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molecules
Article
GC-MS Analysis of the Composition of the Extracts
and Essential Oil from Myristica fragrans Seeds
Using Magnesium Aluminometasilicate as Excipient
Inga Matulyte 1, Mindaugas Marksa 2, Liudas Ivanauskas 2, Zenona Kalv˙
enien˙
e1,3,
Robertas Lazauskas 4and Jurga Bernatoniene 1, 3, *
1Department of Drug Technology and Social Pharmacy, Medical Academy, Lithuanian University of Health
Sciences, Kaunas LT-50161, Lithuania; inga.matulyte@lsmu.lt (I.M.); zenona.kalveniene@lsmuni.lt (Z.K.)
2
Department of Analytical and Toxicological Chemistry, Medical Academy, Lithuanian University of Health
Sciences, Kaunas LT-50161, Lithuania; mindaugas.m.lsmu@gmail.com (M.M.);
Liudas.ivanauskas@lsmuni.lt (L.I.)
3Institute of Pharmaceutical Technologies, Medical Academy, Lithuanian University of Health Sciences,
Kaunas LT-50161, Lithuania
4Institute of Physiology and Pharmacology, Medical Academy, Lithuanian University of Health Sciences,
Kaunas LT-50161, Lithuania; Robertas.lazauskas@lsmuni.lt
*Correspondence: jurga.bernatoniene@lsmuni.lt
Academic Editor: Igor Jerkovi´c
Received: 13 February 2019; Accepted: 14 March 2019; Published: 18 March 2019
Abstract:
Myristica fragrans (f. Myristicaceae) seeds are better known as a spice, but their chemical
compounds may have a pharmacological effect. The yield of their composition of extracts and
essential oils differs due to different methodologies. The aim of this study was to evaluate
an excipient material—magnesium aluminometasilicate—and to determine its influence on the
qualitative composition of nutmeg extracts and essential oils. Furthermore, we wanted to compare
the yield of essential oil. The extracts were prepared by maceration (M) and ultrasound bath-assisted
extraction (UAE), and the essential oil—by hydrodistillation (HD). Conventional methods (UAE,
HD) were modified with magnesium aluminometasilicate. The samples were analyzed by gas
chromatography-mass spectrometry (GC-MS) method. From 16 to 19 chemical compounds were
obtained using UAE with magnesium aluminometasilicate, while only 8 to 13 compounds were
obtained using UAE without an excipient. Using our conditions and plant material, for the
first time eight new chemical compounds in nutmeg essential oil were identified. Two of these
compounds (
γ
-amorphene and cis-
α
-bergamotene) were obtained with the use of excipient, the other
six (
β
-copaene, bergamotene, citronellyl decanoate, cubebol, cubenene, orthodene) by conventional
hydrodistillation. Magnesium aluminometasilicate significantly increased the quantity of sabinene
(from 6.53% to 61.42%) and limonene (from 0% to 5.62%) in essential oil. The yield of the essential oil
from nutmeg seeds was significantly higher using magnesium aluminometasilicate; it increased from
5.25 ±0.04% to 10.43 ±0.09%.
Keywords:
Myristica fragrans; nutmeg; essential oil; extract; magnesium aluminometasilicate;
hydrodistillation
1. Introduction
Since ancient times, nutmeg (Myristica fragrans, f. Myristicaceae) has been used as a spice.
Its aphrodisiac effect is often mentioned [
1
]. In traditional medicine, nutmeg is used to treat
rheumatism, pain, nausea, stomach cramps, and other illnesses [
2
]. A lot of research is being done in
order to test the pharmacological effects of nutmeg. Scientific sources say that nutmeg has antibacterial,
Molecules 2019,24, 1062; doi:10.3390/molecules24061062 www.mdpi.com/journal/molecules
Molecules 2019,24, 1062 2 of 12
insecticidal, antioxidant, anticancer, antidepressant, hepatoprotective, and many other effects [
3
–
6
].
The most common studies about essential oils are of nutmeg, which is rich in terpenes, phenols,
various organic acids, and other compounds. Using different solvents, methods, and conditions, the
composition of extracts and essential oils variations were studied [
3
,
7
–
9
]. Nutmeg essential oil is
colorless to pale-yellow with a specific odor. The essential oil yield may be more than 10% [
10
] and
varies by 5–15% [
11
]. Myristica fragrans essential oil has predominantly monoterpene hydrocarbons
(
α
-thujene,
β
-pinene, sabinene), sesquiterpene hydrocarbons (D-germacrene, trans-
β
-bergamotene),
monoterpene alcohols (linalool,
α
-terpineol, cis-p-menth-2-en-1-ol, terpinen-4-ol), esters (
α
-terpinyl
acetate, cis-sabinene hydrate acetate, citronellyl acetate), aromatics (eugenol, myristicin, elemicin,
methoxyeugenol), and other compounds [5,7–9,12].
Traditional methods such as maceration, percolation, infusion, decoction, and Soxhlet method are
commonly used to produce extracts [
8
,
13
]. These methods are cheap but less efficient, which wastes
a lot of time and raw materials [
14
]. One of the more recent methods for extracts is extraction in
an ultrasound-assisted bath, it is an effective method, in which it is possible to choose the desired
conditions (i.e., temperature, duration), and it saves on materials and time [
2
,
15
]. Ultrasound-assisted
extraction (UAE) has many modifications with solvents, and extra material can be used for extraction;
for example: surfactants [
16
], polysaccharides [
17
], salts [
18
], acids [
19
], and other excipients, which can
increase the efficiency of extraction.
The most popular methods for extracting essential oils are distillation with water
(hydrodistillation) and distillation with steam or steam and water [
12
,
14
,
20
]. Other methods are
cold or hot-pressing, supercritical fluid extraction, enfleurage, microwave, and ultrasound-assisted
methods, solvent extraction, and aqueous infusion [18,21].
Hydrodistillation is a method used for extracting essential oils. It is cheap because it mostly uses
water as a solvent. The equipment is not expensive, most commonly a Clevenger-type apparatus or
similar modifications are used [
14
,
20
]. In different sources, hydrodistillation is carried out for various
times and solid:solvent ratios. Hydrodistillation is carried out using water, and few studies have been
carried out using extra material for extraction such as sea water [
22
], non-ionic surfactants [
23
] or 5%
NaCl salt solution. [
14
]. According to the information found, it can be said that excipient substances
are rarely used for hydrodistillation.
Excipients are used for improving extraction and changing environmental conditions.
For example, salts that change ion voltage, surfactants, emulsifiers (sorbitan esters (Spans
®
),
polysorbates (Tweens
®
)), and pH-adjusting substances are used [
9
,
24
,
25
]. The magnesium
aluminometasilicate will be used for the first time as an excipient for chemical compound extractions,
previously it was used as an excipient only in solid dosage forms [
26
,
27
]. It is a white amorphous
powder with high surface area, practically insoluble in water. The magnesium aluminometasilicate
has the ability to absorb materials, which is characterized by water absorption capacity [26,28].
The purpose of this study is to evaluate excipient material—magnesium aluminometasilicate—
and to determine its influence on the qualitative composition of nutmeg extracts and essential
oils. Furthermore, to compare the yield of essential oil using conventional hydrodistillation and
hydrodistillation with magnesium aluminometasilicate.
2. Results
Using the traditional maceration method (conditions are mentioned in Table 1, see Materials and
Methods) myrislignan and elemicin were established at 22.59% and 13.99%, respectively, (Table 2).
Maceration as a method was modified with magnesium aluminometasilicate (0.5%, 1%, and 2%
excipient), but the results were not significant. The magnesium aluminometasilicate did not increase the
quantity of chemical compounds, so these conditions and results were not mentioned in the research.
Molecules 2019,24, 1062 3 of 12
Table 1. Extraction conditions used for each experiment.
Method Sample
Code Temperature Time (h) Solvent Solvent:
Nutmeg Ratio
Magnesium
Aluminometasilicate:
Nutmeg Ratio
Maceration M1 ambient 72 purified water 20:1 -
Ultrasound-Assisted
Extraction
UAE1
25 ◦C0.5
ethanol 50% 20:1 -
UAE2 ethanol 70% 20:1 -
UAE3 ethanol 96% 20:1 -
UAE4
ethanol 70% + 0.5% magnesium
aluminometasilicate 20:1 10:1
UAE5 ethanol 70% + 1% magnesium
aluminometasilicate 20:1 5:1
UAE6 ethanol 70% + 2% magnesium
aluminometasilicate 20:1 2.5:1
Hydrodistillation
HD1
100 ◦C4
purified water 20:1 -
HD2
purified water + 0.5%
magnesium
aluminometasilicate
20:1 10:1
HD3
purified water + 1%
magnesium
aluminometasilicate
20:1 5:1
HD4
purified water + 2%
magnesium
aluminometasilicate
20:1 2.5:1
Using other extraction techniques (i.e., UAE, HD), myrislignan and elemicin were not found.
The extract (M1) has 10 different active compounds: α-phellandrene, β-myrcene, γ-asarone, elemicin,
and others (Table 2).
Analyzing the influence of ethanol concentrations on the extraction of compounds, it was found
that most of the different compounds were extracted using 70% ethanol (fifteen compounds), or at
least 50% ethanol (eight compounds). The higher the ethanol concentration, the better the extracts
were extracted, for example with
α
-pinene, 2.4%, 8.3%, and 14.67%, respectively (Table 2). In contrast,
when weaker ethanol concentrations were used more isolemicin was extracted (62.24%, 23.63%). In the
extract of 96% ethanol isolemicin was not found.
The data of the extracts with magnesium aluminometasilicate, using 70% ethanol as a solvent
(UAE4, UAE5, UAE6, Table 2) showed that when 1% magnesium aluminometasilicate was used, the
utmost quantity of compounds was identified (90.92%). Magnesium aluminometasilicate helped extract
sabinene (from 29.9 to 32.69%, in extracts without magnesium aluminometasilicate sabinene was not
found). The following compounds were also detected in extracts with magnesium aluminometasilicate
exposure: myrcene, isoterpinolene, isogermacrene, limonene, cis-sabinene hydrate,
γ
-terpinene,
γ-amorphene, and β-myrcene (Table 2).
The composition of the essential oils, which were obtained using hydrodistillation (HD1) and
hydrodistillation with excipients from HD2 to HD4 (Table 1), showed that 1% of magnesium
aluminometasilicate significantly improved amount of
α
-pinene and sabinene compared to
conventional hydrodistillation, respectively 54.95% and 106.32%. Using 0.5% and 2% of magnesium
aluminometasilicate the amount of the
α
-pinene and sabinene was higher than HD1 but lower than
HD3 (Figure 1).
Molecules 2019,24, 1062 4 of 12
Table 2. Chemical composition of nutmeg extracts and essential oils.
Compound
Sample Code a
Essential Oil Compounds Extracts Compounds
RI dHD e1 (%) HD2 (%) HD3 (%) HD4 (%) Mf1 (%) UAE g1 (%) UAE2 (%) UAE3 (%) UAE4 (%) UAE5 (%) UAE6 (%)
4-Carene, trans -(+)- 911 7.77 - - - 3.37 1.19 6.56 13.22 - - -
α-Thujene 928 0.99 1.04 0.93 0.99 - 2.63 0.72 1.42 0.84 0.92 0.81
α-Pinene 934 8.27 10.04 15.05 11.5 - 2.4 8.3 14.67 9.65 8.13 9.94
Camphene 944 - 2.8 0.11 0.11 - - - - - - 0.15
Orthodene b949 0.51 - - - - - - 0.59 - - -
3-Carene 956 0.53 - - - - - 0.42 0.78 - - -
α-Phellandrene 957 - - - - 13.04 - 1.41 2.38 - - -
β-Myrcene 958 - - - - 4.6 - - - 9.47 7.26 9.19
2-Carene 960 1.99 - - - - - 0.75 1.15 - - -
Sabinene 969 6.53 49.64 61.42 47.1 - - - - 29.9 31.74 32.69
β-Pinene 970 26.61 4.03 4.32 3.84 - - - - - - -
Myristicin 981 3.26 2.34 2.15 2.16 - - - - 1.79 1.65 1.96
γ-Terpinene 993 - 0.78 0.68 0.73 - - - - 0.68 1.65 0.71
α-Terpinene 999 1.23 1.23 0.94 1.23 - - 0.6 1.07 0.67 1.18 0.69
3,7,7-trimethylcyclohepta-1,3,5-triene 1005 0.18 0.47 0.33 0.44 - - - - 0.37 - -
Limonene 1009 - 5.62 4.2 5.23 - - - - 4.2 4.46 4.63
Isoterpinolene 1030 - 2.11 1.18 2.07 - - - - 1.19 2.02 1.22
Cis-sabinen hydrate 1037 7.76 0.58 0.3 0.83 - - - - 1.3 2.32 1.46
γ-Terpineol 1043 - 0.04 0.04 0.04 - - - 0.14 - - 0.09
α-Terpinolene 1051 - 0.7 0.38 0.7 - - - - - - 0.54
Sylvestrene 1059 - - - - 1.57 - - - - - -
Isomethyleugenol 1062 0.2 - - - 6.38 - - - - - -
Cis-p-menth-2-en-1-ol 1076 2.02 0.38 0.37 0.38 0.43 3.27 5.88 3.25 1.32 2.42 1.62
4-Propenyl syringol 1083 - - - - - 2.16 1.55 - - - -
Citronellyl Decanoate b1110 0.3 - - - - - 0.43 0.16 - - -
1,1-dimethyl-2-[(1E)-3-methylbuta-1,3-dienyl
cyclopropane 1116 - - - - - 8.4 29.99 47.32 - - -
Bicyclogermacrene 1125 0.29 - - - - - - - - - -
4-Terpineol 1152 0.74 0.34 0.03 0.35 - - 0.77 0.3 0.47 0.47 0.47
Cubebol b1174 0.05 - - - - - - - - - -
Cubenene b1177 0.07 - - - - - - - - - -
Piperitol 1189 0.09 - - - - - - - - - -
Isoelemicin 1217 5.98 - - - - 62.24 23.-63 - 21.5 21.5 18.85
Copaene 1228 - 0.25 0.01 0.25 - - - - - - -
β-Copaene b1233 0.25 - - - - - - - - - -
γ-Amorphene b1245 - 0.75 0.03 0.78 - - - - 0.43 0.92 0.08
Cis-α-bergamotene b1283 - 0.09 0.08 0.1 - - - - - - -
Isogermacrene 1311 - 0.99 0.01 1.15 1.61 - - - 0.86 2.44 0.82
Licarin B 1328 - - - - - - 0.72 - 0.71 - -
Bergamotene b1339 0.07 - - - - - - - - - -
γ-Asarone 1361 3.69 1.21 1.22 3.51 0.79 0.84 2.07 - 0.82 1.84 1.25
Elemicin 1542 - - - - 13.99 - - - - - -
Myrislignan 2946 - - - - 22.59 - - - - - -
Total Identified Compounds % c79.38 85.43 93.78 83.6 68.37 83.13 83.8 86.45 86.1 90.92 87.17
All data are given with an 0.05 accuracy.
a
Sample code presented in Table 1.
b
First time chemical compounds extracted from nutmeg.
c
Quantity of all of the identified compounds.
d
Retention indices using RTX-5MS column and n-alkanes (C9–C22) as references. eHD–hydrodistillation. fM–maceration. gUAE–ultrasound-assisted extraction.
Molecules 2019,24, 1062 5 of 12
Figure 1.
Magnesium aluminometasilicate’s influence on the amount of
α
and
β
-pinene, limonene,
and sabinene in the essential oils of nutmeg. Component quantity in essential oils, * p< 0.05 versus
hydrodistillation without magnesium aluminometasilicate. aSample code can be seen in Table 1.
In HD1, essential oil consisted of
β
-copaene, bergamotene, bicyclogermacrene, cubebol, cubenene,
and piperitol, and these compounds were not identified in other extracts. Ten compounds using
magnesium aluminometasilicate were obtained, and in comparison to the essential oil of HD1,
their amount was not large but it was about 13.05
±
3.54% of the quantity of all of the identified
compounds in essential oil.
Comparing water and ethanol as solvents, and maceration with ultrasound-assisted extraction
methods, it was found that water is better in extracting
α
-phellandrene (13.04%) than 70% ethanol
(1.41%), but worse in
γ
-asarone (0.79% versus 2.07%), cis-p-menth-2-en-1-ol (0.43% versus 5.88%),
4-carene, trans-(+)-(3.37% versus 6.56%) (Table 2).
By using hydrodistillation from nutmeg (HD1), 0.79
±
0.04 g of essential oil was obtained
(Figure 2).
Molecules 2019, 24, x 6 of 12
The composition of the essential oils, which were obtained using hydrodistillation (HD1) and
hydrodistillation with excipients from HD2 to HD4 (Table 1), showed that 1% of magnesium
aluminometasilicate significantly improved amount of α-pinene and sabinene compared to
conventional hydrodistillation, respectively 54.95% and 106.32%. Using 0.5% and 2% of magnesium
aluminometasilicate the amount of the α-pinene and sabinene was higher than HD1 but lower than
HD3 (Figure 1).
Figure 1. Magnesium aluminometasilicate’s influence on the amount of α and β-pinene, limonene,
and sabinene in the essential oils of nutmeg. Component quantity in essential oils, * p < 0.05 versus
hydrodistillation without magnesium aluminometasilicate. a Sample code can be seen in Table 1.
In HD1, essential oil consisted of β-copaene, bergamotene, bicyclogermacrene, cubebol,
cubenene, and piperitol, and these compounds were not identified in other extracts. Ten compounds
using magnesium aluminometasilicate were obtained, and in comparison to the essential oil of HD1,
their amount was not large but it was about 13.05 ± 3.54% of the quantity of all of the identified
compounds in essential oil.
Comparing water and ethanol as solvents, and maceration with ultrasound-assisted extraction
methods, it was found that water is better in extracting α-phellandrene (13.04%) than 70% ethanol
(1.41%), but worse in γ-asarone (0.79% versus 2.07%), cis-p-menth-2-en-1-ol (0.43% versus 5.88%), 4-
carene, trans-(+)-(3.37% versus 6.56%) (Table 2).
By using hydrodistillation from nutmeg (HD1), 0.79 ± 0.04 g of essential oil was obtained (Figure
2).
Figure 2. Essential oils’ mass and yield from nutmeg using HD and HD with magnesium
aluminometasilicate. Mass and yield of essential oil, * p < 0.05 versus hydrodistillation without
magnesium aluminometasilicate.
Figure 2.
Essential oils’ mass and yield from nutmeg using HD and HD with magnesium
aluminometasilicate. Mass and yield of essential oil, * p< 0.05 versus hydrodistillation without
magnesium aluminometasilicate.
At least this amount of mass was gained in all of the samples. Then we used the magnesium
aluminometasilicate, and the essential oil was obtained in higher quantities—from 1.19
±
0.09 g to 1.57
Molecules 2019,24, 1062 6 of 12
±
0.09 g (p< 0.05). The highest yield of essential oil (HD4, 10.43%) was obtained using 2% magnesium
aluminometasilicate (Figure 2).
3. Discussion
In this study, the extracts, essential oil, and the influence of excipient were analyzed.
The composition of the aqueous (M1) and ethanolic (UAE1–UAE6) extracts and essential oil (HD1–HD4)
of nutmeg were determined (Table 1). Our results show that the extract which was obtained using
maceration with water had 10 different chemical compounds of which the most commonly found are
myrislignan (22.59%), elemicin (13.99%), and
α
-phellandrene (13.04%). We did not find any research
done with aqueous extract of nutmeg. In ethanolic extract, which was made by using maceration,
the quantity of elemicin (5.7%) was 2.45 times lower, and the quantity of
α
-phellandrene was 21.73
times higher [
8
]. Elemicin has antibacterial effects [
29
] and
α
-phellandrene, anti-inflammatory [
30
],
so using maceration with water is more effective if a bigger effect is needed. Myrislignan has
anti-inflammatory [
31
] and anti-cancer [
32
] effects. This compound was not extracted using ethanol [
8
].
In our study, when 96% ethanol was used (ultrasound extraction, sample UAE3), the
α
-pinene
content was obtained at 14.67%, in other study using the same method, 6.75
±
0.92% was obtained [
8
].
In one of the studies, the compound of cis-p-menth-2-en-1-ol was obtained at about 0.1% [
8
]. However,
in our study using 96% ethanol the compound was obtained at 3.25%, which is 32.5 times more than in
the previously mentioned study, and if we compare this with extraction using 70% ethanol, the yield of
cis-p-menth-2-en-1-ol was obtained at 58.8 (5.88%) times more in our research (Table 2). Using nutmeg
seeds material from Grenada we did not find any isoelemicin (ultrasound extraction, 96% ethanol), the
other researchers found 0.8
±
0.46% [
8
], but we used 50% and 70% ethanol and determined the quantity
of isoelemicin, which was 62.24% and 23.63%, respectively. The excipients are used to improve the
extraction of the active compounds. Surfactants used in experiments improve the extraction of certain
compounds, such as Sodium dodecyle sulfate (SDS), Triton X 100, PEG 2000, Brij 35, Tween, etc. [
25
].
In our study we used magnesium aluminometasilicate as excipient, and our purpose was to research
and determine the effect of magnesium aluminometasilicate on the extraction of active compounds.
Previously, this substance has not been used in studies with extracts and essential oils. Magnesium
aluminometasilicate is used as excipient in solid drug form formulations [
26
,
27
,
33
]. Using this material,
we have relied on its mechanism of action. It adsorbs high loads of oil [
34
], so we predicted that it
could extract more essential oil components. The results of GC-MS analysis showed the influence of
the excipients on gaining different quantities of certain compounds. Magnesium aluminometasilicate
significantly improved the amount of sabinene. If we compare it to the essential oils that were made by
using simple hydrodistillation (Table 2), we see that 0.5% and 2% of magnesium aluminometasilicate
increased sabinene by 7.41
±
0.2 times (49.6% and 47.1%, traditional hydrodistillation—6.53%), the
1% of magnesium aluminometasilicate increased sabinene by 9.41 times (61.42%). These results are
significant. Dupuy [
7
] reported 6.00–36.08% quantity of sabinene, and the nutmeg seeds were from
Indonesia. Also, magnesium aluminometasilicate had influence on limonene (Figure 1) and
γ
-terpinene
extraction. The other samples made without the excipient did not have these compounds (M1, HD1,
UAE1-UAE3) (Table 2). Sabinene has anti-inflammatory, antifungal, and antioxidant effects [
35
,
36
],
limonene has low-toxicity and anticancer effect [
37
], and
γ
-terpinene characterizes as an antimicrobial
and antioxidant agent [
38
]. Magnesium aluminometasilicate’s influence on
α
-pinene and
β
-pinene was
different. The 1% of magnesium aluminometasilicate increased the quantity of
α
-pinene by 45% (from
8.27% to 15.05%) and decreased
β
-pinene by 84% (from 26.61% to 4.32%). Piaru [
9
] results showed that
Myristica fragrans seeds’s essential oil from Malaysia contained 8.5% α-pinene and 3.5% β-pinene.
After analyzing many studies with nutmeg essential oil we found a few compounds which were
not mentioned in publications. Our study showed that essential oil HD1 from nutmeg (Grenada)
contains
β
-copaene (0.25%), bergamotene (0.07%), citronellyl decanoate (0.3%), cubebol (0.05%),
cubenene (0.07%), and orthodene (0.51%) (Figure 3).
Molecules 2019,24, 1062 7 of 12
Molecules 2019, 24, x 8 of 12
contains β-copaene (0.25%), bergamotene (0.07%), citronellyl decanoate (0.3%), cubebol (0.05%),
cubenene (0.07%), and orthodene (0.51%) (Figure 3).
Figure 3. First time extracted and identified chemical compounds in essential oil of nutmeg.
In other research we found that the oleoresins of Myristica fragrans (Egypt) have α-copaene
(0.63%) [8] and trans-α-bergamotene (0.1%) [39]. The compound of cubebol was found in Piper
Cuberica Linn. essential oil and oleoresins [40] and Chromolaena odorata essential oil (8.6%) [41].
Cubenene was obtained in Caryopteris incana essential oil (9.7%) [42]. The γ-amorphene (0.03–0.78%)
and cis-α-bergamotene (0.08–0.1%) were obtained by using magnesium aluminometasilicate in
hydrodistillation (HD2–HD3) (Figure 3).
Volatile compound composition in extracts which have been produced using magnesium
aluminometasilicate have similarities with essential oil. Seventy-one percent of the chemical
compounds in the extract correspond to the composition of essential oil. However, only extracts with
magnesium aluminometasilicate extracted β-myrcene and isoelemicin, and these compounds are not
present in essential oil.
Our experimental data showed that the essential oil yield increased if we used magnesium
aluminometasilicate as excipient which facilitated volatile compounds extraction. Essential oil yield
from Myristica fragrans was 5.25 ± 0.04%; however, the magnesium aluminometasilicate was used for
the yield of the essential oil and it was from 7.9 ± 0.09% to 10.43 ± 0.09%. Using sea water, the yield
of oil-bearing rose increased statistically insignificantly by 0.045%, but the sea water significantly
decreases the citronellol rate from 41.49% to 33.56%, and significantly increases geraniol (from 17.58%
to 27.44%) [22]. Using non-ionic surfactants for extraction of sage essential oil it was found that Tween
20 increased the yield of oil at 1.83% and Span 80 or Span 20 had no effect [23]. Using 5% NaCl salt
solution for hydrodistillation, the yield of lavender oil was 5.01 ± 0.45% in comparison with only
water, where the yield was 4.55 ± 0.14% [14]. All essential oil samples with magnesium
aluminometasilicate have 21 chemical compounds, although essential oil made by simple
hydrodistillation has 24 compounds but that is not significantly important. The yield of nutmeg
essential oil significantly increases with magnesium aluminometasilicate as excipient. The essential
oil yield from nutmeg seeds (Nigeria) was 1.46% [43], from other nutmeg seeds (China) it was 2.4%
[44]. Nutmeg seeds from Indonesia were hydrodistillated and the yield of essential oil was obtained
at 5.84%.
Figure 3. First time extracted and identified chemical compounds in essential oil of nutmeg.
In other research we found that the oleoresins of Myristica fragrans (Egypt) have
α
-copaene
(0.63%) [
8
] and trans-
α
-bergamotene (0.1%) [
39
]. The compound of cubebol was found in Piper
Cuberica Linn. essential oil and oleoresins [
40
] and Chromolaena odorata essential oil (8.6%) [
41
].
Cubenene was obtained in Caryopteris incana essential oil (9.7%) [
42
]. The
γ
-amorphene (0.03–0.78%)
and cis-
α
-bergamotene (0.08–0.1%) were obtained by using magnesium aluminometasilicate in
hydrodistillation (HD2–HD3) (Figure 3).
Volatile compound composition in extracts which have been produced using magnesium
aluminometasilicate have similarities with essential oil. Seventy-one percent of the chemical
compounds in the extract correspond to the composition of essential oil. However, only extracts
with magnesium aluminometasilicate extracted
β
-myrcene and isoelemicin, and these compounds are
not present in essential oil.
Our experimental data showed that the essential oil yield increased if we used magnesium
aluminometasilicate as excipient which facilitated volatile compounds extraction. Essential oil yield
from Myristica fragrans was 5.25
±
0.04%; however, the magnesium aluminometasilicate was used
for the yield of the essential oil and it was from 7.9
±
0.09% to 10.43
±
0.09%. Using sea water, the
yield of oil-bearing rose increased statistically insignificantly by 0.045%, but the sea water significantly
decreases the citronellol rate from 41.49% to 33.56%, and significantly increases geraniol (from 17.58% to
27.44%) [
22
]. Using non-ionic surfactants for extraction of sage essential oil it was found that Tween 20
increased the yield of oil at 1.83% and Span 80 or Span 20 had no effect [
23
]. Using 5% NaCl salt solution
for hydrodistillation, the yield of lavender oil was 5.01
±
0.45% in comparison with only water, where
the yield was 4.55
±
0.14% [
14
]. All essential oil samples with magnesium aluminometasilicate have
21 chemical compounds, although essential oil made by simple hydrodistillation has 24 compounds
but that is not significantly important. The yield of nutmeg essential oil significantly increases with
magnesium aluminometasilicate as excipient. The essential oil yield from nutmeg seeds (Nigeria) was
1.46% [
43
], from other nutmeg seeds (China) it was 2.4% [
44
]. Nutmeg seeds from Indonesia were
hydrodistillated and the yield of essential oil was obtained at 5.84%.
Molecules 2019,24, 1062 8 of 12
4. Materials and Methods
4.1. Materials
The dried seeds of Myristica fragrans (nutmeg) were from Grenada. The seeds were identified by
Jurga Bernatoniene, Medical Academy, Lithuania University of Health Sciences, Kaunas, Lithuania.
A voucher specimen (I 18922) was placed for storage at the Herbarium of the Department of Drug
Technology and Social Pharmacy, Lithuanian University of Health Sciences, Lithuania. The seeds were
a brown-beige color, had a characteristic odor, and strong, bitter, and spicy flavor. Dried nutmeg seeds
were ground into a powder (particles smaller than 0.5 mm) with a laboratory mill. The samples were
kept in an airtight container in a dark environment and in ambient temperature.
Magnesium aluminometasilicate (Neusilin
®
US2, Fuji Chemical Industries Co., Ltd., Toyoma,
Japan) was used as excipient. Ethanol 96% (Vilniaus degtine, Lithuania) was used as solvent for
extraction. Distilled water was used throughout the experiment.
4.2. Maceration
The nutmeg seeds’ powder was extracted using a maceration technique with water in ambient
temperature while occasionally stirring. The process lasted 3 days. Powder and solvent ratio were 1:20
(w/v). The extracts were filtered with paper filter (0.22
µ
m pore size) and stored in refrigerator at 4
◦
C
until further analysis. Conditions are mentioned in Table 1.
4.3. Ultrasound Assisted Extraction
Extracts were also prepared by using ultrasound-assisted extraction. This method was used as a
simple and effective method for nutmeg extracts. The 1 g material and 20 mL solvent were used. We
used different concentrations of ethanol for extraction (96%, 70%, and 50%). The flask with substance
was put in an ultrasound bath (Sonorex Digitec DT 156 BH, Germany). Extracts were prepared on
25
◦
C temperature for 30 min. After that, the extracts were filtered with paper filter and stored in a
refrigerator at 4 ◦C until further analysis.
Some samples were modified and made with magnesium aluminometasilicate. The extracts were
made in the same conditions which were listed earlier. But for the solvent only a certain concentration
of ethanol (70%) was used and the excipient was added in the mixture. The excipients’ concentration
was from 0.5% to 2%. Magnesium aluminometasilicate (g) was based on ethanol quantity. Extracts
were filtered and stored. Conditions are mentioned in Table 1.
4.4. Hydrodistillation
Samples were prepared from nutmeg seeds and purified with water. The solid material
was 15 g and the water was 300 mL. A Clevenger-type apparatus (European pharmacopoeia) for
hydrodistillation was used. Hydrodistillation was carried out for 4 h. A colorless essential oil with
specific odor was obtained. The essential oil was collected with water in airtight bottle and stored in a
refrigerator at 4 ◦C until needed.
The hydrodistillation method was modified and magnesium aluminometasilicate as excipient was
used. The amount of magnesium aluminometasilicate was based on water quantity, its concentration
was from 0.5% to 2%. Fifteen grams of nutmeg seed powder were used and all of the sample ratios
of solvent were the same (20:1). The essential oil was obtained and stored in the same conditions.
Conditions are mentioned in Table 1.
4.5. Gas Chromatography-Mass Spectrometry Analysis
Gas chromatography-mass spectrometry analysis was performed on a GCMS-QP2010 system
(Shimadzu, Tokyo, Japan). Twenty microliters of sample (extract or essential oil) was diluted to 1 mL
with hexane (
≥
99%, Sigma–Aldrich, Germany). The column used was a 30 m
×
0.25 mm i.d.
×
0.25
Molecules 2019,24, 1062 9 of 12
µ
L film thickness RTX-5MS column. Flow rate of helium (99.999%, AGA Lithuania) carrier gas was
set at 1.23 mL/min. The oven temperature was maintained at 40
◦
C for 2 min after injection and then
programmed at 3
◦
C/min to 210
◦
C, at which the column was maintained for 10 min. The split ratio
was 1:10. The mass detector electron ionization was 70 eV. Identification of volatile compounds was
carried out using mass spectra library search (NIST 14) and compared with the mass spectral data
from literature [45].
4.6. Statistical Analysis
Data are presented as mean
±
SEM. Statistical analysis was preformed using Student’s t-test.
The results were significant when p< 0.05.
5. Conclusions
Extracts and essential oils from Myristica fragrans seeds (Grenada) were prepared using three
different methods (M, UAE, HD). The volatile compounds were analyzed by GC-MS. The results
showed the samples’ quality and quantity of the chemical compounds composition. During
the experiment magnesium aluminometasilicate was used for extraction in the hydrodistillation
process. Previously, this excipient was used for the production of solid dosage forms. Ten chemical
compounds were obtained using maceration, from eight to thirteen compounds were obtained using
ultrasound-assisted extraction with different ethanol concentrations and without excipient, using UAE
with magnesium aluminometasilicate, 16–19 compounds were obtained. Hydrodistillation with
magnesium aluminometasilicate lead to 21 compounds, and finally, 24 compounds were obtained
without the use of excipient. Although using hydrodistillation with the excipient fewer chemical
compounds were obtained, some of the compounds’ quantities were bigger than using hydrodistillation
without excipient. Magnesium aluminometasilicate as excipient improved the extraction of
α
-pinene,
limonene, isoterpinolene, and sabinene, although it reduced the quantity of
β
-pinene. For the first
time, eight chemical compounds in nutmeg essential oil were identified. Two of these compounds
(γ-amorphene and cis-α-bergamotene) were obtained by the use of magnesium aluminometasilicate.
The yield of the essential oil from nutmeg seeds was significantly higher using magnesium
aluminometasilicate. The data shows that essential oil yield which was obtained by hydrodistillation
with water was 5.25
±
0.04% and it increased using hydrodistillation with water and 2% of magnesium
aluminometasilicate to 10.43
±
0.09%. Using the 2% of magnesium aluminometasilicate the yield of
essential oil increased by almost twice.
The use of magnesium aluminometasilicate in the extraction process improved the yield of
essential oil and the number of compounds in the extracts.
Author Contributions:
I.M., Z.K., and J.B. conceived and designed the experiments; L.I. and M.M. performed
experiments with GC-MS; I.M., M.M., R.L. and J.B. analyzed the date; I.M. and J.B. wrote the paper.
Acknowledgments:
The authors would like to thank Open Access Centre for the Advanced Pharmaceutical
and Health Technologies (Lithuanian university of Health Sciences) and for the opportunity to use modern
infrastructure and perform this research.
Conflicts of Interest:
The authors declare that there are no competing interests regarding the publication of
this paper.
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).