Dodecanal Caryophyllene trans-alphaFingered citronene (E)-betaFamesene Humulene Germacrene D Germacrene beta-Bisabolene
CAS#
Similarity (%)
3.3. Antibacterial activity analysis of FEO
Area percentage (%)
2867-05-2 80-56-8 3387-41-5 127-91-3 123-35-3 2867-05-2 29050-33-7 99-87-6 5989-27-5 3338-55-4
97 97 97 97 97 96 96 94 95 99
2.28 4.44 0.76 4.35 4.44 0.18 1.06 0.79 46.0 3.20
99-85-4
97
21.4
586-63-0 78-70-6 2385-77-5
97 97 98
2.42 0.30 0.31
562-74-3 98-55-5 112-31-2 106-25-2 106-22-9 106-26-3 106-24-1 141-27-5 112-12-9 112-44-7 150-84-5
96 97 96 94 95 98 96 98 95 97 95
0.29 0.65 0.26 0.21 0.14 0.32 0.28 0.42 0.10 0.25 0.10
141-12-8 105-87-3 112-54-9 87-44-5 13474-59-4
96 97 97 96 96
0.14 0.12 0.20 0.73 0.79
18794-84-8
96
0.07
6753-98-6 23986-74-5 15423-57-1 495-61-4
96 94 92 96
0.09 0.60 0.31 1.25
The antibacterial activities of the three kinds of different essential oils were assessed by inhibition zone diameter using agar disc diffusion method. And the inhibition zone could reflect the antibacterial activity of the essential oils. It can be seen from the data shown in Supplement Table 6 that the essential oils all had antibacterial activity on the tested strains, but the antibacterial activities of the essential oils varied greatly. The Gold fingered citron essential oil had better antibacterial activity on Staphylococcus aureus and Streptococcus pneumoniae and the inhibition zone diameters were all larger than 10 mm. The Cantonese fingered citron essential oil had a significant inhibitory effect on Enterococcus faecalis, Escherichia coli and Streptococcus pneumoniae, and the inhibition zones all reached approximately 11 mm. The essential oil of Sichuan fingered citron effectively inhibited Enterococcus faecalis and Escherichia coli, especially the growth of E. coli, and the average diameter of the inhibition zone reached 12.79 mm. Further MIC assays were performed on the bacteria that were inhibited. Essential oils at different concentrations were prepared with petroleum ether. The essential oils began to exhibit antibacterial activity when the inhibition zone was larger than 6 mm, and the minimum inhibitory concentration (MIC) was recorded. The greater the MIC value was, the weaker the antibacterial activity was, the smaller the MIC value was, the stronger the antibacterial activity was. The results showed that the MIC of the Gold fingered citron essential oil against Streptococcus pneumoniae was 24 mg mL−1, and the MIC against Staphylococcus aureus was 12 mg mL−1 (Table 5), illustrating that the antibacterial activity against Staphylococcus aureus was better than that against Streptococcus pneumoniae. The Cantonese fingered citron essential oil had similar antibacterial activity against Escherichia coli and Streptococcus pneumoniae, both of which began to be inhibited at the concentration of 48 mg mL−1. In contrast, the Cantonese fingered citron essential oil had the best antibacterial effect against Enterococcus faecalis, with the MIC of 12 mg mL−1 reflects the highest susceptibility of this strain when compared with the Escherichia coli and Streptococcus pneumoniae (Table 5). According to the data (Table 5), the Sichuan fingered citron essential oil had the strongest antibacterial activity against Escherichia coli, and the MIC was 12 mg mL−1, which was significantly better than that against Enterococcus faecalis, with the MIC of 48 mg mL−1. Cantonese fingered citron essential oil had obvious inhibitory effects on the three tested strains to a certain extent, indicating that it could be used as a raw material for the development of natural antibacterial drugs. The compositions and the contents of compounds and antibacterial activities of the three fingered citron essential oils were different, indicating that their antibacterial activity had a certain correlation with the composition and content. The antibacterial activity of the essential oils was influenced by the chemical composition, the content of the components, and the interactions between the compounds (Marino et al., 2016). D-limonene was the main component of the Gold fingered citron essential oil, which could significantly inhibit the growth of Staphylococcus aureus. A previous study also showed that monoterpenes could inhibit the growth of Staphylococcus aureus. The intensity of inhibition was related to the content of monoterpene. (Rahman et al., 2011). D-limonene could destroy the integrity of the microbial plasma membrane and inhibit the activity of respiratory enzymes, thereby inhibiting the growth of bacteria (Rhayour et al., 2003). According to the data with Escherichia coli, the activity of Escherichia coli inhibition by Sichuan and Cantonese fingered citron essential oils may be positively correlated with the content of oxygenated monoterpenes in essential oil components (Rashed et al., 2017). The content
followed by gamma terpinene with a content of over 20 % (Table 1). The results also indicated that the addition of NaCl adjuvant does not significantly affect the composition of citron essential oil. The main component of Cantonese fingered citron essential oil was also alkenes (Table 3), the content of D-limonene was the highest, reaching 40 %, but was significantly lower than that in the Gold fingered citron essential oil. The γ-terpinene content was approximately 22.8 %. In addition, the Cantonese fingered citron essential oil also contained a certain amount of aldehydes, particularly a high citral content. Similarly, it was found that the essential oil extracted from the Sichuan fingered citron was still mainly composed of D-limonene (27.6 %). It also contained a certain amount of ethanol and esters (Table 4). In summary, GC–MS analysis results showed that the main component of the essential oils extracted from the three kinds of fingered citrons was D-limonene; the high percentage of the D-limonene contained in the three kinds of fingered citron essential oil is consistent with the results obtained in other studies (Sun, 2018; Wang, 2014). However, the content was somewhat different among the three types of essential oils. The strongest fragrance of Gold fingered citron essential oil may be related to the alkenes (Hayouni et al., 2008). The Cantonese fingered citron essential oil contained more aldehydes, and the Sichuan fingered citron oils contained more esters and ethanol, which may be related to its unique pharmacological activity. Therefore, analyzing the 5
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Table 3 GC–MS qualitative analysis results of the Cantonese fingered citron essential oil (with NaCl). Number
Retention time (min)
Component name
CAS#
Similarity (%)
Area percentage (%)
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
10.799 11.105 12.598 12.787 13.300 13.847 14.258 14.588 15.136 15.467 16.156 16.836 17.263 18.952 19.908 20.383 21.298 21.375 21.747 22.082 22.660 25.171 25.714 27.026 27.328 28.658 29.035 29.290
alpha-Thujene alpha-Pinene 4(10)-Thujene beta-Pinene beta-Myrcene 3-Thujene (+)-4-Carene p-Cymene D-Limonene beta-cis-Ocimene gamma-Terpinene Isoterpinolene Linalool (R)-(+)-Citronellal Terpinen-4-ol alpha-Terpineol cis-Geraniol Citronellol beta-Citral Geraniol alpha-Citral Nerol acetate Geranyl acetate Caryophyllene trans-alpha-Fingered citronene Germacrene D Bicyclogermacrene beta-Bisabolene
2867-05-2 80-56-8 3387-41-5 127-91-3 123-35-3 2867-05-2 29050-33-7 527-84-4 5989-27-5 3338-55-4 99-85-4 586-63-0 78-70-6 2385-77-5 562-74-3 98-55-5 106-25-2 106-22-9 106-26-3 106-24-1 141-27-5 141-12-8 105-87-3 87-44-5 13474-59-4 23986-74-5 24703-35-3 495-61-4
97 97 97 96 97 95 95 95 95 99 97 97 98 97 96 97 95 94 98 96 98 96 97 96 96 94 92 96
2.50 4.67 0.66 4.56 3.89 0.19 1.27 1.34 39.0 2.59 22.8 2.75 0.35 0.26 0.58 0.78 0.51 0.15 2.83 0.25 3.73 0.44 0.27 0.91 0.66 0.80 0.23 1.01
Table 4 GC–MS qualitative analysis results of the Sichuan fingered citron essential oil (with NaCl). Number
Retention time (min)
Component name
CAS#
Similarity %
Area percentage (%)
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
10.785 11.081 12.792 13.288 14.260 15.080 15.423 16.053 16.786 17.318 18.952 20.003 20.538 21.468 21.770 22.229 22.630 23.630 24.938 25.267 25.804 27.035 27.346 28.673 29.313 31.134 32.361 33.724 39.953 42.522
2867-05-2 80-56-8 127-91-3 123-35-3 29050-33-7 5989-27-5 3338-55-4 99-85-4 586-63-0 78-70-6 2385-77-5 20126-76-5 98-55-5 106-22-9 106-26-3 106-24-1 141-27-5 499-75-2 150-84-5 141-12-8 105-87-3 87-44-5 13474-59-4 23986-74-5 495-61-4 6750-60-3 88395-46-4 515-69-5 57-10-3 61447-89-0
97 97 96 97 96 96 99 97 97 98 92 97 98 93 98 96 98 94 94 97 97 96 96 94 96 94 95 95 96 90
0.95 2.81 3.57 1.48 0.81 27.6 0.45 14.0 1.52 2.43 0.47 4.36 6.55 5.31 2.31 4.33 1.82 0.63 0.59 4.23 3.40 0.60 0.87 0.99 1.62 0.62 0.64 0.76 2.74 0.62
31
43.198
alpha-Thujene alpha-Pinene beta-Pinene beta-Myrcene (+)-4-Carene D-Limonene beta-cis-Ocimene gamma-Terpinene Isoterpinolene Linalool (R)-(+)-Citronellal (-)-4-Terpinenol alpha-Terpineol Citronellol beta-Citral Geraniol alpha-Citral Carvacrol Citronellol acetate Nerol acetate Geranyl acetate Caryophyllene trans-alpha-Fingered citronene Germacrene D beta-Bisabolene (+)-Spathulenol Isospathulenol alpha-Bisabolol n-Hexadecanoic acid 6-Methyl-4,6-bis(4-methyl-pent-3-en-1-yl) cyclohexa −1,3-dienecarbaldehyde cis, cis-Linoleic acid
60-33-3
91
0.89
of linalool in the Sichuan fingered citron essential oil was higher than that in the Cantonese fingered citron, and the inhibitory activity of the Sichuan fingered citron essential oil on Escherichia coli was stronger than that of the Cantonese fingered citron essential oil. Previous studies have shown that the linalool in the essential oil of Ocimum basilicum was
high (11.2–43.8 %) and had good antibacterial activity against Escherichia coli (Sharopov et al., 2016). In addition, it has been reported that monoterpene ethanol has a hydrophilic property, which disrupts the cell membrane of gram-negative bacteria (Hayouni et al., 2008). The inhibitory effect of the Cantonese fingered citron essential oil 6
Industrial Crops & Products 147 (2020) 112172 8.93 ± 0.10 10.83 ± 0.24 7.77 ± 0.22 9.14 ± 0.22
8.21 ± 0.17 6.00 ± 0.00
6.00 ± 0.00 7.14 ± 0.24 6.00 ± 0.00 6.00 ± 0.00
6.00 ± 0.00 6.00 ± 0.00
6.00 ± 0.00 6.00 ± 0.00
on Enterococcus faecalis was stronger than the inhibitory effect on Escherichia coli and Streptococcus pneumoniae. It was probably caused by aldehydes such as citral. Previous research showed that perillaldehyde had a stronger inhibitory effect on gram-positive bacteria than gramnegative bacteria (Dahmane et al., 2017). The similar antibacterial activity on Escherichia coli and Streptococcus pneumoniae may be caused by the activity of the test strain. Recently, related literature found that fingered citron flavonoids not only had a great effect on human hyperlipidaemia and atherosclerosis (Cappello et al., 2016), but also had significant anti-inflammatory and antioxidant effects (Daniela et al., 2016). The essential oil composition was close to the corresponding pharmacological activities, and the differences among the three essential oil components reflected their distinct biological activities, which further reflected the diverse value in practical application. In addition, other pharmacological activities of fingered citron essential oil, such as antioxidation effects (Cosentino et al., 2014) and anti-depression effects (Saiyudthong and Mekseepralard, 2015) are also worth further exploration. However, the antinociceptive and antiallodynic effects are very important in complementary medicine (Rombolà et al., 2016). Another significant discovery was that fingered citron essential oil has lipid-lowering activity, so it can be used to replace statins with side effects (Giglio et al., 2016). Based on previous comprehensive reports and the results of the antibacterial activity test in this experiment, the differences in the compositions of the three essential oils and their different biological activities indicated that the compositions were closely related to the pharmacological activities, reflecting the diverse value of fingered citron essential oil in practical applications. Fingered citron essentiol oil exhibited resistances on many bacterial strains (Cirmi et al., 2016). We only tested its resistances on five bacteria strains. There are still many representative strains that deserve further research. These results may help us develop antibacterial and anti-inflammatory drugs and bring hope for the solutions of drug resistance.
7.29 ± 0.37 9.02 ± 0.14 7.56 ± 0.24 8.77 ± 0.24 8.21 ± 0.17 8.93 ± 0.10 48 96
6.00 ± 0.00 7.14 ± 0.24 24
Note： “-” indicate that the essential oil have no inhibitory activity on the tested strain at this concentration.
– – – – __ 9.99 ± 0.14 10.82 ± 0.20
– –
8.99 ± 0.68 9.81 ± 0.20
6.00 ± 0.00 –_ –_ 8.65 ± 0.17
–_
8.01 ± 0.28
6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.66 ± 0.24 – –_ – –_ – –_ 6.00 ± 0.00 6.00 ± 0.00 6 12
6.00 ± 0.00 6.00 ± 0.00
6.00 ± 0.00 3
6.00 ± 0.00
– – _ – _ – –
6.00 ± 0.00 7.93 ± 0.10
–_ –_ 6.00 ± 0.00
–_
6.00 ± 0.00
6.00 ± 0.00
– _ – _ – – _ – _ – – _ 6.00 ± 0.00 –_ –_ 6.00 ± 0.00 0
6.00 ± 0.00
– _ –
6.00 ± 0.00
–_
6.00 ± 0.00
Cantonese FEO Gold FEO Cantonese FEO Gold FEO Sichuan FEO Cantonese FEO Gold FEO Gold FEO
Cantonese FEO
Sichuan FEO
Staphylococcus aureus Streptococcus pneumoniae Concentr-ation (mg/mL)
Table 5 The MIC values of three kinds of fingered citron essential oils on the tested strains.
Enterococcus faecalis
Sichuan FEO
Escherichia coli
Sichuan FEO BEO
F. Wang, et al.
3.4. Statistical analysis Design-Expert software was used to study the effects of the material liquid ratio, reflux time and content of NaCl on the exaction rate. The data were analyzed by GraphPad Prism7. All data were reported as a source of variance squared and the degree of freedom, mean square, F value and P value.
4. Conclusion In this study, we found that ultrasonic pretreatment with 9 % NaCl solution was the best auxiliary method to extract fingered citron essential oil. The most abundant component of the essential oil from three areas of China was D-limonene. However, contents of the compositions were significantly different in three kinds of fingered citron. In addition, the strong fragrance of Gold fingered citron may be related to the high content of olefins in essential oil. The Cantonese fingered citron essential oil contained more aldehydes, and the Sichuan fingered citron essential oil contained more esters and ethanol, which may be related to its unique pharmacological activity. Three kinds of essential oils had different antimicrobial strengths on diverse strains. The MIC revealed that Streptococcus pneumoniae, and Enterococcus faecalis were less susceptible than Staphylococcus aureus to the Gold fingered citron essential oil. The Enterococcus faecalis showed the highest susceptibility to the Cantonese citron essential oil compared to Streptococcus pneumoniae and Escherichia coli. The Sichuan citron essential oil had the strongest antibacterial activity against Escherichia coli. These findings could help us to develop citron essential oils as natural antibacterial agents, new drugs, food additives and other products.
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Credit author statement I solemnly declare the manuscript is the result of independent research. Except for the cited results, this manuscript does not include manuscripts published or written by others or groups. The legal consequences of this statement shall be borne by myself. Declaration of Competing Interest Authors have no conflict of interest to declare. Acknowledgments We were appreciated the financially supported by National Natural Science Foundation of China (81673536, 81973415, 31570370, 31871694), and Open Fund of Shanghai Key Laboratory of Plant Functional Genomics and Resources (PFGR201901). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112172. References Bayala, B., Bassole, I.H., Scifo, R., Gnoula, C., Morel, L., Lobaccaro, J.M., Simpore, J., 2014. Anticancer activity of essential oil and their chemical components - a review. Am. J. Cancer Res. 4, 591–607. Belsito, E.L., Carbone, C., Di, G.M., Leggio, A., Liguori, A., Perri, F., Siciliano, C., Viscomi, M.C., 2007. Comparison of the volatile constituents in cold-pressed fingered citron oil and a volatile oil isolated by vacuum distillation. J. Agric. Food Chem. 55, 7847–7851. Cappello, A.R., Dolce, V., Iacopetta, D., Martello, M., Fiorillo, M., Curcio, R., Muto, L., Dhanyalayam, D., 2016. Fingered citron (Citrus bergamia Risso) flavonoids and their potential benefits in human hyperlipidemia and atherosclerosis: an overview. J. MiniRev. Med. Chem. 16, 619–629. Cirmi, S., Bisignano, C., Mandalari, G., Navarra, M., 2016. Anti-infective potential of Citrus bergamia Risso et Poiteau (fingered citron) derivatives: a systematic review. J. Phytother. Res. 30, 1404–1411. Cosentino, M., Luini, A., Bombelli, R., Corasaniti, M.T., Bagetta, G., Marino, F., 2014. The essential oil of fingered citron stimulates reactive oxygen species production in human polymorphonuclear leukocytes. J. Phytother. Res. 28, 1232–1239. Dahmane, D., Dob, T., Krimat, S., 2017. Chemical composition, antioxidant and antibacterial activities of the essential oil of medicinal plant Ammodaucus leucotrichus from Algeria. J. Essent. Oil Res. 29, 48–55. Dai, W., Gong, R., Hu, J., Zhou, L.M., 2014. Thiophene capture by an oxidation-modified activated carbon derived from fingered citron. J. Sep. Sci. Technol. 49, 367–375. Daniela, I., Marika, C., Michela, C., Enrico, G., Emanuela, E., Filippo, B., Salvatore, C., Michele, N., 2016. Anti-inflammatory and antioxidant effects of Flavonoid-Rich Fraction of fingered citron Juice (BJe) in a Mouse Model of Intestinal Ischemia/ Reperfusion Injury. J. Front. Pharm. 7. https://doi.org/10.3389/fphar.2016.00203.
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