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ISOLATION OF ESSENSIAL OIL OF NUTMEG (Myristica fragrans Houtt) and ANTIOXIDANT ACTIVITY TEST WITH DPPH

The essential oil of the nutmeg plant (Myristica fragrans Houtt) has been obtained from roots, bark, fruit, mace and seeds using Stahl steam distillation. Each essential oil have tested for antioxidant activity with DPPH. Antioxidant activity of essential oil from each nutmeg plant to DPPH with concentration 25, 50 and 100 ppm. Each essential oil did not show strong antioxidant activity but the result of nutmeg isolation had strong antioxidant activity with IC50 that was 80,555 ppm. Based on GC-MS analysis of essential oil of nutmeg isolate obtained myristicin compound with 96.52% area and time Retention 22.127. Myristicin is one of the main components of essential oils of nutmeg plants that play an important role as antioxidants.


INTRODUCTION
Indonesia is a prosperous country in various types of plants that can be used as medicines (herbal medicine). Active compounds in plants are generally contained in the form of secondary metabolites such as alkaloids, steroids, terpenoids, flavonoids, coumarins, tannins, saponins and so forth. Secondary metabolites can serve as a self-defense against plants from outside attacks such as viruses, fungi and bacteria [1]. One of the plants that could potentially be medicinal is the nutmeg plant (Myristica fragrans Houtt). Nutmeg plants grow a lot in the region of West and South Aceh. Nutmeg is known as a spice plant that has economic value and multipurpose because every part of the plant can be utilized in various industries. Besides being used as flavoring spice, nutmeg is also used as a medicinal material because it has properties as antioxidants, antimicrobial [2] and antifungals [3]. Nutmeg plant is one of the essential oil producing plants that known as nutmeg oil [4]. Essential oil is one of the multi benefit vegetable oils [5]. The basic material of this oil is obtained from various parts of plants such as leaves, flowers, fruits, seeds, seed shell, stems, roots or rhizomes. One of the main characteristics of volatile oil is its volatile and distinctive flavor. The essential oil of nutmeg known in the world market is oil which is processed from mace and nutmeg [6]. The nutmeg oil contains 20 types of compound components with main composition is β-pinen 22.69%, α-pinen 14.06% and α-thujen 13.93% and shows antimicrobial and anticancer activity [7]. The essential oil of nutmeg seed has also been reported to have antioxidant, antimicrobial and antifungal activity [2] and has a strong antioxidant properties [6] the presence of antioxidant activity from the nutmeg mace [8], mace and nutmeg flowers can also be used as a powerful antibacterial and antioxidant [9,8]. Antioxidants are compounds that can inhibit the rate of oxidation or neutralize free radicals. All of these radicals have in common that have one or more unpaired electrons and potentially cause damage to living cells [10]. These unpaired electrons cause free radicals to be highly reactive compounds to the cells of the body by binding to electrons of cell molecules.
In everyday life, we can not be clear from the free radical compounds. Foods that are either fried or burnt, excessive sun exposure, cigarette smoke, motor vehicle fumes, certain drugs, toxins and air pollution are some sources of free radical compounds. In a previous study reported  _ 12 that the extract ethyl acetateof nutmeg leaves had antibacterial activity against Staphilococcus aureus and Escherichia coli, while the leaf nutmeg methanol extract is inactive [11]. Further phytochemical test of methanol extract of nutmeg leaf contains alkaloid, flavonoid, terpenoid and tannin, while ethyl acetate extract contains flavonoid compound and has antifungal activity against Candida albicans [12]. The isolation result of methanol extract of nutmeg contains flavonoid compound type dihydrocaemferol or 3,5,7,4'-tetrahydroxy dihydroflavonol which is active as an antioxidant with IC 50 9,75 ppm [13] and also has anticancer with activity IC 50 5,3209 µg/mL [14]. Research on essential oils on the stem and root of the nutmeg especially from Aceh Province has not been reported. Likewise, the isolation of chemical components and antioxidant activity test part of the plant. Therefore it is necessary to isolate parts of nutmeg plants such as fruit, mace, seeds, roots and stem bark by steam distillation method and activity test of antioxidants using the free radical DPPH method. Essential oils of the most actively planted parts were isolated and determined by structure based on data analysis Gas Chromatography -Mass Spectrometry (GC-MS).

Distillation of essential oils from plant samples
A total of 500 g of nutmeg samples were fed into distillation flask, aquadest added 1000 ml until all the flesh of nutmeg submerged then string up the distillation equipment (Stahl equipment) and oil temperature 120 o C. Distilled for 3 h is calculated after boiling water, the oil obtained is accommodated into the erlemeyer and then added Na 2 SO 4 anhydrous to separate water and oil. The same treatment is done to obtain essential oils on bark, roots, seeds and mace. Essential oils are then tested for antioxidant activation. The essential oil obtained is divided into two parts for the antioxidant activity test and analysis of its components with GC-MS.

Antioxidant test
Antioxidant activity test can be done by making solution of DPPH 0,4 mM. DPPH powder (BM 394.32 g/mol) was weighed as much as 7.9 mg, subsequently dissolved with methanol in 50 mL flask, then homogenized. To make a variation of the total concentration of each essential oil of nutmeg plants (root, bark, fruit, fuli and seeds) previously made main solution 500 ppm by dissolving each essential oil 5 mg into methanol until volume reaches 10 mL. Furthermore, from the main solution be made variations in solution concentration of 25, 50 and 100 ppm (Table 1).  _ 13 Essential oil at 25 ppm concentration of 250 μL, 50 ppm concentration of 500 μL and 100 ppm concentration of 1000 μL, each added 1 mL of DPPH 0.4 mM and volume of 5 mL with methanol. Further homogenized using a vortex mixer and incubated for 30 min at 37°C then read the uptake on  = 517 nm [16]. IC 50 value is the concentration of antioxidants in ppm (µg/mL) which is able to inhibit 50% of free radicals. IC 50 value obtained from the intersection of the line between 50% inhibitory power with the concentration axis, then included in the equation Y = a + bX which is Y = 50 and X value shows IC 50 . The percentage of inhibition is calculated by the Eq (1).
Essential oil is declared active if IC 50 value is less than 100 ppm (μg / mL).

III. RESULT DAN DISCUSSION
The method that can be used to obtain essential oil of nutmeg is generally by water distillation method. Water distillation has the advantage of being used to distill powdered material, as it can coagulate if distilled by steam distillation [17]. A wet nutmeg cultivated sample of 500 g distilled water steam to extract essential nutmeg oil components contained in the sample. The advantage of water steam distillation is the presence of hydrodifusi events where water vapor will enter into the cellular tissue of the plant resulting in the rupture of the cell wall of the plant so that the oil contained in it will be pushed out. The mixture of water steam and essential oil will flow into the condenser so that condensation occurs and distillate is produced. Essential oils that still contain water molecules are dried by adding anhydrous Na 2 SO 4 . The function of addition of anhydrous Na 2 SO 4 to bind the water still contained in the oil, to obtain essential oil of nutmeg no longer contain water.

Phytochemical test
Fresh part of the nutmeg cultivars was analyzed phytochemically to determine the secondary metabolite content contained in the plant, while terpenoid test was performed. The results of phytochemical tests on seeds, mace, fruit, bark and root of nutmeg plants contain secondary terpenoid metabolite compounds. The main chemical components contained in essential oils are generally monoterpen compounds. Secondary metabolites or chemical components in a part of the plant are generally present in other parts of the plant [18].

Results of Antioxidant Activity Test
Antioxidant activity is expressed by % DPPH inhibition of antioxidant ability to damp free radical of DPPH. The antioxidant activity test was performed on  max that is 517 nm. The results of antioxidal activity of root essential oils, bark, fruit, mace and nutmeg seeds can be seen in Table 3. The result of antioxidant activity test in Table 3 shows that essential oil of fuli and fruit has IC 50 value stronger than other nutmeg parts such as seeds, roots and bark. The IC 50 value from each essential oil of mace and fruit is 185,943 ppm and 221,036 ppm, respectively. The world's known essential oil is derived from nutmeg seed and nutmeg mace, while the nutmeg flesh is rarely processed into essential oil [19]. Then it can be proposed that the fruits essential oil can be isolated further because not many have isolated. The curve of inhibition relationship to the concentration of root essential oils, bark, fruit, mace and nutmeg seeds can be seen in the curve of Figure 1.    Terpinene-4-ol has strong antimicrobial activity, anti-inflammatory and antifungal activity. These compounds are used as flavors, perfumes, and medications (such as treating sinus, bronchial, and throat infections, and (others).

Isolation and Purification of Nutmeg Fruits
Fractionation of nutmeg essential oil to separate its components was done by column chromatography using silica gel 60 mesh silicone phase and n-hexane motion phase: diethyl ether (9:1) gradient elution. The Isolation of Essensial Oil of Nutmeg (Myristica fragrans Houtt) and Antioxidant Activity… (Ratna Maira, Binawati Ginting, Mustanir, Hira Helwati, Lydia Septa Desiyana, Rohmat Mujahid) _ fractions obtained were analyzed by Thin Layer Chromatography (TLC) to see the stain patterns of each fraction on the TLC plate with eluent nhexane: diethyl ether (9:1). The same stain and Rf patterns serve as a basis for combining multiple fractions. Of the 140 fractions, 13 fractions were obtained as in Table 5. Based on the results of GC-MS data from MFMABu 2 containing compound components that is Myristcin. Myristicin, isoeugenol and eugenol compound have strong antioxidant activity [2], therefore the compounds having antioxidant activity in the MFMABu 2 fraction are thought to contain the compound. The curve of inhibition % relationship to the concentration of root essential oils, bark, fruit, mace and nutmeg seeds can be seen in Figure 2. The result of the fraction test of MFMABu 2 has IC 50 value that is 80,555 ppm which has strong antioxidant activity and has a single stain pattern, then purified by means of preparative TLC using n-hexane and diethyl ether, to obtain a pure compound weighing 25.9 mg. Furthermore, MFMABu 2 compound was done two-dimensional TLC ( Figure 3). The TLC results can show that the fraction of MFMABu 2 is purely visible from one resulting stain. The fraction of MFMABu 2 obtained was a pale yellow liquid weighing 25.9 mg. MFMABu 2 Fraction.  [20,21,2]. The myristicin compounds contained in the active isolate of persimmon leaf (Diospyros kaki Thunb.) has antioxidant activity with IC 50 value 100,0 µg/mL [22]. Myristicin has activity as antioxidant [23], myristicin is classified in flavonols from flavonoid compounds which are phenolic compounds that functionate as antioxidants [24]. One important component in nutmeg is myristicin which has activity as a hepatoprotector [25]. Myristicin is a psychoactive drug, functionate as an anticholinergic [26]. The myristicin compound is a volatile compound and has a peculiar smell of nutmeg and has great assassinate power against insect larvae and can increase mental activity or as a psychoactive or psychotropic substance.