{"id":42173,"date":"2021-12-30T11:22:51","date_gmt":"2021-12-30T11:22:51","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=42173"},"modified":"2022-01-04T07:16:37","modified_gmt":"2022-01-04T07:16:37","slug":"the-anticancer-activity-of-phytoconstituents-of-the-stem-of-bouea-macrophylla","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol14no4\/the-anticancer-activity-of-phytoconstituents-of-the-stem-of-bouea-macrophylla\/","title":{"rendered":"The Anticancer Activity of Phytoconstituents of the Stem of Bouea macrophylla"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

The B<\/em>. macrophylla<\/em> (Anacardiaceae) is a high fruit-producing plant rich in antioxidant compounds. It is common in Indonesia, especially on the islands of Sumatra, Java, Kalimantan and Maluku.1, 2<\/sup> The methanol extract of B. macrophylla<\/em> fruit has antioxidant activity with an\u00a0IC50<\/sub> value of 16.29 mg\/mL.2<\/sup> The methanol extract and the fruit skin as well as the fruits of B. macrophylla<\/em> has been reported to be active as antioxidant.3-7<\/sup> The fruit of B. macrophylla<\/em> contains compounds of flavonoid class with an antioxidant activity value of IC50<\/sub> 2.43 \u03bcg\/mL,\u00a0using in vitro 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) radical scavenging activity method.1, 7<\/sup> The antioxidant activity has an important correlation with anticancer activity. Kulsum et al.<\/em> (2018) reported that the activity of antioxidants is proportionally correlated with anticancer\u00a0activity in the test of amla and ginger extract with a probability value under 0.05.8<\/sup> Their results indicated that compounds in the extract with an excellent antioxidant activity also have good anticancer activity. Gandaria has been reported to show a good antioxidant activity; thus it\u00a0makes gandaria has possibility to show a\u00a0 good anticancer activity, too.<\/p>\n

Andina and Musfirah reported that the ethanol extract of B. macrophylla<\/em> leaves has strong antioxidant activity with an IC50<\/sub> value of 55.83 \u03bcg\/mL.9<\/sup> They also demonstrated that the antioxidant activity of the ethanol extract of the stem bark of B. macrophylla<\/em> with an IC50\u00a0\u00a0<\/sub>value of 20.03 mg\/mL is greater than that of the leaf ethanol extract with an IC50 value of 55.83 mg\/mL.9 According to Rudiana et al. (2018)10 the ethyl acetate extract from gandaria stems (B. macrophylla) has the best antioxidant activity compared to n-hexane and methanol extracts\u00a0 with an IC50<\/sub> value of 4.89 \u00b5g\/mL.<\/p>\n

The seed extract of B. macrophylla<\/em> has been reported to have anticancer activity against \u00a0human hypopharyngeal FaDu (HTB-43), MCF-7 and MDA-MB-231 cells with IC50<\/sub> values \u200b\u200bof 34.36; 59.07; 28.65 \u03bcg\/mL, respectively.11<\/sup> The seed extract of B<\/em>. macrophylla<\/em> contains\u00a0 pentagalloyl glucose and ethyl gallate compounds, which can inhibit MCF-7 cells through the apoptotic pathway.11-13<\/sup> Besides that, the seed extract of B. macrophylla<\/em> can inhibit the growth of leukemia and lung cancer cells with IC50<\/sub> values \u200branging from 3 to 45 \u03bcg\/mL.11, 12<\/sup><\/p>\n

The exploration of pure phytoconstituents isolation of the stem of B. macrophylla<\/em> has not been investigated. In our previous work, two compounds, luteolin and naringenin, have been identified in the ethyl acetate extract of B. macrophylla<\/em> stem using liquid\u00a0chromatography-mass spectrometry. Still, they were not isolated.14<\/sup> The previous works on B. macrophylla<\/em> mostly focused on the chemical content of their extracts.10-14 <\/sup>\u00a0The present work aims to study the isolation of of secondary metabolites in the stem of B. macrophylla<\/em> and\u00a0determined the anticancer activity of the compounds isolated against MCF-7, HCC-1954, MDA-MB-231, and A549 cell lines.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Plant Material<\/strong><\/p>\n

The stem of B. macrophylla<\/em> was obtained from Serang District, Banten Province of Indonesia and identified at the Herbarium Bogoriense, a Center for Biological Research,\u00a0Indonesian Institute of Research, Cibinong with voucher specimen number of 1068\/IPH.1.01.\/If.07\/VI\/2018.<\/p>\n

General Experiment<\/strong><\/p>\n

Thin layer chromatography analysis was carried out using silica gel on an aluminum layer (Merck Kieselgel 60 F254<\/sub>), monitoring TLC under UV lamps 254 and 365 nm. The vacuum liquid chromatography was performed using silica gel 60 G (Merck) as the stationary\u00a0phase and silica gel 60 (Merck) in chromatography gravity column. The chemical structure of the isolates was determined using spectroscopic techniques including mass spectroscopy (Waters UPLC-MS\/MS H-Class TQD), and 1<\/sup>H- and 13<\/sup>C-NMR spectroscopy which were\u00a0obtained with JEOL ECA 500 with frequencies at 500 MHz and 125 MHz, respectively.<\/p>\n

E<\/strong>xtraction and Isolation<\/strong><\/p>\n

The stem powder of B. macrophylla<\/em> (7.6 kg) was macerated in stages with n<\/em>-hexane, ethyl acetate, and methanol (Technical, Pha Che, Indonesia) for 3 x 24 hours each using similar procedure available in the literatures.15, 16<\/sup> Each extract was tested anticancer activity against\u00a0MCF-7, HCC 1954, MDA-MB 231, and A549 cell lines. The n<\/em>-hexane extract (21 g) was separated by VLC using the mobile phase n<\/em>-hexane: ethyl acetate: methanol: acetone: methanol in a 10% polarity gradient in such a way that the A-B fraction was obtained. Fraction A was\u00a0purified by CC using n<\/em>-hexane, ethyl acetate as the stationary phase to produce compound 1 (28 mg).<\/p>\n

The ethyl acetate extract (19.90 g) was separated by VLC using methylene chloride: ethyl acetate: ethanol as the mobile phase to produce the A-K fraction. The G fraction (594.70 mg) was purified by CC using n<\/em>-hexane: MTC: ethyl acetate as the mobile phase to produce compound 2 (5.1 mg). The methanol extract (15 g) was separated by VLC using n<\/em>-hexane:\u00a0ethyl acetate: methanol in 10% gradient as the mobile phase to obtain the A-J fraction. The G fraction (778.2 mg) was purified by CC using n<\/em>-hexane: ethyl acetate as the mobile phase to produce compound 3 (4.7 mg). Fraction J (7 g) was purified by CC using n<\/em>-hexane: ethyl acetate: methanol in a polarity gradient to give compound 4 (20 mg).<\/p>\n

Anticancer<\/strong> Activity<\/strong><\/p>\n

The anticancer activity of MCF-7 breast adenocarcinima (ATCC HTB-22) and A549 Lung Carcinoma (ATCC CCL-185) cell lines were analysed using the MTS assay method which were carried out at The Biological Activity Laboratory, the Central Laboratory, Universitas Padjadjaran, Bandung, Indonesia: the cells were cultured on RPMI media (Sigma-Aldrich) containing 10% FBS and antibiotics, and trypsin-EDTA was added and incubated for 5 minutes. When the growth of cells reached confluent level where the numbers of cell lines were minimum 70%, they were then transferred to 96-microtube well plates, and each of the\u00a0microtube was added with samples of various concentrations and incubated for 48 hours. The mixture was then added with presto blue gluing as cell staining and incubated for 1-2 hours until a discoloration was observed. PrestoBlue\u00ae reagent is reduced by the blue compound\u00a0resazurin to resorufin, which is pink and very fluorescent. The absorbance measurements were carried out at 570 nm (resorufin) and 600 nm (resazurin) wavelengths using a multimode reader, and cisplatin was used as a positive control and DMSO as a negative control.17<\/sup><\/p>\n

The anticancer activity against HCC 1995 and MDA-MB 231 cell lines was analyzed using the MTT assay method which were carried out at The Culture Cell and Cytogenitics Laboratory, Medical Faculty, Universitas Padjadjaran, Bandung, Indonesia. The HCC-1954 and MDA-MB-231 cells were cultured in RPMI 1640 media (Sigma-Aldrich) containing 10% fetal calf\u00a0serum, antibiotics, and streptomycin. The cells and media were incubated for 24 hours, and then the cells were then added with samples with various concentrations and phosphate buffer saline. Furthermore, the mixture was then incubated again for 24 hours, and 3-(4,5-\u00a0Dimetiltiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT) compound was added to each well containing 15,000 cells and incubated for 2 hours. The MTT reaction was stopped using n<\/em>-hexane, and the absorbance of the reaction was measured using an ELISA reader at a wavelength of 550 nm.18<\/sup><\/p>\n

Results and Discussion<\/strong><\/p>\n

The Isolated Compounds<\/strong><\/p>\n

Four compounds were successfully isolated. The separation is guided by spot pattern. The compounds were as follows: Stigmasterol (1) (Figure 1): colourless crystal, 1<\/sup>H-NMR (in CDCl3<\/sub>, 500 MHz) \u03b4<\/em>H<\/sub> (ppm) 1.83 (2H, m<\/em>, H-1); 1.51 (2H, t<\/em>, H-2); 3.53 (1H, m<\/em>, H-3); 2.26 (2H,\u00a0d<\/em>, J<\/em>=6.5 Hz, H-4); 5.33 (1H, d<\/em>, J<\/em>=4.9, H-6); 1.97 (2H, m<\/em>, H-7); 1.47 (1H, m<\/em>, H-8); 0.95 (1H, m<\/em>, H-9); 1.50 (2H, m<\/em>, H-11); 1.97 (2H, m<\/em>, H-12); 1.05 (1H, m<\/em>, H-14); 1.52 (2H, m<\/em>, H-15); 1.23 (2H, m<\/em>, H-16); 1.15 (1H, m<\/em>, H-17); 0.66 (3H, s<\/em>, H-18); 0.99 (3H, s<\/em>, H-19); 1.97 (1H, m<\/em>, H-20);\u00a01.00 (3H, d<\/em>, J=9.7 Hz, H-21); 5.13 (1H, m<\/em>, H-22); 5.12 (1H, m<\/em>, H-23); 1.47 (1H, m<\/em>, H-24); 1.81 (1H, m<\/em>, H-25); 0.81(3H, d<\/em>, J=\u00a0 Hz, H-26); 0.82 (3H, m<\/em>, H-27); 1.15 (2H, m<\/em>, H-28) and 0.78 (1H, s<\/em>, H-29). 13<\/sup>C-NMR (in CDCl3<\/sub>, 125 MHz): \u03b4<\/em>C<\/sub>\u00a0 (ppm): 36.3 (C-1); 32.5 (C-2); 71.9\u00a0(C-3); 42.3 (C-4); 140.9 (C-5); 120.7 (C-6); 32.0 (C-7); 32.0 (C-8); 50.3 (C-9); 36.5 (C-10); 21.4 (C-11); 39.2 (C-12); 42.4 (C-13); 56.9 (C-14); 24.5 (C-15); 29.2 (C-16); 56.2 (C-17); 12.2 (C-18); 19.5 (C-19); 39.9 (C-20); 23.2 (C-21); 138.5 (C-22); 129.4 (C-23); 51.4 (C-24); 31.8\u00a0(C-25); 20.0 (C-26); 21.2 (C-27); 25.6 (C-28) and 12.2 (C-29). UPLC-QTOFMS m\/z 411.24 [M-] (calculated (calcd.) for C29<\/sub>H48<\/sub>O, m\/z 412.69).<\/p>\n

Fustin (2) (Figure 1): yellow amorphous, 1<\/sup>H-NMR (in acetone-d6<\/sub>, 500 MHz) \u03b4<\/em>H<\/sub> (ppm): 6.40 (1H, d<\/em>, J <\/em>= 2.5 Hz, H-8); 6.62 (1H, dd<\/em>, J <\/em>= 8.5 and 2 Hz, H-6); 6.86 (1H, dd<\/em>, J <\/em>= 8 and 2.5 Hz, H-5\u2019); 6,92 (1H, td<\/em>, J <\/em>= 8 and\u00a0 2 Hz, H-6\u2019); 7.07 (1H, d<\/em>, J <\/em>= 2.5 Hz, H-2\u2019); 7.72 (1H, d<\/em>, J\u00a0= 8.5 Hz, H-5); 4.98 (1H, d<\/em>, J <\/em>= 12 Hz, H-2) and 4.53 (1H, d<\/em>, J <\/em>= 11.5 Hz, H-3). 13<\/sup>C-NMR (in acetone-d6<\/sub>, 125 MHz): \u03b4C<\/sub>\u00a0 (ppm): 74.0 (C-3); 85.0 (C-2); 103.1 (C-8); 111.8 (C-6); 115.8 (C-2\u2019); 113.1 (C-10); 115.9 (C-5\u2019); 120.9 (C-6\u2019); 129.8 (C-5); 130.1 (C-1\u2019); 145.8 (C-4\u2019); 146.6\u00a0<\/em>(C-3\u2019); 164.6 (C-9); 165.8 (C-7); and 193.2 (C-4). UPLC-QTOFMS m\/z 287.28 [M–<\/sup>] (calcd. for C15<\/sub>H12<\/sub>O6<\/sub>, m\/z 288.25).<\/p>\n\n\n\n
\"Vol14No4_The_Tar_fig1\"<\/a><\/td>\nFigure<\/strong> 1: Structure of the isolated compound; Stigmasterol (1), Fustin (2), Garbanzole (3), and Methyl gallate (4)<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Garbanzole (3) (Figure 1): yellow needle crystal. 1<\/sup>H-NMR (in acetone-d6<\/sub>, 500 MHz) \u03b4<\/em>H<\/sub> (ppm): 7.70 (1H, d<\/em>, J<\/em>= x, H-5); 6.60 (1H, dd<\/em>, H-6); 6.37 (1H, d<\/em>, H-8); 7.41 (2H, d<\/em>, H-2\u2019 and 6\u2019); 6.87 (2H, d<\/em>, H-3\u2019 and H-5\u2019) 5.02 (1H, d<\/em>, H-2); and 4.45 (1H, dd<\/em>, H-3). 13<\/sup>C-NMR (in\u00a0acetone-d6<\/sub>, 125 MHz): \u03b4<\/em>C<\/sub> (ppm): 84.9 (C-2); 73.9 (C-3); 193.3 (C-4); 129.8 (C-5); 111.8 (C-6); 165,9 (C-7); 103.7 (C-8); 164.6 (C-9); 113.1 (C-10); 129.4 (C-1\u2019); 130.4 (C-2\u2019 and C-6\u2019); 115.9 (C-3\u2019 and C-5\u2019); and 158. 9 (C-4\u2019). UPLC-QTOFMS m\/z 273.0559 [M+2] (calculated\u00a0for C15<\/sub>H12<\/sub>O5<\/sub>, m\/z 272.0559).<\/p>\n

Methyl gallate (4) (Figure 1): yellow needle crystal. 1<\/sup>H-NMR (in acetone-d6<\/sub>, 500 MHz) \u03b4H<\/sub> (ppm): 3.75 ppm (3H, s,<\/em>-OCH3<\/sub>); 7.07 (2H, s<\/em>, H-2 and H-6); and 8.19 (1H, s, <\/em>-OH). 13<\/sup>C-NMR (in acetone-d6<\/sub>, 125 MHz): \u03b4C<\/sub> (ppm): 166.3 (C-7); 145.2 (C-3 and C-5); 137.9 (C-4);\u00a0120.9 (C-1); 108.9 (C-2 and C-6); and 51,1 (-OCH3<\/sub>). UPLC-QTOFMS m\/z 183.09 [M-] (calcd. for C15<\/sub>H118<\/sub>O5<\/sub>, m\/z 184.15).<\/p>\n

Compound 1 is a colorless crystal. The UPLC-QTOFMS spectrum for compound 1 has the molecular formula C29<\/sub>H48<\/sub>O, m\/z 412.69. The 1<\/sup>H-NMR spectrum of isolate 1 is similar to the reference compound reported by others19<\/sup> and it showed a typical signal for the group of\u00a0steroid compound in which the signal accumulated in the area below 2 ppm \u03b4H<\/sub> was typical for steroids. Four signals in the \u03b4<\/em>H<\/sub> 1.00 region (3H, d<\/em>, J<\/em> = 9.74 Hz); 0.81 (3H, s<\/em>); 0.82 (3H, m<\/em>); and 0.79 (1H, s<\/em>) ppm indicated the presence of a methyl signal bound to C-21, C-26, C-27 and\u00a0C-29, respectively. Furthermore, it is believed that 9 signals indicated the presence of methylene protons in the \u03b4<\/em>H<\/sub> region of 1.82 (2H, m<\/em>); 1.51 (2H, m<\/em>); 2.26 (2H, d, J<\/em> = 6.5 Hz); 1.97 (2H, m<\/em>); 1.50 (2H, m<\/em>); 1.97 (2H, m<\/em>); 1.52 (2H, m<\/em>); 1.23 (2H, m<\/em>); 1.15 (2H, m<\/em>) ppm, which\u00a0is binds to C-1, C-2, C-4, C-7, C-11, C-12, C-15, C-16 and C-28 respectively. The proton signal from methine is believed to appear at \u03b4<\/em>H<\/sub> 1.47 (1H, m<\/em>); 0.95 (1H, m<\/em>); 1.05 (1H, m<\/em>); 1.15 (1H, m<\/em>); 1, 97 (1H, m<\/em>); 1.47 (1H, m<\/em>); and 1.81 (1H, m<\/em>) ppm bound to C-8, C-9, C-14, C-17, C-20,\u00a0C-24, and C-25, respectively. There is a typical signal for olefinic protons at \u03b4<\/em>H<\/sub> 5 ppm and there is a signal for oxygenated protons at \u03b4<\/em>H<\/sub> 3 ppm, which is commonly reported in the class of the steroid compound.19, 20<\/sup> The signal was detected as an oxygenated proton at \u03b4<\/em>H<\/sub> 3.51 (1H,\u00a0m<\/em>) ppm. The signal in the region \u03b4<\/em>H<\/sub>13 (1H, d<\/em>); 5.12 (1H, m<\/em>) and 5.15 (1H, m<\/em>) ppm are from the olifinic methine double bond protons attached to C-6, C-20 and C-21, respectively.<\/p>\n

Rings A, B, and C consist of six carbon or cyclohexane atoms, and ring D consists of five or cycloheptane. Furthermore, most of the steroids have properties, which includes the oxygen functional group (as = O or OH) at C-3, and contains side groups at C-17, many of\u00a0which contain double bonds at C-4 – C-5 or C-5 – C-26. The carbon signal that appears in the area above \u03b4<\/em>C<\/sub> is 100 ppm (140.9; 120.7; 138.5; and 129.4 ppm), which are at C-5, C-6, C-22, and C, respectively. C-23 was confirmed by the presence of two double bonds in the analyzed\u00a0compound. The double bond signal is reported in the \u03b4<\/em>C<\/sub> 140.9 region; 120.7; 138.5; and 129.4 ppm. The methyl signal is believed to be in the \u03b4<\/em>C<\/sub> 12.2 (C-18) region; 19.5 (C-19); 23.2 (C-21); 20.0 (C-26); 21.2 (C-27) and 12.2 (C-29) ppm. The signal from the methylene group is\u00a0believed to be present in the region: \u03b4<\/em>c 36.3 (C-1); 32.5 (C-2); 42.3 (C-4); 32.0 (C-7); 21.4 (C-11); 39.2 (C-12); 24.5 (C-15); 29.2 (C-16) and 25.6 (C-28). In addition, the signal in the region 32.0 (C-8); 50.3 (C-9); 56.9 (C-14); 56.2 (C-17); 39.9 (C-20); 51.4 (C-24), and 31.8 (C-25)\u00a0ppm were believed to indicate the presence of a methine group.<\/p>\n

The quaternary carbon group containing 3 signals was predicted in the \u03b4<\/em>c 140.9 (C-5) region; 36.5 (C-10) and 42.4 (C-13) ppm. Figure 1 shows that the methyl proton at position C-29 \u03b4<\/em>H<\/sub> – 0.78 correlates with C-26 (\u03b4<\/em>c – 20.0) and C-28 (\u03b4<\/em>c – 25.6). The methyl proton at position\u00a0C-18 (\u03b4<\/em>H – 0.66) correlates with C-12, C-13, C-14 and C-17 with each value \u03b4<\/em>c, namely 39.2; 42.4; 56.9; and 56.2 ppm. The methyl proton at position C-19 (\u03b4<\/em>H<\/sub> – 0.99) correlates with C-1, C-5, C-9, and C-10 with values \u200b\u200bof \u03b4<\/em>c – 36.3, \u03b4<\/em>c – 141 \u03b4<\/em>c – 50.1 and \u03b4c \u2013 37. 9, respectively. The\u00a0methyl protons at position C-26 (\u03b4<\/em>H<\/sub> – 0.81) and position C-27 (\u03b4<\/em>H<\/sub> – 0.82) correlate with C-25 (\u03b4<\/em>c – 31.8). In the methyl proton \u03b4<\/em>H<\/sub> – 0.79, the C-29 position correlates with C-28 and C-24 with \u03b4<\/em>c values \u200b\u200bof 25.6 and 52.4 ppm, respectively. The position of the hydroxyl group is\u00a0strengthened and proven by the correlation at C-3 with methylene protons at positions C-2 (\u03b4<\/em>H<\/sub> -1.51) and C-4 (\u03b4<\/em>H<\/sub> – 2.26) with carbon C-3 with values \u200b\u200b(\u03b4<\/em>c – 71, 9). The position of the proton group (\u03b4<\/em>H<\/sub> – 2.26) at C-4 also strengthens the position of the hydroxyl group, which correlates\u00a0with C-3, C-5, C-2 and C-10 with the respective values \u200b\u200bof \u03b4<\/em>c 71.9, 140, 9, 32.5 and 36.5 ppm. The important HMBC correlation of 1 is shown in Figure 2.<\/p>\n\n\n\n
\"Vol14No4_The_Tar_fig2\"<\/a><\/td>\nFigure 2: HMBC <\/strong>correlation <\/strong>of stigmasterol<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Compound 2 is a yellow amorphous solid. The 6-proton aromatic signal appears at \u03b4<\/em>H<\/sub> 6.40 (1H, d, J<\/em> = 2.5 Hz) and 6.62 (1H, dd, J<\/em> = 8.5 Hz; 2 Hz), \u03b4H<\/sub> 6.86 (1H, dd, J<\/em> = 8 Hz; 2.5 Hz), 6.92 (1H, td, J<\/em> = 8 Hz; 2 Hz), and 7.07 (1H, d, J<\/em> = 2.5 Hz), 7.72 (1H, d, J<\/em> = 8.5 Hz). This\u00a0 \u00a0characterization is in agreement to reported values [21]. According to Mabry et al.<\/em>,22<\/sup> the proton signal in the range of 6 to 8 ppm with a pattern of ortho <\/em>and meta<\/em> coupling values \u200b\u200bindicated that the isolates obtained are in the flavonoid group. The proton signal in the range 6.0 to 6.7\u00a0ppm appears up to 2 signals at \u03b4<\/em>H<\/sub> 6.40 (1H, d, J<\/em> = 2.5 Hz) and 6.62 (1H, dd, J<\/em> = 8.5 Hz; 2 Hz), which indicates the characteristics of the H-8 and H-6 protons in ring A. Furthermore, one proton signal appears at a shift of 7.72 ppm (1H, d, J<\/em> = 8.5 Hz). The spectral patterns showing\u00a0the proton matchmaking characteristics of the AB system were observed at \u03b4<\/em>H<\/sub> 6.62 (1H, dd, J<\/em> = 8.5; 2 Hz) and 7.72 (1H, d, J<\/em> = 8.5 Hz) indicated the specificity for protons at H-6 and H-5. Protons H-6 and H-5 have ortho<\/em> correlation with each other. In the range of 6.7 to 7.7 ppm\u00a0there were 3 signals, namely at \u03b4<\/em>H<\/sub> 6.86 (1H, dd, J<\/em> = 8 Hz; 2.5 Hz), 6.92 (1H, td, J<\/em> = 8 Hz; 2 Hz), and 7.07 (1H, d, J<\/em> = 2.5 Hz).<\/p>\n

The proton characteristics of the ABX system are shown in this shift. The three signals indicate the proton signal for ring B with each signal representing H-5 ‘, H-6′, and H-2’ and the three signals are ortho and meta correlated with each other. The H-3 proton on the flavone\u00a0 produced a single sharp signal at a shift of about \u03b4<\/em>H<\/sub> 4.53 ppm. H-2 signal appear at a shift of approximately \u03b4<\/em>H<\/sub> 4.9 ppm. This signal appears in pairs with the H-3 proton signal that appears at a shift of about \u03b4H<\/sub> 4.2 ppm.22<\/sup> The spectrum of isolate 2 shows the flavonoid pattern of the\u00a0dihydroflavonoid type where the signal appears at \u03b4<\/em>H<\/sub> 4.98 (1H, d, J<\/em> = 12 Hz) as H-2 and \u03b4<\/em>H<\/sub> 4.53 (1H, d, J<\/em> = 11.5 Hz) as H- 3. The results of the analysis on the 13<\/sup>C-NMR spectrum showed that isolate 2<\/strong> had 15 carbon signals. The results of the DEPT 135o<\/sup> NMR analysis showed 8 signals of methine carbon (\u03b4<\/em>C<\/sub> 74.0; 85.0; 103.1; 111.8; 115.8; 115.9; 120.9; 129.8 ppm) and\u00a0there were 7 carbon signals quaternary (\u03b4<\/em>C<\/sub> 113.1; 130; 1; 145.8; 146.6; 164.6; 165.8; 193.2 ppm). The 13<\/sup>C-NMR spectrum shows a signal at a shift below 100 ppm, namely at \u03b4<\/em>C<\/sub> 85.0 and 74.0 ppm as a characteristic of saturated carbon sp3<\/sup><\/em> which binds to electronegative atoms such\u00a0as oxygen.23<\/sup> The carbon signal at 193.2 ppm shift is characterized by carbonyl carbon, which has a shift range between 185-220 ppm.23<\/sup> The 2D NMR COSY spectrum of 1<\/sup>H-1<\/sup>H correlation shows that there is a correlation between H-3 (\u03b4<\/em>H<\/sub> 4.53 ppm) with H-2 (\u03b4<\/em>H<\/sub> 5.00 ppm) and H-5\u00a0(\u03b4<\/em>H<\/sub> 7.72 ppm) with H-6 (\u03b4H<\/sub> 6.62 ppm). This confirms that the basic structure of the isolate is a flavonoid with 2 protons in ring A and ring B, each of which is correlated. The results of the 2D NMR HMQC spectrum analysis showed that there were 8 correlations between the proton\u00a0and the carbon signal. The correlation shows a direct bond between protons and carbon, namely the proton signal \u03b4<\/em>H<\/sub> 4.53 (H-3); 5.00 (H-2); 6.40 (H-8); 6.62 (H-6); 6.86 (H-5′); 6.92 (H-6′); 7,07 (H-2′); 7.72 (H-5) ppm, respectively correlated with carbon at \u03b4<\/em>C<\/sub> 74.0 (C-3); 85.0 (C-2);\u00a0103.6 (C-8); 111.8 (C-6); 115.8 (C-5′); 120.9 (C-6′); 115.9 (C-2′); and 129.8 (C-5) ppm. The important HMBC correlation of 2 was shown in Figure 3.<\/p>\n\n\n\n
\"Vol14No4_The_Tar_fig3\"<\/a><\/td>\nFigure <\/strong>3: HMBC <\/strong>correlation of fustin<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The 1<\/sup>H-NMR spectrum of isolate 3 showed that there were 5 proton signals in the aromatic region of 6-8 ppm. The isolates are believed to be a flavonoid class compound as the data obtained are close to the reported values.24, 25<\/sup> The proton signal \u03b4<\/em>H<\/sub> 6.60 ppm was assumed\u00a0to be H-6, ortho<\/em> and meta<\/em> coupling correlating with the proton signal \u03b4<\/em>H<\/sub> 7.70 (H-5) and 6.37 ppm (H-8), respectively. The proton characterization of ABX is shown in the typical shear of ring A of the flavonoid framework for H-6, H-5 and H-8. Meanwhile, the proton signal \u03b4<\/em>H <\/sub>7.41\u00a0is assumed to be H-2’\/ H-6′, which has an ortho<\/em> coupling correlating with the proton signal \u03b4H <\/sub>6.87 that has an ortho<\/em> coupling value (H-3’\/H-6′). The 13<\/sup>C-NMR and DEPT 135 analysis showed that there were 7 methine carbon signals (\u03b4<\/em>C<\/sub> 74.0; 84.9; 103.7; 111.8; 115.9; 129.8 and\u00a0\u03b4<\/em>c 130.4 ppm) and 6 quaternary carbon signals (\u03b4<\/em>C<\/sub> 113.1; 129.4; 158.9; 164.6; 165.9 and \u03b4<\/em>c 193.3 ppm). In the spectrum, a signal appears at 193.3 ppm, which indicates the type of carbonyl carbon (C = O). The 13<\/sup>C-NMR spectrum shows that there are 10 carbon signals, which\u00a0ranges from 100-167 ppm that are believed to be aromatic carbon. Furthermore, HMQC 2D spectrum shows a direct correlation between protons and carbon. The correlation signal appears up to 7 signals at \u03b4<\/em>H<\/sub> 4.55 (H-3); 5.03 (H-2); 6.37 (H-8); 6.60 (H-6); 6.87 (3’\/5′); 7.41 (2’\/6′);\u00a0and 7.70 (H-5) ppm with carbon at \u03b4<\/em>C<\/sub> signal 73.9 (C-3), respectively; 84.9 (C-2); 103.7 (C-8); 111.8 (C-6); 115.9 (C-3’\/5′); 130.4 (C-2’\/6′); and 129.8 (C-5) ppm. 2D HMBC analysis was performed, and the structure of the isolate was believed to be a flavonoid. This is reinforced by\u00a0the signal that appears at \u03b4<\/em>C<\/sub> 5.04 ppm (C-2), which correlates with \u03b4<\/em>C<\/sub> 73.9 (C-3); 193.3 (C-4); 129.4 (C-1′); 130.4 (C-2’\/6′). This signal shows the correlation between the C and B rings of flavonoids. The important HMBC correlation of 3 was shown in Figure 4.<\/p>\n\n\n\n
\"Vol14No4_The_Tar_fig4\"<\/a><\/td>\nFigure <\/strong>4: HMBC <\/strong>correlation of garbanzol<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Based on the 1<\/sup>H-NMR spectrum of compound 4, there is CH3<\/sub> (-OCH3<\/sub>), which is oxygenated at \u03b4<\/em>H<\/sub> 3.75 ppm (3H, s<\/em>). A typical aromatic signal appears at \u03b4<\/em>H<\/sub> 7.07 (2H, s<\/em>) with a symmetrical plane, hydroxy proton (-OH) appears at \u03b4<\/em>H<\/sub> 8.19 (1H, s). Seven carbon signals that are a C = O signal at \u03b4<\/em>C<\/sub> 167.9 ppm (indicating the presence of carbon ester), one signal indicates\u00a0the presence of aromatic carbon (C-OH) at \u03b4<\/em>C<\/sub> 145.2 ppm, at \u03b4<\/em>C<\/sub> 137.9 ppm indicates aromatic carbon (C-OH), \u03b4<\/em>C <\/sub>120.9 contains aromatic carbon (C-C), \u03b4<\/em>C<\/sub> 108.9 ppm contains aromatic carbon (C-H), at \u03b4<\/em>C<\/sub> 51.1 indicates the presence of \u2013O-CH3<\/sub>. These values are in agreement with\u00a0reported values available in the literature.26<\/sup><\/p>\n

Anticancer Activity of Extracts and Compounds 1-4<\/strong><\/p>\n

The anticancer activity against MCF-7 and A549 was measured using the MTS assay method, while the anticancer activity of HCC-1954 and MDA-MB 231 cells was measured\u00a0using the MTT assay method and the results of the anticancer activity test are shown on the Table 1 and the comparison of their IC50 <\/sub>values are shown in Figure 5.<\/p>\n\n\n\n
\"Vol14No4_The_Tar_fig5\"<\/a><\/td>\nFigure 5: The comparison of anticancer activity test for all compounds isolated<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 1:\u00a0Toxicity of extract and compound 1-4<\/strong><\/p>\n\n\n\n\n\n\n\n\n
No<\/strong><\/td>\nSample<\/strong><\/td>\nIC50<\/sub> (\u00b5g\/mL)<\/strong><\/td>\n<\/tr>\n
MCF-7<\/strong><\/td>\nA549<\/strong><\/td>\nHCC-1954<\/strong><\/td>\nMDA-MB-231<\/strong><\/td>\n<\/tr>\n
1<\/td>\nStigmasterol (1<\/strong>)<\/td>\n1209.25 \u00b1 47.92<\/td>\n5757.60 \u00b1 4173.23<\/td>\n853.53 \u00b1 103.87<\/td>\n553.60 \u00b1 121.35<\/td>\n<\/tr>\n
2<\/td>\nFustin (2<\/strong>)<\/td>\n377.94 \u00b1 9.17<\/td>\n359.31 \u00b1 3.58<\/td>\n134.35 \u00b1 44.62<\/td>\n5704.28 \u00b1 289.08<\/td>\n<\/tr>\n
3<\/td>\nGarbanzole (3<\/strong>)<\/td>\n248.16 \u00b1 83.12<\/td>\n568.77 \u00b1 98.13<\/td>\n427.05 \u00b1 122.71<\/td>\n233.41 \u00b1 91.57<\/td>\n<\/tr>\n
4<\/td>\nMethyl gallate (4<\/strong>)<\/td>\n351.75 \u00b1 3.80<\/td>\n388.84 \u00b1 9.75<\/td>\n153.69 \u00b1 12.54<\/td>\n311360.41 \u00b1 262865.2<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

All compounds 1 \u2013 4 were assayed for their anticancer property against MCF-7, A549, HCC-1954 and MDA-MB231 cell lines. All isolated compounds exhibited moderate anticancer activity against almost cell lines tested.\u00a0 Compounds 2 and 4 were more active on HCC-1954\u00a0cell with IC50<\/sub> values of 134.35 \u00b1 44.62 and 153.69 \u00b1 12.54 \u00b5g\/mL, respectively than other isolated compounds. It presumably that the anticancer activity increase with the absence or with the decreasing number of hydroxyl groups.27<\/sup> Additionally, among all isolated compounds,\u00a0compound 3 gave the most active in the anticancer activity test against MDA-MB-231 cell line with IC50<\/sub> value of 233.41 \u00b1 91.57 \u00b5g\/mL. While compounds 2 and 3 demonstrated respectable anticancer activity against MCF-7, A549, and HCC-1954 cell lines with IC50<\/sub> values ranging\u00a0from 134.35 \u00b1 44.62 to 568.77 \u00b1 98.13 \u00b5g\/mL. The compounds 2 and 3 are flavanones containing a chiral carbon on chroman-4-one ring unit which is flexible. According Woo et al.<\/em>, 28<\/sup> the wide range of the flavanone bioactivity may be due to its chiral structure. In addition, the\u00a0carbonyl group at C-4 on the flavan skeleton is very important for anticancer activity.27<\/sup><\/p>\n

However, the structure-activity relationship study is required to provide better understanding of their anticancer activity. The results of the anticancer test for the compounds isolated in this work are lower compared to other compounds reported by others both in the\u00a0synthetic compounds such as organotin (IV) carboxylates29, 30<\/sup> or other isolated compounds from other plants31, 32<\/sup> although the cell lines used were different. However, the results reported in this work are believed still very important results in attempts to find new candidate for\u00a0anticancer drugs.<\/p>\n

Conclusions<\/strong><\/p>\n

Four compounds namely stigmasterol (1), fustin (2), garbanzol (3), and methyl gallate (4) were successfully isolated from the stem of B. macrophylla<\/em>. These compounds were well characterized and the characterization data obtained were similar to the known compounds \u00a0previous published. These compounds were tested for their anticancer activities against 4 cell\u00a0lines. The result showed based on the IC50<\/sub> values of compounds 2 and 4 were more active on HCC-1954 cell with IC50<\/sub> values of 134.35 \u00b1 44.62 and 153.69 \u00b1 12.54 \u00b5g\/mL, respectively. The compound 3 was the most active against MDA-MB-231 cell line with IC50<\/sub> value of 233.41 \u00b1 91.57 \u00b5g\/mL.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest.<\/p>\n

Funding Sources<\/strong><\/p>\n

This research was funded by the Ministry of Research and Technology-National Agency for the Research and Innovation of the Republic of Indonesia through a higher education collaboration research scheme with the contract number 226\/SP2H\/LT\/DRPM\/2019 and No. 011\/LT-AMAND\/LP3M-UNMA\/2020.<\/p>\n

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    \n    CrossRef<\/a><\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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