z

Available online at http://www.journalcra.com INTERNATIONAL JOURNAL OF CURRENT RESEARCH International Journal of Current Research Vol. 8 , Issue, 11, pp.40897-40903, November, 2016

ISSN: 0975-833X RESEARCH ARTICLE

FTIR SPECTRAL ANALYSIS OF THE PHYTOCONSTITUENTS IN THE MACE OF FRAGRANS FOR THE DETECTION OF POSSIBLE ADULTERATION WITH THE MACE OF AND MYRISTICA BEDDOMII

*Ajith Kumar, K.G., Sunil Kesava Deth, G., Pratheepkumar, V., Suresh Kumar, K. A. and Dinesh Babu, K. V.

Department of , Govt. College for Women, Thiruvananthapuram, Kerala, India

ARTICLE INFO ABSTRACT

Article History: Myristica fragrans is an evergreen commercially cultivated for their valuable essential oils present in the mace and . India is a leading producer and exporter of these spices. Received 29th August, 2016 Received in revised form Unfortunately, the Indian market and world market markets have been flooded with the mace and 10th September, 2016 nutmeg obtained from their wild relatives with inferior qualities. There should be some checks for the Accepted 23rd October, 2016 detection of adulterants from the original one. The present investigation explores the possibility of Published online 30th November, 2016 utilizing FTIR spectral analysis for detecting the possible adulteration. The results indicate that a set of biochemical markers which are unique in the mace of Myristica fragrans can be used for the Key words: detection of original mace from adulterant.

Myristica fragrans, Myristica malabarica, Myristica beddomii, FTIR, Mace, Molecular fingerprinting, Adulteration.

Copyright©2016, Ajith Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Citation: Ajith Kumar, K.G., Sunil Kesava Deth, G., Pratheepkumar, V., Suresh Kumar, K.A. and Dinesh Babu, K.V. 2016. “Ftir spectral analysis of

the phytoconstituents in the mace of Myristica fragrans for the detection of possible adulteration with the mace of Myristica malabarica and Myristica

beddomii”, International Journal of Current Research, 8, (11), 40897-40903.

INTRODUCTION Mace aril contains more vitamin A, vitamin C, calcium, copper, iron and magnesium than nutmeg. Mace oil has been Myristica fragransis an evergreen tree belonging to the family used in dentistry for toothache relief and it is used a local Myrtaceae, indigenous to the Moluccas in but it has massage to reduce muscular pain and rheumatic pain of joints. been successfully grown in other Asian countries. The species Indonesia and are the dominant producers and has been cultivated in many parts of the world for their nutmeg exporters of nutmeg and mace and India is also a leading and mace rich in valuable essential oils. Nutmeg contains about producer. However, Indian market and world markets have 10% essential oils chiefly composed of terpene hydrocarbons been flooded with adulterants of nutmeg and mace. The Indian (sabinene and pinene), myrcene, phyllandrene, camphene, mace is adulterated with the mace of Myristica malabarica, limonene, terpinene, pcymene and other terpnene derivatives commonly called Bombay mace. While in other countries, the (Qiu et al., 2004; Yang, 2008; Jaiswal, 2009). The various mace of is used as an adulterant of the mace pharmacological activities like hepatoprotective, anti-oxidant, of Myristica fragrans. Fourier-Transform Infra-Red memory enhancing, cytotoxicity, aphrodisiac, anti-diabetic, Spectrometry (FTIR) is an analytical technique is used for the anti-depressant, anti-microbial, anti-inflammatory, anti- quality analysis in agro-food products (Downey, 1998). It carcinogenic, hypolipidemic and hypocholesterolemic effect focuses on the MIR region of (4000-400cm-1) of the are due to the presence of these secondary compounds in electromagnetic spectrum and monitors the fundamental nutmeg and mace. Mace is technically the dry, outer aril that vibrational and rotational stretching modes of molecules, which firmly enveloping around the nutmeg kernel. It is a strong, produce a chemical profile of the sample (Sun, 2009). The aromatic substance and warm in taste. Nutmeg and mace are biological molecules can be identified based on the two separate spice products of same nutmeg fruit. However, characteristic vibration of their functional groups. Most of the mace has higher concentration of certain essential oils with relevant information that is used to interpret MIR spectra is intense aroma than nutmeg. usually obtained from the functional group region. This

*Corresponding author: Ajith Kumar, K.G. molecular fingerprinting can be used as a reliable technique for Department of Botany, Govt. College for Women, Thiruvananthapuram, the detection of adulteration in food products (Brindet et al., Kerala, India. 40898 Ajith Kumar et al. Ftir spectral analysis of the phytoconstituents in the mace of myristica fragrans for the detection of possible adulteration with the mace of Myristica malabarica and Myristica beddomii

1996; Herringshaw, 2009). The present investigation explores RESULTS AND DISCUSSION the possibility of utilizing FTIR for detecting the possible adulteration in the mace of Myristica fragrans. The biomass and moisture content of mace showed significant differences among Myristica fragrans, Myristica malabarica MATERIALS AND METHODS and Myristica beddomii (Table 1). The biomass was higher in Myristica malabarica (72.39%) followed by Myristica The mature fruits of Myristica fragrans, Myristica malabarica beddomii (57.16%) and Myristica fragrans (52.06%). The and Myristica beddomii were collected from their natural mace was reddish in Myristica fragrans but yellow in Myristica habitats and the mace (aril) was collected from the seeds. The malabarica and yellowish brown in Myristica beddomii. aril was surface sterilized with 2.5% sodium hypochlorite for Considerable differences have been observed in the seed shape 30 minutes and washed extensively with distilled water.A in which the seeds were small and round in Myristica fragrans sample of ten replicates of mace from each treatment was taken while medium seized and round in Myristica beddomii and for dry weight and moisture content analysis. 5g of mace from oblong in Myristica malabarica. The FTIR spectral analysis of each treatment was chopped and ground in organic solvents phytochemicals in the mace of Myristica fragrans, Myristica such as chloroform, methanol, methanol (50%) using mortar malabarica and Myristica beddomii has been shown (Fig. 1-9; and pestle. The homogenized sample was centrifuged and the Table 2-3). The chloroform extract of the mace of Myristica supernatant was taken. The organic solvents were evaporated in fragrans showed the presence of alkanes, aromatic rings, a rotary evaporator and the sample was dispersed in dry aldehydes, quinones, esters and lactones, carboxylic acids and potassium bromide. The mixture was mixed in a mortar and salts, ammonium salts, alcohols, primary/secondary/tertiary pressed at pressure of six bars to form a KBr thin disc. The disc alcohols, phenols, carbohydrates, ethers, primary/secondary was placed in a sample cup of a diffuse reflectance accessory. amines, nitriles, sulphides, disulphides, sulphur oxy The IR spectra were obtained using IRPrestige-21, Shimadzu compounds, peroxides, organic phosphates, aromatic Infrared spectrophotometer. The sample was scanned from 400 phosphates, aliphatic phosphates, fluoro/chloro/bromo/iodo to 4000cm-1 for sixteen times to increase the signal to noise compounds, silicon oxy compounds, aromatic nitro compounds ratio. Samples were run in triplicate and data were collected and aliphatic nitro compounds. However, the chloroform within one day (Manigandan et al., 2015; Marimuthu and extract of the mace of Myristica malabarica showed the Gurumoorthi, 2013). presence of alkanes, aldehydes, esters and lactones, carboxylic acids and salts, alcohols, carbohydrates, ethers, Statistical analysis primary/secondary amines, sulphides, sulphur oxy compounds, peroxides, organic phosphates, aromatic phosphates, aliphatic All statistical analyses were carried out using SPSS 16.0 (SPSS phosphates, fluoro/chloro/bromo/iodo compounds, silicon oxy Inc., Chicago, IL, USA). compounds, aromatic nitro compounds and aliphatic nitro compounds.

Plate 1. Left - Mature fruit of Myristicamalabrica, Right – Fruit splits open to expose arillated seed. Yellow aril (mace) is an adulterant of common valuable mace of Myristicafragrans

Plate 2. Oblong seeds of Myristicamalabarica, an adulterant of common nutmeg of Myristicafragrans 40899 International Journal of Current Research, Vol. 08, Issue, 11, pp.40897-40903, November, 2016

Plate 3. Mature fruit of Myristicabeddomii

Plate 4. Fruit splits open to expose the arillated seed of Myristicabeddomii

140

%T 120

100

80

2308.79 2353.16 534.28 584.43 607.58

60 767.67 856.39 819.75 927.76 653.87 1689.64 678.94 962.48 1504.48 1028.06 40 1147.65 1452.40 1593.20 1739.79 1267.23 1332.81 1236.37 3116.97

3047.53 3080.32 3508.52 3541.31 3429.43 3182.55 3462.22 3201.83 3404.36 3213.41 3375.43 3275.13 3259.70 3292.49 20 2854.65 2926.01

0 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 31 ajith bot 1/cm Fig. 1. FTIR analysis of mace of Myristica fragrans (chloroform extract)

The chloroform extract of the mace of Myristica beddomii The mace of all these species showed similarity in most of showed the presence of alkanes, aromatic rings, quinones, phytochemicals such as alkanes, esters and lactones, carboxylic esters and lactones, carboxylic acids and salts, ammonium acids and their salts, alcohols, carbohydrates, ethers, salts, alcohols, carbohydrates, ethers, primary/secondary primary/secondary amines, sulphur oxy compounds, organic amines, nitriles, sulphur oxy compounds, organic phosphates, phosphates, aromatic phosphates, aliphatic phosphates, aromatic phosphates, aliphatic phosphates, fluoro/chloro fluoro/chloro compounds, silicon oxy compounds, aromatic compounds, silicon oxy compounds, aromatic nitro compounds nitro compounds and aliphatic nitro compounds. The mace of and aliphatic nitro compounds. Myristica fragrans showed difference with the mace of 40900 Ajith Kumar et al. Ftir spectral analysis of the phytoconstituents in the mace of myristica fragrans for the detection of possible adulteration with the mace of Myristica malabarica and Myristica beddomii

100

%T

90

921.97 858.32 650.01

80 677.01 819.75 1734.01 2740.85 964.41 1508.33 3539.38 1429.25 3512.37

1452.40 3109.25 3043.67 3458.37 3068.75 1138.00 1126.43

70 3431.36 1591.27 3165.19 3406.29 3207.62 3377.36 3230.77 1332.81 1029.99 3342.64

3263.56 3321.42 3296.35 3286.70 1265.30 1234.44

2852.72

60 2929.87

50

40

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 32 ajith bot 1/cm

Fig. 2. FTIR analysis of aril of Myristica fragrans (50%methanol extract)

120

%T

105

90

2351.23 2306.86 1552.70 2384.02 75 3971.43 1681.93 894.97 792.74 601.79 765.74 819.75 860.25 651.94 678.94 1498.69 721.38

1512.19 962.48 60 1598.99 2735.06 1031.92 1124.50 1332.81 1456.26 1207.44

1263.37 1236.37 1159.22 1147.65 3107.32 3086.11 3070.68 3537.45 3169.04 3512.37 3188.33 3205.69 3491.16 3469.94 3454.51 3431.36 3406.29 3265.49 3375.43 3284.77

45 3344.57 3321.42 3300.20 3003.17

1743.65

30 2852.72 2924.09

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 33 ajith bot 1/cm Fig. 3. FTIR analysis of mace of Myristica fragrans (methanol extract)

150

%T

125

100

75 599.86 646.15

549.71

50 2686.84 788.89 2736.99 960.55 3431.36 3402.43 3348.42 3379.29 864.11 1504.48 823.60 723.31 25 1039.63

3003.17 1114.86 1585.49 1342.46 1211.30 0 1460.11 1236.37 1165.00 1745.58 2852.72 2924.09 -25 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 19 ajith bot 1/cm Fig. 4. FTIR analysis of mace of Myristica malabarica (chloroform extract) 40901 International Journal of Current Research, Vol. 08, Issue, 11, pp.40897-40903, November, 2016

150 %T 125

100

75 2310.72 2349.30 2411.02 2382.09 619.15 648.08 50 659.66 900.76

867.97 3109.25 2679.13 2733.13 25 968.27 792.74 3531.66 1062.78 1514.12 1018.41 721.38 1039.63 3332.99 1097.50 3350.35 3450.65 1631.78

0 3371.57 1600.92 3425.58 3388.93 3406.29

1114.86 1344.38 3003.17 1710.86 1278.81 1452.40 1165.00 1238.30 1226.73 1743.65 2852.72 2922.16 -25

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 21 ajith bot 1/cm

Fig. 5. FTIR analysis of mace of Myristica malabarica (methanol extract)

105 %T

90

75 781.17 588.29 864.11 545.85 2314.58 821.68 368.40 678.94 623.01 646.15 725.23

60 1508.33 1112.93

45 1041.56

1155.36 1344.38 1236.37 1458.18 30 1593.20

3001.24

1743.65

15 3294.42 3329.14 3315.63

2854.65 0 2924.09 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 20ajith bot 1/cm Fig. 6. FTIR analysis of mace of Myristica malabarica (50%methanol extract)

120

%T

105

90

75 3400.50 1095.57 2675.27 2731.20 1598.99

1116.78 1631.78 3319.49 1039.63 3300.20 3282.84 3261.63 1413.82 3223.05 3201.83 3188.33 721.38 3163.26 3145.90 3128.54 3105.39 3086.11 1344.38 3068.75 1195.87

60 1163.08

1278.81 1240.23 1226.73 3005.10

1452.40

45 1745.58

1712.79

30 2852.72 15 2922.16

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 7 bot gcw 1/cm Fig. 7. FTIR analysis of mace of Myristica beddomii (chloroform extract) 40902 Ajith Kumar et al. Ftir spectral analysis of the phytoconstituents in the mace of myristica fragrans for the detection of possible adulteration with the mace of Myristica malabarica and Myristica beddomii

105 %T 102.5

100

97.5

437.84 437.84 95

538.14 92.5 599.86 1714.72 682.80 858.32 1112.93 1514.12 723.31 90 798.53 3008.95 87.5 3084.18 1039.63

3163.26 1348.24 3205.69 3292.49 85 3257.77 1448.54 82.5 2854.65 1238.30

1593.20 80 2926.01

77.5 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 8 bot gcw 1/cm Fig. 8. FTIR analysis of mace of Myristica beddomii (50%methanol extract)

110 %T 100

90 538.14

601.79 1510.26 2312.65 80 2355.08

3624.25 796.60 862.18 933.55 1095.57 1039.63 1116.78 1625.99 3539.38 1600.92 3512.37 70 723.31 3454.51 1344.38 3431.36

2677.20 1168.86 1278.81 3286.70 3253.91 60 1230.58 3184.48 1460.11 3136.25

1743.65

50 3003.17 1710.86

40

2852.72 2924.09 30

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 9 bot gcw 1/cm Fig. 9. FTIR analysis of mace of Myristica beddomii (methanol extract)

Myristica malabaricain the presence of certain phytochemicals aromatic nitro compounds and aliphatic nitro compounds (fig such as aromatic rings, quinones, ammonium salts, primary/ 1-9; table 2-3). A specific set of phytochemicals such as secondary/tertiary amines, phenols and primary/secondary alkenes, aromatic rings, aldehydes, amides, sulphides and amines. However, it showed difference with the mace of polysulphides which can be taken as a unique fingerprint of the Myristica beddomii in the presence of aldehydes, mace of Myristica fragrans so that the adulteration can be primary/secondary/tertiary alcohols, phenols, sulphides, detected. disulphides, peroxides and bromo/iodo compounds. The results indicate that the mace of Myristica fragrans is characterized by Conclusions a unique set of biochemical markers such as aromatic rings, aldehydes, quinones, ammonium salts, phenols, The following conclusions are drawn from the present primary/secondary/tertiary alcohols, nitriles, sulphides, investigation disulphides, peroxides and bromo/iodo compounds which can be taken as a fingerprint for the identification of possible  FTIR spectral analysis can be used for molecular adulteration. The methanolic extract of the mace of Myristica fingerprinting of food stuff so as to detect possible fragrans showed the presence of alkanes, alkenes, aromatic adulteration. rings, quinones, esters and lactones, carboxylic acid and their  The mace of Myristica fragrans can be identified from salts, ammonium slats, amides, phenols, carbohydrates, ethers, the mace of adulterants due to their colour and biomass. primary/secondary amines, nitriles, sulphides, disulphides,  The mace of Myristica fragrans is characterized by a polysulphides, sulphur oxy compounds, peroxides, organic unique set of biochemical markers such as aromatic phosphates, aromatic phosphates, aliphatic phosphates, rings, aldehydes, quinones, ammonium salts, phenols, fluoro/chloro/bromo/iodo compounds, silicon oxy compounds, primary/secondary/tertiary alcohols, nitriles, sulphides, 40903 International Journal of Current Research, Vol. 08, Issue, 11, pp.40897-40903, November, 2016

disulphides, peroxides and bromo/iodo compounds Jaiswal, P., Kumar, P., Singh, V.K., Singh, D.K. 2009. which can be taken as a fingerprint for the identification Biological effects of Myristica fragrans. Annu Rev Biomed of original sample from the adulterants. Sci., 11:21-9. Manigandan, S., Shanmugapackiam, S., Ramamoorthy, R. Acknowledgment 2015. Preliminary phytochemical screening and FTIR studies of soursop (Annona muricata L.) bark. Global The authors greatly acknowledge the financial assistance and Journal for Research Analysis., Vol 4., 5. the support provided by the Kerala State Council for Science, Marimuthu, M., Gurumoorthi, P., 2013. Phytochemical Technology and Environment (KSCSTE), Thiruvananthapuram, screening and FTIR studies on wild and common South Kerala for the successful completion of the work. Indian legumes. Asian Journal of Pharmaceutical and Clinical Research, vol 6., 2: 141-144. REFERENCES Qiu, Q., Zhang, G., Sun, X., Liu, X. 2004. Study on chemical constituents of the essential oil from Myristica fragrans Brindet, R., Kemsley, E.K., Wilson, R.H. 1996. Approaches to Houtt. by supercritical fluid extraction and steam adulteration detection in instant coffees using IR distillation. Zhongyaocai, 27:823-6. spectroscopy and chemomatrix. Journal of the Science of Sonavane, G.S., Sarveiya, V.P., Kasture, V.S., Kasture, S.B. Food and Agriculture, 71, 359-366. Anxiogenic activity of Myristica fragrans seeds. Pharmacol Downey, G. 1998. Food and Food ingredients authentication by Biochem Behav. mid-IR spectroscopy and chemometrics. Trends in Sun, D.W. 2009. IR spectroscopy for food quality analysis and Analytical Chemistry, 17(7), 418-424. control. First Edn. Pp146-173., Elsevier Inc. Herringshaw, S. 2009. Application of IR spectroscopy Yang, X.W., Huang, X., Ahmat, M. 2008. New neolignan from chemomatics for the authentication of organic butter and the seed of Myristica fragrans. Zhongguo Zhongyao Zazhi, determination of of sugars in tomatoes. Master thesis, pp1- 33:397-402. 54, The Ohio State University.

*******