Accepted Manuscript

Influence of molecular distillation on antioxidant and antimicrobial activities of rose

essential oils

Fengping Yi, Jing Sun, Xiaoli Bao, Baodi Ma, Min Sun

PII: S0023-6438(18)31109-5
DOI: https://doi.org/10.1016/j.lwt.2018.12.051
Reference: YFSTL 7712

To appear in: LWT - Food Science and Technology

Received Date: 27 July 2018

Revised Date: 12 December 2018
Accepted Date: 16 December 2018

Please cite this article as: Yi, F., Sun, J., Bao, X., Ma, B., Sun, M., Influence of molecular distillation
on antioxidant and antimicrobial activities of rose essential oils, LWT - Food Science and Technology
(2019), doi: https://doi.org/10.1016/j.lwt.2018.12.051.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT
Influence of molecular distillation on antioxidant and
antimicrobial activities of rose essential oils

Fengping Yia, Jing Suna, Xiaoli Baoa,*, Baodi Mab, Min Suna
a
School of Perfume and Aroma Technology, Shanghai Institute of

PT
Technology, Shanghai 201418, P. R. China

RI
b
School of Chemical and Environmental Engineering, Shanghai Institute

SC
of Technology, Shanghai 201418, P. R. China

U
AN
M
D
TE

*To whom correspondence should be addressed.

E-mail address: bxlsit@163.com (Xiaoli Bao)

C EP
AC
 1 ABSTRACT
ACCEPTED MANUSCRIPT
2

3 Chemical compositions, antioxidant and antimicrobial activities of the essential oils

4 (REOs) of roses including Rosa damascene (RD), Rosa centifolia (RC), Rosa pomponia

5 (RP) and Rosa chinensis Jacq “Crimson Glory” H. T. (CG), and their fractions obtained

PT
6 through molecular distillation (MD) were investigated. The major constitutes were:

7 β-citronellol and nonadecane in RDEO, RCEO and RPEO, 1-nonadecene and nonadecane

RI
8 in CGEO. After MD, a larger proportion of more volatile compounds existed in the

SC
9 distillates relative to their original oils and residues. The REOs and fractions exhibited not

U
10 only moderate antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH),
AN
11 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and reducing power

12 assays, but also obvious broad-spectrum antimicrobial activities towards the seven tested
M

13 microorganisms. Notably, the higher antioxidant and antimicrobial activities were observed
D

14 for the distillates of RDEO, RCEO and RPEO. Moreover, the active antioxidant constitutes
TE

15 in the REOs were characterized as eugenol and methyl eugenol, and the potential

antimicrobial components were linalool, phenethyl alcohol, β-citronellol, geraniol, eugenol

EP

16

17 and methyl eugenol. Results demonstrated that MD could be used to obtain fractions from
C

18 REOs with higher antioxidant and antimicrobial activities, and the distillates of RDEO,
AC

19 RCEO and RPEO have potential to act as novel natural antioxidant and antimicrobial

20 agents.

21 Keywords: Rose essential oils; Molecular distillation; Antioxidant activity; Antimicrobial

22 activity

1
23
ACCEPTED MANUSCRIPT
24 1. Introduction

25 Plants are the important sources of natural compounds with diverse bioactivities,

26 enabling their use for functional food production and medicinal purposes (Petrović et al.,

27 2017). Especially, numerous aromatic plants and spices are highly prized in the

PT
28 pharmaceutical, food and cosmetic industries, due to their fragrance, antioxidant and

29 antimicrobial capacities (Valderrama & Ruiz, 2018). Along with the rise of the “getting

RI
30 back to nature” trend, the functionality of plants and their secondary metabolites in the

SC
31 body has become one of the most significant topics among nutrition and health

U
32 professionals, as well as consumers (Franz, Chizzola, Novak, & Sponza, 2011).
AN
33 Among the multiple types of natural compounds derived from plants, essential oils

34 derived from aromatic and herbal plants are receiving particular attention in virtue of their
M

35 radical-scavenging capacities (Barros et al., 2015). A free radical is a molecule that has one
D

36 or more unpaired electrons generally formed in the body during metabolism. Excess free
TE

37 radical has deleterious effects, such as the per-oxidation of membrane lipids, the attack on

enzymes, protein tissues and carbohydrates (Husain, Cillard, & Cillard, 1987). As a
EP

38

39 consequence, there are several relevant pathologies such as arthritis, cataracts, heart disease
C

40 and cancer, in which free radicals are probably the cause or aggravating factor of the
AC

41 general situation (Halliwell, Gutteridge, & Cross, 1992). Previous reports have

42 demonstrated a direct protective role of dietary antioxidants on intestinal mucosa through

43 local antioxidant and anti-inflammatory abilities (Falé et al., 2013). Therefore, it becomes

44 very important to seek for antioxidative substances since they are capable of stabilizing or

2
45 deactivating free radicals before they attack targets in biological cells and tissues (Barros et
ACCEPTED MANUSCRIPT
46 al., 2015). Besides, various human and plant pathogens have developed resistance owing to

47 the indiscriminate use of commercial antimicrobial agents commonly available in the

48 treatment of infectious diseases. The increasing antibiotic resistance provides the demand

49 to search for novel antimicrobial agents, particularly from natural origins (Said et al., 2016).

PT
50 In practice, the essential oils concerned with a variety of aromatic plants have been

51 revealed to exhibit potent antibacterial and antifungal properties in natural medicine

RI
52 development (Burt, 2004; Ćavar, Maksimović, Vidic, & Parić, 2012; Kordali et al., 2009).

SC
53 The genus Rosa (Family Rosaceae) consists of more than 200 species with 18,000

U
54 cultivated varieties, but only a few of them have found industrial scale application for their
AN
55 fragrance and flavoring properties (Nedeltcheva-Antonova, Stoicheva, & Antonov, 2017).

56 With honey flavor and aroma, the rose, well known for its relaxing effects, stands outs for
M

57 its culinary and herb tea uses to combat anxiety, depression and stress-related conditions
D

58 (Naquvi, Ansari, Ali, & Najmi, 2014). It was reported in the literature that this plant has
TE

59 been widely used in medicine for the treatment of abdominal and chest pain, digestive

problems, menstrual bleeding, nervous stress, and skin problems due to its analgesic,
EP

60

61 anti-inflammatory, antispasmodic, hypnotic and anticonvulsant activities (Gholamhoseinian,

C

62 Shahouzehi, Joukar, & Iranpoor, 2012; Shohaye, Abdel-Hameed, Bazaid, & Maghrabi,
AC

63 2014). Rose essential oil is an exceptionally rich source of different monoterpenoids and

64 sesquiterpenoids, which are mostly responsible for the high bioactivities of the oil exploited

65 widely in traditional and modern medicines (Patrascu & Radoiu, 2016). Antioxidant,

66 laxative, hypolipidemic, antidiabetic, antimicrobial and anticancer activities of rose

3
67 essential oil (REO) justify its use in a broad range of applications including perfumery,
ACCEPTED MANUSCRIPT
68 cosmetics, nutraceuticals and pharmaceutical industries (Kovacheva, Rusanov, &

69 Atanassov, 2010; Naquvi et al., 2014).

70 Molecular distillation (MD) technology is known as a useful and mild separation means

71 to purify thermolabile substances and low volatile compounds (Mezza et al., 2018; Ruben,

PT
72 Valeria, & Ruben, 2014). This method is characterized by short distance between the

73 evaporator and the condenser, high vacuum in the distillation space and short exposure of

RI
74 the distilled substances to the operating temperature. MD will not damage the essential oil

SC
75 components due to the low pressure and short dwell time. Moreover, without solvents used,

U
76 the process avoids the solvent contamination towards the handled materials (Xiong et al.,
AN
77 2013). This technology has become very useful in the petroleum and oil fields for obtaining

78 fractions enriched in particular compounds (Mezza et al., 2018).

M

79 Although the antioxidant and antimicrobial activities of REOs have been evidenced, the
D

80 relevant studies were mostly limited to the original oils (Kumar, Bhandari, Singh, & Bari,
TE

81 2009; Mileva, Kusovski, Krastev, Dobreva, & Galabov, 2014; Moein, Zomorodian, Almasi,

Pakshir, & Zarshenas, 2017; Senol et al., 2013; Shohaye et al., 2014), and there was little
EP

82

83 information available about the bioactive activities of their MD fractions. Furthermore, it

C

84 was not demonstrated clearly which compounds were responsible for these antioxidant and
AC

85 antimicrobial activities. The current study focused on the preparation of REO fractions by

86 MD, and evaluation on the antioxidant and antimicrobial activities of the fractions to

87 identify the compounds responsible for their use in functional foods and natural medicines.

88

4
 89 2. Materials and methods
ACCEPTED MANUSCRIPT
90

91 2.1. Materials

92

93 The rose essential oils obtained by hydro-distillation, including the Rosa damascene

PT
94 essential oil (RDEO), the Rosa centifolia essential oil (RCEO), the Rosa pomponia

95 essential oil (RPEO) and the Rosa chinensis Jacq "Crimson Glory" H. T. essential oil

RI
96 (CGEO), were provided by Gelle Freres (China). β-Citronellol, geraniol, farnesol,

SC
97 phenylethyl alcohol, linalool, eugenol, and methyl eugenol were obtained from Macklin

U
98 (China). Potato dextrose agar (PDA), Müeller-Hinton agar (MHA) and Müeller-Hinton
AN
99 broth (MHB) were purchased from Hope Bio-Technology Co., Ltd. (China).

100 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis

M

101 (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma (USA). All
D

102 other chemicals utilized were of analytical grade.

TE

103

2.2. Fractions obtained by MD

EP

104

105
C

106 MD was performed with a laboratory wiped-film molecular distillation apparatus (Pope,
AC

107 USA). After melted in water bath at 45°C, each rose essential oil was pipetted into the feed

108 flask and degasified using the short path evaporator to prevent bubbling and splashing

109 during distillation under high vacuum conditions. After the degasifying operation, the

110 distilling temperature, distilling pressure, wiper rolling speed, feed flow rate and cooling

5
111 water temperature were set at 70°C, 50 Pa, 200 rpm, 4 mL/min, and 10°C, respectively.
ACCEPTED MANUSCRIPT
112 When the distilling pressure and temperature were fixed, the feeding valve was turned on,

113 and the degassed feed liquid immediately flowed down by gravity and the scraper quickly

114 spun it into a very thin film spreading over onto the evaporating surface. Depending upon

115 the changes of temperature and pressure at the evaporating surface, a different numbers of

PT
116 volatile components evaporate as the liquid flows down the wall through the heating zone.

117 More volatile components concentrated onto the closely positioned internal condensing

RI
118 surface and became the distillates, while the less volatile components flowed down along

SC
119 the cylinder and became the residues. After distillation, the distillates and residues fractions

U
120 were weighed and transferred to the containers. They were stored at 4°C until analysis.
AN
121

122 2.3. Gas chromatography/mass spectrometry (GC/MS) analysis

M

123
D

124 The compositions of the REOs and their MD fractions were determined by GC/MS using
TE

125 a Hewlett-packard 6890 GC coupled with a Hewlett-packard mass-selective detector

5973N quadropole MS (Agilent Technologies, USA). The samples were analyzed with a
EP

126

127 fused silica capillary column DB-5MS (5% phenyl-methylsiloxane, 60 mm × 0.25 mm,
C

128 film thickness 0.25 µm) and a flame ionization detector (FID). The oven column
AC

129 temperature was programmed to rise from an initial temperature of 50°C to 100°C at a rate

130 of 10°C/min and maintained at 100°C for 10 min, and then ramped to 140°C at a rate of

131 3°C/min and maintained at 140°C for 10 min, and finally ramped to 230°C at a rate of

132 2°C/min and maintained at 230°C for 10 min. The injector temperature was 230°C. The

6
133 carrier gas nitrogen had a flow rate of 0.8 mL/min and the split ratio was 1:30. The
ACCEPTED MANUSCRIPT
134 ionization was obtained by electron impact at 70 eV. The compounds were identified on the

135 basis of retention index (RI, determined with reference to homologous series of n-alkanes,

136 C7-C30), Mass Spectra Library search (NIST 11.0 and Wiley registry of MS data 7n.1), and

137 by comparing with the MS literature data. The relative amounts of individual components

PT
138 were calculated based on GC peak area.

139

RI
140 2.4. Antioxidant activity

SC
141

U
142 The scavenging activities on DPPH were measured as reported previously by Senol et al.
AN
143 (2013). The ABTS radical cation-scavenging activities were determined as described by

144 Khamtache-Abderrahim et al. (2016). The reducing power was evaluated using the method
M

145 described by Schaich, Tian and Xie (2015). Gallic acid and α-tocopherol were chosen as
D

146 the positive controls.

TE

147

2.5. Antimicrobial assays

EP

148

149
C

150 2.5.1. Microbial strains

AC

151

152 The microorganisms used in this study involved four bacteria Gram-positive

153 Staphylococcus aureus (S. aureus) ATCC 25923 and Bacillus subtilis (B. subtilis) ATCC

154 6633, Gram-negative Escherichia coli (E. coli) ATCC 25922 and Pseudomonas aeruginosa

7
155 (P. aeruginosa) ATCC15442, as well as three fungi Aspergillus niger (A. niger) ATCC
ACCEPTED MANUSCRIPT
156 16404, Rhizopus nigricans (R. nigricans) ATCC 6227b and Blastocladia pringsheimii (B.

157 pringsheimii). All the strains were provided by the Biotechnology College of Shanghai

158 Institute of Technology.

159

PT
160 2.5.2. Disc diffusion test

161

RI
162 Antimicrobial activities against the S. aureus, B. subtilis, E. coli, P. aeruginosa, A. nige,

SC
163 R. nigricans and B. pringsheimii were tested by the disc diffusion method according to the

U
164 National Committee for Clinical Laboratory Standards (NCCLS, 2001).
AN
165

166 2.5.3. Minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC)
M

167 and minimum fungicidal concentration (MFC) determination

D

168
TE

169 MIC and MBC values towards the S. aureus, B. subtilis, E. coli and P. aeruginosa were

determined using the serial two-fold dilution method (CLSI, Clinical and Laboratory
EP

170

171 Standards Institute, 2006; M7-A7). MFC and MBC values towards the A. nige, R. nigricans
C

172 and B. pringsheimii were determined by the agar dilution method described by Boubaker et
AC

173 al. (2016).

174

175 2.6. Statistical analysis

176

8
177 All antioxidant and antimicrobial tests and analyses were carried out in triplicate and the
ACCEPTED MANUSCRIPT
178 results were expressed as mean ± standard deviation (SD). Data obtained were subjected to

179 one way analysis of variance (ANOVA). Means were separated by Tukey’s multiple range

180 tests when ANOVA was significant (p < 0.05) (SPSS statistics 24; Chicago, USA).

181

PT
182 3. Results and Discussion

183

RI
184 3.1. Preparation and chemical characterization of the REOs and MD fractions

SC
185

U
186 After distillation, two fractions, a distillate and a residue were obtained for each REO.
AN
187 The percentages of the distillates on RDEO, RCEO, RPEO and CGEO were 61.6%, 66.9%,

188 65.7% and 19.8%, respectively, while the ratios of the respective corresponding residues
M

189 were 32.6%, 26.3%, 28.7% and 76.5%. Other than the CGEO, higher percentages of
D

190 volatile compounds were obtained in the distillates along with the corresponding residues
TE

191 containing fewer volatile compounds.

Table 1 represents the constituents concerned with the REOs and their MD fractions
EP

192

193 analyzed by GC/MS. According to the findings, the major constituents of RDEO were
C

194 identified as β-citronellol (20.76%), nonadecane (20.15%) and heneicosane (9.70%), while
AC

195 the major constituents of both RCEO and RPEO were β-citronellol (28.44% and 27.15%,

196 respectively), nonadecane (15.52% and 16.73%, respectively) and geraniol (12.84% and

197 12.98%, respectively). Notably, 1-nonadecene (27.78%), nonadecane (22.26%) and

198 heneicosane (15.63%) were identified as the main ingredients of CGEO. Numerous studies

9
199 have been carried out on the essential oil compositions of roses. In most studies (Moein et
ACCEPTED MANUSCRIPT
200 al., 2017; Naquvi et al., 2014), the major constituents presenting in the essential oil profile

201 of these plants were monoterpene alcohols which contain citronellol, geraniol, phenethyl

202 alcohol as well as aliphatic compounds which comprise nonadecane and heneicosane. The

203 amount and types of constituents were highly affected by intrinsic or genetic, extrinsic or

PT
204 environmental parameters (Baydar & Baydar, 2005). As in these previous reports, the

205 highest content in RDEO, RCEO and RPEO was also determined to be β-citronellol in the

RI
206 present work, but 1-nonadecene with the highest level was determined in CGEO.

SC
207 After distillation, the main components of the distillates of RDEO, RCEO and RPEO

U
208 were identified as β-citronellol (24.00%, 13.33% and 26.43%, respectively), nonadecane
AN
209 (13.33%, 13.92% and 16.73%, respectively) and geraniol (12.48%, 13.94% and 12.98%,

210 respectively). While 1-nonadecene (19.69%), nonadecane (12.24%) and uncineol (5.87%)
M

211 were main constituents of CGEO distillate. With regard to the residues, nonadecane
D

212 (35.46%, 34.27% and 38.03%, respectively) heneicosane (26.38%, 27.02% and 23.19%,
TE

213 respectively) and 1-nonadecene (8.24%, 8.16% and 10.09%, respectively) were identified

as the main components of RDEO, RCEO and RPEO, while 1-nonadecene (28.89%),
EP

214

215 nonadecane (23.78%) and heneicosane (19.12%) were main components for the residue of
C

216 CGEO. Concerning the fractions obtained from the REOs through MD, different
AC

217 proportions of constituent compounds were concentrated in different fractions. The

218 differences in the compositions between the fractions are based on the differences between

219 boiling points of the molecules that constitute the essential oil (Ruben et al., 2014). As

220 shown in Table 1, a greater proportion of more volatile compounds, such as myrcene,

10
221 4-terpineol and α-terpineol existed in the distillate fractions with respect to the residue
ACCEPTED MANUSCRIPT
222 fractions in which the less volatile compounds including 1-nonadecene and nonadecane,

223 concentrated. Notably, methyl geranate, geranyl acetate, germacrene D and α-selinene were

224 found only in the original oils and their distillate fractions, which indicated that these four

225 compounds can be concentrated in the distillate fractions and separated by MD using the

PT
226 process condition presented in this study.

227

RI
228 3.2. Antioxidant activities of the REOs and MD fractions

SC
229

U
230 Radical scavenging is an accepted mechanism by which antioxidants act to inhibit lipid
AN
231 oxidation. DPPH radical-scavenging test has been used extensively to predict antioxidant

232 capacities due to the relatively short time required for analysis (Kumar et al., 2009). In
M

233 DPPH assay, the antioxidants are able to reduce the stable purple DPPH· to yellow
D

234 diphenyl-picrylhydrazine (Wu, Li, Yang, Zhan, & Tu, 2013). As shown in Fig. 1. A, the
TE

235 DPPH radical-scavenging activities of the REOs and their distillate fractions exhibited

concentration-dependent profiles. RPEO with the IC50 of 0.92 mg/mL was the most active
EP

236

237 original oil, followed by RCEO, RDEO and CGEO with the IC50 values of 0.97, 1.24
C

238 and >20 mg/mL, respectively. Based on lower IC50 values, the distillate fractions of RDEO,
AC

239 RCEO and RPEO were found superior in efficacy than their respective original oils.

240 However, all residue fractions as well as the distillate fraction of CGEO showed very low

241 radical-scavenging activities since the IC50 values were all larger than 20 mg/mL.

242 Moreover, the activities of the REOs and their MD fractions were less than those of the

11
243 positive controls gallic acid (IC50: 0.003 mg/mL) and α-tocopherol (IC50: 0.012 mg/mL).
ACCEPTED MANUSCRIPT
244 ABTS+· is also widely used to determine the radical-scavenging effects of plant extracts.

245 In contact with an H· donor, it leads to the formation of ABTS+ and then to a discoloration

246 of the solution at 734 nm. The decrease in absorbance due to the antioxidant

247 activity reflects a capacity to capture free radical (Khamtache-Abderrahim et al., 2016). As

PT
248 shown in Fig. 1. B, the REOs and their distillate fractions also exhibited dose-dependent

249 ABTS radical-scavenging effects. The IC50 values of RDEO, RCEO and RPEO were all

RI
250 about 0.07 mg/mL. After MD, the distillate fractions of RDEO, RCEO and RPEO also

SC
251 exhibited more potent ABTS+· scavenging activity relative to their respective original oils

U
252 as in the DPPH assay (p < 0.05). The IC50 values of the four residue fractions as well as the
AN
253 distillate fraction of CGEO were all larger than 20 mg/mL, indicating that these fractions

254 exhibited low ABTS radical-scavenging activities. The REOs and their MD fractions also
M

255 showed lower ABTS radical-scavenging capacities than gallic acid (IC50: 0.001 mg/mL)
D

256 and α-tocopherol (IC50: 0.002 mg/mL).

TE

257 The reducing capacity of a compound has been considered to be a significant indicator of

its antioxidant potential (Schaich et al., 2015). Results in this study showed that the
EP

258

259 reducing power of the REOs and derived MD fractions increased with its increasing
C

260 concentrations in the range of 0.25-3.0 mg/mL and the absorbance values ranged from
AC

261 0.212 to 0.858 (Fig. 1. C). Through MD, the distillate fractions of RDEO, RCEO and

262 RPEO also exhibited more significant reducing power than their respective original oils. As

263 in the radical-scavenging assays, the absorbance values of the four residual fractions were

264 all less than 0.025, which also indicated that these fractions exhibited very low reducing

12
265 power. Likewise, the REOs and their MD fractions showed lower reducing power than
ACCEPTED MANUSCRIPT
266 α-tocopherol.

267 To find the potential anoxidant substances in the REOs, the antioxidant activities of

268 seven constituents including linalool, phenethyl alcohol, β-citronellol, geraniol, farnesol,

269 eugenol and methyl eugenol were also evaluated with the three antioxidant assays. Among

PT
270 the components, eugenol showed the most potent radical-scavenging capacity and reducing

271 power with the IC50 values of 0.006 (in DPPH assay) and 0.001 (in ABTS assay), as well as

RI
272 the absorbance value of 1.121 at a lower concentration of 0.1 mg/mL (in reducing power

SC
273 assay), respectively, which even stronger than those of α-tocopherol with IC50 values of

U
274 0.012 (in DPPH assay), 0.002 (in ABTS assay), and the absorbance value of 0.697 at the
AN
275 concentration of 0.1 mg/mL (in reducing power assay). Methyl eugenol also exhibited

276 considerable antioxidant activities in the assays. Particularly, its antioxidant capacities were
M

277 superior to all the REOs and MD fractions based on the lower IC50 values and higher
D

278 absorbance values (p < 0.05) in all the DPPH, ABTS and reducing power assays. However,
TE

279 linalool, phenethyl alcohol, β-citronellol, geraniol and farnesol showed very low activities

both in the radical-scavenging assays with the IC50 values larger than 20 mg/mL and in the
EP

280

281 reducing power assay with the absorbance values less than 0.15. Taken together, eugenol
C

282 and methyl eugenol were main components that contributed the antioxidant activities of the
AC

283 REOs.

284

285 3.3. Antimicrobial activities of the REOs and MD fractions

286

13
287 Antimicrobial activities of the REOs and their MD fractions were evaluated against four
ACCEPTED MANUSCRIPT
288 bacterial and three fungal strains. The results in terms of IZ, MIC, MBC and MFC are

289 present in Table 2 and 3. RCEO and RPEO manifested significant inhibitory activities

290 against all tested strains with the IZs of 8.97-28.01 and 7.3-22 mm, respectively. RDEO

291 obviously inhibited all the tested strains except for the P. aeruginosa with the IZs of

PT
292 11.85-44.73 mm. However, CGEO showed appreciable activity against only the S. aureus

293 and B. subtilis with the IZs of 6.04 and 11.04 mm, respectively. Through MD, other than

RI
294 the distillate fraction of RDEO against the S. aureus and B. subtilis, all the rest distillate

SC
295 fractions of the REOs demonstrated stronger or equal activities towards the tested strains as

U
296 compared to their respective original oils. However, none of the residue fractions showed
AN
297 any inhibition towards the strains. In the medium dillution assays, the MIC, MBC and MFC

298 values of the distillate fractions were all lower than those of their respective original oils,
M

299 while the values of the residue fractions were all larger than 40 mg/mL for all the bacterial
D

300 strains and larger than 100 mg/mL for all fungal ones, which also indicated that most of the
TE

301 distillate fractions showed significant antimicrobial activity, while the residue fractions

showed almost no antimicrobial activity.

EP

302

303 The potential antimicrobial activities of the seven constituents were also tested against
C

304 the seven microorganisms. Results indicated that the inhibitory activities of the REOs and
AC

305 their fractions againt the Gram-positive S. aureus and B. subtilis were attributed to all the

306 seven components, while the inhibition against the Gram-negative E. coli was probably

307 caused by linalool, phenethyl alcohol, β-citronellol, geraniol, eugenol and methyl eugenol,

308 and the inhibitory activities against the P. aeruginosa were primarily attributed to phenethyl

14
309 alcohol and eugenol. The antifungal activities of the REOs and their fractions against the
ACCEPTED MANUSCRIPT
310 fungi A. niger, R. nigricans and B. pringsheimii were all mainly attributed to phenethyl

311 alcohol, β-citronellol, geraniol, eugenol and methyl eugenol. Indeed, the effects of these

312 constituents on the growth of bacteria and fungi have been extensively studied. For

313 example, phenethyl alcohol has been reported as an active agent on the inhibition of N.

PT
314 crassa, E. coli, and B. cinerea (Qadri et al., 2015). Moreover, the fungal growth on the

315 surface of phenethyl alcohol-treated strawberry during storage at 4°C was significantly

RI
316 lower than that of the control group during the designed experimental period (Mo and Sung,

SC
317 2007). In the literature, geraniol and citronellol showed pronounced antifungal efficacy

U
318 against T. rubrum strains. The antifungal activity of geraniol against Fusarium oxysporum,
AN
319 Penicillium digitatum, Aspergillus spp. and Candida spp, as well as the antifungal capacity

320 of citronellol against Aspergillus spp, Fusarium spp, Penicillium spp. and richophyton spp.
M

321 were also demonstrated (Kim & Park, 2012). The antimicrobial effects observed for the
D

322 constitute compounds of the REOs in this study were basically consistent with these
TE

323 reported results.

The variety of chemical structures of constitutes allows for the existence of different
EP

324

325 modes of action underlying the potential antimicrobial activities of plant essential oils,
C

326 including disturbance of the cytoplasmic membrane that results in disrupting the proton
AC

327 motive force, electron flow, active transport and coagulation of cell contents (Dannenberg

328 et al., 2016; Zeng et al., 2011).

329

330 4. Conclusions

15
331
ACCEPTED MANUSCRIPT
332 A greater proportion of more volatile compounds existed in the distillates derived from

333 MD of the REOs relative to their respective original oils and residues. Most distillates

334 exhibited significantly higher antioxidant and antimicrobial activities than their respective

335 original oils and residues in the assays, with the IC50 values of 0.735-0.948 and

PT
336 0.0653-0.0707 mg/mL in the radical-scavenging assays, the absorbance values of

337 0.212-0.858 in the concentration range of 0.25-3.0 mg/mL in the reducing power assay, the

RI
338 IZs of 6.93-32 mm and the MICs of 0.625-1.25 mg/mL for the bacteria and 6.25-12.5

SC
339 mg/mL for the fungi in the antimicrobial tests. Moreover, eugenol and methyl eugenol were

U
340 characterized as the active antioxidant constitutes, and linalool, phenethyl alcohol,
AN
341 β-citronellol, geraniol, eugenol and methyl eugenol were proven to be the potential

342 antimicrobial components in the REOs.

M

343 In conclusion, MD allowed for a different proportion of chemical components of rose

D

344 essential oils to be concentrated in different fractions, and it constituted an alternative to

TE

345 separate fractions from the essential oils with higher antioxidant and antimicrobial

capacities that might be used as novel plant-based pharmaceutical and healthcare

EP

346

347 ingredients.
C

348
AC

349 Acknowledgement

350

16
351 ACCEPTED
This work was supported by the Education MANUSCRIPT
Commission of Shanghai Municipality (No.

352 ZZZZyyx16012) and the Science and Technology department of Shanghai Institute of

353 Technology (No. A06-(39120K189028)ZQ2018-28).

354

355 References

PT
356

357 Barros, A. D. S., Morais, S. M. D, Ferreira, P. A. T., Vieira, Í. G. P., Craveiro, A. A.,

RI
358 Fontenelle, R. O. D., Menezes, J. E. S., Silva, F. W. F., & Sousa, H. A. D. (2015). Chemical

SC
359 composition and functional properties of essential oils from Mentha species. Industrial

Crops and Products, 76, 557-564.

U
360 AN
361 Baydar, H., & Baydar, N. G. (2005). The effects of harvest date, fermentation duration and

362 tween 20 treatment on essential oil content and composition of industrial oil rose (Rosa
M

363 damascena Mill.). Industrial Crops and Products, 21, 251-255.

D

364 Boubaker, H., Karim, H., El Hamdaoui, A., Msanda, F., Leach, D., Bombarda, I., Vanloot,
TE

365 P., Abbad, A., Boudyach, E. H., & Ait Ben Aoumar, A. (2016). Chemical characterization

366 and antifungal activities of four Thymus species essential oils against postharvest fungal
EP

367 pathogens of citrus. Industrial Crops and Products, 86, 95-101.

C

368 Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in
AC

369 foods-a review. International Journal of Food Microbiology, 94, 223-253.

370 Ćavar, S., Maksimović, M., Vidic, D., & Parić, A. (2012). Chemical composition and

371 antioxidant and antimicrobial activity of essential oil of Artemisia annua L. from Bosnia.

372 Industrial Crops and Poducts, 37, 479-485.

17
373 Clinical Laboratory Standards Institute, (2006). Methods for dilution antimicrobial
ACCEPTED MANUSCRIPT
374 susceptibility tests for bacteria that grow aerobically. Approved standard, M7-A7 (7th ed.),

375 CLSI, Wayne, PA.

376 Dannenberg, G. D. S., Funck, G. D., Mattei, F. J., Silva, W. P. D., & Fiorentini, Â. M.

377 (2016). Antimicrobial and antioxidant activity of essential oil from pink pepper tree

PT
378 (Schinus terebinthifolius Raddi) in vitro and in cheese experimentally contaminated with

379 Listeria monocytogenes. Innovative Food Science & Emerging Technologies, 36, 120-127.

RI
380 Falé, P. L., Ferreira, C., Rodrigues, A. M., Cleto, P., Madeira, P. J. A., Florêncio, M. H.,

SC
381 Frazão, F. N., & Serralheiro, M. L. M. (2013). Antioxidant and anti-acetylcholinesterase

U
382 activity of commercially available medicinal infusions after in vitro gastrointestinal
AN
383 digestion. Journal of Medicinal Plants Research, 7, 1370-1378.

384 Franz, C., Chizzola, R., Novak, J., & Sponza, S. (2011). Botanical species being used for
M

385 manufacturing plant food supplements (PFS) and related products in the EU member states
D

386 and selected third countries. Food & Function, 2, 720-730.

TE

387 Gholamhoseinian, A., Shahouzehi, B., Joukar, S., & Iranpoor, M. (2012). Effect of Quercus

infectoria and Rosa damascena on lipid profile and atherosclerotic plaque formation in
EP

388

389 rabbit model of hyperlipidemia. Pakistan Journal Biological Sciences, 15, 27-33.
C

390 Halliwell, B., Gutteridge, J. M., & Cross, C. E. (1992). Free radicals, antioxidants, and
AC

391 human disease: where are we now? Journal of Laboratory & Clinical Medicine, 119,

392 598-620.

393 Husain, S. R., Cillard, J., & Cillard, P. (1987). Hydroxyl radical scavenging activity of

394 flavonoids. Phytochemistry, 26, 2489-2491.

18
395 Khamtache-Abderrahim, S., Lequart-Pillon, M., Gontier, E., Gaillard, I., Pilard, S.,
ACCEPTED MANUSCRIPT
396 Mathiron, D., Djoudad-Kadji, H., & Maiza-Benabdesselam, F. (2016). Isoquinoline

397 alkaloid fractions of Fumaria officinalis: Characterization and evaluation of their

398 antioxidant and antibacterial activities. Industrial Crops and Products, 94, 1001-1008.

399 Kim, E., & Park, Il-K. (2012). Fumigant antifungal activity of myrtaceae essential oils and

PT
400 constituents from Leptospermum petersonii against three Aspergillus species. Molecules, 17,

401 10459-10469.

RI
402 Kordali, S., Cakir, A., Akcin, T. A., Mete, E., Akcin, A., Aydin, T., & Kilic, H. (2009).

SC
403 Antifungal and herbicidal properties of essential oils and n-hexane extracts of Achillea

U
404 gypsicola Hub-Mor. and Achillea biebersteinii Afan. (Asteraceae). Industrial Crops and
AN
405 Products, 29, 562-570.

406 Kovacheva, N., Rusanov, K., & Atanassov, I. (2010). Industrial cultivation of oil bearing
M

407 rose and rose oil production in Bulgariaduring 21st century, directions and challenges.
D

408 Biotechnology & Biotechnological Equipment, 24, 1793-1798.

TE

409 Kumar, N., Bhandari, P., Singh, B., & Bari, S. S. (2009). Antioxidant activity and

ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry

EP

410

411 for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana
C

412 and Rosa brunonii. Food and Chemical Toxicology, 47, 361-367.
AC

413 Mezza, G. N., Borgarelloa, A. V., Grossob, N. R., Fernandezc, H., Pramparoa, M. C., &

414 Gayola, M. F. (2018). Antioxidant activity of rosemary essential oil fractions obtained by

415 molecular distillation and their effect on oxidative stability of sunflower oil. Food

416 Chemistry, 242, 9-15.

19
417 Mileva, M. M., Kusovski, V. K., Krastev, D. S., Dobreva, A. M., & Galabov, A. S. (2014).
ACCEPTED MANUSCRIPT
418 Chemical composition, in vitro antiradical and antimicrobial activities of Bulgarian Rosa

419 alba L. essential oil against some oral pathogens. International Journal of Current

420 Microbiology and Applied Sciences, 3, 11-20.

421 Mo, E. K., & Sung, C. K. (2007). Phenylethyl alcohol (PEA) application slows fungal

PT
422 growth and maintains aroma in strawberry. Postharvest Biology & Technology, 45,

423 234-239.

RI
424 Moein, M., Zomorodian, K., Almasi, M., Pakshir, K., & Zarshenas, M. M. (2017).

SC
425 Preparation and analysis of Rosa damascena essential oil composition and antimicrobial

U
426 activity assessment of related fractions. Iranian Journal of Science & Technology
AN
427 Transactions A Science, 41, 87-94.

428 Naquvi, K. J., Ansari, S. H., Ali, M., & Najmi, A. K. (2014). Volatile oil composition of
M

429 Rosa damascena Mill. (Rosaceae). Journal of Pharmacognosy and Phytochemistry, 2,

D

430 177-181.
TE

431 National Committee for Clinical Laboratory Standards. (2001). Performance standards for

antimicrobial susceptibility testing: Eleventh informational supplement, M100-S11,

EP

432

433 NCCLS, Wayne, PA, USA.

C

434 Nedeltcheva-Antonova, D., Stoicheva, P., & Antonov, L. (2017). Chemical profiling of
AC

435 Bulgarian rose absolute (Rosa damascena Mill.) using gas chromatography-mass

436 spectrometry and trimethylsilyl derivatives. Industrial Crops and Products, 108, 36-43.

20
437 Patrascu, M., & Radoiu, M. (2016). Rose essential oil extraction from fresh petals using
ACCEPTED MANUSCRIPT
438 synergetic microwave & ultrasound energy: chemical composition and antioxidant activity

439 assessment. Journal of Chemistry and Chemical Engineering, 10, 136-142.

440 Petrović, S., Ušjak, L., Milenković, M., Arsenijević, J., Drobac, M., Drndarević, A., &

441 Niketić, M. (2017). Thymus dacicus as a new source of antioxidant and antimicrobial

PT
442 metabolites. Journal of Functional Foods, 28, 114-121.

443 Qadri, M., Deshidi, R., Shah, B. A., Bindu. K., Vishwakarma, R. A., & Riyaz-Ul-Hassan, S.

RI
444 (2015). An endophyte of Picrorhiza kurroa Royle ex. Benth, producing menthol,

SC
445 phenylethyl alcohol and 3-hydroxypropionic acid, and other volatile organic compounds.

U
446 World Journal of Microbiology & Biotechnology, 31, 1647-1654.
AN
447 Ruben, O., Valeria, N., & Ruben, G. N. (2014). Antioxidant activity of fractions from

448 oregano essential oils obtained by molecular distillation. Food Chemistry, 156, 212-219.
M

449 Said, Z. B. S., Haddadi-Guemghar, H., Boulekbache-Makhlouf, L., Rigou, P., Remini, H.,
D

450 Adjaoud, A., Khoudja, N. K., & Madani, K. (2016). Essential oils composition,
TE

451 antibacterial and antioxidant activities of hydrodistillated extract of Eucalyptus globulus

fruits. Industrial Crops and Products, 89, 167-175.

EP

452

453 Schaich, K. M., Tian, X., & Xie, J. (2015). Hurdles and pitfalls in measuring antioxidant
C

454 efficacy: A critical evaluation of ABTS, DPPH, and ORAC assays. Journal of Functional
AC

455 Foods, 14, 111-125.

456 Senol, F. S., Orhan, I. E., Kurkcuoglu, M., Khan, M. T. H., Altintas, A., Sener, B., & Baser,

457 K. H. C. (2013). A mechanistic investigation on anticholinesterase and antioxidant effects

458 of rose (Rosa damascena Mill.). Food Research International, 53, 502-509.

21
459 Shohaye, M., Abdel-Hameed, E. S., Bazaid, S. A., & Maghrabi, I. (2014). Antibacterial and
ACCEPTED MANUSCRIPT
460 antifungal activity of Rosa damascena MILL. essential oil, different extracts of rose petals.

461 Global Journal of Pharmacology, 8, 1-7.

462 Valderrama, F., & Ruiz, F. (2018). An optimal control approach to steam distillation of

463 essential oils from aromatic plants. Computers & Chemical Engineering, 117, 25-31.

PT
464 Wu, Z., Li, H., Yang, Y., Zhan, Y., & Tu, D. (2013). Variation in the components and

465 antioxidant activity of Citrus medica L. var. sarcodactylis essential oils at different stages

RI
466 of maturity. Industrial Crops and Products, 46, 311-316.

SC
467 Xiong, Y., Zhao, Z., Zhu, L., Chen, Y., Ji, H., & Yang, D. (2013). Removal of three kinds of

phthalates from sweet orange oil by molecular distillation. LWT-Food Science and

U
468 AN
469 Technology, 53, 487-491.

470 Zeng, W. C., Zhu, R. X., Jia, L. R., Gao, H., Zheng, Y., & Sun, Q. (2011). Chemical
M

471 composition, antimicrobial and antioxidant activities of essential oil from Gnaphlium affine.
D

472 Food and Chemical Toxicology, 49, 1322-1328.

TE
C EP
AC

22
 ACCEPTED MANUSCRIPT

Figure Caption

Fig. 1. Antioxidant activities of the REOs and their MD fractions in the

DPPH, ABTS and reducing power assays. O1: RDEO; D1: The distillate

fraction of RDEO; O2: RCEO; D2: The distillate fraction of RCEO; O3:

PT
RPEO; D3: The distillate fraction of RPEO; O4: CGEO; D4: The

RI
distillate fraction of CGEO.

U SC
AN
M
D
TE
C EP
AC

1
 ACCEPTED MANUSCRIPT

Table 1

Chemical compositions of the REOs and their MD fractions.

Percentageb (%)
RIa

PT
No. Constituent
O1 D1 R1 O2 D2 R2 O3 D3 R3 O4 D4 R4
Monoterpene hydrocarbons

RI
1 α-Pinene 937 0.81 0.23 0.14 0.23 0.04 0.02 0.11 0.03 0.01 0.02 0.07 ND
2 β-Myrcene 985 0.11 0.13 0.05 ND 0.02 ND 0.06 0.07 0.01 ND ND ND

SC
3 β-Pinene 986 0.13 0.05 ND 0.04 0.01 0.01 0.02 0.02 ND ND ND ND
Oxygenated monoterpenes

U
4 Linalool 1100 0.90 1.06 0.04 1.81 1.33 0.06 1.54 2.13 0.03 0.09 0.22 ND
5 Rose oxide 1111 0.36 0.33 0.03 0.42 0.17 0.02 0.38 0.20 0.01 0.02 0.04 ND

AN
6 Neroloxide 1151 0.08 0.09 ND 0.10 0.08 ND 0.12 0.11 0.06 ND ND ND
7 4-Terpineol 1186 0.11 0.15 ND 0.04 0.17 ND 0.07 0.15 0.04 ND ND ND

M
8 α-Terpineol 1200 0.37 0.51 0.02 0.69 0.82 0.03 0.83 1.12 0.02 0.02 0.03 ND
9 β-Citronellol 1229 20.76 24.01 1.05 28.44 30.43 1.46 27.15 36.79 1.53 0.79 2.58 0.18

D
10 Z-Citral 1239 0.41 0.57 0.05 0.51 0.44 0.04 0.59 0.81 0.04 0.04 0.12 0.08
11 Geraniol 1252 9.66 12.48 0.48 12.84 13.94 0.64 12.98 18.38 0.71 0.31 1.14 ND

TE
12 E-Citral 1267 0.56 0.95 0.07 0.65 0.85 0.05 0.65 1.18 0.08 0.67 0.55 ND
13 Methyl geranate 1316 0.11 0.19 ND 0.09 0.13 ND 0.09 0.17 ND 0.09 0.13 ND
EP
14 Citronellyl acetate 1340 0.19 0.23 ND 0.26 ND ND 0.24 0.37 ND 0.12 0.24 ND
15 Geranic acid 1347 0.84 0.87 ND ND 0.17 0.03 0.68 0.95 0.19 0.18 0.46 0.09
C

16 Eugenol 1349 1.27 2.77 0.06 2.52 2.99 0.08 2.34 3.39 0.09 ND ND ND
17 Geranyl acetate 1370 0.18 0.30 ND 0.28 0.41 0.01 0.30 0.50 ND 0.12 0.62 ND
AC

18 β-Damascenone 1378 0.03 0.03 ND 0.02 0.03 ND 0.03 0.0 ND ND ND ND

19 Methyl eugenol 1397 0.38 0.65 0.02 0.67 1.04 0.03 0.71 1.12 0.04 0.13 0.46 0.02
Sesquiterpene hydrocarbons
20 β-Elemene 1389 0.25 0.23 ND 0.04 ND ND 0.06 0.07 ND 0.08 0.18 ND
21 Caryophyllene 1422 0.70 1.06 0.04 0.10 0.11 ND 0.09 0.13 ND 0.31 0.80 0.03
1
 ACCEPTED MANUSCRIPT

22 α-Guaiene 1435 0.69 1.11 0.04 0.09 0.12 ND 0.10 0.14 ND 0.02 0.05 ND
23 Germacrene D 1482 0.82 1.29 ND 0.10 0.17 ND 0.10 0.17 ND ND 0.14 ND
24 α-Selinene 1490 0.14 0.29 ND ND ND ND ND ND ND 0.13 0.30 ND
25 α-Bulnesene 1501 0.64 1.10 ND 0.11 0.20 ND 0.13 0.21 0.01 1.46 2.08 0.15

PT
26 α-Cadinene 1517 0.06 0.12 ND 0.03 0.08 ND ND 0.03 ND 0.34 1.42 0.08
Oxygenated sesquiterpenes
27 Nerolidol 1559 0.23 0.27 ND 0.19 0.30 ND 0.15 0.24 0.03 0.16 0.66 ND

RI
28 Uncineol 1631 0.41 0.70 0.03 0.31 0.60 0.02 0.36 0.45 0.14 2.19 5.87 0.49
29 α-Eudesmol 1655 0.57 1.02 0.04 0.37 0.72 0.02 0.21 0.54 0.19 2.15 5.86 1.21

SC
30 Farnesol isomer 1689 0.26 0.49 ND 0.18 0.42 ND 0.17 0.33 0.14 0.13 0.25 ND
31 Farnesol 1712 2.54 3.89 0.77 1.93 3.57 0.41 2.10 2.46 1.93 0.49 1.04 0.41

U
3,7-dimethyl-6-octe
32 1907 0.12 0.06 0.12 0.04 0.06 ND 0.01 0.03 0.27 0.07 ND 0.07

AN
n-1-o butyrate
Methyl
33 1920 0.06 0.07 0.14 0.04 0.05 0.11 0.04 0.02 0.12 ND ND ND
hexadecanoate

M
34 Palmitic acid 1957 0.24 0.07 1.04 0.15 0.08 0.65 0.1 0.07 0.51 1.03 0.04 1.45
Aliphatic compounds

D
35 Pentadecane 1500 0.51 0.73 0.03 0.32 0.55 0.02 0.35 0.57 0.06 0.24 1.73 0.06

TE
36 Hexadecane 1600 0.10 0.17 0.01 0.09 0.17 0.01 0.08 0.16 0.05 0.15 0.63 ND
37 7-Heptadecene 1673 0.27 0.48 0.08 0.25 0.49 0.06 0.20 0.36 0.23 1.12 3.56 0.64
38 Heptadecane 1700 4.54 3.88 1.45 2.27 3.80 1.31 2.04 2.79 3.17 2.04 4.11 1.47
EP
39 5-Octadecene 1771 0.07 0.11 0.07 0.06 ND 0.06 0.08 0.06 0.11 0.18 0.31 0.15
40 Octadecane 1800 0.32 0.43 0.49 0.24 ND 0.46 0.32 0.19 0.62 0.18 0.21 0.16
C

41 1-Nonadecene 1871 4.71 4.34 8.24 4.47 4.90 8.16 5.15 2.27 10.09 27.78 19.69 28.89
AC

42 Nonadecane 1900 20.15 13.33 35.46 15.52 13.92 34.27 16.73 6.88 38.03 22.26 12.24 23.78
43 5-Icosene 1968 0.21 0.14 0.64 0.18 0.17 0.63 0.26 0.04 0.63 0.55 0.23 0.75
44 Eicosane 2000 2.11 0.98 5.69 1.40 1.10 6.25 2.30 0.33 5.42 1.89 0.59 2.48
45 5-Nonadecene 2089 0.89 0.22 2.51 0.65 0.30 1.08 0.91 0.02 2.14 2.38 0.24 1.76
46 Heneicosane 2100 9.70 2.59 26.38 6.55 3.18 27.02 6.60 0.81 23.19 15.63 2.61 19.12

2
 ACCEPTED MANUSCRIPT

47 Phytan 2265 3.32 2.41 0.96 3.95 2.61 1.16 1.09 1.61 1.77 1.33 2.04 2.14
48 Tricosane 2300 1.83 0.36 7.06 1.14 0.23 8.14 2.75 0.63 5.50 3.98 0.59 5.51
Others
49 1-Nonanal 1104 0.03 0.03 0.01 0.06 0.06 0.01 0.06 0.09 ND 0.06 0.12 ND

PT
50 Phenethyl alcohol 1118 2.72 3.62 0.14 4.57 4.25 0.17 5.29 6.62 0.13 0.02 0.03 ND
Total 96.47 91.19 93.45 95.01 95.28 92.50 96.72 95.81 97.34 90.95 74.28 91.17

RI
a
Retention indices relatives to C5-C25 n-alkanes calculated on non-polar HP-5MS™ capillary column; b Percentage based on FID peak area

SC
normalization; O1: RDEO; D1: The distillate fraction of RDEO; R1: The residue fraction of RDEO; O2: RCEO; D2: The distillate fraction

U
of RCEO; R2: The residue fraction of RCEO; O3: RPEO; D3: The distillate fraction of RPEO; R3: The residue fraction of RPEO; O4:

AN
CGEO; D4: The distillate fraction of CGEO; R4: The residue fraction of CGEO; ND: Not detected.

M
D
TE
C EP
AC

3
 ACCEPTED MANUSCRIPT

Table 2

Antibacterial effects of the REOs, their MD fractions and seven constitutes against the S. aureus, B. subtilis, E. coli and P. aeruginosa using disc diffusion

test and broth micro-dilution method.

PT
S. aureus B. subtilis E. coli P. aeruginosa
Sample* Inhibition zones MIC MBC Inhibition zones MIC MBC Inhibition zones MIC MBC Inhibition zones MIC MBC

RI
(mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL)
i g ef
O1 30.11 ± 0.55 2.5 5 44.73 ± 1.10 1.25 2.5 24.75 ± 1.83 1.25 2.5 NA > 40 > 40

SC
D1 18.23 ± 0.45d 1.25 2.5 20.56 ± 0.63de 0.625 1.25 25.32 ± 2.79f 0.625 1.25 6.93 ± 0.27a > 40 > 40

R1 NA > 40 >40 NA > 40 > 40 NA > 40 > 40 NA > 40 > 40

U
440
O2 28.01 ± 1.16g 2.5 5 18.83 ± 0.45d 1.25 2.5 21.16 ± 0.61d 1.25 2.5 8.97 ± 0.60c > 40 > 40

AN
D2 33.20 ± 0.42h 1.25 2.5 20.45 ± 0.75de 0.625 1.25 24.10 ± 0.40ef 0.625 1.25 8.16 ± 0.60bc > 40 > 40

R2 NA > 40 >40 NA > 40 > 40 NA > 40 > 40 NA > 40 > 40

M
O3 22.85 ± 0.76f 2.5 5 14.91 ± 0.36c 1.25 2.5 17.66 ± 0.97c 1.25 2.5 7.30 ± 0.20ab > 40 > 40

D3 29.46 ± 0.60g 1.25 2.5 20.22 ± 0.50de 0.625 1.25 20.04 ± 0.32cd 0.625 1.25 7.92 ± 0.32abc > 40 > 40

D
R3 NA > 40 >40 NA > 40 > 40 NA > 40 > 40 NA > 40 > 40

TE
O4 6.04 ± 0.00a > 40 >40 11.40 ± 0.49a 5 10 NA > 40 > 40 NA > 40 > 40

D4 13.71 ± 0.30b 20 40 22.72 ± 0.45f 2.5 5 9.45 ± 0.31a 2.5 10 NA > 40 > 40
EP
R4 NA > 40 >40 NA > 40 > 40 NA > 40 > 40 NA > 40 > 40

18.27 ± 0.31d 22.16 ± 1.01ef 13.73 ± 0.40b

C

Linalool 10 20 5 10 5 10 NA > 40 > 40

Phenethyl alcohol 24.15 ± 0.40f 22.82 ± 1.25f 25.26 ± 0.46f 28.50 ± 0.50f
AC

5 10 2.5 10 2.5 5 5 10
alcohol
β-Citronellol 19.56 ± 0.56de 2.5 10 19.41 ± 0.78d 5 10 17.80 ± 0.56c 0.625 1.25 8.83 ± 0.40c > 40 > 40

E-Geraniol 18.75 ± 0.40de 1.25 5 19.73 ± 0.09d 1.25 2.5 22.07 ± 1.18de 0.625 1.25 10.17 ± 0.45d > 40 > 40
Farnesol 13.73 ± 0.35b 0.313 0.625 12.66 ± 0.55ab 5 10 8.16 ± 0.31a > 40 > 40 NA > 40 > 40

1
 ACCEPTED MANUSCRIPT

Methyl eugenol 16.45 ± 0.91c 2.5 5 14.86 ± 0.95bc 1.25 2.5 17.7 ± 0.46c 1.25 2.5 NA > 40 > 40

NA : Not active (no inhibition zone); * Samples were diluted to 40 mg/mL with 2% DMSO; Values followed by the same letter within the same column are

not significantly different (p ≧ 0.05); O1: RDEO; D1: The distillate fraction of RDEO; R1: The residue fraction of RDEO; O2: RCEO; D2: The distillate

PT
fraction of RCEO; R2: The residue fraction of RCEO; O3: RPEO; D3: The distillate fraction of RPEO; R3: The residue fraction of RPEO; O4: CGEO; D4:

RI
The distillate fraction of CGEO; R4: The residue fraction of CGEO.

U SC
AN
M
D
TE
C EP
AC

2
 ACCEPTED MANUSCRIPT

Table 3

Antifungal capacities of the REOs, their MD fractions and seven constitutes against the A. niger, R. nigricans and B. pringsheimii using disc

diffusion test and agar microdilution method.

PT
A. niger R. nigricans B. pringsheimii

RI
Sample* Inhibition zones MIC MFC Inhibition zones MIC MFC Inhibition zones MIC MFC

(mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL)

SC
O1 18.16 ± 0.36c 25 25 15.22 ± 0.35cd 12.5 25 11.85 ± 0.30c 25 50

D1 20.83 ± 0.46de 12.5 25 18.83 ± 1.25e 6.25 12.5 18.77 ± 0.76f 12.5 25

U
R1 NA > 100 > 100 NA > 100 > 100 NA > 100 > 100

AN
d 1b de
O2 19.65 ± 0.53 25 25 12.60 ± 0.45 25 50 16.28 ± 0.95 25 50
f g g
D2 23.97 ± 0.47 12.5 25 22.54 ± 1.04 12.5 25 23.83 ± 0.40 12.5 25

M
R2 NA > 100 > 100 NA > 100 > 100 NA > 100 > 100
de ef c
O3 20.50 ± 0.20 12.5 25 19.63 ± 0.81 25 50 10.81 ± 0.71 25 50

D
f g f
D3 23.37 ± 0.91 6.25 12.5 22.44 ± 0.89 12.5 25 19.27 ± 0.35 12.5 25

TE
R3 NA > 100 > 100 NA > 100 > 100 NA > 100 > 100

O4 NA > 100 > 100 NA > 100 > 100 NA > 100 > 100
a a a
D4 7.38 ± 0.27 > 100 > 100 8.27 ± 0.25 > 100 > 100 6.50 ± 0.47 > 100 > 100
EP
R4 NA > 100 > 100 NA > 100 > 100 NA > 100 > 100
b d b
Linalool 11.90 ± 0.44 50 100 15.95 ± 0.35 25 50 8.62 ± 0.42 50 100
C

h h g
Phenethyl alcohol 33.67 ± 0.40 6.25 12.5 27.93 ± 0.76 6.25 12.5 24.30 ± 1.76 12.5 50
AC

f bc d
β-Citronellol 22.75 ± 0.25 6.25 12.5 13.76 ± 0.35 6.25 12.5 15.56 ± 0.36 6.25 100
g e g
E-Geraniol 31.77 ± 0.27 3.13 6.25 18.24 ± 0.45 6.25 12.5 22.38 ± 0.74 6.25 100
a a ab
Farnesol 7.19 ± 0.15 > 100 > 100 7.38 ± 0.21 > 100 > 100 7.31 ± 0.20 > 100 > 100
i i h
Eugenol 42.80 ± 0.50 3.13 6.25 36.40 ± 0.70 3.13 6.25 36.60 ± 0.42 6.25 25
e fg ef
Methyl eugenol 21.31 ± 0.70 3.13 6.25 21.31 ± 0.50 6.25 12.5 18.22 ± 0.30 6.25 12.5

1
 ACCEPTED MANUSCRIPT

NA: Not active (no inhibition zone); * Samples were diluted to 100 mg/mL with 2% DMSO; Values followed by the same letter within the

same column are not significantly different (p ≧ 0.05); O1: RDEO; D1: The distillate fraction of RDEO; R1: The residue fraction of RDEO;

O2: RCEO; D2: The distillate fraction of RCEO; R2: The residue fraction of RCEO; O3: RPEO; D3: The distillate fraction of RPEO; R3:

PT
The residue fraction of RPEO; O4: CGEO; D4: The distillate fraction of CGEO; R4: The residue fraction of CGEO.

RI
U SC
AN
M
D
TE
C EP
AC

2
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

Highlights

►Molecular Distillation was used to process four rose essential oils

(REOs).

►Most distillate fractions exhibited higher antimicrobial and antioxidant

PT
capacities.

RI
►The potential active components in the REOs were specified.

►REO fractions could be used as novel natural antioxidant and

SC
antimicrobial agents.

U
AN
M
D
TE
C EP
AC