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Chemical Composition of the Essential Oils of Nutmeg and Mace by GC-

FID/MS Indigenous to Pakistan and Evaluation of their Biological Activities

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 Latin American Journal of Pharmacy Regular article
(formerly Acta Farmacéutica Bonaerense) Received: July 18, 2016
Revised July 21, 2016
Lat. Am. J. Pharm. 35 (10): 2176-2184 (2016) Accepted: July 22, 2016

Chemical Composition of the Essential Oils of Nutmeg

and Mace by GC-FID/MS Indigenous to Pakistan
and Evaluation of their Biological Activities
Muhammad I. SHAFIQ 1, Mahmood AHMED 2 *, Ayesha RASUL 1, Zahoor Q. SAMRA 1,
Muhammad A. QADIR 2, Sania MAZHAR 3 & Amir ALI 2

1
Institute of Biochemistry and Biotechnology &
2 Institute of Chemistry,

University of the Punjab, University of the Punjab, Lahore-Pakistan, 54590

3 PCSIR Laboratories Complex, Lahore-Pakistan, 54600

SUMMARY. Myristica fragrans Houtt. (nutmeg and mace) has rich historic reports of its medicinal effica-
cy. In vitro effectiveness of essential oils (EOs) of nutmeg and mace has been evaluated against an array of
emerging plant and human pathogenic bacterial and fungal strains. The activity of nutmeg and mace
against three filamentous fungal strains (Alternaria alternata, Fusarium solani and Penicillium digitatum)
tested in this study has not been depicted earlier. Both the EOs did not demonstrate strong inhibitory ac-
tivity against the test bacterial strains except Bacillus subtilis subsp. spizizenii which appeared to be suffi-
ciently sensitive and equally susceptible to both oils with largest zone size (32 mm) and sensitivity compa-
rable to ciprofloxacin. Antifungal efficacy of both essential oils revealed convincing results (zone size
ranged from 18 to ≥ 45 mm) against all the six assayed fungal strains (Aspergillus niger, A. flavus, Fusari-
um oxysporum, F. solani, A. alternata, and P. digitatum). Fungicidal action of both EOs was confirmed at a
concentration of 2 μg/mL against all fungi excluding Fusarium species (6 μg/mL).The broad antifungal
spectrum together with long term inhibitory potential demonstrated by nutmeg and mace essential oils
strongly recommends their use in formulation of novel antifungals as well as in preservation of foods.
RESUMEN. Myristica fragrans Houtt. (nuez moscada y macis) tiene una rica historia de eficacia medicinal. La
eficacia in vitro de los aceites esenciales (EOs) de la nuez moscada y el macis ha sido evaluada contra una gran
variedad de cepas bacterianas y fúngicas patógenas humanas y de plantas. La actividad de la nuez moscada y el
macis contra tres cepas de hongos filamentosos (Alternaria alternata, Fusarium solani y Penicillium digitatum)
ensayados en este estudio no se ha presentado anteriormente. Ninguno de los EOs mostraron actividad inhibidora
frente a las cepas bacterianas de ensayo, excepto sobre Bacillus subtilis subsp. spizizenii, que resultó suficiente-
mente sensible e igualmente susceptible frente a ambos EOs, con mayor tamaño de la zona de inhibición (32
mm) y sensibilidad comparable a la de ciprofloxacina. La eficacia antifúngica de ambos EOs reveló resultados
convincentes (el tamaño de la zona de inhibición varió de 18 a ≥ 45 mm) contra las seis cepas fúngicas ensayadas
(Aspergillus niger, A. flavus, Fusarium oxysporum, F. solani, A. alternata, and P. digitatum). La acción fungici-
da de ambos EOs se manifestó en una concentración de 2 mg/mL frente a todos los hongos con exclusión de es-
pecies de Fusarium (6 mg/mL). El amplio espectro antifúngico junto con el potencial inhibidor a largo plazo de-
mostrado por los EOs de la nuez moscada y el macis hace recomendable su uso en la formulación de nuevos anti-
fúngicos, así como en la preservación de los alimentos.

INTRODUCTION fruits and other plant product while the high

Bacterial activity is considered to be the prin-
cipal root of numerous food borne illnesses as
well as a major cause of several infections in
humans 1. Salmonella enterica infections are
transmitted not only by animal-derived foods
like chicken and eggs but also by vegetables,
count of Staphylococcus aureus, Escherichia
coli, and Pseudomonas aeruginosa are found in
municipal water 2,3. S. aureus and Enterobacter
aerogenes is also responsible for post-operative
wound infections, endocarditis and Bacillus sub-
tilis subsp. spizizenii has major role in food poi-

KEY WORDS: antibacterial, antifungal, antioxidant, essential oils, medicinal plant, Myristica fragrans.
* Author to whom correspondence should be addressed. E-mail: mahmoodresearchscholar@gmail.com

ISSN 0326 2383 (printed ed.)

2176 ISSN 2362-3853 (on line ed.)

soning. Klebsiella pneumoniae is the culprit for
hospital-acquired urinary tract infections, sep-
ticemia, severe pneumonia, meningitis and intra-
abdominal-infections 4-6. Postharvest deteriora-
tion of crops and spoilage of stored food com-
modities by moulds is emerging as a seriously
grueling challenge particularly for the warm and
humid regions of world 7.
Aspergillus, Fusarium and less commonly Al-
ternaria are among those few genera of fila-
mentous fungi that have emerged as infectious
pathogens not only for plants but also for hu-
mans and animals 8. The principal causative
agents of fungal keratitis in humans are also the
Fusarium species 9. Fusarium solani causes ker-
atitis that is not easy to diagnose and treat, often
results in rapid corneal sloughing and serious
vision loss. A. niger and A. flavus can cause oto-
mycosis in healthy individuals 10. In certain ar-
eas of China and South Africa, where the con-
sumption of incurred commodities is high, Al-
ternaria toxins in grains are associated with oe-
sophageal cancer 11. The ever increasing find-
ings concerning herbs and spices as sources of
natural antioxidants have stimulated researchers
to look for natural antioxidants with low cyto-
toxicity 12,13.
Free radicals, which cause oxidative stress,
are believed to play a crucial role in human
health. These free radicals cause damage to the
components of the cell, resulting in cellular and
metabolic injury such as cardiovascular diseases,
inflammation and cancer 14-16. Myristica fra-
grans, a prominent member of the genus Myris-
tica from the family Myristicaceae, is a medium
heighted evergreen aromatic tree native to the
Maluku Islands in Indonesia. This plant has rich
historic reports of its medicinal efficacy and is
still the focus of some recent researches for its
excellent therapeutic properties. The fully ripen
fruit of the plant contains a brown ovoid crum-
pled seed with bright red aril veins around 17,18.
The seed is nutmeg and its exterior casing is
mace and both these intimate relatives are pop-
ular condiments of paramount importance with
common names Jaiphal and Javatri. In Pakistani
culture it is being used as spice in different food
dishes. Detailed anti-fungal studies on essential
oils (EOs) from this plant are quite neglected.
This study aimed to evaluate concurrently the
chemical composition, antioxidant, antibacterial,
and antifungal efficacy of nutmeg and mace es-
sential oils. This is in best of our knowledge,
antimicrobial possession of EOs from nutmeg
Latin American Journal of Pharmacy - 35 (10): 2176-84 (2016)
and mace is not reported indigenous to Pakistan
in such extensive way as we did, although it is
studied from different geographical region.
MATERIALS AND METHODS
Plant material and essential oil extraction
Nutmeg seeds and mace were collected from
Myristica fragrans Houtt. grown in Botanical
Garden, Quaid-e-Azam Campus, Punjab Univer-
sity, Lahore-Pakistan. Fresh essential oils were
extracted from dried plant parts in powdered
form (200 g) by subjecting to modified Cle-
venger’s apparatus 14. The obtained volatile oils
were dried over anhydrous sodium sulphate
(Sigma-Aldrich) and stored at 4°C in sealed vials
until screening. Maximum oil yield for each
sample was calculated.
GC-FID and GC-MS analysis
GC-FID and GC-MS equipped with non-polar
(HP-5MS) and polar (DB-Wax) columns for
chemical characterization of the EOs. GC-MS
coupled to mass spectrometry (Model 7010-Agi-
lent Technologies). HP-5MS capillary columns
(30 m × 0.25 mm × 0.25 µm) were used and us-
ing splitless mode the injector was set at 250 °C.
Analysis was performed in electron impact
mode at 70 eV with a mass range from m/z 40
to 500. Helium was used as the carrier gas at the
linear flow rate of 1 mL/min. The temperature
was initially programmed at 50 °C (held for 2
min) and then raised to 100-250 °C at the rate of
3 °C/min, thereafter held constant at 260 °C for
20 min. DB-Wax fused silica capillary column
(30 m × 0.25 mm × 0.25 µm) was used for sup-
plementary analysis. The operation conditions
for DB-Wax programmed at 40 °C (held for 5
min) and then raised to 100-250 °C at the rate of
3 °C/min, thereafter held constant at 220 °C for
20 min. Under the same operating condition as
described above GC analysis performed on GC
chromatograph (Model 7010-Agilent Technolo-
gies) equipped with FID using both polar (DB-
Wax) and nonpolar (HP-5MS) columns. The res-
olutions of constituents were obtained by inject-
ing 1 µL sample to the column. The relative per-
centages of EO constituents were calculated
based on GC peak areas. Straight-chain hydro-
carbons mixtures C8-C31 (Sigma-Aldrich, USA)
were injected into both polar (DB-Wax) and
nonpolar (HP-5MS) columns under same operat-
ing conditions for oil samples to obtain linear
retention indices (LRI) of constituents present in
oils.
2177
SHAFIQ M.I., AHMED M., RASUL A., SAMRA Z.Q., QADIR M.A., MAZHAR S. & ALI A.
Antimicrobial assay
Bacterial and fungal strains
Seven bacterial strains Bacillus subtilis subsp.
spizizenii ATCC 6633, Staphylococcus aureus
ATCC 25923, Salmonella enterica ATCC 14028,
Escherichia coli ATCC 25922, Enterobacter aero-
genes ATCC 13048, Pseudomonas aeruginosa
ATCC 27853, Klebsiella pneumoniae ATCC
13882 and six indigenous pathogenic fungal iso-
lates have Accession No. Aspergillus niger 1109,
Aspergillus flavus 1110, Fusarium oxysporum
1175, Fusarium solani 1199, Alternaria alterna-
ta 1200, and Penicillium digitatum 1160 were
obtained from Institute of Agricultural Sciences,
University of the Punjab, Lahore, Pakistan. The
bacterial and fungal cultures were revitalized
from the respective fresh isolates and main-
tained on tryptic soy agar (TSA) and potato dex-
trose agar (PDA) respectively slants at 4 °C until
use.
MIC, MBC, MFC and percentage growth
inhibition
Ciprofloxacin and gentamicin were used as
reference (positive control to check the sensitiv-
ity of tested bacterial strains). 1-3 × 108 cfu/mL
of each of gram negative E. coli, P. aeruginosa,
E. aerogenes, K. pneumoniae, S. enterica and
gram positive S. aureus, B. subtilis subsp. spiz-
izenii, were obtained after adjusting the optical
density of inoculum at 0.2-0.3 and 0.3-0.4 (620
nm) respectively 19-22. While fungal suspension
(A. niger, A. flavus, F. oxysporum, F. solani, A.
alternata and P. digitatum) with cell density of
106 cfu/mL (0.1 OD at 700 nm) 22 was studied in
present work and the ketoconazole, nystatin
were used as reference antifungal agent. Agar
wells (6 mm diameter) in triplicate were punc-
tured via sterile cork borer and loaded with 90
µL of pure undiluted sample oils (nutmeg and
mace) in test plates containing agar media by
micropipette (Thermo Scientific, UK) and com-
pared to control plate wells loaded with stan-
dard drugs. The plates were finally incubated at
35 °C (bacterial strains) and 25 °C (fungi strains)
in cold incubator (Sanyo Mir 253, Japan) to al-
low diffusion of oil through agar. Measurements
of growth inhibition zones were made in day
light using digital vernier caliper (Starrett 799A-
6/150, USA) after incubation for 2 days (48 h).
Broth dilution assay was performed for determi-
nation of minimum inhibitory concentration
(MIC) for both EOs against the bacterial and
fungal strains. Two fold serial dilutions of both
EOs were made in methanol from 2.0-0.015
2178
mg/mL for MIC determination for bacterial
strains while 6.0, 4.0, 2.0 and 0.4 µg/mL for MIC
calculation against fungal strains. In order to as-
sess the bactericidal and fungicidal activity of
oils on the test bacteria and fungi, the same
concentration range previously used for evaluat-
ing MIC by broth dilution assay was adopted.
Minimum concentration of EO that entirely
seized the bacterial and fungal growth and did
not allow slightest growth revival in liquid broth
after 48 h of incubation was considered as mini-
mum bactericidal concentration (MBC) and min-
imum fungicidal concentration (MFC). For per-
centage growth inhibition, 100 µL aliquot at a
concentration of MBC and MFC was then sub-
cultured onto sterile plate containing respective
agars (TSA and PDA) then incubated at 35 °C
(bacterial growth inhibition) and 25 °C (fungal
growth inhibition) for 24-48 h. After incubation,
emergent bacterial colony forming units were
marked over each plate and counted as
CFU/mL. Percentage growth inhibition of sam-
ple oils against each test strain was calculated in
comparison to control plate count.
Antioxidant activity
Free radical scavenging activity
DPPH free radical scavenging activity (RSA)
was determined as reported earlier with slight
modification 23,24. DPPH (Sigma- Aldrich, St
Louis, MO, USA) (50 µL, 1 mmol/L) in methanol
was added to 200 µL of various concentrations
of sample in methanol (range 6.25-100 µg/mL)
and the mixture was shaken vigorously and left
at room temperature for 30 min in dark. DPPH
radical scavenging effect was determined by
measuring the absorption at 517 nm. The per-
centage scavenging activity of DPPH radical was
calculated as follows: % RSA = ([A DPPH –
AS ] /ADPPH) × 100, where AS is the absorbance of
sample solution and ADPPH is the absorbance of
the DPPH solution. Butylated hydroxytoluene
(BHT) was used as a reference standard. Extract
concentration giving 50 % inhibition (IC50) was
calculated from the graph of inhibition percent-
age versus concentration of extract. Each sample
assay was carried out in triplicate and data were
presented as a mean of the three values.
RESULTS AND DISCUSSION
Essential oils yields and chemical
composition
On the basis of dry weight of the plant mate-
rial, extraction of essential oils by Clevenger’ ap-
 Latin American Journal of Pharmacy - 35 (10): 2176-84 (2016)

paratus was done and the yields of nutmeg and
mace EOs obtained in the current study were
7.6 and 10.3%, falling in the ranges (5-15%) and
(7-14%), respectively, as reported in the litera-
ture 25. Although numerous investigations have
been made on the chemical analysis of nutmeg
volatile oil, however there are comparatively
few reports on mace. GC analysis of nutmeg
and mace essential oil in this study revealed that
each contains 17 components comprising 98.7
and 94.1 % of the oils correspondingly. Data in
Tables 1 and 2 present the identified con-
stituents from the two EOs with their LRI, con-
tent percentages and m/z values (major frag-
ments) using two different columns HP-5 MS
and DB-Wax. The comparison of composition
of both EOs revealed 9 common constituents. In
agreement with previous investigation 26, the
key components of both oils were terpenes fol-
lowed by terpene-alcohols and phenolics. The
main fraction of nutmeg EO was comprised of
monoterpenes (sabinene:18.9 %, α- pinene:
15.8%, m-cymene: 15.2 %), terpene alcohols
(terpinen-4-ol:11.7 %) and phenylpropenes (el-
emicin:11.5 %, safrole: 6.2) whereas the chief
constituents of mace EO included monoterpenes
(γ -terpinene: 19.1 %, α -pinene: 11.6 %,
sabinene: 11.2%), terpene alcohols (terpinene-4-
ol: 12.7), phenylpropenes (safrole: 18.2 %,
myristicin: 7.4 %). Sabinene was found to be the
key constituent of nutmeg EO and the dominant
aromatic ethers in mace EO were myristicin and
safrole in contrast to nutmeg EO.
Biological evaluation
Nutmeg and mace, the two popular spices
and close relatives from the plant Myristica fra-
grans have remained and are still the focus of

Identified Constituent a LRI b LRI Nutmeg (%) Mace (%)

α-Pinene 932 1015 15.8 11.6

Sabinene 969 1121 18.9 11.2
m-cymene 1023 1266 15.2 -
γ-terpinene 1057 1238 3.5 19.1
α-terpinolene 1089 1274 3.5 3.4
Cis-p-2-Menthen-1-ol 1121 1614 2.2 -
Trans-p-2-Menthen-1-ol 1142 1585 1.2 0.8
cis-β-Terpineol 1147 1641 0.7
Terpinen-4-ol 1177 1606 11.7 12.7
2-pentyl-anisole 1203 - - 1.7
Safrole 1287 1872 6.2 18.2
Eugenol 1363 2167 - 1.7
Iso-safrole 1367 - - 0.2
Geranyl acetate 1386 1744 1.5 -
β-Caryophyllene 1431 1599 1.9 -
α-Caryophyllene 1452 1636 0.8 -
Iso-eugenol 1456 2330 - 0.3
Methyl-eugenol 1460 2030 2.3 3.5
Iso-eugenol methyl ether 1462 - - 0.6
β-Cadinene 1474 - 1.1 0.2
Myristicin 1529 - - 7.5
Elemicin 1554 2239 11.5 -
Methoxy-eugenol 1582 2554 - 0.2
Caryophyllene oxide 1588 1991 0.8 -
Palmitic acid 1968 2910 0.6 -

Total identified 98.7 94.1

Table 1. Composition of essential oils derived from nutmeg and mace with retention indices . a LRI determined
linear retention index on HP-5 MS column; b LRI =determined linear retention index on DB-Wax column.

2179
SHAFIQ M.I., AHMED M., RASUL A., SAMRA Z.Q., QADIR M.A., MAZHAR S. & ALI A.

Identified Constituent Fragments: m/z (%)

α-Pinene M+ 136(16), 105(14), 121(17), 93(100), 77(34), 67(7), 53(7)

Sabinene M+ 136(20), 121(9), 107(4), 93(100), 77(35), 69(13), 63(2), 53(7)
m-cymene M+ 134(33), 119(100), 103(4), 93(43), 77(17), 65(7), 51(4)
γ-terpinene M+ 136(59), 121(42), 105(14), 93(100), 77(34)
α-terpinolene M+ 136(97), 121(100), 105(25), 93(86), 79(32), 67(9), 53(9)
Cis-p-2-Menthen-1-ol M+ 154(8), 139(36), 132(100), 125(7), 117(98), 107(14), 93(67), 81(53), 71(82)
Trans-p-2-Menthen-1-ol M+ 154(3), 139(19), 121(14), 110(55), 95(36), 91(28), 83(12), 79(100), 71(10)
cis-β-Terpineol M+ 154(4), 139(18), 132(100), 117(97),105(11), 93(71), 81(30), 71(73), 63(9), 55(37)
Terpinen-4-ol M+ 154(33), 136(21), 125(5), 111(82), 93(68), 86(27), 71(100), 79(8), 55(22)
2-pentyl-anisole M+ 178(26), 161(4), 136(6), 121(100), 105(7), 95(14), 77(13), 67(4), 55(8)
Safrole M+ 162(100), 131(40), 104(38), 91(6), 77(22), 63(6), 51(11)
Eugenol M+ 164(100), 149(38), 137(20), 131(26), 121(18), 103(26), 91(21), 77(23), 65(8)
Iso-safrole M+ 162(100), 131(39), 104(38), 91(10), 77(19), 69(2), 63(7), 51(9)
Geranyl acetate M+ 136(19), 121(24), 112(27), 93(33), 83(19), 69(100), 53(12)
β-Caryophyllene M+ 204(12), 189(27), 175(14), 161(41), 147(34), 133(100), 120(44), 105(59), 93(94)
α-Caryophyllene M+ 204(11), 161(7), 147(23), 121(35), 107(23), 93(100), 80(31), 67(19), 55(17)
Iso-eugenol M+ 164(100), 149(36), 131(21), 121(15), 103(22), 91(21), 77(21), 65(6), 55(12)
Methyl-eugenol M+ 178(100), 163(33), 147(33), 115(9), 103(22), 91(23), 77(9), 65(6), 51(4)
Iso-eugenol methyl ether M+ 178(100), 163(41), 147(11), 135(8), 115(10), 107(27), 91(22), 77(9), 65(6), 51(4)
β-Cadinene M+ 204(60), 189(19), 161(100), 145(7), 134(53), 119(58), 105(45), 91(31), 81(18), 69(6)
Myristicin M+ 192(100), 177(7), 165(23), 147(13), 131(16), 119(18), 103(8), 91(20), 77(9), 65(10)
Elemicin M+ 208(100), 193(62), 177(13), 165(10), 150(10), 133(16), 118(10), 105(10), 91(10)
Methoxy-eugenol M+ 194(100), 179(14), 167(11), 161(6), 147(13), 131(16), 119(19), 103(8), 91(23)
Caryophyllene oxide M+ 220(10), 205(11), 187(37), 131(58), 121(54), 107(85), 91(100), 79(78), 67(48), 55(61)
Palmitic acid M+ 256(11), 228(25), 208(100), 193(92), 171(16), 159(29), 143(19), 129(49), 115(25)
Table 2. Fragments of identified compounds from essential oils derived from nutmeg and mace.

various investigations for their immense phar-
maceutical worth. Even though several research-
es have documented the antimicrobial potential
of this plant, however a comparison of results is
highly unfeasible presumably because of varia-
tion in choice of test methodology, the type and
source of test strains (clinical isolate/reference
strain), culture medium, incubation time and
temperatures, the nature of antimicrobial sample
(extract/volatile oil/oleoresin), the geographical
location of plant. Initial screening of chosen
EOs (nutmeg and mace) for antibacterial activity
was done using agar well method. The results
are displayed in Table 3.
distinct defined zones; in some cases the zones
were jagged or vague. On the whole, nutmeg
and mace EOs did not demonstrate strong in-
hibitory activity against the tested bacterial
strains by agar well method except B. subtilis
subsp. spizizenii, which appeared to be suffi-
ciently sensitive and equally susceptible to both
oils with largest zone size (32 mm) and sensitiv-
ity comparable to ciprofloxacin. Smallest zones
were obtained against P. aeruginosa for both
oils. P. aeruginosa is well-known for its intrinsic
resistance towards antibiotics that is accredited
to the low permeability of its outer membrane
27. The poor inhibition of this strain can thus be

The oils showed inhibitory activity against
each of the seven test bacterial strains including
two gram positive (B. subtilis subsp. spizizenii
and S. aureus) and five gram negative strains (S.
enterica, E. coli, E. aerogenes, K. pneumoniae
and P. aeruginosa). Inhibition zones size ranged
from 12.0-32 mm. Not all the strains exhibited
related to the restricted diffusion of active EO
constituents through its outer membrane.
The inhibitory activity of nutmeg EO was
found to decrease in the order; B. subtilis subsp.
spizizenii > E. coli > E. aerogenes > S. aureus >
S. enterica > P. aeruginosa. For mace EO, the
order was B. subtilis subsp. spizizenii > S. au-

2180
 Latin American Journal of Pharmacy - 35 (10): 2176-84 (2016)

Nutmeg EO Mace EO Gentamicin Ciprofloxacin

Bacterial strains
(90 µL) (90 µL) (10 µg) (5 µg)

B. subtilis subsp. spizizenii 32.0 ± 0.1 32.0 ± 0.1 26.4 ± 0.1 34.2 ± 0.2
S. aureus 12.5 ± 0.3 16.0 ± 0.2 21.2 ± 0.3 29.8 ± 0.3
S. enterica 12.0 ± 0.2 15.0 ± 0.2 22.3 ± 0.1 35.1 ± 0.1
E. coli 14.1 ± 0.0 15.6 ± 0.1 23.2 ± 0.2 36.1 ± 0.4
E. aerogenes 12. 8 ± 0.2 13.2 ± 0.3 14 ± 0.2 15 ± 0.2
K. pneumoniae NI NI 20.4 ± 0.1 30.1 ± 0.1
P. aeruginosa 10.0 ± 0.2 10.5 ± 0.2 20.1 ± 0.3 30.1 ± 0.1
Table 3. Diameter of zone of inhibition (mm ± SD) of the pure undiluted nutmeg and mace essential oils after
24 h incubation. SD: standard deviation (n = 3); NI: no inhibition.

Nutmeg EO Mace EO Gentamicin Ciprofloxacin

Bacterial
strains MIC/MBC % MIC/MBC % MIC/MBC % MIC/MBC %
mg/mL inhibition mg/mL inhibition µg/mL inhibition µg/mL inhibition

B. subtilis
1/2 99.9 1/2 99.9 0.062/0.125 99.9 1.25/2.50 99.9
subsp. spizizenii
S. aureus 1/2 99.8 2/2 100 0.062/0.125 99.9 1.25/2.50 99.9
S. enterica 1/2 99.9 1/2 100 0.015/0.062 99.9 0.125/2.50 99.9
E. coli 1/1 99.9 1/2 100 0.015/0.062 99.9 0.625/2.50 99.9
E. aerogenes 1/2 99.7 1/2 99.9 0.062/0.250 99.9 0.25/0.50 99.9
K. pneumoniae 1/2 99.9 1/2 99.9 0.015/0.062 99.9 0.625/1.25 99.9
P. aeruginosa 1/2 99.4 1/2 99.2 0.015/0.062 99.9 0.25/0.50 99.9
Table 4. MIC, MBC and percentage inhibition of the pure undiluted nutmeg and mace essential oils.

reus > E. coli > S. enterica > E. aerogenes > P.
aeruginosa. The only strain for which both oils
did not generate any inhibition zone but appre-
ciably reduced the overall growth was K. pneu-
moniae. A likely and reasonable explanation for
the low anti-bacterial action plus the hazy and
indistinct zones observed in screening assay can
be the poor solubility in water and hence the
incomplete, irregular diffusion of EOs through
the solid nutrient agar medium since no solubili-
ty enhancer was added to EO most of which are
supposed to contribute to antimicrobial action.
Moreover, the composition of EO, the interac-
tion between the oil constituents as well as the
cell structure and the intrinsic resistance of each
particular strain also affect the activity of EO.
Excluding P. aeruginosa, E. aerogenes and S.
aureus, the nutmeg oil was found to pose a
strong bactericidal effect against the other four
bacterial strains (B. subtilis subsp. spizizenii, E.
coli, S. enterica and K. pneumoniae) by absolute
sequestering of their growth. The tubes stayed
totally free of turbidity even up to 10 days incu-
bation. For the three strains P. aeruginosa, E.
aerogenes and S. aureus, the oil exhibited static
effect. The growth of P. aerogenosa and E. aero-
genes was inhibited for at least 24 h whereas the
multiplication of S. aureus was restrained by
nutmeg oil up to 48 h after which turbidity was
noticed. The percentage inhibition of each test
bacterial strain in broth by individual oils using
viable count technique is given in Table 4 along
with their MIC’s.
In pure form, both oils were found to inhibit
all test strains no less than 99%. Bactericidal
concentration is defined as the concentration
which kills 99.9 % (or greater) of inoculum. Nut-
meg EO exhibited 99.9% killing action against
B. subtilis subsp. spizizenii, E. coli, S. enterica
and K. pneumoniae illustrating its cidal effect.
For the rest three strains, inhibition was less
than 99.9 % showing the static effect of oil.
Mace EO was cidal in action with 99.9% or
greater killing activity against all strains except
P. aeruginosa for which it showed static effect
(< 99.9 % growth inhibition). A summary of an-
tifungal activity of both fresh essential oil sam-
ples after two and seven days incubation (25

2181
SHAFIQ M.I., AHMED M., RASUL A., SAMRA Z.Q., QADIR M.A., MAZHAR S. & ALI A.

°C) expressed as mean diameter of inhibition
zones is given in Table 5. Both oils were found
effective against all tested strains to varying ex-
tents. Nutmeg oil showed maximum inhibition
for A. alternata (33 mm) followed by A. niger
(30 mm), A. flavus (26.5 mm), F. oxysporum (21
mm), P. digitatum (26.5 mm) and F. solani (18
mm). Mace oil produced clear zones of 45 mm
diameter against all 6 fungal strains when exam-
ined after an incubation of 48 h.
No change in zone size was noticed even af-
ter 7 days incubation for A. niger, A. flavus and
A. alternata, however reduction in zone size
was seen in case of both Fusarium species and
P. digitatum. Both EO’s have higher zone of in-
hibition than the standard drugs (8 µg/mL) used
as reference. On the whole, mace EO demon-
strated higher antifungal potential against all
fungi relative to nutmeg EO. This finding can be
associated with comparatively high content of
phenolic compounds (particularly safrole, myris-
ticin, eugenol, methyl eugenol) in mace EO re-
both oils against two fungal strains; A. niger and
A. alternata that seemed equally sensitive to
them. For the rest four strains, MIC values of
nutmeg EO were equal to 2 µg/mL while mace
EO had the same MIC (0.4 µg/mL) for all tested
fungi. MIC’s along with their percentage inhibi-
tion (Table 6) is presented. Because of absence
of any researches on activity of these oils
against three of the assayed fungal pathogens,
correlation among results is not feasible. For-
merly, a very limited data has been published
upon the anti-fungal action of essential oils of
Myristica fragrans (nutmeg and mace). Poor ac-
tivity of both nutmeg and mace essential oils
from Pakistan, at a fixed concentration of 200
µl/mL against most tested fungi including F.
oxysporum, A. niger and A. flavus by agar dilu-
tion method is described. 71% and 75% inhibi-
tion against A. niger and F. oxysporum respec-
tively by Brazilian nutmeg EO at a concentration
of 0.1% using poison food technique is reported
28,29. The anti-fungal spectrum of freshly extract-

vealed (Table 2). ed essential oils of Myristica fragrans (nutmeg

Phenolic compounds are best-known for and mace) against three of the filamentous fun-
their strong antimicrobial properties. MICs were gal strains (A. alternata, F. solani and P. digita-
found (Table 6) to be the same (0.4 µg/mL) for tum) tested in this study has not been depicted

Fungal Nutmeg EO Mace EO Nystatin Ketoconazole

strains
48 h 168 h 48 h 168 h 48 h 168 h 48 h 168 h

A. niger 30.0 ± 0.8 29.0 ± 0.8 ≥45.0 ± 0.0 ≥45.0 ± 0.0 25.33 ± 0.4 17.0 ± 0.4 12.0 ± 0.0 11.66 ± 0.4
A. flavus 26.5 ± 0.8 25.0 ± 0.8 ≥45.0 ± 0.0 ≥45.0 ± 0.0 25.66 ± 0.4 25.0 ± 0.0 19.66 ± 0.4 18.33 ± 0.4
F. solani 18.0 ± 0.8 15.0 ± 0.8 ≥45.0 ± 0.0 21.3 ± 0.2 22.66 ± 0.4 15.0 ± 0.8 10.0 ± 0.0 10.0 ± 0.0
F. oxysporum 21.0 ± 0.0 18.0 ± 0.0 ≥45.0 ± 0.0 22.5 ± 0.4 17.33 ± 0.4 11.33 ± 0.4 10.0 ± 0.0 **6.0 ± 0.0
P. digitatum 26.5 ± 0.8 22.5 ± 0.0 ≥45.0 ± 0.0 25.3 ± 0.4 26.33 ± 0.4 24.33 ± 0.4 24.66 ± 1.2 23.33 ± 0.4
A. alternata 33.0 ± 2.4 30.3 ± 2.4 ≥45.0 ± 0.0 ≥45.0 ± 0.0 25.33 ± 0.4 23.33 ± 1.2 22.5 ± 01.2 21.0 ± 0.4
Table 5. Diameter of zone of inhibition (mm±SD)* of the pure undiluted nutmeg and mace essential oils * SD:
standard deviation (n=3); ** no inhibition.

Nutmeg EO Mace EO Nystatin Ketoconazole

Fungal
strains MIC/MFC % MIC/MFC % MIC/MFC % MIC/MFC %
(µg/mL) inhibition (µg/mL) inhibition (µg/mL) inhibition (µg/mL) inhibition

A. niger 0.4/2.0 73 0.4/2.0 83 25.0/100 99.9 10.0/40.0 99.9

A. flavus 2.0/2.0 67.3 0.4/2.0 79 12.5/50.0 99.9 5.0/20.0 99.9
F. solani 2.0/6.0 20 0.4/6.0 43 100.0/200.0 99.9 20.0/80.0 99.9
F. oxysporum 2.0/6.0 48 0.4/6.0 50 50.0/100.0 99.9 20.0/80.0 99.9
P. digitatum 2.0/2.0 81.2 0.4/2.0 95 200.0/400.0 99.9 2.5/20.0 99.9
A. alternata 0.4/2.0 70 0.4/2.0 66 200/200.0 99.9 2.5/20.0 99.9
Table 6. MIC, MFC and percentage inhibition of the pure undiluted nutmeg and mace essential oils.

2182

IC50 of DPPH radical-quenching
Sample
activity (µg/mL)
Nutmeg oil 136
Mace oil 112
BHT 21.2
Table 7. Antioxidant activity of the essential oil of
nutmeg oil, mace oil and BHT in DPPH free radical-
scavenging activity BHT, butylated hydroxytoluene.
earlier. It is worth mentioning that excluding
two all previous researches that have been done
for determining antifungal properties of nutmeg
EO have used commercially available EO sam-
ple ignoring the fact that oil composition and
hence activity may vary during storage 30,31.
MBC values of both the oils are presented in
Table 4, nutmeg and mace have same MBC val-
ue for all tested bacterial strains except the E.
coli where 1 mg/mL MBC was noted for nutmeg
essential oil. MFC values of both oils are 6.0
µg/mL for F. solani and F. oxysporum while 2.0
µg/mL is MFC for remaining fungi (Table 6).
Oxygen-quenching, radical scavenging and
reducing power of essential oils is responsible
for their antioxidant potential. The effect of an-
tioxidants of essential oils on DPPH radical
scavenging was determined by their hydrogen-
donating ability. DPPH become stable diamag-
netic molecule after accepting electron or hy-
drogen radical. Different concentrations (6.25-
100 µg/mL) of both the oils were used for scav-
enging effect on DPPH and low IC50 value asso-
ciated with higher DPPH radical scavenging
ability. As shown in Table 7, both essential oils
exhibited significant activity. The antioxidant ac-
tivity of the essential oil of nutmeg (IC50 = 136
µg/mL) was found to be higher than that of es-
sential oil of mace, which showed an IC50 of on-
ly 112 µg/mL.
Relation between composition and bioactiv-
ity of essential oils
In our study, the more pronounced activity
exhibited by mace EO can be justified for the
presence of phenylpropenes in significant
amounts and eugenol as exposed by GC analy-
sis. Eugenol and myristicin have been reported
as effective antifungals. The relatively low in-
hibitory potential of nutmeg EO might be due to
its significantly high terpenes content (known to
be less efficient as antimicrobials) plus the ab-
sence of myristicin and eugenol. Nevertheless,
Latin American Journal of Pharmacy - 35 (10): 2176-84 (2016)
in view of the fact that the oil’s intrinsic antimi-
crobial activity depends upon the interactions
among the various components - the major
components plus the minor components, it
would be unjust to attribute the antifungal po-
tential of oil to a particular constituent. The
components may interact in synergism reinforc-
ing each other’s bioactivity or may act antago-
nistically thereby controlling the overall activity
of volatile oil. Across the world, the control over
pathogenic microbes is attained largely by syn-
thetic chemicals. However, realizing their poten-
tial health hazards, adverse impact on environ-
ment plus the intrinsic and acquired resistivity
towards them by a range of pathogens, these
conventional synthetic chemicals are now tar-
geted by the scientific community to be substi-
tuted with natural products-the green products.
Thus, in the current era while the food security
problems and infections by pathogenic mi-
crobes are becoming a global issue, the demand
for both the manufacture of novel natural antibi-
otics plus the use of harmless natural preserva-
tives in food products has also been remarkably
amplified. Consequently, the interest in inspect-
ing the unique biological properties of natural
products is boosted up.
CONCLUSION
Essential oils from two intimate relatives viz.
nutmeg and mace from the promising medicinal
plant Myristica fragrans Houtt. revealed broad
antifungal spectrum. The results of in vitro in-
vestigations confirmed the high efficacy of these
oils as natural combatants against an array of fil-
amentous fungal isolates. Complete growth ar-
rest of the tested fungal strains at even low dos-
es of these oils recommends their use not only
in the development of novel natural fungicides
In vivo studies should be undertaken to ap-
prove their application in both food matrices
and pharmaceutical industries.
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