Triterpenoids from the Bark of Aglaia glabrata and their In vitro Effects on P-388 Murine Leukemia Cells

Four dammarane-type triterpenoids, dammardienon (1), aglaiabbreviatin E (2), dammar-20, 25-dien-3b,24-diol (3) and dammar-24-en-3b,20-diol (4) were isolated from methanolic extract of the bark of Aglaia glabrata. The structures of all triterpenoids were elucidated by 1D-, 2D-NMR, and comparison with previously reported data. All triterpenoids were applied into in vitro bioassay against P-388 murine leukemia cell. Dammar-24-en-3b,20-diol (4) has cytotoxic activity with IC50 value of 9.45 mM towards P-388 murine leukemia cell lines.


General Experimental Procedure
The melting point values of triterpenoids were obtained using a electrothermal apparatus with uncorrected measurement.A Perkin-Elmer 1760X spectrophotometer was used to obtained infrared spectra of compounds in pellets of potassium bromide.HRTOFMS spectra were recorded on Water, Qtof HR-MS XEVotm mass spectrometer.All NMR spectra are measured on JEOL NMR A-500 MHz with an internal standard of tetramethylsilane.All chromatography techniques, including vacuum liquid chromatography (VLC) and column chromatography (CC), were undertaken on silica gel (SiO 2 , Merck, Darmstadt, Germany).TLC analysis was done on Kieselgel 60 F 254 (Merck) plates; visualization was performed with UV light and plates were sprayed with 10% H 2 SO 4 solution in ethanol and heated.

Plant material
Dried bark of A. glabrata were obtained in August 2016 from Bogor Botanical Garden, Bogor, West Java, Indonesia.A voucher specimen was deposited at the Bogoriense Herbarium (code Bo-1288718).

Cytotoxic Activity
Determination of cytotoxic activities was undertaken based on the MTT assay explained in the previous reports 18,19 .The P-388 murine leukemia cells were seeded into 96-well plates at an initial cell density of approximately 3 x 10 4 cells cm -3 .After 24 h of incubation for cell attachment and growth, varying concentrations of samples were added.The compounds were first dissolved in DMSO (dimethyl sulfoxide) at the required concentration.Six concentrations were prepared using PBS (phosphoric buffer solution, pH = 7.30 -7.65).Control wells received only DMSO.The assay was terminated after 48 h incubation period by adding MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and the incubation was continued for another 4 h in which the MTT-stop solution containing SDS (sodium dodecyl sulphate) was added and another 24 h incubation was conducted.An optical density was read by using a micro plate reader at 550 nm.IC 50 values were taken from the plotted graph of percentage live cells compared to control (%), receiving only PBS and DMSO, versus the tested concentration of compounds (µg/mL).The IC 50 value is the concentration required for 50% growth inhibition.Each assay and analysis was run in triplicate and averaged.

Extraction and Isolation
Dried powder of A. glabrata (2.5 kg) were extracted by maceration with 80% aqueous methanol.The extract was partitioned with respective n-hexane and EtOAc.Fraction C of n-hexane extract was VLC-chromatographed twice to yield dammardienon (1, 13.6 mg) 23 .The second fraction (fraction G) was VLC-chromatographed two times, 0.96, s followed by crystallization to result in aglaiabbreviatin E (2, 12.4 mg) 17 .
Two fractions of EtOAc extract (Fraction J and N) were chromatographed using successive VLC techniques and were followed recrystallization, yielding respective dammar-20,25-dien-3b,24diol (3, 23.4 mg) 24 and dammar-24-en-3b,20-diol (4, 12.4 mg) 25,26 .The structural determination of all compounds was undertaken on the basis of spectroscopic methods (UV, IR, MS, 1D and 2D NMR) and comparison with those reported in the literature.All of these data can be seen in Table 1 and 2.

DISCUSSION
The barks of A. glabrata were macerated with aqueous MeOH at room temperature and the macerated residue was fractionated with respective n-hexane, EtOAc and n-BuOH.Repetitive SiO 2 CC. of the n-hexane and EtOAc fractions afforded four dammarane-type triterpenoids 1-4 (Figure 1).carbon NMR spectra after associated with DEPT analysis, which consisted of seven tertiary methyls, ten methylenes of sp 3 , four methines of sp 3 , four quaternary carbons of sp 3 , one carbonyl (d C 217.6), one sp 2 methylene (d C 106.9) one sp 2 methines (d C 125.1) and two sp 2 quaternary carbons (d C 132.0 and 151.4).Four remaining of hydrogen deficiency index was corresponding to four ring dammarane-type triterpenoid 18,19 .The presence of dammarane-type in 1 was also supported by a combined experiment of 1 H-1 H COSY and HMBC (Fig. 2).Correlations of an olefinic proton signals at d  MS data of compound 1 showed that there was a hydrogen deficiency index of seven.The infrared spectra displayed absorbance bands for an aliphatic moiety (3082 and 2949 cm -1 ), a ketonic carbonyl (1705 cm -1 ) and an olefin (1645 cm -1 ).The proton NMR analysis displayed seven methyl protons at d H 0.87, 0.95, 1.00, 1.05, 1.08, 1.61 and 1.68, which corresponding to tertiary Me-18, Me-19, Me-26, Me-27, Me-28, Me-29, respectively.There are signals of a sp 2 methine proton at chemical shift of d H 5.11 and sp 2 methylene protons at chemical shifts of d H 4.71 and 4.75, found in the spectrum.Thirty carbon signals were read in the S imilar to compound 1, there was a hydrogen deficiency index of seven in compound 2, based on the MS data.The infrared spectrum displayed absorbance band for groups of an alcohol (3431 cm -1 ), an aliphatic (2943 and 2870 cm -1 ), a ketonic carbonyl (1725 cm -1 ), olefine (1642 cm -1 ) and ether moieties (1245 cm -1 ).The molecular weight of 2 has 17 atomic mass unit higher than 1, describing an additional OH group in 2. The NMR spectra of 2 have similarity with spectra of 1.A molecular formula of compound 3 suggested a hydrogen deficiency index of six, based on its MS data.The IR spectrum showed functional group wavenumbers at 3430 cm -1 (hydroxyl), 2949 and 2875 cm -1 (aliphatic), 1610 cm -1 (olefinic), 1450 and 1354 cm -1 (gem-dimethyl) and 1050 cm -1 (ether).The NMR spectral data of 3 were superimposed with spectral data of dammaradienon (1), unless the absence of a ketonic carbonyl (d C 218. ).NMR data of 3 were highly overlapped to those of dammar-20, 25-dien-3b,24-diol 24 , eventually, compound 3 was identified as a dammar-20,25-dien-3b,24-diol.
A hydrogen deficiency index of five was also found for compound 4 based on the MS data.IR wavenumbers of 3369 cm -1 , 2939 cm -1 , 1639 cm -1 , 1458 and 1230 cm -1 , and 1109 cm -1 were analyzed for respective hydroxyl, aliphatic, olefinic, gem-dimethyl, and ether groups.NMR spectral data of 4 were similar to spectral data of dammar-20, 25-dien-3b,24-diol 3, except the absence of one of two terminal double bonds at d H 4.85, 4.90 and one oxygenated methine at d H 4.20 and appearance of newly an olefinic double bond at d H 5.10 and d C 124.8 and 131.7 and a tertiary methyl at d H 1.13, d C 25.5, suggesting that compound 4 was the lack of one of the olefinic moiety and appearance of a hydroxyl group.In the HMBC spectrum, correlation from tertiary methyl at d H 1.13 and sp 3 methine at d H 1.69 to sp 3 oxygenated carbon at d C 75.5, were used to determine the C-20 position of the tertiary hydroxy group.A remaining olefine was positioned at C-24/C-25 that was determined based on the correlation of an olefinic proton at d H 5.10 and tertiary methyl at d H 1.66 to sp 2 tertiary carbon at C-25 (d C 131.7).The NMR spectra data of 4 were superimposed with those of dammar-24-en-3b, 20-diol 25,26 , therefore, compound 4 was characterized as a dammar-24-en-3b,20-diol.The cytotoxic properties of compounds 1-4 were tested towards P-388 murine leukemia cells using a modified method priory described. 18,20,26rtonin E was used as a positive control with IC 50 0.68 µM 27 .The results of cytotoxic assay of isolated compounds 1-4 (Table 3), showed that dammar-24en-3 b,20-diol (4), having two hydroxy groups and one double bond, has the strongest activity among all, whereas dammardienon (1) has the weakest activity, suggesting that the presence of hydroxyl and double bond moieties are significant structural element for the cytotoxic activity.

CONCLUSION
Four dammarane-type triterpenoids, 1-4, were isolated from the bark of A. glabrata, for the first time in this species.These findings support the presence of dammarane triterpenoid in the Aglaia genus.In dammarane-type triterpenoids, hydroxyl and double bond groups are important requirements for the cytotoxic properties.
H 5.11 (H-24) to d C 132.0 (C-25) and d C 28.0 (C-23) and at d H 1.68 (Me-26) and d H 1.61 (Me-27) to d C 132.0 (C-25) strongly supported that double bond was positioned at C-24/C-25.A double bond at terminal C-20/C-21 was deduced from correlations between two protons at d H 4.71 and 4.75 and C-20 (d C 151.4), d C 35.1 (C-22) and d C 48.1 (C-17).Correlations of Me-28 (d H 1.05), 1.92 (H-2a) and 2.40 (H-2b) to carbonyl signal at d C 217.6, indicated the position of carbonyl ketone at C-3. NMR data of 1 were superimposed with those of dammardienon 23 , consequently, compound 1 was determined as a dammardienon.

Fig. 2 .
Fig. 1.The structures of compound 1-4 The main differences are the presence of newly double bond at d H 5.64 and 5.70 and one signal of oxygenated carbon at d C 70.7 instead of a proton signal of olefin at d H 5.11, d C 125.1, showing a double bond in new position and a hydroxyl group in 2. HMBC correlations of olefinic protons at d H 5.64 to d C 37.2 (C-22) and d C 125.6 (C-23) and d H 5.70 to d C 125.6 (C-23) and d C 70.7 (C-25) were used to determine the newly double bond (23,24).Additionally, correlations from two methyls at Me-26 (d C 28.4) and Me-27 (d C 28.4) to an oxygenated carbon (d C 70.7) were used to determine the newly hydroxyl group at C-25.NMR data of 2 were superimposed with those of aglaiabbreviatin E 17 , which strongly recommended that compound 2 was aglaiabbreviatin E.
4) at C-3 and the presence of an oxymethine (d C 76.5), which were confirmed by HMBC analysis through correlations of CH 2 -2 (d H 1.86 and 2.01) and two methyl signals (d H 1.05) to C-3 (d C 76.5).Another difference is the presence of an oxygenated sp 3 methines at d H 4.20 and d C 70.5.A proton signal of oxygenated methine at d H 4.20 was correlated to C-23 (d C 25.9) and quaternary sp 2 carbon at C-25 (d C 141.6), suggesting that a secondary hydroxyl group was located at C-24.Another double bond was assigned to be at position C-25/C-26 based on the correlation of terminal double bond at d H 5.10 and 5.20 as well as a methyl at C-27 (d H 1.70) to sp 2 quaternary carbon at C-25 (d C 141.6