Accepted Manuscript

Rapid Communication$Analytical Methods$Nutritional and Clinical Methods

Rosmarinus eriocalyx:an alter native toRosmarinus officinalisas a sour ce of

antioxidant compounds

Hamdi Bendif, Messaoud Boudjeniba, Mohamed Djamel Miara, Loreta Biqiku,

Massimo Bramucci, Giovanni Caprioli, Giulio Lupidi, Luana Quassinti, Gianni
Sagratini, Luca A. Vitali, Sauro Vittori, Filippo Maggi

PII: S0308-8146(16)31446-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.09.063
Reference: FOCH 19843

To appear in: Food Chemistry

Received Date: 1 June 2016

Revised Date: 7 September 2016
Accepted Date: 8 September 2016

Please cite this article as: Bendif, H., Boudjeniba, M., Djamel Miara, M., Biqiku, L., Bramucci, M., Caprioli, G.,
Lupidi, G., Quassinti, L., Sagratini, G., Vitali, L.A., Vittori, S., Maggi, F., Rosmarinus eriocalyx:an alter native
toRosmarinus officinalisas a sour ce of antioxidant compounds, Food Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.foodchem.2016.09.063

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 1 Rosmarinus eriocalyx: an alternative to Rosmarinus officinalis as a source of antioxidant

2 compounds

4 Hamdi Bendifa,b1, Messaoud Boudjenibaa, Mohamed Djamel Miarab, Loreta Biqikuc, Massimo

5 Bramuccic, Giovanni Capriolic1, Giulio Lupidic, Luana Quassintic, Gianni Sagratinic, Luca A. Vitalic,

6 Sauro Vittoric, Filippo Maggic*

7
a
8 Laboratory of Ethnobotany and Natural Substances, Department of Natural Sciences, Ecole

9 Normale Supérieure (ENS), Kouba, Algiers, Algeria

b
10 Faculty of Sciences, Natural and Life Sciences Department, Mohamed Boudiaf University, M’sila,

11 Algeria
c
12 School of Pharmacy, University of Camerino, Camerino, Italy

13
*
14 Corresponding author: Filippo Maggi, School of Pharmacy, University of Camerino, via S.

15 Agostino 1, 62032 Camerino, Italy. Phone: +390737404506. Fax: +390737637345. Email:

16 filippo.maggi@unicam.it
1
17 These authors equally contributed to the article.

18

19 Abstract

20 Rosmarinus eriocalyx is an aromatic evergreen bush endemic to Algeria where it is used as a

21 condiment to flavour soups and meat and as a traditional remedy. In the present work we have

22 analysed for the first time the phenolic composition of polar extracts obtained from stems, leaves

23 and flowers of R. eriocalyx by HPLC, and determined the antioxidant and antimicrobial effects by

24 DPPH, ABTS, FRAP, ORAC and agar disc diffusion methods, respectively. Results showed that

25 ethanolic extracts of leaves and flowers are a rich source of phenolic compounds, mainly rosmarinic

26 acid, carnosic acid and carnosol that are the main responsible for the noteworthy antioxidant

1
27 activity observed in the assays. This study showed that R. eriocalyx might be a spice to be included

28 in the European food additive list and used as a preservative agent besides R. officinalis.

29

30 Keywords: Rosmarinus eriocalyx, rosmarinic acid, HPLC, antioxidant, essential oil, GC-MS

31

32 1. Introduction

33 Rosemary is a worldwide culinary additive to foods. This spice is obtained mainly by Rosmarinus

34 officinalis L. a Mediterranean shrub cultivated mainly in Spain, Morocco and Tunisia (Flamini,

35 Cioni, Morelli, Macchia & Ceccarini, 2002). Normally, the parts of rosemary used for the

36 preparation of extracts and essential oils are the top flowering aerial parts including leaves, twigs

37 and inflorescences. In the European Union R. officinalis is used in foodstuffs as a preservative agent

38 due to the presence of antioxidant compounds such phenolic acids, flavonoids and diterpenoids (EC

39 Regulation, 2012). For the purpose, various kinds of extracts can be prepared and used as additives.

40 In addition, rosemary leaf, under the name ‘Rosmarini folium’, is included in the European

41 Pharmacopeia and used for medicinal preparations; the plant drug is chemically defined as it is

42 characterized by minimum 3% of hydroxycinnamic derivatives expressed as rosmarinic acid

43 (European Pharmacopeia, 2014).

44 Rosemary is one of the oldest known medicinal plants also in Algeria. Herein, the genus

45 Rosmarinus is considered as an universal remedy under the arabic vernacular name Iklil el djebel

46 (Arnold, Valentini, Bellomaria & Hocine, 1997). Besides the more widespread R. officinalis,

47 another member of the genus occurs in Algeria, i.e. R. eriocalyx Jord. & Fourr. The species,

48 previously known as R. tournefortii (Noë ex Jord. & Fourr.) Jahand. & Maire, is represented by

49 aromatic evergreen bushes endemic to Algeria, Spain and Morocco (Bendeddouche, Benhassaini,

50 Hazem & Romane, 2011). The plant grows widely on rocky ground and pastures in the

51 mountainous areas of eastern Algeria (Arnold et al., 1997; Benbelaïd et al., 2016). R. eriocalyx

52 differs from R. officinalis for its smaller leaves, only 5-15 mm long and less than 2 mm broad, and

2
53 for a more densely hairy flower stems. In this regard, the epithet ‘eriocalyx’ means woolly calyx,

54 alluding to its double hairy calyx, being characterized by one short type and long erect glandular

55 hairs. Another difference with respect to R. officinalis is its prostrate growth and lower height (often

56 under 25 cm and never exceeding 1 m tall) (Fadel et al., 2011).

57 To the best of our knowledge, most of phytochemical and pharmacological studies focused on R.

58 officinalis proving its universal use as a food preservative agent. On the other hand, only a few

59 studies were conducted on R. eriocalyx and concerned mainly its volatile fraction (Soriano Cano,

60 Sotomayor Sánchez, Sánchez Gomez & Garć ia

ia Vallejo,
Vallejo 1993; Dahmane, Eddarir, Aubert,

61 Bouamama & Taourirte, 2010; Fadel et al., 2011; Arnold et al., 1997; Bendeddouche et al., 2011;

62 Benbelaïd et al., 2016) whereas the polar fraction was not investigated in deep, especially the

63 content of bioactive constituents like rosmarinic acid, carnosic acid and carnosol that are

64 responsible for preservative properties of rosemary (EC Regulation, 2012). In order to provide

65 scientific evidences for a possible inclusion of R. eriocalyx in the list of preservative substances

66 used in foodstuffs, in the present work we conducted a comprehensive phytochemical and

67 biological characterization of polar extracts (aqueous and ethanolic) and essential oils obtained from

68 different plant parts (stems, leaves and flowers). For the purpose, polar extracts and essential oils

69 were analysed by HPLC-DAD and GC-MS, while the biological activities, namely antioxidant and

70 antimicrobial activities were evaluated by DPPH, ABTS, FRAP and disc diffusion assay,

71 respectively.

72 2. Materials and methods

73 2.1 Plant material

74 Inflorescences, stems and leaves of R. eriocalyx were separately collected from ten individuals

75 spontaneously growing in Djbel Boutaleb, province of Setif (North-East Algeria, 900-1500 m above

76 sea level, N 35°43’52”; E 5 °11'13”) in March 2015. Botanical determination was performed by Dr.

77 Miara using available literature and a voucher specimen was deposited in the Herbarium

78 Universitatis Camerinensis (CAME, included in the online edition of Index Herbariorum c/o School

3
79 of Biosciences and Veterinary Medicine, University of Camerino, Italy) under the codex CAME

80 27738; it was also archived in the anArchive system for Botanical Data (http://www.anarchive.it).

81 After collection, plant material was dried at room temperature in darkness for one week before

82 undergoing extraction and hydrodistillation.

83 2.2. Reagents and standards

84 The analytical standards of gallic acid, (+)-catechin hydrate, (-)-epicatechin, chlorogenic acid, 3,5-

85 di-O-caffeoylquinic acid, neochlorogenic acid, shikimic acid, rutin, quercitrin, hyperoside,

86 rosmarinic acid, carnosol, carnosic acid were purchased from Sigma-Aldrich (Milan, Italy). The

87 stock standard solution was prepared by dissolving 10 mg of the analyte in 10 ml of methanol and

88 stored in a glass-stoppered bottle at 4 °C in the dark. Standard working solutions, at various

89 concentrations, were daily prepared by appropriate dilution of aliquots of the stock solutions in

90 methanol. HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich (Milan,

91 Italy), while HPLC-grade formic acid 99-100% was bought from J.T. Baker B.V. (Deventer,

92 Holland). For sample preparation and chromatographic analysis, deionized water ≤ 18 MΩ/cm

93 resistivity purified with a Milli-Q system (Millipore, Bedford, USA) was used. All solvents and

94 solutions were filtered through a 0.45-µm PTFE filter from Supelco (Bellefonte, PA, USA) before

95 use. For the identification of the gas chromatographic peaks the analytical standards of α-pinene,

96 camphene, benzaldehyde, β-pinene, 1-octen-3-ol, myrcene, α-phellandren, δ-3-carene, α-terpinene,

97 p-cymene, limonene, 1,8-cineole, γ-terpinene, terpinolene, linalool, trans-pinocarveol, camphor,

98 isoborneol, borneol, terpinen-4-ol, α-terpineol, myrtenol, n-decanal, carvone, bornyl acetate,

99 thymol, carvacrol, eugenol, (E)-caryophyllene, α-humulene, (E)-β-farnesene, caryophyllene oxide,

100 α-bisabolol and linoleic acid were purchased from Sigma Aldrich (I-Milan). A mixture of n-alkanes

101 (C8-C30) was purchased from Supelco (Bellefonte, PA) and used to calculate the linear retention

102 indices of chromatographic peaks. n-Hexane was purchased from Carlo Erba (I-Milan).

103 2.3. Sample preparation and solvent extraction

4
104 Dried R. eriocalyx stems, leaves and flowers were reduced into a powder using a blender MFC

105 DCFH 48 IKA-WERK (D-Staufen) equipped with sieves of 2-mm size in diameter. Five grams of

106 each part were weighed and extracted with 50 mL of two different solvents, i.e. water and ethanol.

107 After 24 h extraction under magnetic stirrer, the extract was dried with rotavapor, freeze-dried and

108 stored in freezer. The yield obtained were 6.98, 14.46 and 14.40% for aqueous extracts, and 4.40,

109 19.60 and 12.60% for ethanolic extracts from stems, leaves and flowers, respectively. The samples

110 were prepared by re-dissolving 10 mg of each extract with water or ethanol. The sample solutions

111 were filtered through a 0.45 µm pore size nylon membrane filter (Phenex, Phenomenex, Torrance,

112 CA, USA) before injection into HPLC-DAD. Each sample was analyzed in triplicate.

113 2.4 Hydrodistillation

114 Stems, flowers and leaves of R. eriocalyx (200 g in all cases) were cut in small pieces, then

115 separately hydrodistilled in a Clevenger-type apparatus using 3.5 l of deionized water. The yellow-

116 colored essential oils were dried over anhydrous sodium sulfate to remove water drops, then filtered

117 and stored in sealed vials protected from the light at −20 °C before chemical and biological

118 investigations. The essential oil yields were calculated on a dry weight basis (w/w).

119 2.5. HPLC-DAD Analysis

120 2.5.1. Analysis of polyphenols and chlorogenic acids

121 HPLC-DAD studies were performed using a Hewlett-Packard HP-1090 Series II (Palo Alto, CA,

122 USA), equipped with a vacuum degasser, a binary pump, an autosampler and a model 1046A HP

123 photodiode array detector (DAD). The chromatographic separation was accomplished on a Synergi

124 Polar-RP C18 (4.6 mm x 250 mm, 4 µm) analytical column from Phenomenex (Chesire, UK). The

125 column was preceded by a security cartridge. The mobile phase for HPLC-DAD (diode array

126 detector) analyses was a mixture of (A) water with 0.1% formic acid (v/v) and (B) acetonitrile with

127 0.1% formic acid, flowing at 0.8 ml/min in gradient conditions: 0 min, 20% B; 0-15 min, 60 % B;

128 15-25 min, 75% B; 25-30 min, 75 % B, 30-35 min, 20%B. The column temperature was set at 30

129 °C and the injection volume was 5 µl. UV spectra were recorded in the range 230-350 nm for the 13

5
130 compounds, where 230 nm was used for quantification of shikimic acid, 256 nm for rutin,

131 hyperoside and quercitrin, 272 nm for gallic acid, 280 nm for catechin hydrate, epicatechin,

132 carnosic acid and carnosol, 325 for chlorogenic acid, neochlorogenic acid and 3,5-di-O-

133 caffeoylquinic acid and rosmarinic acid.

134 2.6. GC-MS Analysis

135 Gas chromatographic analysis of R. eriocalyx essential oils was performed on an Agilent 6890N gas

136 chromatograph equipped with a 5973N mass spectrometer. For separation of volatiles a HP-5 MS

137 (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 µm film thickness; J & W Scientific,

138 Folsom) capillary column was used with the following temperature programme: 5 min at 60 °C,

139 then 4 °C/min to 220°C, then 11 °C/min to 280 °C, held for 15 min, for a total run of 65 min.

140 Injector and detector temperatures: 280 °C; carrier gas: He; flow rate: 1 mL/min; split ratio: 1:50;

141 acquisition range: 29–400 m/z in electron-impact (EI) mode; ionization voltage: 70 eV. The

142 essential oils were diluted 1:100 in n-hexane, then 2 µl were injected into GC-MS. For

143 identification of the essential oil components co-injection with commercial standards (see section

144 2.2.) was used whenever possible, together with correspondence of retention indices (RIs) and mass

145 spectra (MS) with respect to those reported in commercial libraries (Adams, 2007; NIST 08, 2008;

146 FFNSC2, 2012). Semi-quantification of essential oil components was made by peak area

147 normalisation considering the same GC response of the detector towards all volatile constituents.

148 2.7. Evaluation of Antioxidant activity

149 The extracts and the essential oils were tested for their in vitro antioxidant activity using the

150 standard methods.

151 2.7.1. Total phenols content

152 Total phenols quantification on the polar extracts was perfomed following the Folin–Ciocalteu

153 method using a microplate assay as reported by Esparza Rivera et al. (2006). The total phenolic

154 content was expressed as gallic acid equivalents/g product.

155 2.7.2. Free radical scavenging activity (DPPH assay)

6
156 Free radical scavenging activity (DPPH assay) of R. eriocalyx extracts and essential oils were

157 assessed through a microplate analytical assay according to the procedure described by Srinivasan,

158 Chandrasekar, Nanjan, & Suresh (2007). A 50 µl aliquot of the different concentrations of samples

159 (oil 10 mg/ml; extracts: 3 mg/ml) and standards at various concentrations were added to 200 µl of

160 DPPH in a 96-well microtitre plate (Tarson Products (P) Ltd., Kolkota, India). Appropriate blanks

161 were prepared using the solvent in addition to the DPPH reagent to get rid of any inherent solvent

162 activity. After incubation at 37 °C for 20 min, the absorbance of each solution was determined at

163 517 nm using using a SPECTROstar Omega (BMG LABTECH GmbH, Ortenberg, Germany)

164 microplate reader. The free radical-scavenging activity of each solution was then calculated as

165 percent inhibition according to the following equation:

166 % inhibition = 100 (A(blank)-A(sample)) /A(blank)

167 Antioxidant activity of the samples was expressed as IC50, defined as the concentration of the test

168 material required to cause a 50% decrease in initial DPPH concentration. Trolox was used as

169 reference. Results were expressed in µmol

mol TE (Trolox equivalent)/g extract or essential oil.

170 All data of antioxidant activity were expressed as means ± standard deviations (SD) of triplicate

171 measurements. The confidence limits were set at p< 0.05. SD did not exceed 5% for the majority of

172 the values obtained.

173 2.7.3. Total radical scavenging capacity ABTS assay

174 The free radical-scavenging activity was determined by ABTS radical cation decolorization assay

175 (Re, Pellegrini, Proteggente, Pannala, Yang & Rice-Evans, 1999), ABTS solution was prepared by

176 mixing 10 mg/4 ml nanopure H2O of ABTS with 0.5g MnO2 and allowing the mixture to stand in

177 the dark at room temperature for 30 min before use. After incubation, the solution was filtered and

178 it was stable in this form for more than two days when stored in the dark at 4 °C room temperature.

179 Working solution was prepared by filtration and diluting the ABTS solution with MeOH to obtain

180 the absorbance of AU 1±0.03 at 734 nm. A 50 µl aliquot of the different concentrations of samples

181 (oil: 10 mg/ml; extracts: 3 mg/ml) and standards at various concentrations were added to 200 µl of

7
182 ABTS in a 96-well microtitre plate. After 20 min at ambient temperature, absorbance was measured

183 at 734 nm using a SPECTROstar Omega (BMG LABTECH GmbH, Ortenberg, Germany)

184 microplate reader. The radical scavenging activity was estimated by the decrease of absorbance and

185 expressed as the Trolox equivalent (TEAC) ABTS per ml of essential oil or component.

186 I% = ((Abs blank-Abs sample)/Abs blank)*100;

187 Where: A blank = is the absorbance of the control reaction at t=0 (containing all reagents except the

188 test compound), and A sample: is the absorbance of the test compound after 45 min. The results

189 were expressed in in µmol

mol TE/g extract or essential oil. All determinations were performed in

190 triplicate.

191 2.7.4. Ferric reducing antioxidant power (FRAP)

192 Total antioxidant activity (TAA) of oils and extracts were investigated using the ferric reducing

193 antioxidant power (FRAP) assay which is based on the reduction of Fe3+–TPTZ complex, to the

194 blue colored ferrous form, with an increase in absorbance at 593 nm (Müller, Fröhlich & Böhm,

195 2011), FRAP reagent was freshly prepared by mixing 25 ml of acetate buffer (300 mM, pH 3.6), 2.5

196 ml TPTZ solution (10 mM TPTZ (Fluka) in 40 mmol/l HCl) and 2.5 ml of 20 mM FeCl3 water

197 solution in the ratio of 10:1:1 at the time of use. The FRAP reagent was warmed at 37 °C before

198 use. Then we added 50 µl of each sample, blank and standard (Trolox) in tubes at various

199 concentrations to 200 µl of FRAP reagent, that were stirred and, after incubation for 30 min,

200 centrifuged at 5000 rpm for 5 min. Aliquots of 200 µL of each sample, blank and standard were

201 added to each well of the 96-well microplate. Absorbance was measured using plate reader at 595

202 nm. A reference solution of Trolox was run simultaneously and used to generate the calibration

203 curve by linear regression. Results were expressed in µmol

mol TE/g extract or essential oil. Additional

204 dilution was needed if the FRAP value measured was over the linear range of the standard curve.

205 For each sample, experiments were carried out in triplicate.

206 2.7.5. ORAC assay

8
207 The capacity of extracts and essential oils from R. eriocalyx to scavenge peroxyl radicals generated

208 by spontaneous decomposition of 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) was

209 estimated in terms of Trolox Equivalents, using the oxygen radical absorbance capacity (ORAC)

210 assay (Dominguez, Nieto, Marin, Keck, Jeffery & Céspedes, 2005). Fluorescein (FL) was used as

211 the fluorescent probe. Diluted extracts and essential oils samples were placed in a microplate (96

212 well treated plate) and 180 µl of a 10 nM solution of fluorescein (in 10 mM phosphate buffer pH

213 7.4) were added in the well. The microplates were incubated at 37 °C for 30 min without shaking.

214 After incubation, fluorescence measurements (Ex. 485 nm, Em. 520 nm) were taken every 90 sec to

215 determine the background signal with a spectrometer FLUOstar OPTIMA (BMG LABTECH,

216 Offenburg, Germany). After 3 cycles, 25 µl (240 mM) of AAPH were added manually with a multi-

217 channel-pipette. The test was resumed and fluorescent measurements were taken up to 90 min. The

218 decrease of fluorescence over time was quantified as area under the curve (AUC). The samples

219 were measured in triplicate. A calibration curve was prepared using Trolox at different

220 concentrations, with Trolox solution added in the well to give a final concentration in the range

221 12.5–100 µM. This curve was specific for the assay of the samples. ORAC values were calculated

222 following a previous work (Prior et al., 2003). Briefly, the net area under the curve (AUC) of the

223 standards and samples was calculated. The standard curve was obtained by plotting Trolox

224 concentrations against the average net AUC of the two measurements for each concentration. Final

225 ORAC values, expressed as micromole Trolox equivalent per g of sample, were calculated using the

226 regression equation between Trolox concentration and the net AUC. Data were analyzed by

227 Graphpad Prism, Vers. 5 (Graphpad Software, San Diego, CA, USA).

228 2.8. Antimicrobial activity

229 Antimicrobial activity of extracts and essential oils was assayed by disc diffusion test. The panel of

230 microorganisms included Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922,

231 Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Candida

232 albicans ATCC 24433 (American Type Culture Collection, Rockville, MD, USA). Bacterial strains

9
233 were cultured overnight at 37°C on Tryptone Soya Agar (TSA). C. albicans was grown on

234 Sabouraud Dextrose Agar (SDA). Tests were performed by disc diffusion following the Clinical

235 and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2009). Briefly, cells were resuspended

236 in saline (1–2·108 cells/mL for bacteria - 106 cells/mL for the yeast) and spread on the solid media

237 plates using a sterile cotton swab. Sterile paper discs (6 mm in diameter) were placed on the surface

238 of inoculated plates and spotted with 10 µl of the essential oil or extract (1 mg/ml in proper

239 solvents). The plates were incubated 24 h at 35 ± 1 °C (48 h for C. albicans). The activity was

240 determined by measuring the diameter of the growth inhibition zone (inhibition zone diameter, IZD)

241 visible around the paper disc with a caliper. Reported IZDs are inclusive of the paper disc diameter

242 (6 mm). More than 6 mm indicated growth inhibition while 6 mm mean no activity. No zone

243 inhibition was observed using the sole solvent, namely ethanol or water. Positive controls were set

244 using ciprofloxacin (5 µg disc) against S. aureus and E. faecalis, gentamicin (10 µg disc) against P.

245 aeruginosa and E. coli, and Nystatin (100 Units disc) against the yeast C. albicans.

246

247 3. Results and Discussion

248 3.1. HPLC-DAD Analysis of polar compounds

249 The acqueous and ethanolic extracts were obtained from different parts of R. eriocalyx, i.e. flowers,

250 leaves and stems. The highest number of monitored compounds was found in the ethanolic extracts

251 from flowers and stems (13 compounds) followed by leaves (11 analytes), meanwhile the acqueous

252 extracts contained a lower number of target compounds (10, 9 and 7 compounds found in flowers,

253 leaves and stems, respectively) (Table 1, Fig. 1).

254 A higher content of total polyphenols was found in the ethanolic extracts (range 64903-89842

255 mg/kg), with respect to the acqueous ones (range 2661-4316 mg/kg). In this regard, with a total

256 content of 89842 mg/kg the leaves were the richest part, followed by flowers (73758 mg/kg) and

257 stems (64903 mg/kg). On the other hand, aqueous extracts of R. eriocalyx showed a significant

10
258 lower level of polyphenols in all parts, ranging from 17 (flowers) to 24 (stems) folds lower than the

259 respective ethanolic extracts.

260 The major compound present in the ethanolic extracts was rosmarinic acid (range 27811-43359

261 mg/kg) (Fig. 1), that instead was present in poor amounts in the water extracts (n.d.- 62.2 mg/kg).

262 The same trend is showed by carnosic acid (15515-29177 mg/kg in ethanolic extracts vs n.d.-1294

263 mg/kg in aqueous extracts) and carnosol (546-29770 mg/kg in ethanolic extracts vs n.d.-184 mg/kg

264 in acqueous extracts).

265 Leaves were the richest part in terms of rosmarinic acid, carnosic acid and carnosol, with a total

266 amount of these compounds of 86757 mg/kg in the ethanolic extract. These compounds are

267 considered hallmarks of rosemary (R. officinalis) that is used in the European Union as an

268 antioxidant (E 392) in foodstuffs (EC Regulation, 2012). Notably, the European Regulation on

269 Food Additives indicates as quality requirement a total content of carnosic acid and carnosol at least

270 equal or above 5%. Our results showed that the extract from leaves of R. eriocalyx contains 58946

271 mg/kg of carnosic acid/carnosol (corresponding to 5.89% w/w). Therefore, they can be used in

272 foodstuffs as an alternative to R. officinalis as a source of antioxidant compounds. Parallely, these

273 values are consistent with those requested for the content of hydroxycinnamic acids by the

274 European Pharmacopoeia for ‘Rosmarini folium’ (European Pharmacopeia, 2014).

275 Also in ethanolic extracts from flowers the levels of rosmarinic acid, carnosol and carnosic acid

276 were noteworthy (61941 mg/kg). The etanolic extract from stems showed the highest level in

277 rosmarinic acid (43359 mg/kg), but the poorest in carnosic acid (2052 mg/kg) and carnosol (546

278 mg/kg). On the other side, stems acqueous extract did not contain all of these marker compounds.

279 Carnosic acid, carnosol and rosmarinic acid are phenolic antioxidants that are commonly used to

280 improve meat preservation (Ortuño, Serrano Bañón, 2015). Thus, the high concentrations of

281 these constituents may support the use of R. eriocalyx as a natural preservative in foodstuffs.

282 Concerning the family of chlorogenic acids, 3-caffeoylquinic acid (chlorogenic acid) was the most

283 abundant, mainly in the ethanolic extract of stems (10351 mg/kg) and flowers (7823 mg/kg); in

11
284 leaves it was present in scant amounts (335 mg/kg in ethanolic extract and 713 mg/kg in aqueous

285 extract). Recently, cholorogenic acid was proven to be a potential feed additive protecting shrimp

286 from oxidative stress (Wang et al., 2015).

287 Gallic acid was identified in all extracts and it was detected in higher level in the ethanolic extracts

288 of stems (2177 mg/kg) and leaves (1526 mg/kg). This compound was shown as an enhancer of the

289 antioxidative potential, and nutritional and functional qualities of broiler breast meat (Jung, Choe,

290 Kim, Yun, Kruk & Jo, 2010).

291 Also catechin and epicatechin were more abundant in the ethanolic extracts; flowers contained the

292 highest level of the former (1432 mg/kg), while stems were the richest in the latter (2843 mg/kg).

293 Instead, they were completely missing in the aqueous extract of stems. Interestingly, catechin is

294 used in active packaging, incorporated in PVA-starch film, to improve the food shelf life through its

295 antioxidant and antimicrobial activity (Wu, Wang & Chen, 2010).

296 Finally, shikimic acid was identified in all samples but it was found in higher amount in the

297 aqueous extracts (1452-1853 mg/kg). Quercitrin was not found in leaves (both acqueous and

298 ethanolic extracts), meanwhile rutin was missing in all aqueous extracts.

299 To the best of our knowledge, studies on the qualitative and quantitative determination of phenolic

300 compounds in R. eriocalyx have never been reported so far. Most of literature concerns the

301 quantitative determination of polyphenols in R. officinalis. In this regard, Del Baño et al. (2003)

302 analyzed six polyphenols of R. officinalis including rosmarinic acid, carnosol and carnosic acid,

303 finding rosmarinic acid (700-84600 mg/kg) as the most abundant compound in all plant organs.

304 This is in agreement with our result, although the level of these phenolic components in R. eriocalyx

305 were lower. Teruel, Garrido, Espinosa & Linares (2015) analyzed carnosol and carnosic acid in

306 different extracts of R. officinalis (powder-acetone, liquid-methanol, liquid-acetone) from Spain and

307 they found higher levels (46530-179160 mg/kg of carnosic acid and 5840-28880 mg/kg of carnosol)

308 than those detected in our samples.

309 3.2. Essential oil analysis

12
310 Hydrodistillation of various parts of R. eriocalyx gave different essential oil yields on a dry weight

311 basis. The richest part was represented by flowers (0.89%), followed by leaves (0.73%), while

312 stems exhibited a modest essential oil content (0.02%). These values are consistent with those

313 reported for high-quality batches of R. officinalis (Gurbuz, Bagdat, Uyanik & Rezaeieh, 2016),

314 although slightly lower than those previously reported for R. eriocalyx growing in Algeria (Arnold

315 et al., 1997).

316 The composition of the essential oil hydrodistilled from stems, leaves and flowers of R. eriocalyx is

317 reported in Table 2. Qualitatively, stems were the richest part, with 116 components identified,

318 whereas leaves were the poorest, with 57 components found; in the flowers 73 components were

319 identified. These numbers were significantly higher with respect to those reported in previous

320 studies on R. eriocalyx from Spain, Morocco and Algeria (Soriano Cano et al., 1993; Dahmane,

321 Eddarir, Aubert, Bouamama & Taourirte 2010; Fadel et al., 2011; Bendeddouche et al., 2011;

322 Arnold et al., 1997; Bembelaïd et al., 2016) where the total of identified compounds ranged from a

323 minimum of 13 to a maximum of 36. We assume that this relevant difference might depend on the

324 higher sensitivity of instruments employed, together with the combinatory use of up-to-date MS and

325 RI libraries.

326 Overall, the essential oils from the three plant parts were characterized by the monoterpene fraction.

327 Oxygenated monoterpenes were the major group in the essential oil of stems and leaves, accounting

328 for 57.7 and 53.4% of the total composition, respectively, followed by monoterpene hydrocarbons

329 (26.2 and 44.0%, respectively). Sesquiterpenes were poorer in these parts, with oxygen-containing

330 compounds (8.6 and 1.3%, respectively,) more abundant than hydrocarbons (2.0 and 0.8%,

331 respectively). Flowers showed a slightly different volatile profile. With respect to leaves and stems

332 they showed monoterpene hydrocarbons (42.4%) more abundant than oxygenated monoterpenes

333 (37.4%), and higher levels of sesquiterpen hydrocarbons (11.6%).

334 Overall, the major volatile components (relative percentage > 10%) in the three parts were

335 monoterpenoids such as camphor (29.7% in flowers, 36.9% in leaves and 41.2% in stems), α -pinene

336 (7.8% in stems, 15.1% in flowers and 17.8% in leaves), camphene (10.0% in stems, 13.1% in

13
337 flowers and 15.6% in leaves) and 1,8-cineole (3.5% in flowers, 5.8% in stems and 10.2% in leaves).

338 Other minor monoterpenes were represented by β -pinene (0.7% in leaves, 2.2% in stems and 7.7%

339 in flowers), limonene (2.7% in stems, 3.0% in flowers and 4.1% in leaves) and borneol (1.7% in

340 leaves, 1.8% in flowers and 4.2% in stems). Finally, (E)-caryophyllene and epi-α -bisabolol were the

341 most representative compounds among sesquiterpenoids, accounting for 4.6 and 5.1% in flowers,

342 respectively.

343 The reported chemical profile for different parts of R. eriocalyx growing in Algeria was quite

344 consistent with those previoysly reported in literature, that are relative to populations growing in

345 Spain (Soriano Cano et al., 1993), Morocco (Dahmane et al., 2010; Fadel et al., 2011) and Algeria

346 itself (Arnold et al., 1997; Bendeddouche et al., 2011; Benbelaïd et al., 2016). Also in these works

347 the major essential oil constituents were the monoterpene ketone camphor that ranged from 17.3%

348 in samples from Morocco (Dahmane et al., 2010) to 37.8% in samples from Algeria (Benbelaïd et

349 al., 2016), the hydrocarbons camphene that ranged from 13.3% (Benbelaïd et al., 2016) to 20.0%

350 (Arnold et al., 1997) in both samples from Algeria, and α -pinene that ranged from 12.4% in samples

351 from Spain (Soriano Cano et al., 1993) to 18.2% in samples from Algeria (Arnold et al., 1997), and

352 the oxide 1,8-cineole that ranged from 6.5% in samples from Morocco (Fadel et al., 2011) to 13.5%

353 in samples from Spain (Soriano Cano et al., 1993). Concerning the essential oil composition of

354 Algerian populations previously investigated, the work of Bendeddouche et al. (2011) showed a

355 slightly different profile, being rich of p-cymene-7-ol (7.8%) and poor of camphene (0.7%) and α -

356 pinene (1.8%).

357 On the above, the essential oil of R. eriocalyx can be classified into the camphor chemotype that is

358 the only one recognized for this species so far. On the other hand, different chemotypes are already

359 recognized for R. officinalis on the basis of the most abundant component among α -pinene, 1,8-

360 cineole, camphor and verbenone (Flamini et al., 2002).

361 Camphor is a flavouring substance for use in foodstuffs (Category B) in the Blue Book, Volume I

362 (Council of Europe, 1992) and is listed in the EU-Register of chemically-defined flavouring

363 substances (EEC, 1999). In the USA, this compound is recognized as GRAS by FEMA and is

14
364 approved by the FDA for food use. Camphor oil has also GRAS status. Recently, the EFSA Panel

365 on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food was asked to

366 advise on the implications for human health of the presence of camphor in the diet (European Food

367 Safety Authority, 2008). The Panel concluded that there is no safety concern regarding chronic

368 toxicity and that it is unlikely that acute effects may occur in relation to consumption of foods.

369 Finally, the Panel suggested that maximum limits should be set to ensure that exposure to camphor

370 does not exceed 2 mg/kg bw on a single day in any age group.

371 3.3. Antioxidant activity

372 Essential oils and extracts of aromatic plants are known to have antioxidant properties and this

373 offers the possibility of using these products as natural preservatives for food and cosmetics

374 (Bakkali, Averbeck, Averbeck & Idaomar, 2008). In the present study we investigated the

375 antioxidant activity of essential oils, ethanolic and water extracts of R. eriocalyx and results are

376 reported in Tables 3 and 4. The essential oils showed moderate antioxidant activity as reported in all

377 the assays where Trolox was used as reference (Table 3). The antioxidant activity of essential oil

378 from stems resulted three times higher than the other two oils examined (stems>leaves >flowers)

379 and about 230 and 800 times lower than that of Trolox in ABTS and DPPH assay, respectively.

380 Comparing the antioxidant activity measured by DPPH radical scavenging and ferric reducing

381 power (FRAP), all the essential oils exhibited similar trends in both methods and showed minor

382 reducing activity. From data reported in Table 1 major components as camphor, 1-8-cineole and α-

383 pinene represent more than 55% of the total content and earlier studies on 1,8-cineole/camphor/α-

384 pinene-type rosemary oils revealed stronger radical scavenging activity than that found in our

385 samples (Erkan, Ayranci & Ayranci, 2008). The greater antioxidant activity observed in different

386 rosemary essential oils (Sacchetti et al., 2005; Wang, Wu, Zu & Fu, 2008) could be attributed also

387 to other compounds present or to synergistic effects between oil components. As a matter of fact,

388 Dawidowicz & Olszowy (2014) reported that in general isolated essential oil components have

389 lower antioxidant activity than the whole essential oil, suggesting that the antioxidant properties are

390 significantly affected not only by the major constituents but also by other minor components.

15
391 In the ORAC method, a hydrogen atom transfer reaction-based assay, the R. eriocalyx essential oils

392 revealed to be effective as chain breaking antioxidants, showing TEAC values in the range 562.1-

393 680.1 µmol

mol TE/g
TE/g (Table 3). These results may be justified by the presence of 1,8-cineole and α -

394 pinene, that are effective scavengers against AAPH-derived peroxyl radicals (found commonly in

395 lipid peroxidation processes) (Porres-Martiínez, González-Burgos, Carretero & Gómez-Serranillos,

396 2015).

397 Recent researches have shown that rosemary extracts have a variety of pharmacological activities,

398 such as antioxidant, antimicrobial, cancer chemoprevention, anti-diabetic, hepatoprotective. Thus,

399 rosemary is considered as one of the most effective herbs for treating headaches, poor circulation,

400 and inflammatory diseases (Rocha et al., 2015). In our work we evaluated the antioxidant activity of

401 R. eriocalyx water and ethanol extracts and the results are reported in Table 4. In general, the

402 antioxidant activity of plant extracts is due primarily to phenolic compounds and in rosemary

403 extracts, the presence of different groups of phenolics such as diterpenoids, flavonoids and phenolic

404 acids are responsable for the antioxidant properties observed. The major components of R. eriocalyx

405 extracts, i.e. the diterpenes carnosic acid and carnosol, and the hydroxycinnamic acid ester

406 rosmarinic acid are considered the most important antioxidant compounds (Troncoso, Sierra,

407 Carvajal, Delpiano & Gunther, 2005). The total phenolic content (TPC) (expressed as gallic acid

408 equivalents) is often used as an approximately measurement of the antioxidant power of a plant

409 extract. The TPC values reported for R. eriocalyx aqueous and ethanol extracts, which were

410 determined by the Folin–Ciocalteu assay, are reported in Table 4. From data, we can observe slight

411 differences in TPC between ethanol and water extracts, with the former exhibting higher values.

412 Comparing different parts, the leaves showed the highest TPC among ethanolic extracts (57.96 mg

413 GAeq/g), while flowers were the richest among aqueous extracts (37.05 mg GAeq/g). These results

414 are consistent with the concentrations of the major antioxidant compounds determined by HPLC

415 (Table 1). The DPPH, ABTS+ and FRAP assays have been widely used to evaluate the free radical

416 scavenging capacity of extracts. All the three methods proved that R. eriocalyx extracts had a higher

16
417 antioxidant activity than essential oils (Tables 3-4). The ethanolic extracts showed stronger free

418 radical scavenging effectiveness than water extracts and both were about 30/40 times more active

419 than essential oils in all the assays. The ethanolic extracts were about 6 times less active, while

420 aqueous extracts were about 24 times weaker than Trolox in the scavenging activity (Table 4).

421 Recently, in vivo studies indicated the effectiveness of treatments with rosemary extracts, as a way

422 to eliminate the free radicals generated, reinforce the antioxidant system and prevent oxidative

423 stress (Zohrabi, Ashtiyani, Hajihashemi, Akbar Hassanpoor & Hosseini, 2012). Rosemary extract

424 can protect against histological injury and functional impairment due to its ability to decrease

425 malondialdehyde (MDA) and increase the ferric reducing antioxidant power (FRAP) (Zohrabi et al.,

426 2012). In our work, the two types of extracts showed a very high reducent power with ethanolic

427 extracts (2561.2-6146.2 µmol

mol TE/g) being 4 times more effective than aqueous extracts (1278.5-

428 1704.7 µmol

mol TE/g) (Table 4). The FRAP values obtained with R. eriocalyx extracts were better than

429 those obtained by Ibarra et al. (2010) on different R. officinalis leaf extracts. Notably the same

430 authors reported that rosmarinic acid had higher antioxidant capacity on FRAP than carnosic acid.

431 From Table 1 we can observe that the ethanolic extracts of R. eriocalyx contained high quantity of

432 both compounds and this can explain the higher FRAP activity observed. Aqueous extracts were

433 characterized by low concentrations of rosmarinic acid, but contained appreciable values of

434 carnosic acid that in this extract may contribute to the ferric reducing power observed.

435 In the ORAC assay, both ethanolic and aqueous extracts of R. eriocalyx revealed to be more

436 effective as chain breaking antioxidants than the essential oils samples (Table 4). Notably, the

437 aqueous and ethanolic extracts from stems exhibited the highest ctivity, with TEAC values of

438 2078.5 and 2045.5 µmol

mol TE/g,
TE/g, respectively.
respectively. On
On the
the other
other hand,
hand, ethanolic extracts from
ethanolic extracts from flowers
flowers and
and

439 leaves (TEAC values of 1642.5 and 1493.9 µmol

mol TE/g,
TE/g, respectively) were more active than the

440 aqueous ones (TEAC values of 803.7 and 1106.0 µmol

mol TE/g,
TE/g, respectively).
respectively). It is important to note

441 that when comparing our results with those obtained from R. officinalis extracts using the ORAC

442 assay (Ibarra et al., 2010; Piedrahita, Peñaloza, Cogollo & Rojano, 2015), we observed a slightly

17
443 lower antioxidant capacity, thus confirming the exploitation of R. eriocalyx as a preservative agent

444 in foodstuffs.

445 Overall, the R. eriocalyx polar extracts seem to act as antioxidants by donating a hydrogen atom or

446 an electron. Such results were already expected because of the major components such as

447 rosmarinic acid, carnosic acid and carnosol that are endowed with high antioxidant capacity (Rocha

448 et al., 2015; Piedrahita et al., 2015). However, also the contribution to the overal activity by other

449 and minor constituents occurring in the extracts may be taken into account as showed by the

450 noteworthy antioxidant capacity displayed by the stem aqueous extract.

451 3.4. Antimicrobial activity

452 In this study, we screened the antimicrobial activity of the essential oils and extracts of R. eriocalyx

453 against four bacterial strains belonging to four different species and against one yeast strain by disk

454 diffusion. Table 5 summarizes the results. Discs loaded with standard antibiotics ciprofloxacin,

455 gentamicin and nystatin were used for comparison.

456 Overall, the results indicated that 10 µl of 10 mg/ml extracts and 10 µl of pure oil had not a

457 considerable antimicrobial activity. Essential oils from the flowers and leaves exhibited a very low

458 activity against S. aureus, E. faecalis, E. coli, and C. albicans (Table 5), while they did not affect

459 the growth of P. aeruginosa. Overall, the essential oils showed the highest activity against C.

460 albicans (7 to 10 mm). In the experimental conditions used, ethanolic extracts were not very much

461 active (IZD = 7.3-9 mm) and independent of the part of the plant they were extracted from.

462 Conversely, E. faecalis and E. coli were not susceptible at all. Aqueous extracts were not active

463 against all the microbial species tested, including the yeast C. albicans.

464 The antimicrobial activities of the ethanolic extracts of R. eriocalyx could be due to its greater

465 complexity (Table 1). Overall, the inhibition zones were fairly lower than those obtained by

466 reference antibiotics. Our results on various extracts from R. eriocalyx show that the measured low

467 activity is in agreement with the findings by Dahmane et al. (2010), but not with those by Benbelaïd

468 et al. (2016). Previous investigations on R. officinalis extracts by Gachkar, Yadegari, Rezaei,

18
469 Taghizadeh, Astaneh & Rasooli (2007) also concluded on a similarly low antimicrobial activity

470 against the same bacterial species. The apparent discrepancy among results by different works is not

471 uncommon and may be ascribed to many variables, such as the method of extraction from the plant,

472 the amount and volume of extract spotted onto the disc, the incubation time (Marzouk et al., 2006),

473 and the specific strains employed to assess susceptibility.

474 4. Conclusions

475 R. eriocalyx ethanolic extracts are a rich source of polyphenols, especially carnosic acid, carnosol

476 and rosmarinic acid, that are phytochemicals endowed with important biological activities. Their

477 abundance explained the relevant antioxidant activities displayed by ethanolic extracts and may

478 justify a future inclusion of this species in the European list of natural additives to be used in

479 foodstuffs for their preservative properties.

480

481 Aknowledgements

482 This work was financed by the FAR 2014-2015 (FPI000044, Fondo di Ateneo per la Ricerca,) of

483 the University of Camerino.

484

485 Conflict of Interest

486 The authors declare that there are no conflicts of interest.

487

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617

24
Figure 1

mAU 8
250 A)
2
200

150

9
100 7
6
3 13
12
10
50 11
4
5
1
0

5 10 15 20 25 30 35 min
mAU 11

B)
400

350

300

250

200

150
13
8
100 9 10
67
3
50 2 4 12
1 5
0

5 10 15 20 25 30 35 min
mAU 11
700
C)
600

500

400 3

300
7 8
200 12
4 6 9 13
10
5
100 2
1
0
5 10 15 20 25 30 35 min
mAU
500 D)
11

400

300

200

4 9
100 2 6 7
3 12 13
8 10
1
5
0

5 10 15 20 25 30 35 min
Figure 1. HPLC-DAD chromatograms reported only at 280 nm for
sake of clarity and corresponding to A) standard mixture solution at
concentration of 25 mg/l containing the analytes investigated, B)
leaves ethanolic extract, C) stems ethanolic extract, D) flowers
ethanolic extract. List of compounds: 1 shikimic acid, 2 gallic acid, 3
5-O-caffeoylquinic acid, 4 3-O-caffeoylquinic acid, 5 catechin, 6
epicatechin, 7 rutin, 8 hyperoside, 9 quercitrin, 10 3,5-di-O-
caffeoylquinic acid, 11 rosmarinic acid, 12 carnosic acid, 13
carnosol.
618 Table 1. Chemical constituents of ethanolic and aqueous extracts from different parts of Rosmarinus eriocalyx
619 growing in Algeria as determined by HPLC-DAD.

Extracts (mg/kg DW)

RT
Compound Flowers Leaves Stems
(min)
ETOH Water ETOH Water ETOH Water
shikimic acid 3.4 292±11.8 1680±80.3 53±4.1 1452±70.8 121±10.6 1853±354.1
gallic acid 4.9 68±6.3 72±5.4 1526±2.2 200±22.4 2177±40.3 75±9.3
5-O-caffeoylquinic acid 5.3 467±6.4 n.d. 283±3.5 70±6.4 395±8.1 19±5.3
3-O-caffeoylquinic acid 6.7 7823±175.4 n.d. 335±5.3 713±12.8 10351±32.4 62±7.4
catechin 7.5 1432±35.9 1053±17.9 355±35.9 n.d. 723±17.9 n.d.
epicatechin 7.9 878±83.2 282±3.5 141±8.7 201±6.9 2843±69.3 n.d.
rutin 8.2 424±5.9 n.d. 315±5.9 n.d. 1107±35.6 n.d.
hyperoside 9.2 255±2.1 99±28.9 78±2.8 173±19.3 779±9.6 241±2.5
quercitrin 10.3 95±7.1 56±0.0 n.d. n.d. 175±19.6 81±17.3
3,5-di-O-caffeoylquinic acid 10.8 84±2.4 61±8.5 n.d. n.d. 276±8.2 329±17.6
rosmarinic acid 11.8 32002±772.9 26±3.2 27811±278.5 62±5.7 43359±92.8 n.d.
carnosic acid 23.0 15515±727.9 779±187.2 29177±291.2 1294±83.2 2052±197.6 n.d.
carnosol 23.5 14424±255.9 184±9.3 29770±46.5 151±0.0 546±18.6 n.d.
Total 73758 4292 89842 4316 64903 2661
620 Results are expressed as mean ± standard deviations (SD) of three determinations; n.d., not detected.

621

25
622 Table 2. Composition of the essential oil from stems, leaves and flowers of Rosmarinus eriocalyx growing in Algeria.

%d IDe
a b c
N. Component RI exp. RI lit.
Stems Leaves Flowers

1 (2E)-hexenal 850 846 trf tr tr RI,MS

2 n-hexanol 863 863 tr RI,MS

3 n-heptanal 903 901 tr RI,MS

4 tricyclene 916 921 0.4 0.7 0.6 RI,MS

5 α -thujene 921 924 tr tr RI,MS

6 α -pinene 926 932 7.8 17.8 15.1 Std

7 camphene 939 946 10.0 15.6 13.1 Std

8 thuja-2,4(10)-diene 945 953 tr tr RI,MS

9 benzaldehyde 954 952 tr Std

10 n-heptanol 965 959 tr RI,MS

11 β -pinene 968 974 2.2 0.7 7.7 Std

12 1-octen-3-ol 976 974 0.1 0.2 Std

13 3-octanone 986 979 tr RI,MS

14 myrcene 989 988 0.3 0.6 0.4 Std

15 α -phellandrene 1003 1002 0.1 0.3 0.3 Std

16 δ -3-carene 1008 1008 tr tr Std

17 (2E,4E)-heptadienal 1013 1005 tr RI,MS

18 α -terpinene 1014 1014 0.2 0.4 0.5 Std

19 p-cymene 1022 1020 1.9 2.7 0.8 Std

20 limonene 1025 1024 2.7 4.1 3.0 Std

21 1,8-cineole 1027 1026 5.8 10.2 3.5 Std

22 (Z)-β -ocimene 1037 1032 0.1 0.3 tr RI,MS

23 benzene acetaldehyde 1042 1036 0.2 tr RI,MS

24 (E)-β -ocimene 1047 1044 tr 0.1 RI,MS

25 γ -terpinene 1055 1054 0.2 0.3 0.7 Std

26 (2E)-octen-1-al 1058 1049 tr RI,MS

27 cis-sabinene hydrate 1064 1065 tr RI,MS

28 acetophenone 1065 1059 tr RI,MS

26
29 n-octanol 1072 1063 0.2 RI,MS

30 camphenilone 1081 1078 tr RI,MS

31 terpinolene 1085 1086 0.2 0.2 0.3 Std

32 p-cymenene 1086 1089 0.1 0.1 tr RI,MS

33 6-camphenone 1089 1095 tr RI,MS

34 trans-sabinene hydrate 1096 1098 tr RI,MS

35 linalool 1100 1095 0.1 0.1 tr Std

36 n-nonanal 1105 1100 0.2 RI,MS

37 endo-fenchol 1110 1114 0.1 0.1 0.1 RI,MS

38 cis-p-menth-2-en-1-ol 1119 1118 0.1 RI,MS

39 α -campholenal 1123 1122 0.1 0.1 tr RI,MS

40 limona ketone 1130 1137 tr tr RI,MS

41 trans-pinocarveol 1134 1135 0.4 tr Std

42 camphor 1139 1141 41.2 36.9 29.7 Std

43 camphene hydrate 1142 1145 0.4 0.2 0.1 RI,MS

44 isoborneol 1151 1155 tr Std

45 trans-pinocamphone 1156 1158 tr RI,MS

46 pinocarvone 1158 1160 0.2 tr tr RI,MS

47 borneol 1160 1165 4.2 1.7 1.8 Std

48 δ -terpineol 1164 1162 0.1 0.1 tr RI,MS

49 cis-pinocamphone 1170 1172 0.1 RI,MS

50 terpinen-4-ol 1173 1177 1.9 1.5 0.6 Std

51 p-methyl-acetophenone 1182 1179 tr RI,MS

52 p-cymen-8-ol 1183 1179 0.1 0.1 tr RI,MS

53 trans-p-mentha-1(7),8-dien-2-ol 1184 1187 tr RI,MS

54 α -terpineol 1187 1188 1.4 2.0 0.4 Std

55 myrtenol 1192 1194 0.3 tr tr Std

56 n-decanal 1206 1201 0.1 Std

57 trans-carveol 1217 1215 tr RI,MS

58 isobornyl formate 1222 1235 tr RI,MS

59 cumin aldehyde 1236 1238 0.1 RI,MS

27
60 carvone 1241 1239 tr Std

61 piperitone 1252 1249 tr RI,MS

62 (2E)-decenal 1262 1260 tr RI,MS

63 n-decanol 1274 1266 0.1 RI,MS

64 nonanoic acid 1277 1267 tr RI,MS

65 bornyl acetate 1282 1287 0.5 0.2 1.1 Std

66 thymol 1289 1289 0.1 0.1 tr Std

67 carvacrol 1297 1298 0.1 0.1 tr Std

68 p-vinyl guaiacol 1311 1309 tr RI,MS

69 (2E,4E)-decadienal 1315 1315 tr RI,MS

70 α -cubebene 1344 1345 tr RI,MS

71 eugenol 1355 1356 0.4 tr tr Std

72 α -ylangene 1364 1373 tr tr 0.1 RI,MS

73 α -copaene 1368 1374 0.1 0.1 0.6 RI,MS

74 decanoic acid 1374 1364 0.1 RI,MS

75 α -bourbonene 1376 1387 tr tr RI,MS

76 methyl eugenol 1406 1403 tr tr tr RI,MS

77 (E)-caryophyllene 1409 1417 0.4 0.3 4.6 Std

78 β -copaene 1420 1430 tr 0.1 RI,MS

79 2-methoxy-naphthalene 1438 1445 tr RI,MS

80 α -humulene 1443 1452 0.1 tr 1.0 Std

81 geranyl acetone 1453 1453 0.2 tr RI,MS

82 (E)-β -farnesene 1456 1454 tr 0.1 Std

83 trans-cadina-1(6),4-diene 1466 1475 0.1 RI,MS

84 γ -muurolene 1470 1478 0.3 0.1 0.9 RI,MS

85 α -amorphene 1473 1483 tr RI,MS

86 β -selinene 1476 1489 0.1 RI,MS

87 ar-curcumene 1479 1479 tr tr RI,MS

88 α -selinene 1486 1498 tr 0.1 RI,MS

89 γ -amorphene 1487 1495 tr 0.2 RI,MS

90 α -muurolene 1494 1500 0.1 tr 0.3 RI,MS

28
91 δ -amorphene 1500 1511 tr RI,MS

92 γ -cadinene 1506 1513 0.3 0.1 0.8 RI,MS

93 sesquicineole 1509 1515 tr 0.1 RI,MS

94 trans-calamenene 1516 1521 0.2 tr 0.4 RI,MS

95 δ -cadinene 1517 1522 0.3 0.1 1.7 RI,MS

96 trans-cadina-1,4-diene 1525 1533 0.2 RI,MS

97 eugenol acetate 1528 1521 tr RI,MS

98 α -cadinene 1530 1537 0.1 RI,MS

99 α -calacorene 1535 1544 0.1 tr 0.1 RI,MS

100 caryophyllene oxide 1572 1582 1.4 0.1 0.6 Std

101 humulene epoxide II 1598 1608 0.3 0.1 RI,MS

102 benzophenone 1617 1626 0.1 RI,MS

103 1-epi-cubenol 1619 1627 0.3 tr 0.1 RI,MS

104 muurola-4,10(14)-dien-1-β -ol 1620 1630 0.3 RI,MS

105 caryophylla-4(12),8(13)-dien-5-olg 1626 1639 0.3 0.1 RI,MS

106 epi-α -cadinol 1632 1638 0.2 tr 0.2 RI,MS

107 epi-α -muurolol 1633 1640 0.2 0.2 RI,MS

108 α -muurolol 1639 1645 0.2 tr RI,MS

109 (Z)-methyl jasmonate 1645 1648 0.1 0.1 RI,MS

110 α -eudesmol 1643 1652 0.1 RI,MS

111 α -cadinol 1646 1652 0.3 0.1 0.3 RI,MS

112 α -bisabolol oxide B 1647 1656 0.3 RI,MS

113 cis-calamenen-10-ol 1660 1660 tr RI,MS

114 cadalene 1666 1675 0.1 tr RI,MS

115 epi-α -bisabolol 1679 1683 3.9 0.9 5.1 RI,MS

116 α -bisabolol 1681 1685 0.8 0.2 0.8 Std

117 10-nor-calamenen-10-one 1692 1702 tr RI,MS

118 eremophilone 1727 1734 tr RI,MS

119 (Z)-lanceol 1757 1760 0.1 tr RI,MS

120 n-tetradecanoic acid 1765 1667 0.1 RI,MS

121 n-nonadecane 1900 1900 tr Std

29
 122 (5E,9E)-farnesyl accetone 1916 1913 tr RI,MS

123 hexadecanoic acid 1967 1959 1.2 Std

124 abietatriene 2037 2039 0.2 RI,MS

125 n-heneicosane 2100 2100 tr Std

126 linoleic acid 2135 2132 0.3 Std

127 trans-ferruginol 2316 2317 0.3 RI,MS

Identified compounds 116

Total identified (%) 98.4 99.8 99.1

Oil yield (%) 0.02 0.73 0.89

Grouped compounds (%)

Monoterpene hydrocarbons 26.2 44.0 42.4

Oxygenated monoterpenes 57.7 53.4 37.4

Sesquiterpene hydrocarbons 2.0 0.8 11.6

Oxygenated sesquiterpenes 8.6 1.3 7.5

Others 3.9 0.3 0.2

a b
623 Compounds are listed in order of their elution from a HP-5MS column. Linear retention index on HP-5MS column,
c
624 experimentally determined using homologous series of C8-C30 alkanes. Linear retention index taken from Adams (2007)
d
625 and/or NIST 08 (2008). Relative percentage values are means of three determinations with a RSD% in all cases below 10%.
e
626 Identification methods: std, based on comparison with authentic compounds; MS, based on comparison with WILEY,
627 ADAMS, FFNSC2 and NIST 08 MS databases; RI, based on comparison of calculated RI with those reported in ADAMS,
f g
628 FFNSC 2 and NIST 08. Tr, % below 0.1%. Correct isomer not identified.

629

630

30
631 Table 3. Antioxidant activity of Rosmarinus eriocalyx essential oils .

DPPH ABTS FRAP ORAC

Rosmarinus eriocalyx Plan part TEACa IC50b TEACa IC50b TEACa TEACa
µmol TE/g µg/ml µmol TE/g µg/ml µmol TE/g µmol TE/g
stems 4.9±0.2 1511±18 16.98±0.2 367±5 31.55±0.0 ndc
Essential oils leaves 2.7±0.1 2775±10 5.18±0.1 1201±10 11.68±0.6 562.1±27.9
flowers 1.84±0.1 4040±30 3.37±0.0 1850±30 6.08±0.9 680.1±42.9

Positive control Trolox 1.87 ±0.09 1.56±0.04

a b c
632 TEAC: Trolox equivalent (TE) antioxidant concentration. IC50: The concentration giving a reduction of 50%. nd, not
633 determined.
634

31
635 Table 4. Antioxidant activity of Rosmarinus eriocalyx water and ethanol and extracts.

Rosmarinu Plan Polyphenol DPPH ABTS FRAP ORAC

s eriocalyx part s mg
GAeq/g
TEACa IC50b TEACa IC50b TEACa TEACa µmol
µmol µg/ml µmol µg/ml µmol TE/g TE/g
TE/g TE/g
Ethanolic stems 47.1±1.13 384.2±6. 31.6±0. 585.0±19. 24.6±0.4 6146.2±0.1 2045.5±97.7
extracts 9 9 2
leaves 58.0±3.6 474.5±2. 25.6±0. 522.6±33. 27.6±1.3 5122.2±21. 1493.9±65.8
7 4 2 3
flower 44.2±5.1 220.9±1. 55.0±0. 342.7±14. 41.3±0.3 2561.2±10. 1642.5±118.
s 4 9 1 4 1
Aqueous stems 29.7±2.1 201.8±5. 60.5±0. 288.8±6.6 49.8±0.3 1491.6±32. 2078.5±119.
extracts 3 7 7 7
leaves 33.1±2.1 130.3±0. 93.2±0. 220.3±1.3 65.3±0.7 1278.5±12. 1106.0±57.5
9 3 1 1
flower 37.1±2.9 185.8±0. 65.2±0. 257.9±6.8 55.7±0.5 1704.7±10. 803.7±22.7
s 8 2 2
Positive Trolox 3.04 3.60±0.0
control ±0.03 6
a b
636 TEAC: Trolox equivalent (TE) antioxidant concentration. IC50: The concentration giving a reduction of 50%.
637

638

32
 Table 5. In vitro antimicrobial activity of Rosmarinus eriocalyx essential oils and extracts by the disc diffusion method.
Compound Inhibition zone diameter (mm ± SDa)
S.aureus E. faecalis E.coli P.aeruginosa C.albicans
Essential oils Flowers 7.3 ± 0.6 7.0 ± 0.0 8.0 ± 1 n.a.b 9.0 ± 0.0
Leaves 8.0 ± 0.0 7.0 ± 0.0 9.7 ± 0.6 n.a. 10.0 ± 0.0
Stems n.a. n.a. n.a. n.a. 7.0 ± 0.0
Ethanolic extracts Flowers 7.5 ± 0.7 n.a. n.a. 8.0 ± 0.0 8.0 ± 0.0
Leaves 7.3 ± 0.6 n.a. n.a. 7.5 ± 0.7 8.0 ± 0.0
Stems 9.0 ± 0.0 7.0 ± 0.0 n.a. 7.5 ± 0.7 8.0 ± 0.0
Aqueous extracts Flowers n.a. n.a. n.a. n.a. n.a.
Leaves n.a. n.a. n.a. n.a. n.a.
Stems n.a. n.a. n.a. n.a. n.a.
c
Std. antibiotics Ciprofloxacin 20.3 ± 0.6 24.0 ± 2.6 n.r. n.r. n.r.
Gentamicin n.r. n.r. 19.7 ± 0.6 21.7 ± 0.6 n.r.
Nystatin n.r. n.r. n.r. n.r. 17.0 ± 0.6
Negative control EOH n.a. n.a. n.a. n.a. n.a.
Negative control water n.a. n.a. n.a. n.a. n.a.
Carnosic acid 10 µL 840 ppm n.a. n.a. n.a. n.a. n.a.
Rosmarinic acid 10 µL 940 ppm n.a. n.a. n.a. n.a. n.a.
Carnosol 10 µL 980 ppm n.a. n.a. n.a. n.a. n.a.
a
SD Standard deviation of three determinations.
b
n.a, no activity (no IZD).
c
n.r., not recommended.
639

640
641 Highlights

642 • Rosmarinus eriocalyx is a spice used in Algeria as a food additive

643 • Ethanolic extracts are rich of rosmarinic and carnosic acids and carnosol

644 • Essential oils belonged to camphor chemotype

645 • Ethanolic extracts exhibited relevant antioxidant capacity

646 • Antimicrobial activity was moderate

647

34