Comparative antioxidant, anticancer and antimicrobial activities of essential oils from Semen Platycladi by different extraction methods

Industrial Crops & Products 146 (2020) 112206

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Comparative antioxidant, anticancer and antimicrobial activities of essential oils from Semen Platycladi by different extraction methods

T

Jing-Jing Zhua, Jing-Juan Yangb, Guo-Jie Wuc, Jian-Guo Jianga,* a

College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China Department of Food and Nutrition, Yunnan University of Chinese Medicine, China c School of Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Essential oils Semen Platycladi Steam distillation extraction Heating reflux extraction Soxhlet extraction

Semen Platycladi is a highly valued seed tonic from the Cupressaceae Platycladus orientalis (L.) Franco, which contains abundant essential oils exhibiting antioxidant, antimicrobial, sedative and hypnotic potency. The study was formulated to estimate the potential antioxidant, anticancer and antibacterial activities of its essential oils as well as its correlation with chemical constituents. The comparison of three isolation methods including steam distillation extraction, heating reflux extraction, and Soxhlet extraction of ethanol was carried out to isolate essential oils whose chemical composition were identified and evaluated by Gas Chromatography-Mass Spectrometer. It turned out that the yield of essential oils obtained through heating reflux extraction and Soxhlet extraction was almost 40 % higher than those extracted by steam distillation extraction in nearly one-quarter of a time used by the steam distillation extraction. A total of 76 compounds were identified, including ten alkanes, 14 alkenes, 13 alcohols, one aldehyde ketone, three ethers, one acid, 17 esters, five aromatic compounds, and 12 others. The essential oils extracted by steam distillation extraction were proved to have the best antioxidant and antimicrobial activity although with the lowest yield. Scanning electron microscopic observation displayed that the of S. aureus revealed serious disruption of cell membranes resulting in the loss of cell viability. Therefore, it’s vital to adopt a proper method to obtain the target ingredients recommended as nutraceuticals and/or phytomedicines.

1. Introduction Semen Platycladi is the dry and ripe kernel of the Cupressaceae Platycladus orientalis (L.) Franco that is a common tree species used for horticulture and afforestation in agriculture (Lin et al., 2016). Essential oil of Semen Platycladi is considered to be highly edible, which is of marked significance for the study of its components and potential bioactivities. In addition to its antioxidant and antimicrobial effects (Emami et al., 2011; Hassanzadeh et al., 2008), Semen Platycladi essential oil has been authenticated to have various bioactivities, including sedative and hypnotic potency (Lai et al., 1994). Various extraction methods such as steam distillation extraction (SDE), heating reflux extraction (HRE), coldpressing, soxhlet extraction (SE), supercritical carbon dioxide extraction and dual-cooled solventfree microwave extraction (SFME) have been used to prepare essential oils (Jiang et al., 2011; Krulj et al., 2016). Among them, the SDE is one of the most mature and conventional methods owing to its simple operation and low cost. The extraction method of HRE and SE are based

⁎

on the principle of "like dissolves like" of solvent extraction (Zhang and Jiang, 2014). Supercritical fluid extraction and SFME requires high operating costs. As we know, no studies have been conducted to compare and estimate the biological activities of Semen Platycladi essential oils extracted by different methods. Different preparation techniques attach great importance in individual advantages but also have some special limitations. The purpose of the study was to estimate the influence of different extraction methods (SDE, HRE, SE) on essential oil components and their functional bioactivities by using Gas ChromatographyMass Spectrometer (GC–MS) to investigate the chemical composition of essential oils of Semen Platycladi, the antioxidant performance to DPPH, ABTS, FRAP, the antibacterial activities against pathogenic bacteria, as well as the cytotoxicity with cancer cells, in order to identify promising industrial-related purposes and bioresources.

Corresponding author. E-mail address: [email protected] (J.-G. Jiang).

https://doi.org/10.1016/j.indcrop.2020.112206 Received 29 October 2019; Received in revised form 30 January 2020; Accepted 3 February 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.

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2. Materials and methods 2.1. Plant material Semen Platycladi was purchased at Qingping TCM market in Guangzhou, China. The samples were dried in air at 60℃ for 24 h and ground in grinder to obtain the raw material powder, which was then stored in a sealed container for further use. 2.2. Chemicals 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sigma Aldrich (St. Louis, Missouri, USA). All other chemicals used were analytically pure. The solvent used in GC–MS was HPLC grade 2.3. Preparation of essential oil 2.3.1. Steam distillation extraction According to the regulations of Chinese Pharmacopoeia 2005 (Commission, 2005), the material was extracted in the volatile oil extraction device by hydrodistillation for 6 h at the solid-liquid ratio of 1:10. The essential oil was then extracted from the oil-water mixture by diethyl ether and dehydrated with anhydrous Na2SO4. The yield was calculated by dividing the mass of the oil obtained by the mass of the raw material of Semen Platycladi. The following two methods were both calculated in this way. 2.3.2. Heating reflux extraction The extraction was carried out by heating and reflux with petroleum ether at 80℃for 4 h with a solid-liquid ratio of 1:10. The solvent was then removed by vacuum filtration (SHZ-D Rotary Evaporator, Shanghai, China) (Pudziuvelyte et al., 2018).

Fig. 1. Total ion chromatograms of GC/MS of essential oils from the Semen Platycladi by three methods (a, b, c.). (RT retention time a SDE. b HRE. c SE).

homologous series of n-alkanes (C8–C40) under same experimental conditions (Adams, 2007; Tohidi et al., 2017).

2.3.3. Soxhlet extraction of ethanol The material was extracted with ethanol solvent utilizing a Soxhlet extractor at 60 °C for 6 h. The ethanol was then removed by a rotary evaporator to obtain a yellow essential oil for subsequent analytical testing (Yang et al., 2009).

2.5. Antioxidant activity 2.5.1. DPPH assay The scavenging capacity against DPPH of the sample was determined by 96-well microplate method with slight modification (Reis et al., 2012). Sample solution of 20 μL with the dose of 12.5−200 μg/ mL was mixed with 180 μL of 150 μmol/L DPPH solution. After that, the reaction wells were shaken evenly and allowed to stand in the dark at room temperature for 30 min. The optical density at 517 nm (OD517) was then immediately read using a microplate reader (BioTek Instruments, Inc.). Anhydrous ethanol was used instead of the sample as a blank control. The free radical scavenging rate of DPPH was calculated as follows:

2.4. GC–MS analysis The chemical constitution of essential oils was analyzed by GC (Agilent 19091S-431UI) equipped with HP-5 MS capillary column (15 m × 250 μm × 0.25 μm) in series with MS (Agilent 5975). The sample was automatically injected at a flow velocity of 1 mL/min and a split ratio of 1:5, using He as a carrier gas. The temperature programming was as below: the start temperature was 50 °C for 3 min, rising to 150 °C at the speed of 8 °C/min, and maintaining the constant temperature for 2 min. The temperature was then retained at 170 °C for 2 min after the temperature-rise at the speed of 4 °C/min. Finally, the oven temperature was set to rise from 170 °C to 250 °C at 10 °C/min and held at 250 °C for 5 min. Other settings were interface temperature of 300 °C and ion source temperature of 230 °C. The mass spectrum was recorded in electron impact ionization (EI) of 70 eV and analyzed in the mass range of 35−450 m/z with an emission current of 34.6 VA and an electron multiplying voltage of 1392 V. The main components of essential oils extracted by three methods SDE, HRE and SE were analyzed and identified, wherein the total ion current was obtained under the above GC/MS conditions. The main components of the sample were analyzed and identified, wherein the total ion current was obtained under that above GC–MS conditions. The identification of individual peaks was accomplished by comparison of their mass spectra with those from available MS libraries (NIST/NBS/Wiley) and their experimental retention indices (AMDIS) with data from the literature determined by a

Scavenging rate(%) =

ODcontrol − ODsample × 100% ODcontrol

(1)

Wherein ODsample and ODcontrol were the OD values of the essential oil samples or absolute ethanol interaction with DPPH solution, respectively 2.5.2. ABTS assay The scavenging ability of sample against ABTS was measured by spectrophotometry (Hoon et al., 2015). The ABTS stock solution was prepared by mixing the 7 mmol/L of ABTS mother liquor with 4.9 mmol/L of K2S2O8 aqueous solution at an equal volume, and then kept away from light at 4℃ overnight. Then it was further diluted with methanol to obtain an absorbance at 734 nm. ABTS standard solution with absorbency of 0.7 ± 0.05 was selected as ABTS working solution at 734 nm. Afterwards, 20 μL sample solution with different concentrations (12.5−400 μg/mL) was blended with 180 μL diluted ABTS 2

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Table 1 Chemical composition of essential oils extracted by three methods from the Semen Platycladi identified by GC/MS. Nr

Retention time (RT)

Compounda

CAS nr

Molecular formula

Molecular weight

Relative percent content/% SDE

HRE

SE

11.71 ± 0.13 5.36 ± 0.07

4.00 ± 0.07

3.52 ± 0.05 –

0.75 ± 0.02 –

0.87 ± 0.03 – 3.00 ± 0.06 – – 1.09 ± 0.02 – 1.56 ± 0.02 – 2.19 ± 0.02 –

– – – – – – 2.60 ± 0.03 – 2.09 ± 0.02 – –

–

–

5.35 ± 0.05 – – 2.89 ± 0.03 – – – 3.90 ± 0.03 – – – 1.21 ± 0.02 – – 1.22 ± 0.02 12.76 ± 0.18 – 7.60 ± 0.09 4.43 ± 0.05 – – 1.51 ± 0.03 2.27 ± 0.05 – – – – 2.61 ± 0.04 – – – – 1.28 ± 0.04 –

– 4.62 ± 0.04 – 1.35 ± 0.02 – 1.89 ± 0.02 – 3.21 ± 0.04 – – – – 1.03 ± 0.03 0.58 ± 0.01 – 10.50 ± 0.14 1.78 ± 0.03 7.13 ± 0.08 – 3.77 ± 0.05 – – 1.79 ± 0.02 – – – 0.57 ± 0.01 – – – – – – –

– 3.58 ± 0.06 – 2.02 ± 0.04 – 1.65 ± 0.03 3.50 ± 0.05 – – 0.52 ± 0.01

– 5.80 ± 0.07 – 0.79 ± 0.01 – 1.06 ± 0.03 3.34 ± 0.04 – – 0.72 ± 0.01

– 2.06 ± 0.04 2.91 ± 0.03

– – –

1

3.133

Cyclobarbital

52-31-3

C12H16N2O3

236

0.37 ± 0.02

2

4.047

–

C22H36O5

380

-b

3 4

4.883 6.368

Succinic acid, tridec-2-yn-1-yl tetrahydrofurfuryl ester (E)-10-Heptadecen-8-ynoic acid methyl ester 3-Carene

16714-85-5 13466-78-9

C18H30O2 C10H16

278 136

5 6 7 8 9 10 11 12 13 14 15

6.407 6.662 7.038 7.316 7.65 7.8 8.108 8.108 8.289 8.289 8.362

Methyl hexadeca-4,7,10,13-tetraenoate α-Fenchene cis-5,8,11,14,17-Eicosapentaenoic acid ψ-Limonene Furan, 2-pentyl2-Octen-1-ol, 3,7-dimethyl-, isobutyrate, (Z)2-Azido-2,4,4,6,6-pentamethylheptane Z,Z,Z-1,4,6,9-Nonadecatetraene Carbonic acid, 2-ethylhexyl undecyl ester Carbonic acid, 2-ethylhexyl nonyl ester β-Cymene

873108-81-7 471-84-1 10417-94-4 499-97-8 3777-69-3 – – – – – 535-77-3

C17H26O2 C10H16 C20H30O2 C10H16 C9H14O C14H26O2 C12H25N3 C19H32 C20H40O3 C18H36O3 C10H14

262 136 302 136 138 226 211 260 329 300 134

16

8.45

D-Limonene

5989-27-5

C10H16

136

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

8.514 9.027 9.027 9.085 9.139 9.701 9.931 9.936 11.138 11.48 11.637 11.734 12.057 12.061 12.208 12.609 12.858

4-Thujanol 2,2,4,4,6,8,8-Heptamethylnonane 4,5-Dimethylnonane γ-Terpinene Valeric acid, 2-ethylhexyl ester 3,4-Dimethylbenzyl alcohol 1-Fluorododecane Linalool Silane, cyclohexyldimethoxymethylTerpinen-4-ol Thymol L-α-Terpineol 2,4,4,6-Tetramethyl-6-phenylheptane 2-Azido-2,4,4,6,6,8,8-heptamethylnonane Cyclononane, 1,1,4,4,7,7-hexamethyl4-Methyldocosane Benzene, 1,3-bis(1,1-dimethylethyl)-

546-79-2 4390-4-9 17302-23-7 99-85-4 5451-87-6 6966-10-5 334-68-9 78-70-6 17865-32-6 562-74-3 89-83-8 10482-56-1 – – 149331-19-1 25117-30-0 1014-60-4

C10H18O C16H34 C11H24 C10H16 C13H26O2 C9H12O C12H25F C10H18O C9H20O2Si C10H18O C10H14O C10H18O C17H28 C16H33N3 C15H30 C23H48 C14H22

154 226 156 136 214 136 188 154 188 154 150 154 232 267 210 325 190

– 14.48 ± 0.10 – 0.27 ± 0.01 – 2.27 ± 0.04 2.30 ± 0.03 – – – – – 10.27 ± 0.14 19.17 ± 0.17 0.26 ± 0.01 – – 5.66 ± 0.06 – 2.87 ± 0.03 – 1.13 ± 0.02 0.26 ± 0.00 4.57 ± 0.05 0.74 ± 0.01 0.76 ± 0.02 – – – – 0.49 ± 0.02

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

12.961 12.961 13.254 13.254 13.459 13.494 14.046 14.535 15.058 15.434 15.477 15.619 15.649 15.752 15.786 15.962 16.348 16.353

– 14905-56-7 17312-74-2 54833-48-6 5655-61-8 3386-33-2 540-97-6 8007-35-0 – 2387-78-2 25117-30-0 959269-58-0 469-61-4 87-44-5 546-28-1 470-40-6 – –

C22H44O3 C17H36 C13H28 C21H44 C12H20O2 C18H37Cl C12H36O6Si6 C12H20O2 C16H28O C15H24 C23H48 C17H32O2 C15H24 C15H24 C15H24 C15H24 C17H30O2 C15H24

357 240 184 297 196 289 445 196 236 204 325 268 204 204 204 204 266 204

– – – – 1.75 ± 0.03 – – 3.29 ± 0.03 0.20 ± 0.00 0.28 ± 0.01 – – 1.16 ± 0.02 0.98 ± 0.01 0.50 ± 0.02 0.96 ± 0.02 – 0.64 ± 0.01

52 53 54 55 56 57 58 59 60 61

16.832 16.968 16.978 17.037 17.408 17.476 17.941 19.182 19.358 19.637

Carbonic acid, decyl undecyl ester 2,6,10-Trimethyltetradecane 5-ethyl-5-methyldecane Heptadecane, 2,6,10,15-tetramethylL-α-bornyl acetate 1-Chlorooctadecane Cyclohexasiloxane, dodecamethylα-Terpinyl acetate Farnesol (E), methyl ether Cyperene 4-Methyldocosane 7-methyl-(Z)-8-tetradecen-1-ol acetate α-Cedrene Caryophyllene β-Cedrene cis-Thujopsene 2-(7-Dodecyn-1-yloxy)tetrahydro-2H-pyran 1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Zα-Curcumene Dotriacontane, 1-iodoα-Acorenol 1,1′-[1,3-Propanediylbis(oxy)]bisoctadecane Cuparene 2,4-Di-tert-butylphenol Disulfide, di-tert-dodecyl β-Costol Allocedrol Cedrol

644-30-4 62154-83-0 28296-85-7 17367-38-3 16982-00-6 96-76-4 27458-90-8 515-20-8 50657-30-2 77-53-2

C15H22 C32H65I C15H26O C39H80O2 C15H22 C14H22O C24H50S2 C15H24O C15H26O C15H26O

202 577 222 580 202 206 403 220 222 222

62 63 64

19.788 21.674 21.938

Isolongifolol Heptacosane 2,6,10,14-Tetramethylhexadecane

1139-17-9 593-49-7 638-36-8

C15H26O C27H56 C20H42

222 381 283

0.50 ± 0.01 – 0.85 ± 0.02 – 0.38 ± 0.01 – – 1.16 ± 0.02 1.29 ± 0.02 14.05 ± 0.16 0.88 ± 0.02 – –

5.41 ± 0.05

(continued on next page) 3

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Table 1 (continued) Nr

Retention time (RT)

Compounda

CAS nr

Molecular formula

Molecular weight

Relative percent content/% SDE

HRE

SE – 0.92 ± 0.03 – 1.45 ± 0.02 0.71 ± 0.01 – 1.33 ± 0.02 – – 21.05 ± 0.23 – 10.46 ± 0.14 32.45 ± 1.01 28

65 66 67 68 69 70 71 72 73 74

22.149 23.008 24.089 24.093 26.747 27.29 27.964 28.41 29.793 29.802

3-(6,6-Dimethyl-5-oxohept-2-enyl)-cycloheptanone tert-Hexadecanethiol cis-13-Eicosenoic acid Acetic acid, 2-chloro-,octadecyl ester (3E,12Z)-1,3,12-Nonadecatriene-5,14-diol Palmitic acid, methyl ester 2,3-Dimethyl-5-(trifluoromethyl)-1,4-benzenediol Hexadecanoic acid, ethyl ester 8,11-Octadecadienoic acid, methyl ester Dasycarpidan-1-methanol acetate

– 25360-09-2 17735-94-3 5348-82-3 – 112-39-0 – 628-97-7 56599-58-7 55724-48-6

C16H26O2 C16H34S C20H38O2 C20H39ClO2 C19H34O2 C17H34O2 C9H9F3 C18H36O2 C19H34O2 C20H26N2O2

250 259 311 347 294 270 206 284 294 326

– – – – – 0.36 ± 0.01 – 0.62 ± 0.01 0.80 ± 0.01 2.22 ± 0.03

0.41 ± 0.01 – 1.69 ± 0.02 – – – – – – 5.34 ± 0.04

75 76

29.876 30.594

6,9,12-Octadecatrienoic acid, methyl ester Ethanol, 2-(9,12-octadecadienyloxy)-, (Z,Z)-

2676-41-7 17367-08-7

C19H32O2 C20H38O2

292 311

1.26 ± 0.03 –

– –

The yield of essential oils (%, W/W)

0.16 ± 0.01

Have detected species of compounds

37

44.23 ± 1.37 30

Identified by compared MS databases (matching similarity ≥ 90 %) and Kovats’ retention time relative to n-alkanes on HP-5 column; if no related retention indices data in the literatures just tentative identified by MS databases. b “-” = Not found or not exist. a

2.6.2. Cytotoxicity assay The in vitro toxicity of essential oils to tumor cells was evaluated by colorimetric MTT method (Ibrahim et al., 2013; Piaru et al., 2012). Cells were treated with or without different concentrations of essential oil (6.25, 12.5, 25, 50, 100, 200 μg/mL) and incubated in a 96 well plates of 100 μL medium with a density of 1 × 104 cells per well for 24 h. The MTT working solution (20 μL; 5 mg/mL in PBS) and DMEM medium (180 μL) were added to each well to incubate for 4 h. Each well was added with 150 μL of DMSO after removing the MTT solution, and then rotated and shaken for 10 min to dissolve the obtained purple crystals and scanned by a microplate reader (BioTek Instruments, Inc.) at 490 nm. The classical anticancer drug 5-Fluorouracil (5-F) was used as a positive control. LO2 cells were used to screen the safe concentration of essential oil samples. Growth inhibition was calculated as follow: Fig. 2. Chemical compositions of essential oils extracted from the Semen Platycladi by three methods (SDE, HRE, SE).

Cell viability(%) =

working solution. The inhibition rate of ABTS radicals was the same as that of the DPPH test.

ODsample × 100% ODcontrol

Cell inhibition rate(%) =

ODcontrol − ODsample × 100% ODcontrol

(2) (3)

wherein ODsample and ODcontrol were the OD values of the cells treated with or without essential oil samples, respectively.

2.5.3. FRAP assay The FRAP value of the sample was based on the method of Shen et al. (Shen et al., 2017) Sodium acetate solution of 0.3 mol/L, TPTZ solution of 10 mmol/L and FeCl3 solution (prepared when using) of 20 mmol/L were mixed and shaken to form 0.83 mmol/L TPTZ working solution. The 96-well plate was added with 20 μL ferrous sulfate solution of different concentration (diluted with 1 mmol/L ferrous sulfate mother liquor) and 180 μL TPTZ working solution. The absorbance was measured at 596 nm after light avoidance reaction at 37℃ for 10 min to establish absorbance-concentration standard curve of ferrous sulfate. The same mM concentration as the ferrous sulfate absorbance was expressed as the FRAP value of the sample.

2.7. Antimicrobial activity 2.7.1. Microbial strains Microbial strains including Staphylococcus aureus ATCC 25923 S. aureus), Escherichia coli ATCC 25922 E. coli) and Candida albicans ATCC 10231 C. albicans) were purchased from Guangdong culture collection center. 2.7.2. Determination of minimum inhibitory concentrations (MIC) The MIC was measured by microdilution using Luria-Bertani (LB) broth (Nejad Ebrahimi et al., 2008). MIC was measured by microdilution using Luria-Bertani (LB) broth. The essential oil samples were dissolved in dimethyl sulphoxide (DMSO) and then dilute with normal saline. A standard suspension (106 CFU/mL) of each microorganism was added to each well containing essential oils (64−1 mg/mL) of different final concentration or standard antimicrobial agent chloramphenicol (CAP) (1000–15.625 μg/mL). The micropores were incubated in a constant temperature shaker at 37 °C for 24 h (Tsukatani et al., 2012). Using BioTek instruments, Inc. microplate system to

2.6. Anticancer activity 2.6.1. Human cell lines and culture LO2, MCF-7, A549 cells were cultured in DMEM medium replenished with 10 % (v/v) FBS, 0.2 % (v/v) penicillin and streptomycin and then cultivated in an incubator with 5 % CO2 at 37 °C. The logarithmic growth phase cells were used in the experiment. 4

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Fig. 3. Main chemical structures of the compounds in the essential oils.

2.8. Statistical analysis

measure the absorbance of 600 nm to evaluate the growth inhibition of the strain by the sample. The lowest concentration of test essential oils to prevent bacteria and fungi growth was defined as MIC.

All experiments were performed in triplicate. The data were displayed as mean ± standard deviation. SPSS 11.5 software was used to statistically analyze the significant differences by one-way ANOVA. P < 0.05 means statistically significant, while P < 0.01 was used for judging highly statistical significance.

2.7.3. Disc diffusion test Luria-Bertani (LB) agar diffusion method was used to evaluate the inhibition zones so as to estimate the antimicrobial potency of essential oils in vitro (Viuda Martos et al., 2008). The microbial suspension (106 CFU/mL) prepared from LB broth was coated on LB solid medium plate. The sterile filter paper disc (diameter 7 mm) impregnated with samples of different concentrations was gently placed on the inoculation medium. In order to avoid the volatilization of essential oil samples, all the plates were kept at 4℃ for 2 h, followed by incubation at 37 °C for 24 h. The diameter of bacteriostatic ring was measured by a vernier caliper.

3. Results and discussion 3.1. Identification and comparative analysis of essential oil of components The components of essential oil from Semen Platycladi were analyzed for the first time. There were significant differences in the number of components (SDE 37, HRE 30, SE 28) and the yield (SDE 0.06 ± 0.01 %, HRE 44.23 ± 1.37 %, SE 32.45 ± 1.01 %), among which the extraction rate of SDE was the least but it contained the most compounds. The main advantages of SDE are reduced usage of organic solvents with a potential risk of storage and the applicability for extraction compounds with higher volatility. HRE and SE with the superior extraction yield was a faster and more effective extraction technique to obtain the abundant oil resource. Meanwhile, it showed that the oleaginousness of this traditional Chinese medicine was as high as 44.23 % through the SDE extraction method (Fig. 1). The relative percentage content of each component was calculated by area integration method (Table 1). A total of 76 compounds were identified in the variety, including 10 alkanes, 14 alkenes, 13 alcohols, 1 aldehyde ketone, 3 ethers, 1 acid, 17 esters, 5 aromatic compounds, and 12 others. Fig. 2 displayed that the key component of essential oils extracted by SDE method was alkenes; while the other two method HRE and SE of which were esters and others. The four highest content of SDE are DLimonen (19.17 %), 3-Carene (14.48 %), Cedrol (14.05 %), and βCymene (10.27 %) (Table 1), among which the D-Limonen contributes to the anticancer and anti-microbial activity (Schmandke, 2003), and

2.7.4. Scanning electron microscope (SEM) Morphological variations of bacteria affected by essential oils were observed by SEM on the basis of the method from Meng et al. (2016) S. aureus was incubated in LB medium at 37℃ for 8 h, and then the suspensions (approximately 107 CFU/mL) were prepared and treated with 1 × MIC of essential oils respectively except the control which was added with the equivalent amount of sterile distilled water. The bacterial suspension was incubated at 37℃ for 8 h, and then centrifuged at a speed of 8000 r/min for 5 min. The precipitated bacteria was washed twice with PBS 7.4 and fixed in 2.5 % glutaraldehyde for 8 h. Afterwards, the suspensions were occupied with two more washes with PBS 7.4 solution to remove excess glutaraldehyde. The samples were dehydrated for each 10 min with different gradients of ethanol (50 %, 70 %, 80 %, 90 %), and finally dehydrated twice with 100 % ethanol for 15 min each.

5

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3.2. Antioxidant activity Antioxidants are substances that are defined as being able to strive against other oxidizable substrates and delay or prevent their oxidation (Nikolić et al., 2014). DPPH and ABTS are dark radicals that accept electrons or hydrogen radicals to form antimagnetic molecules with a discoloration effect (Schaich et al., 2015). Under acidic conditions, Fe3+ forms a complex with TPTZ, in which the trivalent iron ion is reduced to divalent iron ion under the action of reductant, then the solution turns from yellow to bright blue (Bolanos de la Torre et al., 2015). Fig. 4 displayed that essential oils existed free radical-scavenging potency in a not obvious dose-dependent manner. Among the three extraction methods, there was a perceptibly predominance of essential oils extracted from SDE against DPPH radical along with the free radical inhibition rate exceeding 20 % at 50 μg/mL, which distinctly overtopped than VC at the same concentration, while HRE validated the strongest capability to reduce ABTS (50 μg/mL) followed by SDE, but the SE samples had a very weak ability to scavenge free radicals. In addition, essential oils obtained by the three methods are all of extremely small FRAP value, demonstrating that three essential oil samples almost have no reducing capacity to Fe3+. The discrepancies could also be elucidated by the antioxidant mechanism of free radicals. The antioxidant capacity of the test samples depended on the interaction between the used stressing agent and antioxidant (Hu et al., 2014). In addition, the stereoselectivity of free radicals or the solubility of essential oils in different test systems may also affect the reactivity and quenching ability of individual essential oils with different free radicals (Yu et al., 2002). As shown in Fig. 4, essential oils extracted from SDE were dominant in radical scavenging potential followed by HRE, but the SE samples had a very weak ability to scavenge free radicals. Table 1 demonstrated that the SDE sample contained some compounds that didn’t exist in SDE and SE samples, such as thymol, citral, γ-terpinene, and L-α-terpineol, all of which possessed good free radical scavenging activities in previous reports (Yi et al., 2018). In this study, the major components of SDE-essential oil were alkenes while the major compounds of HRE and SE-essential oil were esters and others; the chemical composition discrepancies of three essential oils could accordingly explain the differences in biological activity of the three isolation methods. Furthermore, the monoterpenes detected in the essential oils had been shown to be as ascendantly potential antioxidants (Bayala et al., 2014). 3.3. Anticancer activity Our study reported the antitumor activity of Semen Platycladi essential oils for the first time. Cytotoxicity was assessed by the MTT method, mainly by detecting the viability of a cell enzyme in which the MTT was reduced to insoluble formazan (Sinha et al., 2014). The maximum safety concentration of essential oil samples screened by acting on human normal liver LO2 cells turned out to be 200 μg/mL where the cell viability of all the samples was above 80 %. Thus, essential oils samples of increasing doses ranging from 6.25–200 μg/mL continued for 48 h to cancer cell lines, which observably restrained the growth of the cells (Fig. 5). When the dosage was 25 μg/mL, the inhibitory rate of SDE-extracted essential oil on MCF cancer cells (31.53 %) exceeded 80 % of 5-F (27.63 %). At the same dose, the essential oils isolated by HRE and SE also displayed a certain inhibitory action against the growth of cancer cells, both of which suppression rate (21.32 % and 20.71 % respectively) had been run up to 80 % of the positive control 5-F (27.59 %). The abundant content of alkenes (Table 1 and Fig. 2) such as citral (Maruoka et al., 2018), γ-terpinene (Bayala et al., 2014), and D-Limonene (Zhang et al., 2014) had been observed in SDE-essential oils as they attached significant importance in many cell processes to induce a variety of biological effects, including cell cycle control, transmembrane signal transduction and cellular metabolic pathways, which

Fig. 4. Antioxidant activity of essential oils extracted from the Semen Platycladi by three methods (SDE, HRE, SE). Results are mean ± SD. *P < 0.05, **P < 0.01, statistically significant in comparison with control.

Cedrol has a certain sedative effect in previous studies (Dayawansa et al., 2003; Jiang et al., 2007). However, the essential oils extracted through the HRE and SE don’t vary in their components and contents. More esters, alkanes and others were detected in the essential oils obtained from HRE and SE (Fig. 2), wherein the content of HRE-cyclobarbital reached 11.71 % which was a potential barbiturate sedative hypnotic agent (Table 1). The difference in the composition of these volatile oils might be based on diverse separation principles. The SDE method differs depending on the boiling point of the compound and its solubility in water. HRE and SE are extracted by a lipophilic organic solvent in accordance with the rule of "like dissolves like " (da Silva et al., 2016; Molnar et al., 2017). The structure of primary compounds in all essential oils was shown in Fig. 3. 6

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Fig. 5. Cytotoxic activity of essential oils extracted from the Semen Platycladi by three methods (SDE, HRE, SE). Results are mean ± SD. *P < 0.05, **P < 0.01, statistically significant in comparison with control.

SDE (mg/mL)

HRE (mg/mL)

SE (mg/mL)

CAP (μg/mL)

antioxidant activity show anticancer properties. The interaction of various substrates in cells might explain this correlation. ROS could interact and modify with cell proteins, lipids and DNA, resulting in altered feature of target cells. The bioaccumulative oxidative damage was connected with severe or chronic cell damage, which might be involved in the generation of cancer (Greten et al., 2004).

64 8 16

128 32 32

128 32 32

31.25 31.25 31.25

3.4. Antimicrobial activity

Table 2 MIC of Semen Platycladi essential oils collected by three methods against selected fungal and bacterial strains. Microorganisms

E.coli S.aureus C. albicans

Sample/antibiotic

The results for antibacterial potential of essential oils prepared by SDE, HRE and SE and standard antibiotics performed using MIC are reported in Table 2. The actions of Semen Platycladi essential oils against the pathogenic microorganisms have already been reported (Hassanzadeh et al., 2008). The actions of Semen Platycladi essential oils

SDE = steam distillation extraction, HRE = heating reflux extraction, SE = soxhlet extraction, CAP = chloramphenicol.

perhaps partially explained the pronounced potency of SDE-essential oils (Cury-Boaventuraa et al., 2006). All essential oils with an 7

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transport and solidification of cellular contents (Dannenberg et al., 2016). The antimicrobic potential of essential oils detected by MIC and disc-diffusion assay both showed that essential oil extracted by SDE was more effective against S.aureus, thus the morphological changes of S.aureus treated with SDE essential oil were evaluated by SEM analysis (Fig. 6). The untreated S. aureus cells (Fig. 6, A) were spherical, wellranged, uniform in size and shape and retained normal cell membranes with smooth surface. On the contrary, S.aureus cells treated with the SDE essential oil suffered the severe damage, becoming irregular, sunken and malformed, with the significantly decreasing of the number of cells. Obviously, S. aureus cells (Fig. 6, B, C) revealed serious disruption of cell membranes, which indicated that the antimicrobial effect mode was probably due to changes of the structure in cell membrane and resulting in the loss of cell viability (Zhang et al., 2017).

Table 3 Antimicrobial activity of essential oils taken from Semen Platycladi using disc diffusion assay. Microorganism

E. coli SDE HRE SE S. aureus SDE HRE SE C. albicans SDE HRE SE

Inhibition zone diameter (mm) 128 mg/mL

64 mg/mL

32 mg/mL

16 mg/mL

9.9 ± 0.4 7.6 ± 0.6 7.9 ± 0.4

7.7 ± 0.6 7.3 ± 0.6 NA

NA NA NA

NA NA NA

13.9 ± 0.5 9.3 ± 0.6 9.6 ± 0.3

9 ± 0.4 8.5 ± 0.2 8.8 ± 0.4

8.5 ± 0.2 8.3 ± 0.3 8.2 ± 0.4

8.7 ± 0.4 NA NA

11.4 ± 0.8 9.8 ± 0.5 9.3 ± 0.8

9.3 ± 0.5 8.4 ± 0.2 8.9 ± 0.6

8.6 ± 0.3 7.8 ± 0.6 NA

NA NA NA

4. Conclusion a

The values represent the average (standard deviations) for triplicate analyses. b SDE = steam distillation extraction, HRE = heating reflux extraction, SE = soxhlet extraction, NA = active.

The yields of three essential oils seperated from Semen Platycladi were 0.16 % (SDE), 44.23 % (HRE), and 32.45 % (SE) (w/w), respectively. A total of 76 compounds were identified, including 10 alkanes, 14 alkenes, 13 alcohols, 1 aldehyde ketone, 3 ethers, 1 acid, 17 esters, 5 aromatic compounds, and 12 others. The essential oils extracted by SDE were proved to have the best antioxidant and antimicrobial activity that were allowed to be used as a natural preservative in the pharmaceutical and cosmetic industries. The essential oils isolated by three methods all validated considerable cytotoxicity to cancer cell lines, indicating that it’s vital to adopt a proper method to obtain the target ingredients recommended as nutraceuticals and/or phytomedicines. The different essential oils extraction methods are confirmed to be an important factor in influencing the chemical composition of volatile constituent. Future study may be directed toward the investigation underlying the relationship between the specific components and the various bioactivities to provide new insights, highlighting the possibility to expand the use of this plant in industrial field.

on pathogenic microorganisms were reported as early as 2008. Coherence with the anterior reports, in this study, essential oils did not markedly suppress the growth of sensitive bacterial and fungal strains only if a relatively larger dose was administered. Generally, the grampositive bacteria S.aureus appear to be more sensitive compared with Gram-negative E. coli and fungal strains C. albicans, with MICs of 8 mg/ mL for SDE, 32 mg/mL for HRE and SE respectively, which could be explained by their different cell organization. Overall, the essential oils extracted by SDE were more active against the pathogenic microorganisms than that extracted by HRE and SE. It turned out that the inhibitory action of SDE against pathogenic microorganisms was mainly attributed to D-Limonene (Zahi et al., 2017), γ-Terpinene (Miladi et al., 2017), linalool (Zhou et al., 2016), and thymol (Reyes-Jurado et al., 2019), which not existed in essential oils extracted by HRE and SE. However, there is also reason to believe that it is difficult to attribute their total antimicrobial activity to one or several principles due to the complexity of essential oils. It is necessary to further explore the relationship between chemical constituents and antimicrobial activities, clearly accounting for their activity. The essential oil extracted by SDE had stronger inhibition on E. coli, S.aureus and C. albicans, and the average inhibition circles were 9.9 ± 0.4, 13.9 ± 0.5, and 11.4 ± 0.8 mm (Table 3), respectively, being the same as the result of MICs. Compared with CAP, essential oils showed a little lower inhibition, whereas they existed broad-spectrum antimicrobic activity. The differences in antimicrobial activity between different method extracted essential oils could be explicated by the compositional diversity of each essential oil and the type of target microorganism (Marinoa et al., 2001; Martins Mdo et al., 2014). The structural diversity of essential oil had diverse action and mechanism on its antibacterial activity, including the interruption of electron flow caused by the interference of cell plasma membrane, the active

Credit author statement Jing-Jing Zhu: Experiment, Methodology, Writing - original draft, Jing-Juan Yang: Investigation, Guo-Jie Wu: Data curation, Jian-Guo Jiang: Supervision, Writing - review & editing. Funding This project was funded by Science and Technology Project of Guangzhou City (201704020055). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 6. The photography of SEM of S. aureus, untreated (A), treated with SDE at MIC (B), and the magnification of B (C). 8

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