Chemical Composition, A Ntimicrobial, Antioxidant and Cytotoxic Activities and of Essential Oil from Actinidia Arguta

Chemical composition, antimicrobial, antioxidant, and cytotoxic properties of essential oil from Actinidiatg arguta (AEO) were evaluated. Gas chromatography-mass spectrometry analysis identied 56 chemical compounds, with the most abundant being Squalene (23.08%), γ-sitrostorol (8.10%), and β-Tocopherol (7.01%). Whereas the AEO had signicant antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae, it showed mild ecacy against Bacillus subtilis and Microsporum canis . On the contrary, the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, were not susceptible to the AEO pressure. On the other hand, the AEO exhibited strong antioxidant activity against DPPH, β-carotene, and hydroxyl radicals, having an IC 50 values of 117.60, 73.60 and 35.15 μg/mL, respectively. Additionally, compared to the PC-3 or HT-29 cell lines, the A549 cells were more susceptible to the AEO (IC 50 ; 6.067 mg/mL). Besides, the confocal laser scanning microscopy imaging showed that 16 mg/mL of the AEO induced apoptosis in the A549 cell lines. Our data indicate that the AEO might be useful in the food and pharmaceutical industry.


Introduction
There is a growing interest in the exploration of naturally-occurring bioactive compounds for industrial use. This development has been necessitated by the fact that there is increased resistance to a wide spectrum of commercial antibiotics (Fair and  Actinidia arguta (Sieb. Et Zucc.) Planch. ex Miq. var. are small grape-sized fruits with edible green or redcolored skin, belonging to the Actinidia genus. It originated and widely cultivated in northern China. The A. arguta has a delicious taste and immense health bene ts. It bears fruits rich in vitamins, polysaccharides, phenolics, avones, alkaloids, as well as other essential minerals (Zhu et al. 2019). A. arguta is one of the richest sources of lutein (up to 0.93 mg/100 g FW) and vitamin C (up to 430 mg/100g fresh weight FW), myo-inositol (up to 982 mg/100g FW), and is considered as the most nutritious fruits (Latocha 2017). The rich nutritional value has prompted researchers to interrogate its anti-microbiology, antioxidant, antitumor or anti-in ammatory potentials (Latocha et  Whereas some studies have reported the chemical composition and antimicrobial activity associated with the AEO (Matich et al. 2003), data on the antioxidant or antitumor activities of the AEO remain scant.
Our study embarked on determining the chemical composition of the AEO as well as evaluation of its antimicrobial, antioxidant and cytotoxic properties. Our ndings have set the basis for the use of the AEO in food, pharmaceutical or cosmetic industries.

Plant material
The A. argutafruits were collected from the experimental farm of the Shenyang Agricultural University in September 2019.

Microbial strains
The AEO were tested against six microorganisms, including two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) as well as two fungal strains (Saccharomyces cerevisiae and Microsporum canis). The susceptibility of the bacteria and the fungi to the essential oils were carried out using the disk diffusion method. All the strains were obtained from the Agricultural Culture Collection of China. The chemical composition of the essential oil was analyzed by GC/MS using Agilent 5973 EI mass selective detector coupled with Agilent GC6890, equipped with a HP-5MS fused capillary column (5% phenyl Methyl Silox) (30m×0.25mm, 0.25μm lm thickness). Helium (99.999%) was used a carrier gas with a ow rate of 1.0 mL/min. The initial temperature was programed at 40°C, then increased 3°C/min up to 80°C, then by 5°C/min up to 280°C. The temperature was maintained at 280°C for 20min, just as the injector and detector temperatures. The quadruple mass spectrometer was scanned over a range of 35-500amu at 1 scan per second, with a temperature of 150°C, ionizing voltage of 70 eV, and an ionic source temperature of 230°C. 2.0 μL of the AEO was injected with a split ratio of 10:1. Individual components of the AEO were identi ed on the basis of their retention indices (RI), and the compared with reference data using the Wiley7n.l library.
Antimicrobial activity assay

Agar diffusion method
The effect of the AEO on the bacteria was determined according to Marjana (Radunz et al. 2019), with few modi cations. The bacterial cells were cultured in liquid Luria-Bertani media overnight at 37°C, while the fungal strains were cultured in Sabouraud dextrose broth at 28°C for 48h. The microbial suspensions were diluted to 10 8 CFU/mL, while the fungal cells were diluted to 10 6 CFU/mL. The microbial suspension (150 μL) were evenly spread on solid media. Thereafter, sterile 6mm diameter lter disks were placed on the media seeded with the microorganisms (3 disks per plate) and then AEO was dropped onto each paper disk (40 μL per disk). The treated plates were rst kept at 4°C for 1h, then incubated at 37°C for 24h (bacteria), or at 28°C for 48h (fungi) (Lu et al. 2007). The antimicrobial activity was evaluated by measuring the diameter of growth inhibition zone surrounding the disks. All tests were performed in triplicates.

MIC and MBC/MFC
The AEO was dissolved in 1% (v/v) DMSO and then diluted to different concentrations (0.78-12.5 mg/mL). Minimum inhibitory concentration (MIC) value was measured following a protocol described by (Zhao et al. 2018), with slight modi cations. Brie y, 10 μL from each of the incubated suspensions were transferred into the corresponding media and incubated at 37°C for 24h (bacteria) or at 28°C for 48h (fungi). The minimum concentration that inhibited the growth of the microorganisms was recorded as MBC (minimum bactericidal concentration) or MFC (minimum fungicidal concentration) (Ksouda et al.

Antioxidant activity
The antioxidant activity of the AEO was tested using the following spectrophotometric methods: 1,1diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging assays as described by (Lu et al. 2018), as well as the β-carotene bleaching test by Wang et al., 2008, with minor modi cations. The AEO samples were tested at concentrations of 12.5 to 800 μg/mL and in triplicates. Butylated hydroxytoluene (BHT) was used as the positive control. IC 50 values were de ned by linear regression analysis and depicted as means ± SD of the triplicates.

Cytotoxicity assay
Determination of IC 50 MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay ) was used to evaluate the cytotoxicity of the AEO. Brie y, 1×10 6 cells were seeded in 96 well plates for 24 h. The cells were treated with different concentrations of the AEO samples (1-32 mg/mL) for 48 h. Untreated cells were used as the negative control. Up to 0.5 mg/mL of MTT was added into the cells and incubated for 4 h. Thereafter, the medium was replaced by 100 μL of DMSO to dissolve formazan crystals. Absorbance was detected on a StateFax-3200 microplate reader (AEARNESS, CA, USA) using a wavelength of 570 nm and a reference wavelength of 630 nm. The IC 50 was calculated by a liner regression analysis with 95% con dence limits. The inhibition of cell proliferation was approximated using the following formula: where A t is absorbance of the test sample, A c is the absorbance of negative control.
Confocal laser scanning microscopy (CLSM) assay A total of 1×10 3 A549 cells were seeded in a 6 well plate for 24 h (37℃, 5% CO 2 ). 16 mg/mL of the AEO samples was added and incubated for 48 h. Untreated cells were used as the negative control. The medium was removed, washed twice in cold PBS, and then the cells were digested with 0.25% typsin. The cells were recovered by centrifugation at 3000×g, washed twice in cold PBS, followed by nal centrifugation at 3000×g for 5min. The density of the cells was adjusted to 1×10 3 /mL with cold PBS.
1mL of acridine orange (1 mg/mL) was added into the cells and then incubated at room temperature and in darkness for 5 min. A laser confocal microscope was used to image the DNA morphology of the cells.

Statistical analysis
Each of the experiments was performed in triplicate. The mean value was calculated, and the experimental results were expressed as the mean ± standard deviations (SD). Besides, one-way ANOVA in SPSS Statistics 22.0 software (IBM, USA) was used to analyze the signi cant differences between the data sets. Signi cance was set at p<0.05.

Antimicrobial activity analysis
The in vitro antimicrobial activity of the AEO against Gram-positive, Gram-negativebacteria as well as fungal organisms was assessed. As shown in Table 2, the AEO exerted signi cant activity against S. aureus and S. cerevisiae (Inhibition zone; 19.5 mm±0.54 and 20.5 mm±0.48, respectively) but mild activity against B. subtilis (17.2 mm±0.35) and M. canis (16.8 mm ± 0.57). However, the E. coli and P. aeruginosa did not show any susceptibility to the AEO pressure (Inhibition zone; 8.5 mm ± 0.12 and 10 mm ± 0.21, respectively). Also, our study has shown that the AEO exhibited bactericidal effect against B.

Hydroxyl radical scavenging assay
As the strongest free radical in reactive oxygen species, hydroxyl (·OH) can react rapidly with almost all biological macromolecules in cells (Radunz et al. 2019). Our data showed that the scavenging activity of the AEO on the hydroxyl was concentration dependent as shown in Fig. 2c. 800 μg/mL of the oil resulted in the highest scavenging activity (98.76%±2.42%) and the IC 50 value was determined as 35.15 μg/mL.
However, compared to the AEO, BHA exhibited higher hydroxyl radical scavenging ability, with the highest scavenging activity of 99.45±2.31% μg/mL and an IC 50 value of 6.06 μg/mL.

Cytotoxicity activity analysis
Determination of IC 50 MTT assay was used to determine the cytotoxicity of 1-32 mg/mL of the AEO on HT-29, PC-3 or A549 cell lines, exposed for 48h. The AEO inhibited 78.63%, 60.42% or 57.31% proliferation of A549, HT-29 or PC-3 cells respectively as shown in Fig.4. The IC 50 values were 6.067 mg/mL, 11.905 mg/mL or 13.646 mg/mL for the A549, HT-29 or PC-3 cell lines respectively (Table 3), compared to the control group (P<0.05). This nding indicate that whereas the effect was cell-speci c, the AEO had a signi cant inhibitory effect on tumor growth.
Confocal laser scanning microscopy (CLSM) assay DNA damage of the A549 cells was studied by CLSM. Fluorescence staining by acridine orange showed that whereas the control cells had normal morphology (Fig. 3a), the A549 cells that were subjected to the AEO for 48 h (Fig. 3b) or 72 h (Fig. 3c) exhibited typical apoptotic characteristics. There was presence of dense yellow-green staining in the nucleus or cytoplasm, formation of cell membrane vesicles, lysed nuclei as well as apoptotic bodies. The CLSM results con rmed the ability of the AEO to induce apoptosis in A549 tumor cells, thus anti-tumor activity.

Discussion
The extraction yield and composition of essential oil are various according to extraction methods and reagents. In this study, we have reported for the rst time the presence ofγ-sitrostorol, Stigmast-7-en-3-ol and β-Tocopherol in the AEO. On the contrary, whereas we identi ed 0.96% of ethyl butyrate in the essential oil, Yang et al., 2006 showed a high (86.89%) relative content of ethyl butyrate in volatile components of A. arguta. Besides, Xin et al. (G. Xin 2009) studied the aroma from A. arguta fruits and found that the ole n content accounted for 51.71% of the volatile oil, but the acid was not detected. These chemical differences may be due to variability in varieties, regional differences, maturity stages or extraction and detection methods.
Research on the development of natural antibacterial, antifungal agents has attracted much attention (Fair and Tor 2014). As different solvent extracts possess different concentration and extent of bioactive principles, their antibacterial activity is also variates (Rajput et al. 2021). In this study, AEO had signi cant antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae, mild e cacy against Bacillus subtilis and Microsporum canis. However, the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, were not susceptible to the AEO pressure.This phenomenon suggests that the volatile oil might be acting on the peptidoglycan layer in the cell wall of Gram-positive bacteria. Also, the antimicrobial activity was mainly attributed to squalene, which was not only in higher amounts in the extracted oil but also has been proved to have strong antibacterial activity (Popa et al. 2015). Our data demonstrates that the AEO possesses antimicrobial activity, thus, might be useful in the phasrmaceutical and food industry. Antioxidant assay showed AEO exhibited high antioxidant activities probably due to the diverse constituents that might be working in synergy. The compounds such as squalene, γ-sitosterol and β-tocopherol have been reported to have antioxidant activities (Hidayathulla et

Conclusions
The essential oil extracted from A. argutamainly comprised of squalene, γ-sitrostorol and β-Tocopherol.The AEO exhibited potential antimicrobial, antioxidant and cytotoxic activities, which might be a function of synergy among the compounds. Besides, the activity might be regulated by other secondary components which play a signi cant role in de ning the aroma, density, texture, color, cellular penetration, lipophobia, and hydrophilicity of the AEO (Emami et al. 2016). Therefore, the AEO harbors huge potential that can be used in the phyto-pharmaceutical and food industry.