1

1 Article

2 Development and chemical characterization of

3 fragrances from essential oils.
4 Ricardo C.Moraes Júnior1, Angélica F. Gomes1, Juliano G. Amaral1, and Mateus F. Leite2*.
5 1
Instituto Multidisciplinar em Saúde – Universidade Federal da Bahia, Vitória da Conquista, Bahia, Brazil.
6 2
Universidade Federal de Alfenas
7 * Correspondence: mateusfl@uol.com.br; Tel.: +55- -xxx-xxxx

8 Academic Editor: name

9 Received: date; Accepted: date; Published: date

10 Abstract: The present work aimed the development and chemical characterization of a fragrance
11 for use in perfumery from natural and synthetics sources. For the fragrance development, 21
12 different formulations were tested starting from combining aromatic compounds obtained by
13 hydrodistillation in Clevenger and/or synthetics. The chosen formula was obtained in 7% of
14 concentration of a fragrance using ethanol as vehicle, propylene glycol, triclosan and galaxolide as
15 humectant, preservative and fixer, respectively. The characterization of the product was made by
16 analysis in gas chromatography-mass spectrometer. The identification of the substances was made
17 starting by determination of retention index and comparison to the literature and database from
18 Wiley library. The chosen formula was composed from combination of Pogostemon cablin, Amyris
19 balsamifera, Citrus limon, Baccharis dracunculifolia oils and synthetic essence of passion fruit. In its
20 analysis were identified as major constituents of the product valerianol (12.36%), limonene
21 (7.12%), β-caryophyllene (7.7%) and an unidentified compound with retention time equal to
22 55.731m (5.80%). The successful results confirmed the necessity of more exploration and research
23 of natural sources of aroma compounds, especially those from Brazilian biodiversity.

24 Keywords: Essential oils, fragrances development, GC-MS.

25 PACS: J0101
26

27 1. Introduction

28 Innovation is the first transaction involving a new product, process or system [1],
29 currently being an essential tool in the differentiation strategies and competitiveness in a
30 growing number of companies and business [2]. In this context, the sector of flavors and
31 fragrances in its various applications is a source of continuous development and constant
32 innovation, representing a multibillionaire market, of which Brazil is presented as a
33 prominent character [3], being the world leader in fragrances sales in 2011, according to
34 data from Euromonitor consultancy [4].
35 A fragrance is defined as a composition of odorous raw materials from natural or
36 synthetic sources prepared by a perfumist creative talent [5]. Fragrance olfactory properties
37 demonstrate to carry important sensory information, which may influence how humans
38 interpret informations based in vision as, for example, the facial perception [6]. The
39 perfumery history mingles with human history itself. For over 4000 years, the
40 Mesopotamians used incense as the first form of perfume in rituals dedicated to gods and

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41 ancestors. In the sixteenth century, the fragrance was associated with fashion through its
42 sale along with leather and gloves. In the nineteenth and twentieth centuries, it took place
43 the perfume houses establishment as enterprises and the extracting processes improvement
44 for thermosensitive aromatic compounds [7].
45 With the modern chemistry advancement in the twentieth century, the perfumes
46 production began to be made with synthetic ingredients. However, the natural sources of
47 fragrances still arouse the perfumery industry interest for the wide variety of untapped
48 potential substances. Among the isolated compounds from natural sources stand out the
49 terpenoids, phenylpropanoids, aldehydes, among others classes [7]. This study aimed to
50 develop a fragrance from natural and / or synthetic sources and its chemical
51 characterization.

52 2. Results and Discussion

53 2.1. Fragrance development

54 In the fragrance development first stage it was individually evaluated the odoriferous
55 characteristics of the oil and its combination possibilities, along with the information
56 present in the literature. The oils used present sensory features which allow them to be
57 classified into odorous groups such as, for instance, the woody (A. balsamifera) Citrus (C.
58 limon) and herbaceous (L. hybrida) [8].
59 The development followed based on theoretical assumptions [9, 10]. Were created 21
60 test-formulas whose constituent proportions are shown in Table 1. It is observed in the first
61 formulations an attempt to associate the C. rotundus, C. citratus and J. communis oils,
62 without success. From this point, it was tried to soften the formula woody sensorial with the
63 inclusion of C. limon, C. sinensis and L. hybrida oils and passion fruit synthetic essence. In
64 this strategy, fragrances with more satisfying sensorial were obtained, being formula 15
65 chosen for the product development. The final product formula is shown in Table 2. The
66 physicochemical characterization, whose goal is to provide specifications for the quality
67 control analysis is shown in Table 3.
68
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81 Table 1. Composition of the tested formulas on the fragrance development

Formulas (%)
Essential Oils F. 1 F. 2 F. 3 F. 4 F.5 F.6 F.7 F.8 F.9 F. 10 F. 11
A. balsamifera 27,0 31,3 33,3 36,4 38,5 40,0 37,0 37,3 35,0 35,0 38,9
C. rotundus 19,0 21,8 20,0 18,2 19,2 12,0 29,6 29,9 28,0 28,0 31,1
J.communis 27,0 31,3 40,0 43,6 38,5 40,0 29,6 29,9 17,5 17,5 19,5
C. citratus 13,5 7,8 6,7 1,8 3,8 - 1,9 1,0 1,0 0,3 -
L. hybrid - - - - 8,0 1,9 1,9 1,7 1,7 -
B. dracunculifolia - - - - - - - - - -
C. limon 13,5 7,8 - - - - - - - - -
C. sinensis - - - - - - - - 16,8 17,5 9,7
S. sclarea - - - - - - - - - - 0,8
Passion Fruit
- - - - - - - - - - -
Syntethic Essence
P. cablin - - - - - - - - - - -

82

83 Table 1. Composition of the tested formulas on the fragrance development -

84 Continuing

Formulas (%)
Essential Oils F. 12 F.13 F.14 F. 15 F.16 F.17 F.18 F.19 F.20 F. 21
A. balsamifera - - 27,8 35,3 - - - -
C. rotundus 84,0 88,5 - - 68,0 38,5 35,4 35,1 35,1 43,0
J.communis - - - - - - - - - -
C. citratus - - - - - - - - - -
L. hybrida 2,5 2,6 - - - - - - 1,8 1,0
B. dracunculifolia - - 1,4 1,8 - - - - - -
C. limon 3,6 3,5 13,9 17,7 5,8 19,2 22,1 21,9 26,2 27,0
C. sinensis 4,1 4,4 - - 19,4 - 1,8 2,6 - 3,2
S. sclarea - - - - - 1,9 3,5 3,5 - 4,3
Passion Fruit
5,8 1,0 1,4 1,8 6,8 1,9 1,8 1,8 1,8
Syntethic Essence
P. cablin - - 55,5 43,4 - 38,5 35,4 35,1 35,1 21,5

85

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87

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89

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90 Table 2. Composition of the tested formulas on the fragrance development.

Compound Concentation (%)
Fragrance 7,00
Fixative 1,00
Preservative 1,00
Ethyl alcohol q.s. 100,00%

91

92 Table 3. Product physicochemical characterization.

Parameter Specification
Density (g/mL) 0,9499 ±0,0005
pH 7,43 ± 0,01

93

94 2.2. Chemical characterization of the fragrance

95 The volatile and non-polar property of the perfume constituents makes the technique of
96 gas chromatography (GC) and gas chromatography coupled to mass spectrometer (GC-MS)
97 assist the process of a perfume formulation, since many times the raw materials used in
98 perfumery, whether synthetic or natural, can be separated and identified by these techniques
99 [11].
100 Figure 1 shows the chromatogram obtained by GC-MS analysis of the formula 15
101 product. 37 areas of peaks were registered with retention times ranging between 5.86 and
102 56.20 minutes and peak area ranging from 0.11 to 14.87%. The peak with retention time of
103 approximately 41.11 correspond to the fixative Galaxolide. Table 4 discriminates all the
104 identified substances.
105

106

107 Figure 1. Chromatogram obtained by analyzing the product by GC-MS.

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108 Table 4. Compounds identified and quantified in the analysis by GC-MS. RIobs =
109 Retention index observed in DB-5 capillary column; RI ref = Retention index observed in
110 reference [23].
Peak Compound tR RIobs (RIref) Sample
Area (%)
1 α-pinene 5,87 932 (939) 0,17
2 Sabinene 6,96 971 (976) 0,13
3 β-pinene 7,14 977 (980) 1,06
4 Mircene 7,43 988 (991) 0,13
5 p-cimene 8,65 1024 (1023) 0,29
6 Limonene 8,83 1028 (1031) 7,12
7 ϒ-Terpinene 9,87 1056 (1062) 0,89
8 Terpinolene 10,91 1084 (1088) 0,11
9 Linalool 11,47 1099 (1098) 0,13
10 Terpineol 15,49 1194 (1189) 0,35
11 α-copaene 23,25 1373 (1376) 0,34
12 β-patchoulene 23,61 1381 (1380) 0,12
13 β-elemeno 23,85 1387 (1391) 0,29
14 α-cedrene 24,39 1413 (1409) 0,45
15 β-Caryophyllene 25,11 1417 (1418) 7,07
16 Thujopsene 25,72 1432 (1429) 3,12
17 α-humulene 26,57 1452 (1454) 1,21
18 α-patchoulene 26,78 1457 (1456) 0,49
19 ϒ-curcumin 27,53 1475 (1480) 0,19
20 α-curcumin 27,67 1478 (1483) 1,59
21 α-zingiberene 28,24 1492 (1495) 1,15
22 α –bulnesene 28,48 1497 (1505) 1,31
23 β-dihydro-agarofuran 28,59 1501 (1496) 0,61
24 β-bisabolene 28,76 1505 (1507) 1,12
25 β-Sesquiphellandrene 29,39 1521 (1524) 1,15
26 Selina-3,7(11)-dien 30,04 1537 (1542) 0,96
27 Elemol 30,35 1545 (1549) 3,59
28 Germacrene-B 30,70 1554 (1556) 0,31
29 Guaiol 32,24 1592 (1595) 1,67
30 α-cedrol 32,62 1601 (1596) 1,11
31 10-epi-ϒ-eudesmol 33,20 1618(1619) 3,20
32 ϒ-Eudesmol 33,57 1627 (1630) 2,68
33 Valerianol 34,44 1650 (1655) 12,36
34 10-epi-α-eudesmol 34,67 1657 (1658) 3,30
35 Bulnesol 34,82 1661 (1666) 2,04
36 Patchouli Alcohol 34,98 1665 2,95
37 Unidentified compound 55,73 2307 5,80

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111
112 The fragrance presented four major compounds. The valerianol, a sesquiterpene
113 identified in the Valeriana officinalis essential oil and present in the A. balsamifera
114 essential oil is the substance with higher quantity in the product (12.36%), followed by
115 limonene (7.12%), a monocyclic monoterpenoid with citric and fresh flavor present in C.
116 limon and B. dracunculifolia and β-caryophyllene (7.07 %), a monoterpene present in the
117 P. cablin oil [12-16] (figure 2). It was not possible to identify the major substance with the
118 retention time of 55.73 min (5.80%).
119

120

121 Figure 2. Chemical structures of the major components identified in the fragrance: Valerianol (1),
122 Limonene (2), β-caryophyllene (3).

123 The analysis of the identified substances showed that 26.53% of the total peak area was
124 composed by substances belonging to the essential oil of A. balsamifera as valerianol, 10-
125 epi-γ-eudesmol, γ-eudesmol and 10-epi-α-eudesmol [13]. The P. cablin oil contributed with
126 12.54%, being patchulol and β-caryophyllene the most relevant substances [16, 17].
127 Constituents described only in the B. dracunculifolia oil accounts for 4.81% of the
128 fragrance percentage, spread among guaiol, α-curcumene, α-humulene and α-copaene [15].
129 α-pinene, limonene and Β-pinene correspond to 8.35% of the product composition and have
130 contributions from B. dracunculifolia and C. limon [14-15]. The latter also participates in
131 the fragrance with sabinene, myrcene, p-cymene, γ-terpinene, terpinolene, terpineol,
132 linalool and thujopsene substances [14] with of 5.02% percentage of the fragrance.
133 The fragrance also presented four substances - α-cedrene, γ- curcumene α-cedrol and
134 bulnesol - which are not described in any of the oils used. It was reported in 1962 the
135 synthesis of patchulol and guaiol from bulnesol [18], so it is reasonable to think that it may
136 be an intermediate in the patchulol synthesis in P. cablin.
137 The volatility, along with the molecular weight, is the most important factor in the
138 substance retention in the gas chromatography [19]. This physicochemical property is of

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139 utmost importance in perfumery, allowing the substances of a fragrance to be classified into
140 three groups: head notes or top (high-volatility substances, responsible for the fragrance
141 first impression); body notes (medium volatility substances that characterize the scent) and
142 base notes (low-volatility substances that are felt after head and body notes dissipation) [10,
143 20]. The retention times analysis of the identified substances allows us to suggest the
144 substances classification present in the of C. limon oil as top notes. The constituents of the
145 B. dracunculifolia oil are inserted within the body notes and the P. cablin and A.
146 balsamifera oils can be classified as base notes (figure 3).

147
148 Figure 2. Figure 3. Probable pyramidal structure of the developed perfume.

149

150 4. Materials and Methods

151 3.1. Raw materials

152 The raw materials used in this work were essential oils from different plant species.
153 Lavandin (Lavandula hybrida) and sage (Salvia sclarea) oils were obtained from
154 BioEssencia®. Lemongrass (Cymbopogon citratus) and Field Rosemary (Baccharis
155 dracunculifolia) were obtained from By Samia® and Lazlo®, respectively. Sicicilian lemon
156 (Citrus limon), Juniper (Juniperus communis), Patchouli (Pogostemon cablin) and
157 Sandalwood (Amiris balsamifera) were obtained from Oficina de ervas®. The passion fruit
158 synthetic essence was obtained from Proquimios®.
159 The sedge rhizome oils (Cyperus rotundus) and Lime (Citrus sinensis) were obtained by
160 vapor drag. 100 g of plant material from those species were added to flat-bottomed flask
161 together with 600 ml of purified water. Oil extraction was done in Clevenger apparatus
162 [21]. All oils were stored under refrigeration in order to maintain stability.

163 3.2. Raw materials

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164 Initially were produced solutions containing 10% of the synthetic essential oils and
165 passion fruit essence in vehicle consisting of 95% of absolute ethanol and 5% of
166 propyleneglycol. These solutions were stored under refrigeration for three days.
167 After this period, the prepared solutions were combined in various mixtures with
168 different constituent proportions until the achievement of one most pleasing to the sense of
169 the researchers. For this, a product was developed with 7% of the selected fragrance and
170 93% of vehicle. In the product development pure raw materials quantities were used
171 complying with the selected combination ratios.

172 3.3. Identification by Gas Chromatography Coupled with Mass Spectrometry (GC-MS).

173 The fragrance components were identified by GC-MS according to the adapted
174 methodology [22]. The identification was made by retention rates determination, compared
175 with data from the literature [23] and databases of Wiley library. Chromatographic analysis
176 was performed on Shimadzu ® QP2010 chromatograph coupled to a mass spectrometer.
177 DB-5MS column (5% phenyl-methylpolysiloxane) with dimensions of 30 m × 0.25 mm ×
178 0.25 mm was used. Helium was used as carrier gas with 1mL/min flow, 57.5 kPa pressure.
179 The furnace operated at a temperature of 60 to 230 º C program at 3 ° C / min. The spectra
180 were obtained by electron impact at 70 eV.

181 3.4. Physicochemical characterization

182 The fragrance had the pH value determined potentiometrically in Tecnopon® pH meter
183 Model MPA-210 calibrated with standard pH 4.0 and 7.0 buffer solutions. The mass-to-
184 volume ratio of the product was determined by the relative density by the pycnometer
185 method using the density of the purified water as the reference. All analyzes were
186 performed in triplicate [24].

187 5. Conclusions
188 This study showed good results, developing a quality and innovative product for use in
189 perfumery. The product in question must go through acceptance studies, in order to confirm
190 its market potential, as well as security.
191 The natural sources of odors, especially essential oils, still constitute a source of
192 untapped substances with potential use in fragrances. In this sense, the success of this work
193 confirms the need for further exploration and research of these sources, especially those
194 from the Brazilian biodiversity.
195
196 Acknowledgments: The authors thank the Fundação de Amparo à Pesquisa do Estado da Bahia (edict
197 005/2011 FAPESB) for financial support in carrying out this project and the trainees of the Laboratory of
198 pharmaceutical technology and cosmetology at the Universidade Federal da Bahia - Campus Anísio Teixeira,
199 for the collaboration.

200 Author Contributions: R. M. J and M.L. proposed the study, designed the experiments, wrote the manuscript
201 and edited the text. J. A. and A.G. performed the chemical identification and characterization. All authors have
202 read the m manuscript and approved the final version.

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203 Conflicts of Interest: The authors declare no conflict of interest.

204

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261 Sample Availability: Samples of the compounds ...... are available from the authors.

262 © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
263 article distributed under the terms and conditions of the Creative Commons by
264 Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

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