Trans-cinnamaldehyde inhibits Penicillium italicum by damaging mitochondria and inducing apoptosis mechanisms

Fangwei Yang, Jiaqi Mi, Fei Huang, Prompong Pienpinijtham,Yahui Guo, Yuliang Cheng, Weirong Yao, Yunfei Xie,*

a School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

b State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

c Qingdao Special Food Research Institute, Qingdao 266109, Shandong, China

Keywords:

trans-cinnamaldehyde

Penicillium italicum

Raman microspectroscopy

Inhibition mechanism

Cytochrome c

Apoptosis

A B S T R A C T

Plant-derived essential oils have excellent antifungal effects and can be used for the preservation of fresh foods such as fruits and vegetables, but the detailed mechanism has not been fully elucidated. In this study, we investigated the inhibitory effects of trans-cinnamaldehyde on Penicillium italicum, a common pollution fungus in citrus, and explored the antifungal mechanism of trans-cinnamaldehyde by detecting fungal oxidative damage, mitochondrial metabolism, and cell apoptosis. These results showed that transcinnamaldehyde made the carboxylic acid cycle deregulated by altering the related enzyme activities (succinate dehydrogenase, malate dehydrogenase) and mid product. Moreover, the level of reactive oxygen species rose sharply while the redox level was out of regulation. The mitochondrial membrane potential collapsed, leading to the leakage of cytochrome c, and then triggering the activation of apoptotic protease, which was further con firmed by the significant increase in caspase-3 activity from (3.6 ± 0.6) U to (8.8 ± 1.1) U (P ＜ 0.05). The cytochrome c in mitochondria was detected by confocal Raman microspectroscopy, the characteristic intensity index (I750/I2944) was decreased, indicating that the cytochrome c in mitochondria was reduced and leakage.Besides, the strong negative correlation between Raman intensity and the amount of cytochrome c leakage was established with the correlation coefficient of -0.981 7. This study revealed that destroying the integrity of the mitochondrial membrane, activating the mitochondrial-mediated apoptosis pathway was the in-depth antifungal mechanism of trans-cinnamaldehyde; and Raman spectroscopy technology provided new ideas to study this process with high sensitivity determination of cytochrome c.

1. Introduction

The usage of natural plant-derived essential oils instead of synthetic antifungal agents to control food spoilage microbial is a hot research topic at present [1]. Nevertheless, the research on the antifungal mechanism of essential oils against food spoilage microorganisms still stays in the shallow layer. Few studies have reported works at the molecular level. The vast majority of researches are focused on the destruction of the cell wall and its synthesis [2,3],damage of cell membrane [4,5], destruction of mitochondria [6,7],and damage of DNA [8-10]. Besides, some researchers studied the inhibition mechanism of essential oils against microorganisms from the perspective of genomics by analyzing the expression of different genes [11].

Trans-cinnamaldehyde is the main ingredient of China cinnamon oil and Ceylon cinnamon oil and can be used as food preservatives,flavors, and fragrances [12].Penicillium italicumcan invade citrus fruits and cause penicillium disease, resulting in a rapid decline in fruit yield and quality, causing serious economic losses [13,14].According to our previous study [12],trans-cinnamaldehyde can inhibit the mycelial growth ofP. italicumbecause it can destroy the cell membrane of mycelium. At the same time, a large number of reactive oxygen species (ROS) were produced inside the cells [12].Mitochondria are one of the main sources of ROS, as it utilizes oxygen for energy production [15]. Therefore, our eyes were placed in mitochondria. Mitochondria are considered to be important targets for exogenous toxicants [16,17]. Damage to mitochondrial structure and its function may be a significant cause of cytotoxicity [18,19].When the exogenous harmful substance acts on mitochondria, the cell will undergo the stress reaction and even apoptosis [20].

Raman spectroscopy is a vibrational spectroscopy that provides high-sensitivity fingerprint information, offers molecular level data, and has minimal water background interference [21]. Raman spectroscopy has been widely used in biological detection [22,23].Among them, it plays an extremely good role to detect cytochromec(Cytc) in isolated mitochondria and provides a very outstanding means for elucidating the antimicrobial mechanism [12,24,25]. Hence,Raman spectroscopy is a powerful tool to investigate the essential oils’ antifungal mechanism at the molecular level. It is possible to detect the subtle changes in the cells and apoptosis mediated by Cytcafter exogenous substances stimulate the fungus [26].

Based on these, the purpose of this work was to reveal the inhibitory mechanism oftrans-cinnamaldehyde againstP. italicumat the mitochondrial level, including the mitochondrial function and whether apoptosis was initiated, which was measured by the apoptosis marker Cytcand further confirmed by the caspase-3 activity.Confocal Raman microspectroscopy technique was also applied to detect the content of Cytcin isolated mitochondria, which was used to diagnose apoptosis.

2. Materials and methods

2.1 Fungal species

P. italicumwas purchased from China General Microbiological Culture Collection Center (CGMCC, Beijing, China). All test strains were preserved on potato dextrose agar (PDA) at 4 °C. Conidial spore concentration was adjusted to 1 × 106CFU/mL with the hemocytometer.

2.2 Chemicals

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),tris(hydroxymethyl)aminomethane (Tris), mannitol, potassium chloride, ethylenediaminetetraacetic acid (EDTA), magnesium chloride, luminol, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, sodium chloride,glucose, horseradish, and sodium hydroxide were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). ATPase testing kit, glutathione (GSH) testing kit, succinate dehydrogenase(SDH) testing kit, malate dehydrogenase (MDH) testing kit, citric acid(CA) content assay kit, cytochromecassay kit, and caspase-3 testing kit were obtained from Nanjing SenBeiJia Biological Technology Co., Ltd. (Nanjing, China).

First, 100 μL sterilized potato dextrose broth was injected into 96-well plates, 100 μL essential oils diluted in ethanol was injected into the first well, 2-fold diluted. Then, 100 μL conidial spore suspension(1 × 106CFU/mL) was poured into each well. Finally, the 96-well plate was incubated at (28 ± 2) °C in the constant temperature incubator for 48 h. The lowest concentration that completely inhibited the growth of the fungus was considered the minimum inhibitory concentration (MIC). And then, the mycelium cultured for two days was treated with different concentrations (0, 0.5, 1.0, 2.0 MIC) oftrans-cinnamaldehyde.

2.3 Determination of ATPase activity

Two-day-old mycelium was filtered, and 500 mg mycelium was added to 500 μL of physiological saline. Then, the mixture was uniformly ground by a high-flux tissue grinder. The mixture was centrifuged at 4 000 ×gfor 10 min at 4 °C. According to the ATPase testing kit purchased from Nanjing SenBeiJia Biological Technology,the supernatant was separated and incubated at 37 °C. ATPase activity was indicated by the amount of phosphorus remaining in the solution,which was measured by the specific absorbance at 636 nm (Molecular Devices, USA).

2.4 Measurement of ROS

The luminol chemiluminescence method was employed for the determination of ROS accumulation. Two-day-old mycelium collected by vacuum filtration was ground to powder with liquid nitrogen.The mycelium powder was then suspended in HEPES buffer. After centrifuged at 4 000 ×gfor 10 min at 4 °C, the supernatant was placed on ice for further uses. 50 μL of the supernatant and 830 μL of HEPES buffer (0.02 mol/L, pH 7.4) were injected into a glass vial.Then, 20 μL of horseradish peroxidase (3 mg/mL) was added under the dark environment, and an MPI-B chemiluminescence analysis testing system (Xi’an Remex Analysis Instruments Co., Ltd., China)was immediately performed. The peak area of testing results was integrated to indicate the accumulation of ROS.

2.5 Measurement of GSH

The determination of GSH content in cells was carried out following the kit instruction of the GSH testing kit described by Nanjing SenBeiJia Biological Technology. Brie fly, dithiodinitrobenzoic acid reacts with sulfhydryl compounds to produce a yellow compound which can be measured by spectrophotometer. The method of extracting GSH was same as the method in section 2.4, after the color development reaction, the absorption at 420 nm was measured by UV-Vis spectrophotometer (SHIMADZU Co., Ltd., Japan).

2.6 Measurement of CA content

The determination of CA content was carried out according to the kit purchased from Nanjing SenBeiJia Biological Technology. 50 mg of the two-day-old mycelium was added into 0.5 mL of physiological saline and then ground with a high-throughput tissue grinder. The mixture was centrifuged at 4 000 ×gfor 10 min. The supernatant was continuously centrifuged at 12 000 ×gfor 10 min. Then, the supernatant was measured according to the instructions of the kit.

2.7 Measurement of the tricarboxylic acid (TCA) cyclerelated enzyme activities

The activities of TCA cycle-related enzyme SDH and the enzyme activities of MDH were determined according to the kit purchased from Nanjing SenBeiJia Biological Technology with enzymelinked immunosorbent assay (ELISA). The method of extraction was essentially the same as the method in 2.4, using purified SDH antibody to coat microtiter plate wells, SDH in sample added and combined homologous antibody which with horseradish peroxidase(HRP) labeled, became antibody-antigen-enzyme-antibody complex, after washing, add 3,3’,5,5’-tetramethylbenzidine substrate solution which turned blue color at HRP enzyme-catalyzed, the reaction was terminated by the addition of a sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm (Molecular Devices, USA). MDH shared the same principle and method with SDH except for the SDH antibody replaced by the MDH antibody.

2.8 Measurement of mitochondrial membrane potential

Rhodamine 123 (R123) method was employed for the determination of mitochondrial membrane potential accumulation.200 μL of R123 (10 μg/mL) was added into 3 mL of mitochondrial extraction media with mitochondrial suspended. The mixture was stored at 37 °C in dark for 20 min and then the fluorescence intensity was measured. The mitochondria considered a fluorescence quencher,so the degree of collapse of the membrane potential was proportional to the fluorescence intensity of the detection solution.

2.9 Determination of Cyt c with ELISA

The leakage of Cytcwas measured by the ELISA kit, which purchased from Nanjing SenBeiJia Biological Technology.Sharing the same principle and method with SDH in 2.7, except for the specific Cytcantibody, the method of extracting Cytcwas similar to the method in section 2.4, measuring the color change by spectrophotometer at the wavelength of 450 nm (Molecular Devices,USA) to indicate the leakage of Cytc.

2.10 Measurement of caspase-3 activity

Caspase-3 testing kit was obtained from Nanjing SenBeiJia Biological Technology. Caspase-3 activity was determined by coupling the sequence-specific polypeptide of caspase-3 to the yellow groupp-nitroaniline. The method of extracting caspase-3 was same as the method in section 2.4. According to the instructions of the kit,after the color development reaction for 4 h at 37 °C, the yellow color intensity was measured at 405 nm wavelength by microplate readers(Molecular Devices, USA).

2.11 Detection of Cyt c using confocal Raman microspectrometer

The mycelium cultured for two days was treated with different concentrations oftrans-cinnamaldehyde, and then vacuum-filtered and washed with saline. After that, the mixture was ground in liquid nitrogen and suspended with the mitochondrial extract media(20 mmol/L HEPES-Tris (pH 7.2), 250 mmol/L mannitol, 10 mmol/L potassium chloride, 5 mmol/L EDTA, and 20 mmol/L magnesium chloride) [27]. The mixture was centrifuged at 4 000 ×gfor 10 min to collect the supernatant. Then, the supernatant was again centrifuged at 11 000 ×gfor 15 min, and the obtained pellet was mitochondria. The mitochondria were placed on a tin foil board and then were scanned by a LabRam-HR800 confocal Raman microspectrometer (HORIBA Jobin Yvon Co., Ltd., France) equipped with Lab-spec 6.5 software and an air-cooled He-Ne laser at 532 nm, and the light transmittance was 75%. The Raman spectra were continuously collected with an accumulation time of 15 s in each case by five parallel accumulations.

2.12 Statistical analysis

All tests were performed in triplicate, and all results were expressed as mean ± standard deviation (SD). One-way ANOVA and Duncan’s multiple range test were used. SPSS version 22.0 was administered to analyze the significance of the difference between the control group and the experimental groups. Raman spectral data were processed by Labspec 6.5 and Origin 9.0.

3. Results and discussion

3.1 Effect of trans-cinnamaldehyde on ATP utilization ability

ATP is the most significant energy-supplying substance in cells. The utilization ability of ATP often reflects whether there is a disorder in living cells [28]. After treating mycelium withtranscinnamaldehyde for 2 h, it was found that the activity of ATPase in the cells decreased continuously (P＜ 0.05), which meant that the ability of cells to utilize ATP were decreased. The specific situation was shown in Fig. 1a. The decrease of ATPase activity indicated that ATP cannot be converted to AMP for providing energy [23]. The more amount oftrans-cinnamaldehyde, the lower the enzyme activity was observed, indicating that the cells were doomed to die due to lack of energy [29].

Fig. 1 (a) Changes in ATPase activity; (b) ROS level; (c) GSH level of P. italicum under different treated concentrations of trans-cinnamaldehyde.

3.2 Effect of trans-cinnamaldehyde on the redox level

After the exogenous substance acts on the fungus, a significant redox disorder occurs inside the cell, which in turn acts to inhibit the fungus [30]. The mycelium was treated withtrans-cinnamaldehyde for 2 h, and the results of the redox level were shown in Fig. 1b.The level of ROS in 0.5 MIC treated group was not significantly increased compared to the control group (P＞ 0.05). The level of ROS in 1 MIC treated group was slightly elevated but there was no significant difference (P＞ 0.05), while the level of ROS in 2 MIC treated group was significantly increased (P＜ 0.05). There was no significant increase in ROS in 0.5 MIC or 1 MIC treatment group,suggesting that the intracellular oxygen pressure was not high and did not cause serious damage to the cells. In 2 MIC treatment group, the accumulation of ROS suggested that oxygen radicals were abundant in the cells. These oxygen radicals did not only damage the respiratory chain, but also caused continuous electron leakage and damage to the mitochondrial membrane. This result suggested that 2 MICtranscinnamaldehyde had a great destructive effect on mycelium and was likely to cause irreversible damage to the cells.

GSH is highly reductive and is an important antioxidant and free radical scavenger in cells. It protects many proteins, macromolecules,and sulfhydryl groups of enzyme molecules. The most important functions in GSH are the physiological functions to remove oxidizing substances [31]. The results of GSH levels aftertrans-cinnamaldehyde treatment of mycelia were shown in Fig. 1c. The GSH content in the 2 MIC treated group was significantly lower than that in the control group (P＜ 0.05), while 0.5 MIC and 1 MIC treated group were not significantly different from the control group (P＞ 0.05), which indicated that the ROS level inside the cells can be self-regulated at 0.5 MIC and 1 MIC treatment, but not at 2 MIC treatment. The ROS level exceeded the load, thus GSH had been unable to remove excessive ROS, resulting in the serious shortage of GSH. It implied that an irreversible reaction eventually led to cell oxidative damage and apoptosis, which was similar to the previous studies [32,33].

3.3 Effect of trans-cinnamaldehyde on the TCA cycle

CA is the first reaction product in the tricarboxylic acid (TCA)cycle, and the change in CA content reflects the level of the TCA cycle. The changes in CA content with time under treatments with different concentrations oftrans-cinnamaldehyde were shown in Fig. 2a. The content of CA in 1 MIC and 2 MIC treatment groups increased significantly at 30 min and showed a downward trend after 60 min. Finally, they were lower than the control group. These results indicated thattrans-cinnamaldehyde can affect the mitochondrial TCA cycle at the beginning of the reaction, resulting in the inability of the TCA cycle to proceed normally.

Fig. 2 Changes in the TCA cycle. (a) content of CA; (b) SDH activity;(c) MDH activity of P. italicum under different treated concentrations of trans-cinnamaldehyde.

SDH is the only multi-subunit enzyme integrated into the membrane in the TCA cycle. It is a marker enzyme for mitochondria and the rate-limiting enzyme of the TCA cycle [18]. The enzyme activities under different treatments were detected, as shown in Fig. 2b. There was no significant change in SDH activity between the control group and the 0.5 MIC treatment group (P＞ 0.05), but the 1 MIC and 2 MIC treatment groups showed a downward trend. At 120 min, the enzyme activity in 2 MIC and 1 MIC both lower than the control group, and 2 MIC was below 1 MIC. This indicated thattrans-cinnamaldehyde reduced the enzyme activity of SDH, blocking pivotal energy supply processes in cells, and affecting the normal function of mitochondria [18,29].

MDH is localized in the mitochondrial matrix, which is a matrix marker enzyme, and is in the tail of the TCA cycle [29]. As shown in Fig. 2c, the MDH enzyme activity of the control group did not change significantly with time (P＞ 0.05), while the experimental groups increased with time. At 60 min, the enzyme activities of both 1 MIC and 2 MIC treatment groups were increased significantly (P＜ 0.05).These results illustrated that the reaction was turbulent at the end of the TCA cycle.

3.4 Effect of trans-cinnamaldehyde on mitochondrial membrane potential

Once the mitochondrial membrane potential is destroyed, it will cause irreversible damage to mitochondria [34]. The results of mitochondrial membrane potential under different treatments were measured, as shown in Fig. 3a, the higher the fluorescence intensity,the more serious the collapse of mitochondrial membrane potential was observed. At 30 min, the membrane potentials of all three treatment groups were increased greatly (P＜ 0.05). At 120 min, the level of the 2 MIC treatment group was close to that of the 1 MIC treatment group, which was much higher than that of the 0.5 MIC treatment group. Even though the 0.5 MIC treatment group was much higher than the control group (P＜ 0.05). It can be known from the slope of the curve first 30 min that the higher the concentration oftrans-cinnamaldehyde, the faster the potential collapsed, and the first 30 min was the critical period for membrane potential of mitochondria to collapse.

Fig. 3 Index of initiation under different treated concentrations of trans-cinnamaldehyde. (a) Change in mitochondria membrane potential;(b) leakage of Cyt c; (c) activity of caspase-3.

3.5 Effect of trans-cinnamaldehyde on the initiation of apoptosis

3.5.1 Release of Cyt c from mitochondria

The release of Cytcplays a critical role in the mitochondriamediated apoptosis. For this purpose, changes in the Cytccontent in the cytoplasm were determined to investigate whether Cytcleaked from mitochondria, and the results were shown in Fig. 3b. At 30 min,1 MIC and 2 MIC treatment groups were significantly higher than the control group (P＜ 0.05). The higher thetrans-cinnamaldehyde concentration, the faster the release of Cytcwas observed, which can be seen from the slope of the curve from 0 to 30 min. However, the Cytccontent of 0.5 MIC treatment group did not change significantly(P＞ 0.05). After 60 min, the content of Cytcin 1 MIC and 2 MIC treatment groups tended to be stable and basically no longer increased.After 60 min, the Cytccontent of 0.5 MIC treatment group was slightly higher than that at 0 min. Cytcaccumulated in the cytoplasm,indicating that the integrity of the mitochondrial membrane was disrupted, and the function of the mitochondrial outer membrane was impeded. Hence, the out flow of Cytccannot be selectively controlled,and then the mold cell was about to initiate the apoptosis program.

3.5.2 Change of caspase-3 activity

Upon release into the cytoplasm, Cytcwill bind to apoptotic protease-activating factor 1 to form a complex that activates caspase-9. Caspase-9 further activates the caspase-3, and the protease cascades to initiate the cell death program [35,36]. To demonstrate this conjecture, we measured the activity of caspase-3. As shown in Fig. 3c, the caspase-3 activity in the control group remained unchanged (P＞ 0.05), but the experimental groups had upward trends. In combination with the release of Cytc, it can be inferred that cell death programs have been initiated.

3.6 Detection of Cyt c inside mitochondria with Raman scattering

In order to detect the change of Cytccontent in the mitochondrial membrane gap, the characteristic intensity of Cytcin mitochondria was detected by Confocal Raman Microspectroscopy. The results of 0, 0.5, 1, 1.5, 2 h treatment with 2 MICtrans-cinnamaldehyde were shown in Fig. 4a. The peak at 750 cm-1was the characteristic absorption peak of the porphyrin ring from Cytc[37,38], while the peak at 2 944 cm-1was attributed to C-H stretching vibration [39].Inside the cell, the total intensity of C-H was relatively stable.Therefore, the peak at 2 944 cm-1can be considered as an internal standard for comparing to peaks of other specific substances in the cell. In this case, the peak intensity ratio between peaks at 750 and 2 944 cm-1(I750/I2944) was employed to detect Cytcinside mitochondria. As can be seen from Fig. 4b, as time went by, the I750/I2944value became smaller, indicating that the Cytcinside mitochondria was decreased. This result corresponded to an increase in the amount of Cytcin the cytoplasm. In addition, the Raman intensity of Cytcinside mitochondria can establish a strong correlation with Cytccontent in the cytoplasm, as shown in Fig. 4c.The strong negative correlation coefficient (r= -0.981 7) indicated that Raman spectroscopy can be well-performed. Detection of the content of Cytcin mitochondria can determine whether apoptosis was initiated, which did not require complex analytical methods such as ELISA. It was shown here that Raman spectroscopy had the potential to diagnose whether cells have undergone apoptosis in isolated mitochondria aftertrans-cinnamaldehyde treatment.

Fig. 4 (a) Raman spectra of Cyt c from isolated mitochondria from 0 h to 2 h with 2 MIC trans-cinnamaldehyde. (b) The plot of the characteristic index(I750/I2 944) of Cyt c against time. (c) Correlation between I750/I2 944 in mitochondria and Cyt c in the cytoplasm of mold cells.

After treatment withtrans-cinnamaldehyde for 2 h, different concentrations oftrans-cinnamaldehyde had dissimilar effects onP. italicum. Raman spectra of samples treated with different concentrations oftrans-cinnamaldehyde were shown in Fig. 5a. Along with the concentration oftrans-cinnamaldehyde increased, the I750/I2944value became smaller, as can be seen from Fig. 5b, which indicated the higher the concentration oftrans-cinnamaldehyde, the lower the content of Cytcinside mitochondria. However, the less Cytcinside mitochondria meant the more Cytcreleased into the cytoplasm to initiate apoptosis. As a result, the mitochondria-mediated apoptosis initiation level was positively correlated with the concentration oftrans-cinnamaldehyde.

Fig. 5 (a) Raman spectra of Cyt c from isolated mitochondria at 2 h under different treatments. (b) The plot of the characteristic index (I750/I2 944) of Cyt c against treated concentrations of trans-cinnamaldehyde. (c) Correlation between I750/I2 944 in mitochondria and Cyt c in the cytoplasm of mold cells.

Correlation analysis between the Cytccontent in the cytoplasm and the intensity of the Raman characteristic intensity of 750 cm-1showed in Fig. 5c. The strong negative correlation (r= -0.951 4)suggested that the content of Cytcin mitochondria can be quickly detected by Raman spectroscopy to determine whether apoptosis was initiated. Thus, complex analytical methods such as ELISA can be replaced preliminarily by this technique.

4. Conclusions

Studying the inhibition mechanism oftrans-cinnamaldehyde againstP. italicumwas helpful to provide basic data for future research. Aftertrans-cinnamaldehyde treatment, the redox in cells was dysregulated, and the level of ROS increased, the level of GSH decreased. Meanwhile, the TCA cycle was disordered. Detailly, the CA content increased firstly and then decreased, the SDH activity decreased, and MDH activity decreased, indicating that the function of mitochondria was severely damaged. Furthermore, mitochondrial membrane potential collapse triggered apoptosis, which may be the mechanism thattrans-cinnamaldehyde inhibitedP. italicum.Confocal Raman microspectroscopy has played a promising role in the detection of Cytcduring apoptosis and has shown a serviceable application in the antifungal analysis.

Declaration of competing interest

The authors declare that there is no conflict of interest.

Acknowledgments

The work described in this article was supported by China Postdoctoral Science Foundation (2020M680064), National Natural Science Foundation of China (32172326), and the Postdoctoral Research Startup Fee of Jiangnan University (1025219032200190).