Artonin O , a Xanthone Compound from Root Wood of Artocarpus rigida

A xanthone derivative, artonin O(1),has successfully been isolated from root wood of Artocarpus rigida grown in Lampung, Indonesia. The structure of this compound has been carefully determined by some spectroscopy techniques and based on physical data. The antibacterial activity test on this compound towards Bacillus subtilis, showed that it has medium activity.


INTRODUCTION
We have previously reported that from the root bark of A. rotunda (Houtt.)Panzer, one of endemic species in Indonesia, obtained from Hutan Raya Bengkulu, has been isolated prenylated flavone, artonin E as well as three xanthone derivatives, artonin O, artonin M and cycloartobiloxanthone, and a geranilated flavone, artoindonesianin L 1 .A. rigida is part of Artocarpus plant which is also endemic in Indonesia, so the chemistry research about this plant is still interesting.Some organic compounds have successfully been isolated from some parts of A. rigida obtained from Keputran Village, Sukoharjo, Pringsewu Lampung Indonesia.From its root bark, artonin E has been isolated and it has been shown to have good anticancer and antimalaria activities 2 .
In this work, we reported artonin O which has been isolated from root wood of A. rigida.The antimicrobial activity of this compound has also been tested against B. subtillis.

General
The melting point was deter mined with Gallenkamp melting poin apparatus and is uncorrected.The UV-Vis spectrum was obtained on Agilent Cary 100 spectrometer and the IR spectrum was measured with Agilent Cary 620 FTIR.The 1 H and 13 C NMR spectra were obtained on Agilent spectrometer with DD2 console system at 500 MHz and 125 MHz, respectively.Mass spectrum was measured with Waters mass spectrometer, LCT Premier XE MS.Vacuum liquid chromatography (VLC) was performed using Merck Si-gel 60, thin layer chromatography (TLC) analysis was carried out on pre-coated Si-gel plates (Merck Kieselgel 60 F254, 0.25 mm).

Plant collections
Samples of the root wood of A. rigida Blume were collected from Keputran Village, Sukoharjo Pringsewu Lampung and were identified at the Herbarium Bogoriense, Research Centre for Biology, Indonesia Institute of Sciences Bogor, Indonesia and a voucher specimen has been deposited at the herbarium.

Extraction and Isolation
The powder of root wood of A. rigida (3 kg) was extracted exhaustively with n-hexane ( 3 x 10 L) and a mixture of methanol-ethylacetate (EtOAc) 1:1 (3 x 10 L).On removal of the solvents by vacuum rotatory evaporator produced methanol/EtOAc extract (127 g).The methanol/EtOAc extract was then fractioned by Si-gel vacuum liquid chromatography (VLC) and was eluted with EtOAc:n-hexane with the ratio from 0 -100%, to produce 6 main fractions (A-F), A 3.2 g; B 0.6 g, E 7.8 g Fraction A was then subjected with VLC using Si-gel with eluent of EtOAc:n-hexane by increasing the polarity from 10-20%, to produce 5 main fractions based on their TLCs.The fifth fraction (0.33 g) was purified with VLC (Si gel 60 GF 254, EtOAc:n-hexane 3-20%) to yield the main compound, artonin O (compound 1) (29 mg).The fractination of fraction B was performed with the same way using eluent of EtOAc:n-hexane 4-20% to yield 3 main fractions.The fractination of the third fraction (0.167 g) with VLC using eluent of EtOAc:n-hexane 3-20%, the red crystal (4.5 mg) was obtained after being recrystallized with EtOAc:n-hexane.This crystal has the same retention time (Rf) with compound 1 based on TLC using three eluent systems.Based on TLC, the total amount of compound 1 obtained was 33.5 mg.

Biological activity
The antibacterial activity test was performed using paper disc method test.

Artonin O
The UV spectrum of compound 1 gave l max at 202, 254, and 317 nm in methanol, indicated the characteristic of flavone compound.The maximum absorbance at l max 317 nm is a characteristic spectrum of flavone which is the electron excitation on cynamoil group in B ring, known as Band I, while the l max at 254 nm is characteristic of electron excitation of benzoyl group on A ring known as band II.
Fig. 1 shows the IR spectrum of compound 1.There is a broad band at 3343 cm -1 which is stretch vibration of hydroxyl group bound to hydrogen.The band at 2918 cm -1 is an indication the presence of C-H aliphatic.The band at 1644 cm -1 indicates the present of carbonyl group (C=O) conjugated with C=C bond.The band at 1467 cm -1 is the present of C=C aromatic, while the bands at 1283, 1226, 1081 cm -1 are indication of stretch of C-O.
The IR spectrum of 1 indicated the similarity with spectrum from standard artonin O.It can be seen by comparing the IR spectra of 1 and the standard in literature (Table 1).
To strengthen the analysis of the structure of the compound isolated, the 13 C and 1 H NMR were taken and the spectra of 1 obtained are presented   The deduction was also supported by the HMBC spectrum as shown in Fig. 4.

Bioactivity test
Artonin O has shown to have antibacterial activity toward the growth of B. subtilis by the formation of inhibition zone at all concentrations of 0.3; 0.4 and 0.5 mg/disc, with inhibition zone diameters were 7, 7.5 and 8.5 mm, respectively.While amoxicilline used as control positive with concentrations of .05;0.10; and 0.15 mg/disc, the inhibition zone diameter measured were 20, 22, and 22.5 mm, respectively.
Based on the data obtained, it is known that the higher the concentration of the sample used, the higher the inhibition zone observed, although in overall, the positive controls have also been observed to inhibit much higher.The functional groups of compound 1 that play role on the antibacterial activities are hydroxyl group, carbonyl and isopropenyl.These groups are able to form hydrogen bond with protein from the bacteria cell showing a specific interaction.According to Davis and Stout 9 , in the antibacterial activity, if the inhibition zone diameter observed is 5 mm or less, it is categorized as weak activity; 5-10 mm as medium activity, 10-19 as strong activity and 20 mm or more categorized as very strong activity.Based on the data obtained in this research, therefore artonin O is categorized as medium activity in the antibacterial activity test.

CONCLUSIONS
From the root wood of A. rigida has successfully been isolated a xanthone compound, artonin O (1) showing medium antibacterial activity towards B. subtillis.

Table 1 : The comparison of IR data of compound
Each carbon signals at d 181.6; 182.9; and 184.6 ppm are assigned as carbon from carbonyl group, where the signal at 181.6 ppm is known as C signal of carbonyl from C-4 of a flavone compound, while the other two carbon signals are assigned as carbon from carbonyl group of quinoid structure.