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Biochemical variability in nutmeg (Myristica fragrans) and related taxa

Biochemical variability in nutmeg (Myristica fragrans) and related taxa
K. M. Maya

2003

Department of Biochemistry, University of Calicut, Kozhikode 673635, INDIA.

ABSTRACT

Myristica fragrans produces two spices, nutmeg and mace. It belongs to the Myristicaceae family with about 18 genera and 300 species. Though it is dioecious, male and female flowers are sometimes seen on the same tree. Under Family Myristicaceae, highly relevant species used for variability study are M. fragrans, M. andamanica, M. malabarica, M. magnifica, M. amygdalina, M. beddomeii, M. prainii and Knema andamanica. They are distinctly different in morphology, flowering pattern and related features.

M. fragrans, Houtt. is a native of the Moluccas in the East Indian Archipelago. M. andamanica, Hook. F is seen in the Andaman Islands with slender horizontal branches. They often have curved stilt-like roots at the base. M. malabarica, Lamk, from the Western Ghats yields fruits which have no odour and taste. They are called Bombay nutmeg and Bombay mace; they are used as adulterants of Myristica fragrans. M. magnifica, Beddome Fl. Sylv. is found in the swampy ground of Western Ghats. Knema andamanica is a moderate sized tree seen in the evergreen forests of Western Ghats.

Nutmeg and mace are used for extracting essential oil by steam distillation. The essential oil is colourless or pale yellow coloured with the characteristic spicy odour. Major constituents are sabinene, β-pinene, dipentene, p-cymene, d-linalool, terpinen-4-01, dl-α-terpineol, geraniol, safrole, eugenol, isoeugenol, myristicin, myristic acid, esters of myristic acid and other fatty acids. Myristicin in the oils of nutmeg and mace is believed to be responsible for the toxicity of nutmeg. Oleoresin consists of both volatile and nonvolatile components.

Nutmeg is a dioecious plant, with the occasional occurrence of bisexual plants that may predominate in either male or female flowers. Sex determination is a problem in the seedling stage. Sex of the plant can be ascertained only after flowering, which takes 6-8 years.

Wide variability is observed between the cultivated and the wild plants of Myristica. Morphologically, leaves of M. fragrans are relatively very small compared to the wild plants. The shape of fruits, nutmeg and mace are distinctly different between wild and cultivated species. M. fragrans leaf yields 0.5 to 2% oil while the wild species yield only negligible oil. Nutmeg and mace of M. fragrans yields good amount (up to 16.5% and 26.1% respectively) of essential oil while the wild fruits are almost devoid of essential oil. The mace of M. fragrans is bright red by virtue of the lycopene present in it; it is not seen in M. beddomeii. Hence variability was observed between the wild and cultivated taxa of Myristicaceae with respect to morphological and levels of primary and secondary metabolites. A study was undertaken to establish the biochemical variability with the following objectives.

1 Evaluation of biochemical variability in nutmeg (Myristica fragrans) germplasm.
2 Differentiation of male, female and bisexual plants based on primary and secondary metabolites in the leaf of Myristica fragrans.
3 Biochemical variability between Myristica fragrans and related taxa.

Based on the study conducted, the results obtained can be illustrated as follows.

Biochemical variability in the nutmeg (Myristica fragrans) germplasm. Among the accessions analyzed, no variability was observed in some of the primary metabolites such as total carbohydrate (30.4% - 33.1% in nutmeg, 42.3% - 45.7% in mace and 17.9% - 19.5 O/O in leaf), starch (26.3% -28.4% in nutmeg, 36.3 % - 38.1 % in mace and 15.8 % - 17.4 % in leaf), reducing sugars (0.15% -0.2 % in nutmeg, 0.27% -0.31 % in mace and 2.26% - 3.9 % in leaf) and protein (5.3 % in nutmeg, 6.1% in mace and 3.5 % in leaf). The fat content ranged from 28.2% - 34.9% and 23.7% - 26.4% in nutmeg and mace respectively. When fatty acid methyl esters (FAMES) from the fat were identified in a Gas Chromatograph, myristic acid was found to be the dominant (55.1 %) fatty acid in nutmeg and palmitic acid (52.56%) dominated in mace fat. Rind had higher content of potassium, calcium and iron when compared with nutmeg and mace. Manganese was more in nutmeg while mace had higher copper content.

Distinct variability was observed in the nutmeg germplasm with respect to the essential oil composition. The essential oil content ranged from 5 % to 16.5% in nutmeg and 7.1% to 26.1% in mace. Some of the nutmeg accessions of llSR such as A9118, A9149 and A1 1/49 were rich in both nutmeg and mace oils. The present study has also revealed that the total yield of nutmegs from a tree do not indicate its oil recovery. However, medium yielders have a slight edge in the oil yield compared to the other groups. Accession A9118, with a medium yield of nutmeg, had the highest oil recovery. Myristicin or methoxy safrole is the major aromatic constituent of nutmeg and mace oils. The study identified many accessions with low and high myristicin. The myristicin content was as low as 1.5 % (A911 02) and as high as 15.1 % (A911 8). Another important observation in the study is the high level of sabinene (45% in A9171) in the oils. Many accessions have been identified which can find specific industrial use. Nutmeg leaf is also a potential source for oil and its constituents. Even though the concentration of various constituents varies in the leaf oil compared to nutmeg and mace oil, all the constituents are found in leaf oil too. Variability in the hallucinogenic principles such as myristicin, elemicin, safrole and the confectionery principle sabinene is also found in leaf oil.

Extracting nutmeg oleoresin with acetone will extract fat also. Study conducted using different solvents indicate that extracting fat first with petroleum ether from the kernel powder followed by extraction with acetone gives the true picture of oleoresin and fat content. The oleoresin content of nutmeg had a range of 3.4 O/O - 11.9% while its fat content ranged from 28.2% - 34.9%. The Non-Volatile Ether Extract (NVEE) in nutmeg and mace was 33.77 and 18.65% respectively. Lycopene is the pigment responsible for the brilliant red colour of mace. It had a range of (82.4 mg % - 273.9 mg %). It is a nutraceutical with potent antioxidant properties (Maya et al., 2002).

Primary and secondary metabolites of leaf from male, female and bisexual plants of Myristica fragrans
Some of the primary metabolites like total carbohydrate (15.9%-18.6%), starch (10.3%-14.8%), reducing sugars (2.52%-3.4%) and proteins (3.5%) did not show much variability within Myristica fragrans. When the amino acid profile was studied by HPLC, it revealed that there is no amino acid specific for male or female plant. Some of the male plants had high histidine and the female plant A9/4 had high phenylalanine. Total free amino acids were found more in the bisexual plant compared to the other two groups. The phenol content ranged from 0.21% - 0.31% within the species.

Among the minerals analysed, copper was found to be higher in the male lines. In contradiction to earlier reports, this study illustrates the fact that oil level or its constituents do not give any sexual identity in nutmeg. In case of phenolic acids, compared to the reported dominance of ferulic and synapic acids in nutmeg and mace respectively, the dominance of coumaric acid in leaf was established. Male, female and bisexual lines, however, did not show any variability. Based on polyphenol oxidase (PPO) and peroxidase isozymes, no variability was found between the cultivated lines of Myristica fragrans.

Biochemical variability among the various taxa. Levels of total carbohydrate, starch, reducing sugars and leaf-protein are all on par in cultivated and wild taxa. Fat content in nutmeg (M. fragrans) is about 40%. Only M. prainii had the nearest fat content compared to M. fragrans. In case of the mace samples, M. fragrans and M. malabarica had high fat compared to M. prainii. Cultivated and wild taxa had the same fat in the rind in all the cases. The rind of M. beddomeii, M. malabarica and M. prainii had crude fibre content similar to that of M. fragrans (21 %).

Essential oil is high in the leaf of M. fragrans when compared to the wild taxa. Among the wild taxa, only M. beddomeii has significant content of oil (0.125%). Gas Chromatographic-Mass Spectral (GC-MS) study of leaf oils of M. fragrans and M. beddomeii revealed some important findings. M. beddomeii is very rich in P-caryophyllene (40%). M. fragrans leaf oil contains 2.6% of β-caryophyllene, 30.85% of myristicin and 1.32% of elemicin. M. beddomeii lacks myristicin, elemicin, α- and β-pinene. Phenylalanine was found to be the dominant amino acid in all the different species except Knema andamanica, which is very rich in threonine and alanine. Its alanine content is very high compared to other wild plants. Total free amino acids ranged from 127.65 μg/100mg to 215.21 μg/100mg. M. prainii and Knema andamanica had the highest amino acid content. Nutmegs of both M. fragrans and M. beddomeii have myristic acid as the predominant fatty acid followed by palmitic acid. In M. malabarica, stearic acid is the predominant fatty acid followed by myristic acid. In contrast to nutmeg, M. fragrans mace has palmitic acid as the predominant fatty acid while the mace of M. malabarica dominates in stearic acid. Among the different species studied, M. malabarica differs from the rest of the group in having stearic acid as the predominant fatty acid in both nutmeg and mace. Among the minerals analyzed, nitrogen, copper, iron and zinc are found to be high in the kernel and mace of M. fragrans. Among the wild taxa, calcium and manganese are found to be high in the kernel of M. beddomeii. The rind of M. fragrans contains significantly high amount of iron and that of M. prainii contains high amount of potassium. The pigment present in the mace of M. fragrans and M. prainii is lycopene while, in M. beddomeii, it did not tally with the lycopene pattern.

From the study, it can be concluded that, biochemically, the cultivated and wild taxa are distinctly different. The dissimilarity is very profound in essential oil content, its profile, fat, minerals, amino acid profile, etc. The study also concludes the uniqueness of male and female lines having uniform oil and amino acid profile.