Seeds and Nutrient Composition of Myristica fragrans GC-MS ......Proximate and mineral analysis (6) Nutrient and mineral composition of M. fragrans seeds was carried out to determine

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Journal of Herbs, Spices & Medicinal Plants

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GC-MS Determination of Bioactive Compoundsand Nutrient Composition of Myristica fragransSeeds

Adaora O. Asika, Olaoluwa T. Adeyemi, God’swill N. Anyasor, Olusola Gisarin& Odutola Osilesi

To cite this article: Adaora O. Asika, Olaoluwa T. Adeyemi, God’swill N. Anyasor, OlusolaGisarin & Odutola Osilesi (2016): GC-MS Determination of Bioactive Compounds and NutrientComposition of Myristica fragrans Seeds, Journal of Herbs, Spices & Medicinal Plants, DOI:10.1080/10496475.2016.1223248

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GC-MS Determination of Bioactive Compounds andNutrient Composition of Myristica fragrans SeedsAdaora O. Asika, Olaoluwa T. Adeyemi, God’swill N. Anyasor, Olusola Gisarin,and Odutola Osilesi

Department of Biochemistry, Benjamin S. Carson (Snr.) School of Medicine, College of Health andMedical Sciences, Babcock University, Ilisan-Remo, Ogun State, Nigeria

ABSTRACTThe chemical constituents in Myristica fragrans seeds were ana-lyzed using proximate analysis, atomic absorption spectroscopic(AAS), phytochemical and gas chromatographic-mass spectro-metric (GC-MS) methods. Results showed the percentage prox-imate composition of M. fragrans seeds as: crude ash < crudeprotein < crude fibre < moisture < crude fat < nitrogen freeextract. The AAS analysis indicated the presence of magnesium(133.10mg kg–1), sodium (423.55 mg kg–1), calcium (1560.09 mgkg–1) and potassium (5139 mg kg–1). The aqueous extract of M.fragrans seeds had higher concentrations of phenols (0.10 mggallic acid equivalent (GAE) g–1) and flavonoids (1.10 mg querce-tin equivalent (QE) g–1) than the dichloromethane extracts thathad phenols (0.03 mg GAE g–1) and flavonoids (0.10 QE mg g–1).The aqueous and dichloromethane extracts contained 6 and 18bioactive compounds respectively.

ARTICLE HISTORYReceived 23 May 2016

KEYWORDSChemical constituent;nutmeg; proximate analysis;spice

Introduction

Chemical substances in food, particularly of plant origin, include phenols,flavonoids, saponins, alkaloids, and minerals (32, 22). These phyto-constitu-ents possess a wide range of nutritional and biological effects (18). Myristicafragrans (Myristicaceae) contains a dried kernel seed inside the fruit, which iscommonly called nutmeg (8). This evergreen tree is native to the Spice Islandsin Moluccas-East Indonesia and is cultivated throughout Malaysia (41). Itgrows in a clay loamy, well-drained, humus-rich soil and thrives well underhumid climates with mean temperatures of 20–30°C (28). The Indians com-monly identify theM. fragrans as Jaiphal and Javitri after its introduction by theBritish during the 18th century (30). The nutmeg seeds are ovoid in shape ofabout 3–3.5 cm long and 2 cm thick, enclosed within a ribbonlike lacinatedaril (28).

CONTACT God’swill N. Anyasor [email protected] or [email protected] Departmentof Biochemistry, Benjamin S. Carson (Snr.) School of Medicine, College of Health and Medical Sciences, BabcockUniversity, Ilisan-Remo, P.M.B. 21244, Ikeja, Ogun State, Nigeria.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/whsm.

JOURNAL OF HERBS, SPICES & MEDICINAL PLANTShttp://dx.doi.org/10.1080/10496475.2016.1223248

© 2016 Taylor & Francis Group, LLC

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The nutmeg seeds have been applied in the culinary, pharmaceutical,and cosmetic industries (7). It is one of the most widely used spices infood industries and in the preparation of ayurvedic drugs (1). In Indiantraditional medicine, the nutmeg seed is a constituent of tonics andelectuaries used to treat malaria, rheumatism, and digestive disorders(1, 13) and as a natural antimicrobial in oral-care products and anantioxidant in periodontitis (17).

M. fragrans fruits contains alkyl benzene derivatives including myristicin,elemicin and safrole, terpenes, alpha-pinene, myristic acid, trimyristicin,volatile oil, fixed oil, and starch (39). The nutmeg seed oil extract containsessential oils (5–15%) and fixed oils (24–40%) (35). This study was designedto identify the nutritional and bioactive compounds present in M. fragransseed and its extracts.

Materials and methods

Plant material

M. fragrans seeds were purchased from a local market, Lagos, Nigeria, inJanuary 2015, and authenticated in the Department of Biosciences andBiotechnology, School of Basic Sciences, Babcock University, Ilisan Remo,Ogun State, Nigeria.

Preparation of dichloromethane extract

The nutmeg seeds were oven-dried at 30°C for 7 d and pulverized with anelectrical blender. Pulverized seeds (50 g) were refluxed in 400 mL dichlor-omethane using a Soxhlet apparatus for 8 h (37) and concentrated in arotatory evaporator (Buchi Rotavapor RE, Switzerland), dried in a SANFADHG-9202 thermostatic hot air oven (Gulfex Medical and Scientific,England) at 45°C, and stored at 4°C in a refrigerator until further use.

Preparation of aqueous extract (37)

Pulverized seeds (50 g) were soaked in 2,000 mL distilled water and heated at100°C for 30 min using a hot plate, filtered using Whatman No. 1 filterpaper, concentrated in a rotatory evaporator, dried in a SANFA DHG-9202thermostatic hot air oven (Gulfex Medical and Scientific, England) at 45°C,and stored at 4°C in a refrigerator until further use.

2 A. O. ASIKA ET AL.

Proximate and mineral analysis (6)

Nutrient and mineral composition of M. fragrans seeds was carried out todetermine the moisture, protein, fat, ash, fiber, and nitrogen free extractcontents using standard methods (6).

Powdered sample (6 g) was weighed into a crucible and heated over a Bunsenburner until it charred. The crucible containing charred sample was transferredinto a muffle furnace set at 600°C to heat the sample until it turned into grayishwhite ash, which was cooled first to room temperature and then transferredinto a desiccator. Subsequently, 5 mL of concentrated HCl was added to the ashand heated for 5 min on a hot plate and transferred into a beaker and thecrucible washed several times with distilled water. The mixture was made up to40 mL and boiled for 10 min over a Bunsen burner, cooled, filtered and rinsedinto a 100-mL flask, and made up to 100 mL. The filtrate was subjected to flameatomic emission spectrophotometry to analyze for sodium (Na) and potassium(K); concentrations of calcium (Ca) and magnesium (Mg) in the solution weredetermined using atomic absorption spectrophotometry.

Quantitative phytochemical analysis

Determination of total polyphenol content (19)Dichloromethane or aqueous extract (0.5 mL, 1 mg.mL–1, respectively) wasmixed with 2.5 mL Folin-Ciocalteu reagent (1:10 v/v) and 2 mL (7.5% w/v)sodium carbonate (Na2CO3). The tube was vortexed for 15 s, incubated for 40min at 45°C for color development, and its absorbance measured at 765 nmusing a double-beam UV/visible spectrophotometer (T80 model, PGInstrument). Similar protocol was performed using standard gallic acid. Acalibration curve was obtained using gallic acid as standard; the total polyphenolcontent was expressed as mg.g–1 gallic acid equivalent using the calibration curve.

Determination of total flavonoid content (26)Dichloromethane or aqueous extract (0.5 mL, 1 mg.mL–1, respectively) wasmixed with 0.5 mL 2% aluminum chloride (AlCl3) prepared in ethanol, andincubated for 60 min at room temperature for yellow color development, whichindicates the presence of flavonoid. Absorbance was measured at 420 nm usingthe double-beam UV/visible spectrophotometer (T80 model, PG Instrument).Similar protocol was performed using quercetin as standard. A calibration curvewas obtained using quercetin as standard, and the total flavonoids content wasexpressed as mg.g–1 quercetin equivalent using the calibration curve.

Determination of tannin content (23)Pulverized M. fragrans seeds (0.2 g) were placed in conical flask followedby the addition of 10 mL 1%HCl in methanol. The flask was capped and

JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS 3

mixed continuously for 20 min. The content was centrifuged at 2,500g for5 min. The supernatant (1 mL) was pipetted into a test tube containing 5mL vanillin-HCl reagent. Absorbance at 450 nm was read using thedouble-beam UV/Visible spectrophotometer (T80 model, PGInstrument) after 20 min of incubation at 30°C. A standard curve wasprepared to express the result as tannic acid equivalent.

Tannin %ð Þ ¼ C� 1� 100=200where C = concentration corresponding to the optical density, 1 = volume ofthe extract (mL); 200 = sample weight (mg).

Determination of saponin content (24)Pulverized nutmeg seeds (5 g) were dispersed in 50 mL 20% v/v ethanol indistilled water. The suspension was heated over a hot water bath for 1 hwith continuous stirring at 55°C. The mixture was filtered and the residuere-extracted with another 50 mL of 20% v/v ethanol. The volume ofcombined extracts was reduced to 20 mL over the hot water bath at 90°C.The concentrated solution obtained was shaken vigorously with 10 mLdiethyl ether in a 250-mL separating funnel; the aqueous layer was collectedand the ether layer was discarded. Butanol (20 mL) was added to the filtrateand washed three times with 10 mL 5% w/v NaCl. The whole mixture washeated to evaporation on a hot water bath and oven-dried to constantweight. The percentage saponins in the sample was calculated as

% saponins ¼ weigh tof final filtrate=weight of sampleð Þ � 100

Determination of alkaloid content (25)Pulverized nutmeg seeds (5 g) were weighed into a 250-mL beaker, followedby the addition of 200 mL 20% acetic acid in ethanol, then covered andallowed to stand for 4 h. This was subsequently filtered and concentrated toone-quarter of its original volume using a hot water bath at 100°C.Concentrated ammonium hydroxide was added drop by drop to the extractuntil precipitation was complete. Precipitates were washed with diluteammonium hydroxide, filtered, and the residue was dried and weighed.The alkaloid content was determined as

% alkaloid ¼ final weight of sample=initial weight of sampleð Þ � 100

Gas chromatography-mass spectrometry (GC-MS) of dichloromethane andaqueous fractions of M. fragrans seed

GC-MS analyses of dichloromethane and aqueous extracts of the nutmegseeds were carried out using an Agilent Technologies model 7890A Gas


Chromatography, 6975C Mass Chromatography Detector, and 7683B AutoInjector. Initial temperature of 80°C was held for 2 min, increasing at 5°C permin to 120°C, held for another 2 min, increasing at 120°C per min, andfinally to 240°C, held for 6 min. One microliter of 0.2-g.mL–1 fraction wasinjected. Temperature of the heater was 250°C, pressure was 3.2652 psi, modetype splitless, column type (HP5MS 30M × 320 μM × 0.25 μM) packed with5% polysaloxane and carrier gas helium (flow rate 1.4963 mL.min–1, averagevelocity 45.618 cm.s–1). The constituent compounds were determined bycomparing the retention times and mass spectra of the authentic samplesobtained by GC with the mass spectra from the NIST version 2.0 databaselibrary, Washington, DC, USA, MS database library.

Statistical analysis

Data were analyzed using Windows Excel (2013) and SPSS analyticalsoftware for Windows (SPSS, Inc., Chicago, V 17.0) to determine differ-ences between mean using Student’s t-test (P < 0.05). Data were reportedas mean ± standard error of the mean (SEM).

Results

The nutrient composition of M. fragrans seed was, in order of increasingmagnitude, crude ash (2.28%) < crude protein (7.02%) < crude fiber (13.8 %)< moisture (14.12%) < crude fat (25.60%) < nitrogen free extract (37.20%).The mineral content in M. fragrans seed indicated the presence of magne-sium (133.1 mg.kg–1), sodium (423.55 mg.kg–1), calcium (1560.09 mg.kg–1),and potassium (5139.00 mg.kg–1).

One-mg.mL–1 aqueous extract (0.10 mg gallic acid equivalent.g–1) hadhigher phenolic content than 1-mg.mL–1 dichloromethane extract (0.03 mggallic acid equivalent.g–1. One-mg.mL–1 aqueous extract (1.10 mg quercetinequivalent.g–1) of M. fragrans seed had a higher flavonoid content comparedto 1-mg.mL–1 dichloromethane extract (0.10 mg quercetin equivalent.g–1).The percentages of tannins, saponins, and alkaloids in M. fragrans seed were9.3%, 13.10%, and 5.50%, respectively.

Six compounds were identified by GC-MS analysis of the aqueous extractof M. fragrans seeds (Table 1 and Figure 1); 3-phenanthrol (5.73%) waspresent in the highest concentration and thiazolo[3,2-a]benzimidazol-3(2H)-one,2-(2-fluorobenzylideno)-7,9-dimethyl (2.28%) was in the lowest.

Eighteen compounds were identified by GC-MS analysis of the dichlor-omethane extract of M. fragrans seeds (Table 2 and Figure 2). Odecanoic acid(1.00%), cis-vaccenic acid (7.46%), and cis-13-octadecenoic acid (7.46%) werein highest concentration, followed by tridecanoic acid (7.20%) and tetrade-canoic acid (6.96%). Guaiol (0.12%) was present at the lowest concentration.


Table 1. Chemical compounds in aqueous extract of Myristica fragrans seeds analyzed by gaschromatographic-mass spectrometric (GC-MS) method.

Compounds (%)

1 Thiazolo[3,2-a]benzimidazol-3(2H)-one, 2-(2-fluorobenzylideno)-7,8-dimethyl 2.282 Phenol, 4-[2, 3-dihydro-7-methoxy-3-methyl-5-(1-propenyl)-2-benzofuranyl]-2-methoxy 2.493 Phenol 2,6-dimethoxy-4-(2-propenyl) 3.314 3-Hydroxy-4-methoxycinnamic acid 3.775 1H-Phenalen-1-one, hydrazine 3.716 3-Phenanthrol 5.73

Figure 1. Chromatogram of Myristica fragrans seed extracts in water.

Table 2. Chemical compounds in dichloromethane extract of Myristica fragrans seeds analyzedby gas chromatographic-mass spectrometric (GC-MS) method.

Compound %

1 Guaiol 0.122 α-Terpineol 1,6,10 dodecatrien-3-ol,3,7,11-trimethyl 0.163 Oleic acid 0.194 Phenol, 2,6-dimethoxy-4-(2-propenyl) (eugenol) 0.195 Pentadecanoic acid, 14-methyl-, methyl ester 0.196 Hexadecanoic acid, methyl ester 0.197 Tridecanoic acid, methyl ester 0.198 10-Octadecenoic acid, methyl ester 0.349 9-Hexadecenoic acid, methyl ester 0.3410 Dodecanoic acid (lauric acid) 1.0011 Phenol 2-methoxy-4-(1-propenyl) 1.1912 Isoelemicin 1.2913 n-Hexadecanoic acid (palmitic acid) 5.0214 Pentadecanoic acid 5.0215 Tetradecanoic acid (myristic acid) 6.9616 Tridecanoic acid 7.2017 cis-Vaccenic acid 7.4618 cis-13-Octadecenoic acid 7.46


Discussion

M. fragrans seeds contained an appreciable amount of crude ash, protein, fiber,andmoisture and could serve as a source of minerals and amino acids in the diet(10). Dietary fibers have been reported to retrogress cardiovascular diseases andarteriosclerosis, and increase intestinal transit time. The crude fiber content inthe seed might account for the use of M. fragrans seed as an abortifacient agent(39). The moisture content of M. fragrans seed indicated that it may a havereduced shelf life (14).

The quantities of crude fat and nitrogen free extract were high relative to theother nutrients in M. fragrans seed. This is in agreement with reports of M.fragrans seeds as a rich source of energy (40), high in nitrogen free extractrelative to other nutrients (34), and as being rich in carbohydrates (21). Thehigh crude fat noted in this study is in agreement with other reports of M.fragrans being rich in fatty acids (29). This suggests that nutmeg seed could bean important source of dietary lipids and natural oil for sundry industrialapplications.

Mineral analysis showed the presence of calcium, potassium, sodium, andmagnesium in M. fragrans seed. These elements play a central role in themaintenance of body homeostasis (27). Calcium and potassium contents inM. fragrans seeds were higher than other elements. Calcium and potassiumare known to participate in the development and maintenance of strongbones, muscular function, synthesis of enzymes, and normal physiologicalfunction of the body (36). Sodium and magnesium are involved in enzymesactivation and biological structure promotion (27).

The presence of phenols and flavonoids in M. fragrans seeds was con-firmed as also reported earlier (40). The aqueous extract of M. fragrans seed

Figure 2. Chromatogram of Myristica fragrans seed extracts in dichloromethane.


contained higher concentrations of phenolic compounds than dichloro-methane extract, suggesting that the aqueous extract of M. fragrans seedmay possess higher free-radical scavenging activity than dichloromethaneextract (42). Phenolic compounds are known to inhibit or terminate pro-inflammatory mediators or deleterious chain reactions triggered by reactiveoxygen species (28).

Other phytochemicals present in the M. fragrans seeds were tannins,saponins, and alkaloids. Tannins are characterized by an astringent taste,which may account for the taste of M. fragrans seed as spice (41). It alsopossesses anti-inflammatory activity and is employed medicinally in anti-diarrheal, hemostatic, and antihemorrhoidal compounds. Saponins possessantibacterial, antiprotozoal, and anticancer activities (15). They exhibit dis-tinct foaming properties, and are added to shampoos, liquid detergents,toothpastes, and beverages as emulsifiers and long-lasting foaming agents(15). Previous studies had shown that alkaloids possess anti-inflammatory,antimicrobial, antifungal, antihelminthic, antimalarial, and antidepressantactivities (5). These findings may account for the ethno-medicinal use ofM. fragrans seed as a source of therapeutic agents.

The aqueous extracts of M. fragrans seeds contained six compounds ofbiological interest: 3-phenanthrol and thiazolo (3,2-a)benzimidazol-3(2H)-one 2-(2-fluorobenzylideno)-7,8-dimethyl were the highest and lowest quantities,respectively. The thiazolo (3,2-a)benzimidazol-3(2H)-one 2-(2-fluorobenzyli-deno)-7,8-dimethyl had been shown to possess antibacterial, antifungal, anti-inflammatory, antiulcer, antiviral, anthelmintic, and anticancer activity (4),phenol, 4-(2, 3-dihydro-7-methoxy-3-methyl-5-(1-propenyl)-2-benzofuranyl)-2-methoxy possesses free-radical scavenging activity (31), phenol 2,6-dimethoxy-4-(2-propenyl) (eugenol) is known to possess anti-septic and anesthetic properties(16), 3-hydroxy-4-methoxycinnamic acid also possesses free-radical scavengingand peroxyl radical inhibitory activity (12), 1h-phenalen-1-one hydrazine isknown to possess antifungal properties (20), and 3-phenanthrol with metalcomplex was effective as an antimicrobial (38).

The dichloromethane extract possessed 18 compounds of biologicalinterest. Oleic acid, cis-vaccenic acid, pentadecanoic acid, pentadecanoicacid 14-methyl,methyl ester, and phenol, 2,6-dimethoxy-4-(2-propenyl)had been reported to exhibit antioxidant and anti-inflammatory activity(31), cis-13-octadecenoic acid and 10-octadecenoic acid methyl ester areknown to possess antifungal, antimicrobial, and antibacterial activity (16),9-hexadecenoic acid methyl ester is known to induce insulin resistance (9),n-hexadecanoic acid (palmitic acid) has anti-inflammatory, antioxidant,hypocholesterolemic, flavor, nematicide, pesticide, and antiandrogenic activ-ity (33); tetradecanoic acid (myristic acid) can act as an antioxidant andcancer preventative, hypocholesterolemic, nematicide, and lubricant (16),hexadecanoic acid methyl ester possesses anti-inflammatory, antioxidant,


hypocholesterolemic, flavor, nematicide, pesticide, and antiandrogenic prop-erties (33); tridecanoic acid methyl ester and tridecanoic acid possess anti-bacterial and antifungal activity (31); guaiol and dodecanoic acid (lauricacid) possess antimicrobial properties, α-terpineol-1,6,10-odecatrien-3-ol,3,7,11-trimethyl exhibits an inhibitory effect on gamma-butyric acid (2),isoelemicin possesses fumigant and antitoxic activity (11) and phenol 2-methoxy-4-(1-propenyl) has anti-septic and anesthetic properties (16).

Acknowledgment

The authors express sincere appreciation to Babcock University administration for thesupport rendered during the course of this research. We also thank Mrs. C. Anyasor fortechnical assistance in preparation of this manuscript.

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AbstractIntroductionMaterials and methodsPlant materialPreparation of dichloromethane extractPreparation of aqueous extract (37)Proximate and mineral analysis (6)Quantitative phytochemical analysisDetermination of total polyphenol content (19)Determination of total flavonoid content (26)Determination of tannin content (23)Determination of saponin content (24)Determination of alkaloid content (25)

Gas chromatography-mass spectrometry (GC-MS) of dichloromethane and aqueous fractions of M. fragrans seedStatistical analysis

ResultsDiscussionAcknowledgmentReferences

Documents

Seeds and Nutrient Composition of Myristica fragrans GC-MS ......Proximate and mineral analysis (6) Nutrient and mineral composition of M. fragrans seeds was carried out to determine