Isolation and identification of secondary metabolites in ethyl acetate extract from the Maja bark ( Aegle marmelos Linn.)

This exploratory study resulted in the isolation and identification of secondary metabolites in ethyl acetate extracts from the Maja bark ( Aegle marmelos Linn.) from Pattappa Village, Pujananting Subregency, Barru Regency, South Sulawesi. The stages of isolation included maceration, fractionation, and purification. Maceration was performed with methanol, fractionation with ethyl acetate, a particular ratio of acetone (9:1) was used, and purification was done by flowing nitrogen gaseous into the E 5 fraction. Furthermore, the classification test and FTIR spectrophotometer were carried out for identification. Pure isolates in the form of white crystals were obtained with a melting point of 242 o C. Reagent tests indicated a brown precipitate on Wagner's test and a white precipitate on Mayer's test. Infrared-spectrophotometric identification provided typical absorption for such functional groups as NH, aliphatic CH, aromatic C=C, and amine CN. The reagent tests and FTIR-spectrophotometer identification confirmed that the secondary metabolites fit well into the class of alkaloids.


INTRODUCTION
changes the taste to be rather sweet, warmer when drinking it as its natural alcohol levels are reasonably safe for the body.
This study sought to show the potential secondary metabolites in the Maja bark similar to the prior work. The earlier mentioned descriptions showed that it is essential to find secondary metabolite in Maja bark to be used as an active ingredient in pharmacy by applying ethyl acetate solvent. The stem of the plant is one part that has a high possibility to be isolated since such part is expected to produce novel secondary metabolites and is often used by the community as traditional medicine.

MATERIALS AND METHOD Material
The equipment used for the research includes FTIR Spectrophotometer Shimadzu Prestige-21. The identification of isolate was taken on a Shimadzu IR Prestige-21 using KBr pellets in the range of 4500−500 cm -1 wavenumber from which functional groups of the compound were obtained.

Test and procedure
As much as 4.5 kg of Maja bark was grounded and macerated with methanol solvent for 3x24 hours (Tobo et al., 2001), and the extract was harvested for 1x24 hours. Prior to weighing, the macerate was filtered through the Büchner funnel with Whatman filter and was evaporated using a rotary evaporator until the methanol extract thickened (Harborne, 1987). After weighing, the methanol extract was partitioned using liquid-liquid extraction with a separatory funnel and ethyl acetate solvent. The resulting filtrate was evaporated to be dried at room temperature and then weighed.
Ethyl acetate extract was fractionated using Vacuum Liquid Chromatography (VLC) method. The TLC's result was analyzed using Thin-Layer Chromatography (TLC) to obtain fractions of the same staining profiles. These fractions were combined and evaporated at room temperature. One of the fractions was selected and fractionated using Pressure Column Chromatography (PCC). The resulting fraction of PCC was, once again, analyzed using TLC, where fractions of the same staining profiles were combined. Fractions that indicated the presence of crystals at this point were recrystallized to obtain pure crystals. The purification of the fractions was subjected to three eluent systems. The compounds' isolation resulted from the qualitative test with suitable reagents, e.g., Lieberman-Burchard, FeCl3, Wagner's, and Mayer's reagents. The melting points were examined by using the Melting Point Meter, and the functional groups were tested using an FTIR spectrophotometer.

RESULT AND DISCUSSION Classification test
The identification of ethyl acetate extract's chemical content was tested using such reagents as FeCl3, Liebermann-Buchard, Mayer, and Wagner. This test aimed to identify the class of secondary metabolites present in ethyl acetate extract. The result was presented in

Fractionation and purification
The fractionation process involved a stationary phase composed of 60 H silica gel and a mobile phase where eluents gradually increased in polarity. Separation was carried out based on color bands found in the stationary phase. It obtained 27 fractions, as shown in Table 2. Fractions that revealed the same staining profiles were combined and grouped into ten fractions. Fraction E that weighed 1.9526 g was further fractionated using PCC, from which 24 fractions were obtained (Firdaus, 2011), (Iwan and Darminto, 2012). Subsequent TLC was required for fraction combinations that consisted of 10 fractions according to the same staining profiles on TLC plates. The chosen fraction subjected to purification was fraction E5 (Table 3) which weighed 0.5208 g. The resulting isolate was recrystallized with chloroform and methanol solvent that developed into a white crystal-forming isolate that weighed 43.1 mg. The purification of the E5 isolate was carried out with repeated recrystallization. This technique aims to separate the isolates from the remaining impurities. The purification product was pure isolate (isolate E5) in white crystals as much as 43.1 mg. The results of classification and spectroscopy tests that were previously reported corroborated this finding. Research conducted by (Nugroho et al., 2011) reported that aegeline (N-2-hydroxy-2(4-methoxyphenyl)ethylcinnamamide), as an alkaloid derived from cinnamic acid, was obtained in the petroleum ether extract and chloroform of Maja plant leaves. Anthraquinone (1-methyl -2-(3'-methyl-but-2'enyloxy), which has anti-fungal activity against Aspergilus fumigatus and Candida albicans, was isolated in Aegle marmelos correa seeds (Mirsha et al., 2010). The results of A. marmelos Correa isolation found a Coumarin, marmarin 7-(6'-7 'dihidroxygeranial-oxy), which actively inhibits leukemia cells (Nugroho et al., 2011). In another research, the highest concentration of marmelosin (108.65 μg / g) was observed in immature Maja fruit and the highest antibacterial activity of marmelosin against E. coli was shown at a concentration of 200 µg / ml by using the HPLC instrument (Svaefullah et al., 2020).

Purification test
The purity of the isolate was tested using three eluent systems where a pure isolate was indicated by a single spot's appearance when developed on a TLC plate. The result of the purity test with three eluent systems, i.e., n-hexane: acetone (3:7), chloroform: ethyl acetate (6:4), and nhexane: ethyl acetate (7:3), appeared as a single spot, thus confirming the purity of the isolates. The exact temperature at which melting point was determined, using melting point apparatus, was 242°C (Firdaus, 2011). The classification of the resulting isolates was tested using these reagents, as shown in Table 4.

-) Steroids
A pure isolate of fraction E5 was tested for purity by TLC three eluent systems. The isolate was visible after the CeSO4 stain was given, and the heating produced a brown color, as shown in Figure  1.

Figure 1. TLC Results of the isolate using Three Eluent Systems (a). Eluent n-hexane : acetone (b). Eluent chloroform : ethyl acetate (c). Eluent n-hexane : ethyl acetate
The isolate was further tested for purity using TLC with three types of eluents that displayed comparisons between different solvents. This aimed to confirm the isolate's purity as the developed TLC plates each showed a single spot. In the wake of the TLC development (Atun, 2010), a single spot appeared on these eluents, i.e., combinations of n-hexane and acetone; combinations of chloroform and ethyl acetate; combinations of n-hexane and ethyl acetate. As for the melting point, the results show that these isolates began to melt at 240 o C and melted completely at 242 o C. This supports the theory that a pure compound will have a sharp melting point, from solid-state to liquid-state, at no more than 2 o C (Firdaus, 2011). The classification test led to positive alkaloid reactions to Wagner's reagent and Mayer's reagent, indicated by a brown-precipitate formation and a white-precipitate formation. This precipitate occurs because alkaloids are compounds from the nitrogenous base group. If the nitrogenous base is reacted with an acid, HCl (hydrochloric acid) will form an insoluble salt so that this salt will form a precipitate. In contrast, the difference in color that occurs is due to the original color of the used reagent. The three tests show that the Maja Bark (Aegle marmelos) extract was tested positive for alkaloids (Ibrahim and Rusdiaman, 2017).

FTIR spectroscopy test
Based on FTIR spectroscopy, the infrared spectrum of isolate E5 was interpreted in terms of the wavenumber, band, intensity, and group, as shown in Figure 2. Based on the IR spectrum interpretation, several functional groups were indicated: amine (N-H), aliphatic (C-H), carboxylate groups (HO-C = O), double-bond aromatic (C = C), CN, and COC. This indicates that isolate E5 is one of the alkaloid compounds group. It was confirmed by the group test results showing that the pure isolates were positive for the alkaloid group.

CONCLUSION
The isolation of secondary metabolites from ethyl acetate extracts from Maja bark indicated the presence of pure, white crystalline isolates of alkaloid type, which melted at 242 o C.