Chemical Composition of the Essential Oils of Nutmegand Mace by GC-FID/MS Indigenous to Pakistanand Evaluation of their Biological Activities

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KEY WORDS:antibacterial, antifungal, antioxidant, essential oils, medicinal plant, Myristica fragrans.

* Author to whom correspondence should be addressed. E-mail: [email protected]

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

1Institute of Biochemistry and Biotechnology &

2Institute of Chemistry,

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

3PCSIR 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 subtilissubsp. spizizeniiwhich 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 Fusariumspecies (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 fragransHoutt. (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 solaniy 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 subtilissubsp. 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

Bacterial activity is considered to be the principal 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,

fruits and other plant product while the high 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. spizizeniihas major role in food poi-

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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 commodities 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 filamentous fungi that have emerged as infectious pathogens not only for plants but also for humans and animals ⁸. The principal causative agents of fungal keratitis in humans are also the Fusariumspecies ⁹. Fusarium solanicauses keratitis that is not easy to diagnose and treat, often results in rapid corneal sloughing and serious vision loss. A. nigerand A. flavuscan cause oto- mycosis in healthy individuals ¹⁰. In certain areas 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 popular 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 essential oils. This is in best of our knowledge, antimicrobial possession of EOs from nutmeg

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 using 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 percentages 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 operating conditions for oil samples to obtain linear retention indices (LRI) of constituents present in oils.

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Antimicrobial assay

Bacterial and fungal strains

Seven bacterial strains Bacillus subtilissubsp.

spizizenii ATCC 6633, Staphylococcus aureus ATCC 25923, Salmonella enterica ATCC 14028, Escherichia coliATCC 25922, Enterobacter aero- genes ATCC 13048, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13882 and six indigenous pathogenic fungal isolates 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 sensitivity of tested bacterial strains). 1-3 × 10⁸ cfu/mL of each of gram negativeE. 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 10⁶cfu/mL (0.1 OD at 700 nm) ²²was 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 standard drugs. The plates were finally incubated at 35 °C (bacterial strains) and 25 °C (fungi strains) in cold incubator (Sanyo Mir 253, Japan) to allow 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 determination 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

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 minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC). For percentage 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 sample 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 percentage scavenging activity of DPPH radical was calculated as follows: % RSA = ([A_DPPH – A_S] /A_DPPH) × 100,where A_Sis the absorbance of sample solution and A_DPPH 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 percentage 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 material, extraction of essential oils by Clevenger’ ap-

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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 constituents from the two EOs with their LRI, content percentages and m/z values (major fragments) 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-

Identified Constituent ^aLRI ^bLRI 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 . ^aLRI determined linear retention index on HP-5 MS column; ^bLRI =determined linear retention index on DB-Wax column.

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 (elemicin: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

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various investigations for their immense pharmaceutical worth. Even though several researches 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.

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

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.

distinct defined zones; in some cases the zones were jagged or vague. On the whole, nutmeg and mace EOs did not demonstrate strong inhibitory 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 sensitivity comparable to ciprofloxacin. Smallest zones were obtained against P. aeruginosa for both oils. P. aeruginosais 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 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-

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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 solubility 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 incubation. For the three strains P. aeruginosa, E.

Bacterial strains Nutmeg EO Mace EO Gentamicin Ciprofloxacin

(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

MIC/MBC % MIC/MBC % MIC/MBC % MIC/MBC %

mg/mL inhibition mg/mL inhibition µg/mL inhibition µg/mL inhibition B. subtilis

subsp. spizizenii 1/2 99.9 1/2 99.9 0.062/0.125 99.9 1.25/2.50 99.9

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.

Bacterial strains

aerogenes andS. aureus, the oil exhibited static effect. The growth of P. aerogenosaand E. aero- geneswas 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 antifungal activity of both fresh essential oil samples after two and seven days incubation (25

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°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 after 7 days incubation for A. niger, A. flavus and A. alternata, however reduction in zone size was seen in case of both Fusariumspecies and P. digitatum. Both EO’s have higher zone of inhibition than the standard drugs (8 µg/mL) used as reference. On the whole, mace EO demonstrated higher antifungal potential against all fungi relative to nutmeg EO. This finding can be associated with comparatively high content of phenolic compounds (particularly safrole, myristicin, eugenol, methyl eugenol) in mace EO revealed (Table 2).

Phenolic compounds are best-known for their strong antimicrobial properties. MICs were found (Table 6) to be the same (0.4 µg/mL) for

Nutmeg EO Mace EO Nystatin Ketoconazole

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

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.

Fungal strains Fungal strains

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 inhibition (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 activity 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 dilution method is described. 71% and 75% inhibition against A. niger and F. oxysporum respectively by Brazilian nutmeg EO at a concentration of 0.1% using poison food technique is reported

28,29. The anti-fungal spectrum of freshly extracted essential oils of Myristica fragrans (nutmeg and mace) against three of the filamentous fungal strains (A. alternata, F. solani and P. digita- tum)tested in this study has not been depicted

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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 sample 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 value 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 antioxidants of essential oils on DPPH radical scavenging was determined by their hydrogen- donating ability. DPPH become stable diamag- netic molecule after accepting electron or hydrogen radical. Different concentrations (6.25- 100 µg/mL) of both the oils were used for scavenging effect on DPPH and low IC₅₀value associated with higher DPPH radical scavenging ability. As shown in Table 7, both essential oils exhibited significant activity. The antioxidant activity of the essential oil of nutmeg (IC₅₀ = 136 µg/mL) was found to be higher than that of essential oil of mace, which showed an IC₅₀of only 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 analysis. Eugenol and myristicin have been reported as effective antifungals. The relatively low inhibitory potential of nutmeg EO might be due to its significantly high terpenes content (known to be less efficient as antimicrobials) plus the absence of myristicin and eugenol. Nevertheless,

Sample IC₅₀of DPPH radical-quenching 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.

in view of the fact that the oil’s intrinsic antimicrobial activity depends upon the interactions among the various components - the major components plus the minor components, it would be unjust to attribute the antifungal potential 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 synthetic chemicals. However, realizing their potential 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 microbes are becoming a global issue, the demand for both the manufacture of novel natural antibiotics 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 investigations confirmed the high efficacy of these oils as natural combatants against an array of filamentous 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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