Two Phenolic Compounds from Chloroform Fraction of Syzygium Polycephalum MIQ . Stem Bark ( Myrtaceae )

Syzygium polycephalum (Kupa) is a plant of the Myrtaceae family which is one of the endemic plants in Indonesia, commonly called as Gowok. The chemical components of the plant have not been reported so far. This study is intended to know the molecular structures of isolated compounds of chloroform fraction from S. polycephalum stem bark. The steam bark of the plant is dried, powdered and macerated with methanol to yield methanolic extract. The methanolic extract was then conducted to fractionation using hexane and chloroform to obtain hexane and chloroform fractions. The chloroform fraction was further subjected to separation using column chromatography to obtain pure isolates and followed by measuring of their spectroscopic evidences. The isolation of chloroform fraction had led to the findings of two pure isolates. Their structures of isolates were elucidated by extensive spectroscopic methods and by comparison with the literature data to gain two phenolic compounds that are gallic acid and 3,4,3’-tri-O-methylellagic acid.


INTRODUCTION
Myrtaceae family has about 130 genera and approximately 3800-5800 species of predominantly tropical and subtropical distribution.The Myrtaceae family is known to possess leaves with high concentrations of terpenes and considerable qualitative and quantitative variation in the types of terpenes, according to taxonomic identity and population and individual levels.These variations have pharmacological potential and many industrial applications (Barbosa, Cleber, Róbson, Renata, & Antônio, 2013).
One important genera of this family is Syzygium, which is one of the larger genera with around 500 species.They are usually trees and shrubs distributed in the tropics of the world from Africa to the West Pacific with major concentration in Malaysia.The genus is popular for the spice plant, i.e.Syzygium aromaticum (L.) Merr.& Perry which is native to Maluku Islands in Indonesia (Mohanan et al., 2015).
Syzygium can be found from sea level on swamp forests, lowland and montane forests to subalpine forests.Their habits are also vary, from canopy-emergent trees to canopy trees, understorey trees, treelets and shrubs.Indonesian Botanic Gardens (Bogor, Cibodas, Purwodadi and Bali) have collected 40 species of Syzygium from all over Indonesia.Purwodadi Botanic Garden has collection of 15 species of Syzygium, 5 of which were from East Java.This number is still quite low compared to the total species of Syzygium in East Java (Ariyanti, Rony, Lia, & Deden, 2012).
The species of syzygium genus is well known for its medicinal properties.S. jambolana (S. cumini), popularly known as Jamun, has been the main ingredient of various medications of the traditional Indian system of medicine.Preclinical studies have shown that the various extracts of S. jambolana possess a range of pharmacological actions, such as antibacterial, antifungal, antiviral, antiulcerogenic, cardioprotective, anti-allergic, hepatoprotective and anti-diarrheal effects, thereby supporting its myriad traditional uses (Baliga, Harshith, Bantwal, Rajesh, & Princy, 2011).Studies in the past one decade have shown that Jamun possess antineoplastic, radioprotective and chemopreventive effects all of which are useful in the prevention and treatment of cancer (Preddy, 2014).
S. aqueum is a species of brush cherry tree.It is commonly known as water apple or water cherry.It is well documented as a medicinal plant, and various parts of the tree have been used in traditional medicine, for instance as an antibiotic.S. aqueum leaf extracts have a significant composition of phenolic compounds, protective activity against free radicals as well as low pro-oxidant capability (Palanisamy et al., 2011).
The Syzygium species having appreciable medicinal properties have drawn the attention of the researchers in recent times included S. polycephalum.S. polycephalum, locally known as gowok or kupa or kepa, is an indigenous tree growth in Indonesia.It has synonyms: Eugenia polycephala Miq., Jambosa cauliflora DC., Jambosa polycephala (Miq.)Miq. and S. cauliflorum (DC.)Bennet.Gowok is indigenous to West and Central Malesia.It is common in Java and Kalimantan in Indonesia.It has been reported the presence of several compounds found in the plant that are ursolic acid, oleanolic acid, squalene, and -sitosterol from S. polycephalum leaves (Ragasa et al., 2014).It could be considered that all of these compounds are non phenolic compounds.
On the other hand, it was reported that the wood extracts (ethyl acetate extract) of S. polychephalum potentially contain anti-fungal compound (i.e.3-O-glucosyl-3',4',5trihydroxyflavonol) to inhibit the growth of S. commune Fr. and Pleurotus sp fungi (Jemi, Syafii, Ferbianto, & Hanafi, 2010).Using the literature searching, there are no reports regarding the phenolic compounds of the stem bark of S. polycephalum.Therefore, it was of great interest to carry out a proper scientific investigation of the stem bark extract of this plant.The present study however, reports for the first time the isolation and structural elucidation of gallic acid (1) and 3,4,3'-tri-Omethylellagic acid (2) from the chloroform fraction of the stem bark of S. polycephalum.

Chemicals and plant materials
The solvents used in this study are hexane, chloroform, ethyl acetate, and methanol that were of pro-analytical Grade (Grade AR) and silica gel obtained from E. Merck (Germany).The stem bark of S. polycephalum (c.a.27 kg) was collected from a local area in Ngawi, East Java, Indonesia in December 2014.The identification of the plant was performed by staff of Herbarium-LIPI, Purwodadi, East Java, Indonesia.A voucher sample is kept in the Herbarium of LIPI with Identification No. 0117/IPH.06/HM/I/2015,January 5, 2015.

Equipment and instruments
The equipment used to do extraction and fractionation (isolation) are filter paper, Buchner funnel, Hirsch funnel, Erlenmeyer flask, pippet, spatula, measuring glass, vials, containers, separating funnel, and vacuum rotary evaporator type BUCHI Rotavapor R-215.The equipment used to measure melting point of isolate is Fisher Scientific.Whereas, chromatographic techniques used to isolate phenolic compounds from chloroform fraction included Vacuum Liquid Chromatography (VLC) (using silica gel 60, 0.040-0.063mm), Gravitational Column Chromatography (GCC) (silica gel 60, 0,063-0,200 mm and 0,200-0,500 mm or 70-230 mesh ASTM), TLC analyses were carried out on silica gel 60 F254 chromaplates with the developing solvent systems.Checking the homogeneity of the compounds were made by TLC on Kieselgel gel 60 F254 pre-coated sheets (E.Merck) and the spots were detected by exposure to UVlamp at 254 nm or 366 nm.
A number of instruments needed to identify and characterize an isolate included spectrophotometer FTIR-8400S SHIMADZU, spectrophotometer UV-1800 SHIMADZU.The 1 H NMR spectra were recorded with a Bruker DRX-600 NMR Spectrometer (600 MHz, CD3OD) instrument and the 13 C NMR spectra were obtained with the same instrument at 150 MHz in CD3OD.Chemical shifts are given in δ (ppm) values relative to those of the solvent signal [CD3OD (δH 3.30; δC 49.0)] on the tetramethylsilane (Sigma) scale.

Preparation of methanolic extract and its fractionation of S. polycephalum stem bark
Stem bark of S. polycephalum (Gowok) was cleaned and cut into small using commercial cutter, dried in under sunlight during c.a. one week and powdered.The powder was then macerated using methanol as extracting solvent for a day.Macerate was filtered and evaporated using vacuum rotary evaporator to yield methanolic extract.The extract was then added with a little of methanol and was fractionated using hexane.The residue of methanol extract was further fractionated again using chloroform and the last extract was subjected to investigate the chemical constituents.

Isolation and characterization of pure isolates
The stem bark of S. polycephalum was macerated in MeOH at room temperature for 24 h and then filtered.The filtrate was concentrated under vacuum to give 349 g of crude residue.The crude (349 g) was suspended in methanol and defatted with hexane to gain hexane extract (22.38 g).This process was also repeatedly carried out by using chloroform to yield chloroform extract (5.66 g).The chloroform extract was then subjected to column chromatography (silica gel, n-hexane, n-hexane-CHCl 3 and MeOH, in order of increasing polarity) yielding 55 fractions that can be grouped to be 5 fractions [A (1-4), B (5-6), C (7-37), D (38-51), and E (52-55)].The fraction A (1-4) was allowed to evaporate at room temperature and yielded a pure isolate as colorless needle crystal (10 mg) with mp.256-257 o C. The crystal was characterized by UV-Vis and FTIR and by comparison with literature data and determined its structure to be gallic acid (1).Then, fraction B (5-6) seemed that the fraction gave simple chromatogram profile and allowed to dry at fume hood yielded a pure enough isolate as off-white amorphous powder (10.3 mg) with mp.267-269 o C. The isolate was then characterized by UV-Vis, FTIR, LCMS and NMR spectroscopies and by comparison with literature data and determined its structure to be 3,4,3'-tri-O-methylellagic acid (2).

RESULTS AND DISCUSSION
The MeOH extract of S. polycephalum stem barks was partitioned successively with hexane and CHCl3.Successive column chromatography of the CHCl 3 extract over silica gel using the various chromatographic techniques yielded compounds 1 and 2.
The 1 H-NMR spectrum (600 MHz, DMSO-d6, ppm) of compound 2 revealed the presence of six significant proton signals that could be explained as follows.Two signals located at δH 7.61 (1H, s) and 7.52 (1H, s) indicated two aromatic protons due to the ellagic acid skeleton.The spectrum of the compound also displayed one signal at δ H 3.31 (1H, br) suggesting the presence of an aromatic hydroxyl group (aryl -OH).In addition, three signals located at δH 4.06, 4.04, and 3.99 (3H, s) showed three methoxyl groups.
There are no previous reports regarding the investigation of chemical components of S. polycephalum stem bark especially for phenolic compounds.In this study, two phenolic compounds were now successfully isolated from chloroform fraction of the plant that are gallic acid (1) and 3,4,3'-tri-Omethylellagic acid (2).Both compounds were found from the plant for the first time.

Phytochemical
investigations of the chloroform fraction of S. polycephalum stem bark led to the isolation of two phenolic compounds: gallic acid (1) and 3,4,3'-tri-Omethylellagic acid (2).Structurally, compound 2 is a derivative compound of ellagic acid in which the acid itself is formed from two gallic acids.This means it is very reasonable that two compounds are equally found in one plant including the plant.wish to thank also deeply thanks to Hyiya Amen for help us to measure NMR and LC-ESI-MS.
13 C-NMR spectrum (150 MHz, DMSO-d6, ppm) of compound 2 displayed seventeen carbon signals that could be described as follows.The spectrum showed 17 signals, of which 14 signals were assigned to the ellagic acid portion and the rest signals were three methoxyl groups.Two carbon signals located at δC 158.52 and 158.32 confirming clearly for two carbonyl groups [C-7(7')] were attributed to ellagic acid lactone carbonyl signals, two carbon signals at δC 153.71 and 153.06 confirmed as benzene ring attached by methoxyl and hydroxyl groups [C-4(4')], and two carbon signals at δC 112.49 and 107.44 indicated benzene ring attached by hydrogen [C-5(5')].Then, two carbon signals located at δC 141.48 and 140.28 with high intensity indicated as benzene ring attached by methoxyl groups [C-3(3')].For a while, two carbon signals on the position of δC 140.95 and 140.77represented benzene ring attached by the respect lactone groups [C-2(2')] and δC 113.44 and 111.84 revealed benzene ring attached by carboxyl groups [C-6(6')].Finally, two carbon signals located at δC 111.76 and 110.89 revealed benzene ring attached by other phenyl group and vice versa [C-1(1')] and assigned as ellagic acid skeleton.