Phenolic compounds from Trigonostemon honbaensis and their cytotoxic activity

Cite this paper: Vietnam J. Chem., 2020, 58(6), 759-764 Article DOI: 10.1002/vjch.202000068 759 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Phenolic compounds from Trigonostemon honbaensis and their cytotoxic activity Ninh Khac Ban 1,2 , Luu Hong Truong 3 , Tran My Linh 1 , Nguyen Chi Mai 1 , Duong Thi Hai Yen 1 , Vu Van Doan 1 , Nguyen Xuan Nhiem 1,2 , Bui Huu Tai 1,2* and Phan Van Kiem 1,2* 1 Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 2 Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 3 Southern Institute of Ecology, VAST, 01 Mac Dinh Chi Street, Ho Chi Minh City, 70000, Viet Nam Received April 30, 2020; Accepted July 15, 2020 Abstract Seven phenolic compounds including (±)-erythro-1-(3,4,5-trimethoxyphenyl)propane-1,2-diol (1), (±)-threo-1- (3,4,5-trimethoxyphenyl)propane-1,2-diol (2), (R)-3-3-(3,4-dimethoxyphenyl)propane-1,2-diol (3), (±)-3-(3,4,5- trimethoxyphenyl)propane-1,2-diol (4), (+)-syringaresinol (5), (7S,8R,8′R)-5,5′-dimethoxylariciresinol (6), juniperoside (7) were isolated from the leaves of Trigonostemon honbaensis. Their chemical structures were determined using ESI- MS, 1D NMR, and 2D NMR spectra and in comparison with the reported literature. Compounds1- 7 were reported from Trigonostemongenus for the first time. At concentration of 30 µM, compounds 1-7 exhibited weak cytotoxic activity with cell viability percentages as low as 73.3±0.87 % and 79.5±0.23 % on CAL-27 and MDA-MB231 cell lines, respectively. Keywords. Trigonostemon honbaensis, phenolic, cytotoxic activity. 1. INTRODUCTION The plants of the Trigonostemon genus (Euphorbiaceae family) comprising about 85 species which are mainly distributed in tropical and subtropical regions of the Asia. Chemical studies on this plant genus have led to the discovery of about 200 structurally diverse compounds in the last two decades, some of which have shown promising biological activities. Of these, Vietnam is considered to be the center of diversity for this genus with 22 species. Recently, the T. honbaensis Tagane&Yahara is recorded as a new and endemic Trigonostemon species, growing at Hon Ba nature reserve of Vietnam. [1] Several parts of the plants from Trigonostemon genus have been traditionally used as folk medicines for treatment of diarrhea, asthma, and skin diseases. Since array of interesting diterpenoids, phenolics, steroids, indole and β- carboline alkaloids reviewed from Trigonostemon genus, the chemical constituent of this plant species rapidly attempted to investigate during the last decade. [2,3] Over 10 plants in this genus have been phytochemically investigated which resulted in isolation and chemical structural elucidation of over 200 compounds. [3-7] Isolated diterpenes (daphnane diterpenoid orthoesters), indole alkaloids (trigonoliimines) and β-carboline alkaloids (trigonoines and trigonostemines) were performed total synthesis because of their interesting chemical structure framework and/or valuable biological activities. [8-10] This paper reports the isolation, structural identification of seven phenolic compounds (1-7) from the leaves of T. honbaensis and their cytotoxic activity on CAL-27 and MDA- MB231 cell lines. 2. MATERIALS AND METHODS 2.1. Plant materials The leaves of Trigonostemon honbaensis Tagane & Yahara were collected at Nui Chua national park, Ninh Thuan province, Vietnam in December 2018 and identified by one of the authors, Dr. Luu Hong Truong. A voucher specimen (No. NCCT-P79) was Vietnam Journal of Chemistry Phan Van Kiem et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 760 kept at the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology. 2.2. General experimental procedures The used characterization equipment’s are the same as described in our previous work. [10] 2.3. Extraction and isolation The dried powdered leaves of Trigonostemon honbaensis (4 kg) was extracted with methanol for three times (each 10L) under sonication (each 60 min) at room temperature. After filtration, filtrated methanol was evaporated under reduced pressure to yield 450 g dark residue. This methanol residue was then suspended with distilled water (3.0 L) and partitioned in turn with dichloromethane and ethyl acetate to give dichloromethane (80.0 g), ethyl acetate (5.0 g), and water-soluble portions. The dichloromethane extract (80.0 g) was chromatographed on a silica gel column eluting with hexane/acetone (gradient from 40/1 to 0/1, v/v) to give 8 fractions as TB1A (10.0 g), TB1B (5.0 g), TB1C (3.0 g), TB1D (3.0 g), TB1E (4.0 g), TB1F (10.0 g), TB1G (0.5 g), and TB1H (2.3 g). The fraction TB1F (10.0 g) was further chromatographed on a YMC column eluting with acetone/water (1/2, v/v) to give five subfractions as TB7A (1.0 g), TB7B (1.0 g), TB7C (3.0 g), TB7D (0.5 g), TB7E (230 mg). TB7B (1.0 g) was further re-chromatographed on a silica gel column eluting with n-hexane/acetone (2/1, v/v) to give TB8A (500 mg), TB8B (200 mg), TB8C (200 mg). The TB8B was purified on HPLC column (J’sphere ODS H-80, 250mm×20mm column) eluting with 20 % acetonitrile (20 %ACN) to yield 1 (5.0 mg) and 2 (6 mg). The TB8C was purified on HPLC column (J’sphere ODS H-80, 250mm×20mm column) eluting with 18 % acetonitrile (18 % ACN) to yield 3 (8.0 mg) and 4 (8 mg). The TB7D (500 mg) was chromatographed on a silica gel column eluting with hexane/acetone (1.5/1, v/v) to give TB13A (100 mg), TB13B (100 mg), TB13C (102 mg). Compound 6 was obtained from TB13C by purifying on HPLC column (J’sphere ODS H-80, 250mm×20mm column) eluting with 30 % acetonitrile. Fraction TB7E (230 mg) was chromatographed on a silica gel column eluting with n-hexane/acetone (1/1, v/v) to give compounds 5 (28.6 mg) and 7 (15 mg). Figure 1: Chemical structures of compounds 1-7 (±)-Erythro-1-(3,4,5-trimethoxyphenyl)propane- 1,2-diol (1): Colorless powder; mp 105-106 o C, c  25D : 0.0° (c 0.1, MeOH), UV (λmax): 212, 263 nm, IR (KBr) ν (cm-1): 3250-3300, 1600, 1404, 1054; ESI-MS m/z241 [M-H] - ; C12H18O5; 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) data, see table 1. (±)-Threo-1-(3,4,5-trimethoxyphenyl)propane- 1,2-diol (2): Colorless powder; mp. 109-110 o C, Vietnam Journal of Chemistry Phenolic compounds from T. honbaensis © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 761  25D : 0.0°(c 0.1, MeOH), UV (λmax): 211, 262 nm, IR (KBr) ν (cm-1): 3240-3300, 1595, 1400, 1035; ESI-MS m/z 241 [M-H] - ; C12H18O5; 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) data, see table 1. (R)-3-3-(3,4-Dimethoxyphenyl)propane-1,2-diol (3): Colorless amorphous; mp 86-87 o C,  25D : +35° (c 0.1, MeOH), UV (λmax): 210, 265 nm, IR (KBr) ν (cm -1 ): 3200-3300, 1610, 14013, 1050; ESI-MS m/z 211 [M-H] - ; C11H16O4; 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) data, see table 2. Kmeriolor (±)-3-(3,4,5- trimethoxyphenyl)propane-1,2-diol (4): Colorlesspowder; mp 96-97 o C,  25D : 0.0° (c 0.1, MeOH), UV (λmax): 213, 258 nm, IR (KBr) ν (cm -1 ): 3220-3310, 1612, 1400, 1054; ESI-MS m/z 241[M- H] - ; C12H8O5. 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) data, see table 2. (+)-Syringaresinol (5): Colorless amorphous; mp 175-176 o C,  25D : +45° (c 0.1, MeOH), UV (λmax): 215, 260 nm, IR (KBr) ν (cm-1): 3240-3300, 1604, 1403, 1036; ESI-MS m/z 417 [M-H] - ; C22H26O10. 1 H- NMR (CD3OD, 500 MHz) and 13 C-NMR (CDCl3, CD3OD, 125 MHz) data, see table 3. (7S,8R,8′R)-5,5′-dimethoxylariciresinol (6): Colorless amorphous; mp 125-126 o C,  25D : +11° (c 0.1, MeOH),UV (λmax): 214, 265 nm, IR (KBr) ν (cm -1 ): 3240-3300, 1595, 1401, 1034; ESI-MS m/z 419 [M-H] - ; C22H28O8. 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) data, see table 3. Juniperoside (7) [9] Light yellow powder; mp 112-116 o C.  25D : -47° (c 0.1, MeOH), ESI-MS m/z 387 [M+H] + . 1 H-NMR (500 MHz, CD3OD) δ (ppm): 6.75 (2H, s, H-2, H-6), 6.65 (1H, d, J = 16.0 Hz, H-7), 6.33 (1H, ddd, J = 16.0, 7.0, 5.5 Hz, H-8), 4.34 (1H, ddd, J = 12.5, 5.5, 1.0 Hz, Ha-9), 4.53 (1H, ddd, J = 12.5, 7.0, 1.0 Hz, Hb-9), 3.87 (6H, s, 2xOCH3, H-10, H-12), 3.87 (3H, s, 11-OCH3), Glc: 4.89 (1H, d, J = 7.5 Hz, H-1′), 3.25 (1H, dd, J = 9.0, 7.5 Hz, H-2′), 3.38 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.30 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.28 (1H, m, H-5′), 3.70 (1H, dd, J = 11.5, 5.5 Hz, Ha-6′), 3.91 (1H, dd, J = 11.5, 2.0 Hz, Hb-6′). 13 C-NMR (125 MHz, CD3OD) δ (ppm): 133.7 (C-1), 105.0 (C-2, C-6), 154.6 (C-3, C-5), 139.0 (C-4), 133.7 (C-7), 126.4 (C-8), 70.7 (C-9), 56.6 (C-10, C- 12), 61.2 (C-11), Glc: 103.4 (C-1′), 75.2 (C-2′), 78.0 (C-3′), 71.7 (C-4′), 78.1 (C-5′), 62.8 (C-6′). Table 1: 1 H- and 13 C-NMR data for compounds 1 and 2 and reference compounds C 1 2 #δC a,bδC a,cδH (mult., J in Hz) @δC a,bδC a,cδH (mult., J in Hz) 1 77.3 79.1 4.45 (d, 5.0) 79.4 80.1 4.45 (d, 5.0) 2 71.3 72.4 3.86* 72.0 72.8 3.86* 3 16.9 18.4 1.15 (d, 6.5) 18.7 19.3 1.02 (d, 6.5) 1 137.0 138.2 - 137.0 138.5 - 2 104.0 105.4 6.72 (s) 104.0 105.5 6.70 (s) 3 153.0 154.2 153.0 154.3 4 137.0 139.6 137.0 139.5 5 153.0 154.2 - 153.0 154.3 - 6 104.0 105.4 6.72 (s) 104.0 105.5 6.70 (s) 3,5-OCH3 56.0 56.6 3.86 (s) 56.0 56.6 3.86 (s) 4-OCH3 60.7 61.1 3.77 (s) 60.7 61.1 3.77 (s) Measured in a)CD3OD, b)125 MHz, c)500 MHz. #δC of (±)-erythro-1-(3,4,5-trimethoxyphenyl)propane-1,2-diol in CDCl3, [4] δC of (±)-threo-1-(3,4,5-trimethoxyphenyl)propane-1,2-diol in CDCl3, [4] *Overlapped signals. 3. RESULTS AND DISCUSSION Compound 1 was obtained as a colorless amorphous powder. The 1 H NMR spectrum of 1 showed signals of two aromatic protons at δH 6.72 (2H, s), three methoxy groups at δH 3.86 (6H, s) and 3.76 (3H, s), two methine carbinol protons at δH 4.45 (d, J = 5.0 Hz) and 3.86 (m), and a doublet methyl signal at δH 1.15 (J = 6.5 Hz). These data suggested that compound 1 was a phenyl propane compounds having three methoxy groups. The 13 C NMR spectrum of 1 exhibited four signals of the 3,4,5- trimethoxyphenyl group at δC 154.2, 139.6, 138.2, 105.4, two methine carbinol carbons at δC 79.1 and Vietnam Journal of Chemistry Phan Van Kiem et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 762 72.4, one methyl group at δC 18.4, and three methoxy groups at δC 56.6 (x 2) and 61.1. The above evidence suggested that compound 1 was 1-(3,4,5- trimethoxyphenyl)propane-1,2-diol, which were further confirmed by comparing NMR data of 1 with those of (±)-erythro-1-(3,4,5- trimethoxyphenyl)propane-1,2-diol [4] and found to match (table 1). In addition, the JH-7/H-8 coupling constant was 5.0 Hz confirming that compound 1 was erythro stereoisomer at C-1 and C-2. [4] From the above evidence and ESI-MS results, compound 1 was identified as (±)-erythro-1-(3,4,5- trimethoxyphenyl)propane-1,2-diol. The NMR spectra of 2 were very similar to those of 1 suggesting that compounds 2 and 1 have the same planar structure (table 1). The ESI-MS of 2 exhibited an ion peak at m/z 242 [M-H] - corresponding to the molecular formula of C12H18O5 as 1. However, comparing the JH-7/H-8 coupling constant between these compounds found that JH-7/H-8 coupling constant of compound 2 was 7.0 Hz, much larger than that of compound 1. This evidence confirmed compound 2 was threo stereoisomer at C-1 and C- 2. [4] Consequently, compound 2 was identified as (±)-threo-1-(3,4,5-trimethoxyphenyl)propane-1,2- diol. Table 2: 1 H- and 13 C-NMR data for compounds 3 and 4 and reference compounds C 3 4 @δC a,bδC a,cδH (mult., J in Hz) #δC a,bδC a,cδH (mult., J in Hz) 1 65.7 66.6 3.47 (dd, 6.0, 11.5) 3.52 (dd, 4.5, 11.5) 66.6 66.6 3.47 (dd, 6.0, 11.5) 3.52 (dd, 4.5, 11.5) 2 72.9 74.5 3.82 (m) 73.0 74.4 3.54 (m) 3 39.2 40.5 2.64 (dd, 7.5, 13.5) 2.79 (dd, 5.5, 13.5) 40.1 41.2 2.63 (dd, 7.5, 13.5) 2.80 (dd, 5.5, 13.5) 1 130.1 133.1 - 136.4 136.4 - 2 111.1 113.2 6.89 (d, 2.0) 106.1 107.8 6.58 (s) 3 147.4 149.0 - 153.1 154.3 - 4 148.7 150.3 - 133.7 137.5 - 5 112.3 114.6 6.87 (d, 8.5) 153.1 154.3 - 6 121.0 122.8 6.80 (dd, 2.0, 8.5) 106.1 107.8 6.58 (s) 3,5- OCH3 55.8 56.6 3.84 (s) 56.0 56.6 3.84 (s) 4-OCH3 55.8 56.4 3.84 (s) 60.8 61.1 3.75 (s) Measured in a)CD3OD, b)125 MHz, c)500 MHz.@δC of (R)-3-3-(3,4-dimethoxyphenyl)propane-1,2-diolin CDCl3 [5], #δC of kmeriol in CDCl3. [6] The 1 H NMR spectrum of 3 showed three aromatic protons with ABX coupling system at δH 6.89 (d, J = 2.0 Hz), 6.80 (dd, J = 8.5, 2.0 Hz), and 6.87 (d, J = 8.5 Hz) suggesting for a 1,3,4- trisubstituted aromatic ring, two methoxy groups at δH 3.84 (3H, s) and 3.75 (3H, s), one methine carbinol proton at δH 3.84 (m), two oxygenated methylene groups at δH 3.47 (1H, dd, J = 11.5, 6.0 Hz) and 3.52 (1H, dd, J = 11.5, 4.5 Hz), and two other protons corresponding to CH2-Ph group at 2.64 (dd, J = 13.5, 7.5 Hz) and 2.79 (dd, J =13.5, 5.5 Hz). The 13 C NMR spectrum of 3 showed six aromatic carbons at δC 150.3, 149.0, 133.1, 122.8, 114.6, 113.2; two methoxy groups at δC56.6 and 56.4, and other signals at δC 74.5 (CH), 66.6 (CH2) and 40.5 (CH2). These carbon data together above proton data were found to match well with the corresponding data of 3-(3,4-dimethoxyphenyl)- propane-1,2-diol [5] (table 2), which were further confirmed by HMBC spectra. The optical rotation of 3 was +35 o (measured in CDCl3, c 1.0) suggesting for R configuration of C-2. [5] The NMR data of 4 were very similar to those of 3 suggesting that they are the same phenyl propane compounds as 3. However, compound 4 had one more methoxy group attaching to the aromatic ring [δH3.84 (6H, s)/δc 56.6 (2 x OCH3), δH3.75 (3H, s)/δc 61.1 (OCH3)]. Only four carbon aromatic signals observed at δC154.3 (C), 137.5 (C), 136.4 (C), 107.8 (CH) confirmed that three methoxy groups were at C-3, C-4, C-5 of the aromatic ring. The ESI-MS of 4 exhibited an ion peak at m/z 241 [M-H] - corresponding to the molecular formula of C12H8O5. Finally, the NMR data of 4 were compared to those of kmeriol or (±)-3-(3,4,5-trimethoxyphenyl)-1,2- propanedioland found to match well. [5] The optical rotation of 4 was almost zero suggesting that 4 was a racemic at C-2 center. Vietnam Journal of Chemistry Phenolic compounds from T. honbaensis © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 763 The 13 C NMR spectra of 5 (in CD3OD) showed ten signals including four aromatic carbon signals at δC 149.4, 136.3, 133.2, 104.6, one methoxy signal at δC 56.8, one oxygenated methine carbon at δC 87.6, one oxygenated methylene carbon at δC 72.8, and one methine carbon at δC 55.5.The 1 H NMR spectra of 5 (in CD3OD) showed one singlet aromatic signal at δH 6.67 and a methoxy singlet signal at δH 3.85 (6H) suggesting that the aromatic ring have two methoxy groups at C-3, C-5ʹ and one hydroxy group at C-4. The ESI-MS of 5 exhibited an ion peak at m/z 417 [M-H] - corresponding to the molecular formula of C22H26O10. The above evidence suggested that compound 5 has a quadratic symmetry structure as (+)-syringaresinol. [7] The NMR data of 5 (in CD3OD and also in CDCl3 as solvent measured for referenced compound) were compared to those of syringaresinol (in CDCl3) as shown in table 3 and found to match well. The NMR spectra of 6 showed two aromatic ring identified by one singlet at δH 6.65 (2H, s) and one other singlet at δH 6.53 (2H, s). Two oxygenated methylene carbons were at δC 73.6 and 60.5, one oxygenated methine was at δC 84.2, one methine and one methylene were at δC 43.8 and 34.2, respectively. The above data suggested that compound 6 was a lignan phenolic compound as (7S,8R,8′R)-5,5′-dimethoxylariciresinol.[8] The NMR data of 6 matched with those of (7S,8R,8′R)-5,5′- dimethoxylariciresinol as shown in Table 3. Furthermore, the ESI-MS spectrum of 6 exhibited an ion peak at m/z 419 [M-H] - corresponding to the molecular formula of C22H28O8. The NMR spectra of 7 confirmed the presence of one aromatic ring having a quadratic symmetry structure [δC154.6, 139.0, 133.7, 105.0/δH 6.75 (2H, s)] one trans double bond [δC133.7/δH 6.65, 126.4/δH 6.33, JH-7/H-8 = 16.0 Hz], one oxygenated methylene group [δC70.7/δH 4.34 and 4.53], and one glucopyranosyl unit identified by a typical set of carbons from C-1ʹ to C-6ʹ as δC 103.4, 75.2, 78.2, 71.8, 78.2, 62.9. The anomeric proton was at δH 4.39 with lager coupling constant of H-1ʹ/H-2ʹ (J = 7.5 Hz) confirmed the β-form of sugar linkage. Finally, all the spectroscopic data of 7 were found to match with the corresponding data of juniperoside. [9] Table 3: 1 H- and 13 C-NMR data for compounds 5 and 6 and reference compounds C 5 6 #δC d,bδC a,bδC a,cδH (mult., J, Hz) @δC a,bδC a,cδH (mult., J, Hz) 1 132.14 132.5 133.2 - 132.9 132.8 - 2 102.77 103.2 104.6 6.73 s 104.6 104.4 6.65 (s) 3 147.18 147.2 149.4 - 149.4 149.3 - 4 134.37 134.8 136.3 - 135.2 135.1 - 5 147.18 147.2 149.4 - 149.4 149.3 - 6 102.77 103.2 104.6 6.73 s 104.6 104.4 6.65 (s) 7 86.07 86.1 87.6 4.77 d (4.5) 84.3 84.2 4.78 (d, 6.5) 8 54.38 54.8 55.5 3.14 m 54.1 54.1 2.41 (m) 9 71.81 72.8 72.7 4.30 d (9.0) 3.93 dd (3.0, 9.0) 60.6 60.5 3.68 (dd, 6.5, 11.0) 3.85* 1′ 132.14 132.5 133.2 - 132.9 132.8 - 2′ 102.77 103.2 104.6 6.73 s 107.3 107.1 6.53 (s) 3′ 147.18 147.2 149.4 - 149.4 149.3 - 4′ 134.37 134.8 136.3 - 135.2 136.0 - 5′ 147.1 147.2 149.4 - 149.4 149.3 - 6′ 102.77 103.2 104.6 6.73 s 107.3 107.1 6.53 (s) 7′ 86.07 86.1 87.6 4.77 d (4.5) 73.6 73.6 3.76 (dd, 2.6, 6.0) 4.03 (dd, 6.0, 8.5) 8′ 54.38 54.8 55.5 3.14 m 43.8 43.8 2.76 (m) 9′ 71.81 72.8 72.7 4.30 d (9.0) 3.93 dd (3.0, 9.0) 34.0 34.2 2.52 (dd, 11.5, 13.0) 2.96 (dd, 5.0, 13.0) 4xOCH3 56.40 53.7 56.8 3.89 (s) 56.9 56.8 3.85 (s) Measured in a)CD3OD, b)125 MHz, c)500 MHz, d)CDCl3, #δC of syringaresinol measured in CDCl3 [7] , @δC of (7S,8R,8R)-5,5-dimethoxylariciresinol in CD3OD. [8] Vietnam Journal of Chemistry Phan Van Kiem et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 764 Table 4: Effect of compounds 1-7 (30 µM) on cell viability of CAL-27 and MDA-MB 231 cancer cell lines Compounds Cell viability (%) CAL-27 (Tongue cancer cells) MDA-MB 231 (Breast cancer cells) 1 89.3±0.98 101.3±0.26 2 102.5±0.92 83.9±0.10 3 84.2±1.10 79.5±0.23 4 81.7±0.87 81.8±0.02 5 73.3±0.87 94.6±0.01 6 85.2±1.00 93.4±0.69 7 95.3±0.88 91.2±0.64 Blank 100.0±0.67 100.0±0.90 MX 12.0±0.12 15.0±0.24 a Mitoxantrone (MX), an anticancer agent, was used as reference compound. Compounds 1-7 were reported from Trigonostemon genus for the first time. Compounds 1-7 were screened their cytotoxicity on CAL-27 and MDA-MB 231 human cancer cell lines using MTT assay. As the results, compounds 1- 7 exhibited weak cytotoxic activity on both CAL-27 and MDA-MB 231 cell lines. In the presence of compounds 1-7 (30 µM), cell viability percentages were obse