Cytotoxic Polyprenylated Benzoylphloroglucinol Derivatives from the Branches of Garcinia schomburgkiana

Abstract

Five undescribed polyprenylated benzoylphloroglucinol derivatives (1 – 5), named garschomcinols A – E, and five known analogues (6 – 10) were isolated from the branches of Garcinia schomburgkiana. Their structures were determined on the basis of 1D and 2D NMR and HRESIMS analyses. The absolute configuration of the bicyclo [3.3.1]nonane core structure of the polyprenylated benzoylphloroglucinols was assigned by comparison of its experimental electronic circular dichroism data with that of related compounds. All isolated compounds were evaluated for their cytotoxicity in vitro against five cancer cell lines. Compound 6 showed potent cytotoxicity against five cancer cell lines including KB, HeLa S3, HT-29, MCF-7, and Hep G2 with IC₅₀ values in the range of 5.05 – 7.03 µM.

Key words

Garcinia schomburgkiana - Clusiaceae - polyprenylated benzoylphloroglucinols - garschomcinols A – E - cytotoxicity

Introduction

The genus Garcinia, belonging to the family Clusiaceae, has been widely studied for their chemical constituents and biological activities and comprises about 20 species in Thailand [1], [2], [3], [4], [5]. Garcinia schomburgkiana Pierre, locally named “Ma dan” in Thai, is an edible plant in Southeast Asia [6]. In Thai folk medicine, its roots, leaves, and fruits are used for the treatment of cough, menstrual disturbances, and diabetes as well as an expectorant and laxative [7]. Previous phytochemical studies of G. schomburgkiana showed the presence of xanthones, phloroglucinols, depsidones, biphenyls, flavonoids, and triterpenoids, some of which exhibited cytotoxic and antimalarial activity [8], [9], [10], [11], [12]. Herein, we report the isolation and structural elucidation of five undescribed polyprenylated benzoylphloroglucinol derivatives, named garschomcinols A – E, and five known analogues from the branches of G. schomburgkiana. The structures of the isolated compounds were determined by spectroscopic analysis, especially 1D and 2D NMR spectroscopy, and comparison with literature data. The configuration of the bicyclo [3.3.1]nonane core structure of the polyprenylated benzoylphloroglucinols was assigned by comparison of the experimental electronic circular dichroism (ECD) data with those of related compounds. The cytotoxicity in vitro of all isolated compounds against five cancer cell lines (KB, HeLa S3, HT-29, MCF-7, and Hep G2) was evaluated by the MTT colorimetric method.

Results and Discussion

Phytochemical investigation of the CH₂Cl₂ crude extract from the branches of G. schomburgkiana led to the isolation of five undescribed polyprenylated benzoylphloroglucinol derivatives, named garschomcinols A – E (1 – 5), and five known analogues (6 – 10) ([Fig. 1]), including oblongifolin C (6) [13], guttiferone K (7) [14], garciyunnanin B (8) [15], oxyguttiferone K (9) [16], and oblongifolin G (10) [17]. The chemical structures of the known compounds were confirmed by NMR spectroscopic data and comparison with previously published data.

Fig. 1 Chemical structures of 1–10.

Garschomcinol A (1) was obtained as a yellow gum [α]_D ²⁰ + 12.5 (c 0.20, MeOH). Its molecular formula was determined as C₄₃H₆₀O₇ from the [M + Na]⁺ ion peak at m/z 711.4233 (calcd. for C₄₃H₆₀O₇Na, 711.4237) in positive HRESIMS. UV absorptions maxima at λ _max 242, 257, and 324 nm revealed aromatic and conjugated carbonyl chromophores. The IR spectrum showed absorption bands at 3425 cm⁻¹ (hydroxyl groups) and 1720 and 1665 cm⁻¹ (carbonyl groups). The ¹H NMR data of 1 ([Table 1]) exhibited signals for a 1,2,4-trisubstituted benzene ring at δ _H 6.69 (1H, d, J = 8.3 Hz, H-15), 6.95 (1H, dd, J = 1.4, 8.3 Hz, H-16), and 7.19 (1H, br s, H-12), a tertiary methyl proton at δ _H 0.81 (3H, s, H-22), a methylene group at δ _H 1.44 and 2.05 (2H, H-7), a methine proton at δ _H 1.74 (1H, m, H-6), and signals attributed to a 2,6-dimethyloct-6-en-2-ol and three 3-methylbut-2-enyl groups. The ¹³C NMR data showed resonances for six aromatic carbons, a conjugated carbonyl group at δ _C 196.5 (C-10), an enolized 1,3-diketone at δ _C 119.2 (C-2), 191.6 (C-3), and 194.8 (C-1), a nonconjugated carbonyl at δ _C 208.9 (C-9), three quaternary carbons at δ _C 51.4 (C-5), 64.0 (C-8), and 69.3 (C-4), a methyl at δ _C 16.2 (C-22), a methylene at δ _C 43.0 (C-7), a methine at δ _C 42.1 (C-6), and 25 signals assignable to five isoprene units. The ¹H and ¹³C NMR spectroscopic data ([Table 1]) of 1 were similar to those of oblongifolin C (6) except for the geranyl group in 6, in which the second double bond was hydrated to a 2,6-dimethyloct-6-en-2-ol group. The structure of the side chain was confirmed by the presence of an oxygenated carbon in the ¹³C NMR spectrum at δ _C 71.4 (C-41), the COSY correlations of H-24/H-25, H-27/H-39 and H-39/H-40, and the HMBC correlations of H-25 with C-6, C-26, C-27, and C-28, H-42 and H-43 with C-40 and C-41, and H-39 with C-26, C-27, C-40, and C-41 ([Fig. 2]). The relative configuration of 1 was determined using ¹H-¹H coupling constants and NOESY correlations. The coupling constant J = 12.8 Hz of H-6/H-7ax and the NOESY interactions between H-7ax/H-24, H-7ax/H-29, H-22/H-17, H-22/H-24, and H-25/H-27 suggested an axial orientation of H-6 and H-22, an equatorial orientation of H-17, H-24, and H-29, and an E-configuration of the Δ ^25,26 double bond ([Fig. 3]). The ¹³C NMR data at δ _C 42.1 supported an equatorial orientation of the C-6 substituent since the C-6 resonance with an axial substituent is reportedly observed at δ _C 46 – 48 [16]. Thus, the structure of 1 was assigned as shown in [Fig. 1].

Table 1 ¹H (400 MHz) and ¹³C (100 MHz) NMR data of compounds 1 – 3 in CD₃OD.

Position	1		2		3
	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C
1		194.8		194.5		194.7
2		119.2		119.3		119.3
3		191.6		191.4		191.4
4		69.3		69.4		69.4
5		51.4		51.5		51.5
6	1.74, m	42.1	1.78, m	42.0	1.79, m	42.0
7eq	2.05, m	43.0	2.07, m	43.1	2.09, m	43.1
7ax	1.44, t (12.8)		1.47, t (12.6)		1.47, t (13.0)
8		64.0		63.9		63.9
9		208.9		208.9		208.9
10		196.5		196.5		196.5
11		130.0		130.0		130.0
12	7.19, br s	117.4	7.21, d (1.8)	117.4	7.21, d (1.5)	117.4
13		146.1		146.1		146.2
14		152.3		152.3		152.3
15	6.69, d (8.3)	115.0	6.70, d (8.3)	115.1	6.71, d (8.3)	115.1
16	6.95, dd (1.4, 8.3)	124.9	6.97, dd (1.9, 8.3)	124.9	6.97, dd (1.5, 8.3)	124.9
17	2.70, m	26.6	2.71, m	26.6	2.72, m	26.6
18	4.87, br s	121.3	4.86, br s	121.3	4.87, br s	121.3
19		134.8		134.9		134.9
20	1.62, s	26.3	1.64, s	26.3	1.65, s	26.3
21	1.69, s	18.4	1.71, s	18.4	1.72, s	18.4
22	0.81, s	16.2	0.83, m	16.2	0.84, m	16.2
23	1.67, m	37.4	1.68, m	37.4	1.69, m	37.4
24	1.76, m, 2.07, m	29.9	1.78, m, 2.09, m	29.9	1.78, m, 2.10, m	29.9
25	5.01, br s	123.7	5.04, m	123.9	5.04, m	123.9
26		138.1		138.0		138.0
27	1.95, m	41.1	1.99, m	40.9	2.00, m	40.9
28	1.56, s	16.4	1.57, s	16.4	1.58, s	16.4
29	2.49, m	31.6	2.51, m	31.6	2.53, m	31.6
30	5.13, br s	120.9	5.13, br s	120.9	5.14, br s	120.9
31		135.4		135.4		135.4
32	1.71, s	26.3	1.72, s	26.3	1.73, s	26.3
33	1.66, s	18.3	1.68, s	18.3	1.69, s	18.3
34	1.97, m	25.2	1.99, m	25.2	1.99, m	25.2
35	5.06, br s	125.5	5.07, m	125.5	5.08, m	125.5
36		132.3		132.4		132.4
37	1.66, s	26.0	1.68, s	25.9	1.69, s	25.9
38	1.59, s	18.0	1.61, s	18.0	1.62, s	18.0
39	1.44, m	23.3	1.39, m	22.7	1.40, m	22.7
40	1.34, m	44.1	1.39, m	39.6	1.40, m	40.0
41		71.4		76.3		76.0
42	1.12, s	29.2	1.11, s	25.5	1.13, s	26.2
43	1.14, s	29.2	1.12, s	25.5	1.13, s	26.2
44			3.13, s	49.4	3.36, m	57.6
45					1.11, m	16.4

Fig. 2 Key HMBC (arrow curves) and COSY (bold lines) correlations of 1–5.

Fig. 3 Key NOESY correlations of 1.

Garschomcinol B (2) was obtained as a yellow gum with [α]_D ²⁰ + 13.5 (c 0.28, MeOH). Its molecular formula of C₄₄H₆₂O₇ was established by the positive HRESIMS [M + Na]⁺ ion peak at m/z 725.4382 (calcd. for C₄₄H₆₂O₇Na, 725.4393). The NMR data ([Table 1]) of 2 and 1 were nearly identical except for the side chain of 2, which showed a signal for a methoxy group at C-41. The presence of the methoxy group was confirmed by the ¹³C NMR resonance at δ _C 49.4 (C-44) and the HMBC correlation of the methoxy protons at δ _H 3.13 (3H, s, H-44) with C-41 (δ _C 76.3) ([Fig. 2]). Finally, the structure of 2 was determined as shown in [Fig. 1].

Garschomcinol C (3) was obtained as a yellow gum with [α]_D ²⁰ + 14.7 (c 0.34, MeOH). Its molecular formula was determined to be C₄₅H₆₄O₇ by HRESIMS ([M + Na]⁺ m/z 739.4524, calcd. for C₄₅H₆₄O₇Na, 739.4550). According to the NMR analysis ([Table 1]), compound 3 possessed the same structure as 2, except that the methoxy group of the side chain was replaced by an ethoxy group. The ¹H and ¹³C NMR spectra showed protons at δ _H 1.11 (3H, m, H-45) and 3.36 (2H, m, H-44), which were correlated in the HSQC spectrum with carbons at δ _C 16.4 and 57.6, respectively. The HMBC spectrum showed cross-peaks between H-44 and C-41 (δ _C 76.0) and C-45 ([Fig. 2]), indicating that the ethoxy group was attached at C-41 in the side chain. Thus, the structure of 3 was characterized as shown in [Fig. 1].

Garschomcinol D (4) was obtained as a yellow gum with [α]_D ²⁰ + 13.1 (c 0.30, MeOH). Its molecular formula of C₃₆H₅₄O₃ was suggested by the positive HRESIMS [M + K]⁺ ion peak at m/z 573.3786 (calcd. for C₃₆H₅₄O₃K, 573.3710). The IR spectrum displayed bands at 1719, 1653, and 1642 cm⁻¹ for carbonyl groups. The ¹H NMR data of 4 ([Table 2]) exhibited signals for two methine protons at δ _H 2.02 (1H, m, H-6) and 5.99 (1H, s, H-2), a methylene group at δ _H 1.35 and 1.93 (2H, H-7), the tertiary methyl protons at δ _H 0.68 (3H, s, H-15), and signals attributed to a 2.2-dimethylpyran ring, a 3,7-dimethylocta-2,6-dienyl, and two 3-methylbut-2-enyl groups. The ¹³C NMR data showed resonances for an enolized 1,3-diketone at δ _C 120.2 (C-2), 175.2 (C-3), and 198.7 (C-1), a non-conjugated carbonyl at δ _C 208.1 (C-9), three quaternary carbons at δ _C 47.4 (C-5), 62.6 (C-4), and 63.2 (C-8), a methyl at δ _C 17.1 (C-15), a methylene at δ _C 39.6 (C-7), a methine at δ _C 36.3 (C-6), and 25 signals assignable to five isoprene units. The NMR data of 4 showed close similarity to those of 6 except for the absence of a benzoyl group at C-2 in 4, which was confirmed by the HMBC correlations ([Fig. 2]) from H-2 to C-1, C-3, C-4, and C-8. In addition, the prenyl group at C-4 in 6 was cyclized to build a pyran ring, which was determined by the HMBC correlations from H-10 [δ _H 1.71, 2.52 (2H, m)] to C-3, C-4, and C-12 (δ _C 83.5), and from H-11 [δ _H 1.27, 1.71 (2H, m)] to C-4, C-12, C-13 (δ _C 29.5), and C-14 (δ _C 26.2). The relative configuration of 4 was assigned using ¹H-¹H coupling constants and NOESY correlations as in 1. The coupling constant J = 13.1 Hz of H-6/H-7ax and the interactions in the NOESY spectrum between H-7ax/H-17, H-7ax/H-22, H-15/H-10, H-15/H-17, and H-18/H-20 suggested an axial orientation of H-6 and H-15, an equatorial orientation of H-10, H-17, and H-22, and the E-configuration of the Δ ^18,19 double bond. Thus, the structure of 4 was assigned as shown in [Fig. 1].

Table 2 ¹H (400 MHz) and ¹³C (100 MHz) NMR data of compounds 4 and 5 in CDCl₃.

Position	4		5
	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C
1		198.7		198.9
2	5.99, s	120.2	5.95, s	119.9
3		175.2		175.4
4		62.6		62.5
5		47.4		47.6
6	2.02, m	36.3	1.87, m	36.0
7eq	1.93, m	39.6	1.92, m	39.5
7ax	1.35, t (13.1)		1.34, t (13.0)
8		63.2		62.8
9		208.1		207.7
10	1.71, m, 2.52, m	18.1	2.47, m	18.0
11	1.27, m, 1.71, m	33.7	1.21, m, 1.67, m	33.5
12		83.5		83.5
13	1.23, s	29.5	1.20, s	29.5
14	1.42, s	26.2	1.39, s	26.0
15	0.68, s	17.1	0.61, s	17.1
16	1.39, m, 1.63, m	37.0	1.31, m, 1.56, m	36.8
17	1.69, m, 1.99, m	29.1	0.89, m, 1.36, m	28.1
18	5.05, m	124.2	1.26, m, 1.73, m	32.2
19		137.1	2.33, m	39.9
20	1.96, m	39.9		181.5
21	1.54, s	16.4	1.13, d (7.0)	17.3
22	2.42, d (6.7)	30.1	2.40, m	30.1
23	4.93, m	120.2	4.86, t (6.6)	119.7
24		133.6		133.7
25	1.69, s	26.2	1.62, s	25.8
26	1.66, s	18.1	1.62, s	18.0
27	1.85, m, 2.04, m	23.0	1.73, m, 1.95, m	23.0
28	4.96, m	124.4	4.92, t (6.1)	124.2
29		131.6		131.5
30	1.66, s	25.9	1.59, s	25.9
31	1.57, s	18.0	1.53, s	17.9
32	2.04, m	26.8
33	5.00, m	122.5
34		133.4
35	1.63, s	26.0
36	1.59, s	18.1

Garschomcinol E (5) was obtained as a yellow gum with [α]_D ²⁰ + 12.5 (c 0.20, MeOH). Its molecular formula was determined as C₃₁H₄₆O₅ from the positive HRESIMS [M + Na]⁺ ion peak at m/z 521.3233 (calcd. for C₃₁H₄₆O₅Na, 521.3243). The NMR data ([Table 2]) of 5 were similar with those of 4 except for the geranyl group at C-6, which was replaced by a 2-methylbutanoic acid group in 5. The structure of the side chain was confirmed by the presence of a carboxylic acid carbon at δ _C 181.5 (C-20) in the ¹³C NMR spectrum, the COSY correlations of H-17/H-18, H-18/H-19 and H-19/H-21, and the HMBC correlations ([Fig. 2]) of H-17 [δ _H 0.89, 1.36 (2H, m)] with C-5 (δ _C 47.6), C-7 (δ _C 39.5), and C-19 (δ _C 39.9), H-18 [δ _H 1.26, 1.73 (2H, m)] with C-6 (δ _C 36.0), C-20, and C-21 (δ _C 17.3), H-19 [δ _H 2.33 (1H, m)] with C-17 (δ _C 28.1), C-20, and C-21, and H-21 [δ _H 1.13 (3H, d, J = 7.0 Hz)] with C-18 (δ _C 32.2), C-19, and C-20. Compound 5 had the same relative configuration as 4 based on the J value and NOESY analysis, while the configuration at C-19 remains undetermined. Finally, the structure of 5 was determined as shown in [Fig. 1].

The absolute configurations of the bicyclo [3.3.1]nonane core in 1 – 5 were assigned by comparison of the experimental ECD data with data reported for related compounds. The experimental ECD curves of 1 – 3 (Fig. 6S, Supporting Information) showed similarities to those of 6 and 7 [9] (positive Cotton effect at 200 – 240 nm, negative Cotton effect at 240 – 310 nm, and positive Cotton effect at 310 – 400 nm), thereby suggesting the 4S, 5S, 6R, 8S absolute configuration of 1 – 3. The experimental ECD curves of 4 and 5 (Fig. 6S, Supporting Information) showed a similar pattern to those of 32-hydroxy-ent-guttiferone M [18] (two negative high-amplitude Cotton effects at 254 and 320 nm, along with a positive Cotton effect at 215 nm), which revealed the 4R, 5S, 6R, 8S absolute configuration of 4 and 5.

Most isolated compounds showed potent cytotoxicity against cancer cell lines, while no cytotoxicity was found for 4 and 5 (IC₅₀ > 100 µM) ([Table 3]). It is worth noting that compound 6 showed cytotoxicity against all five cancer cell lines including KB, HeLa S3, HT-29, MCF-7, and Hep G2 with IC₅₀ values in the range of 5.05 – 7.03 µM. Compounds 1 and 7 exhibited cytotoxicity against four cell lines (KB, HeLa S3, HT-29, and MCF-7) with IC₅₀ values in the range of 4.06 – 7.89 µM. Compounds 2 and 3 were cytotoxic against three cell lines (KB, HeLa S3, and MCF-7) with IC₅₀ values in the range of 6.24 – 9.37 µM. Compound 10 was cytotoxic against KB and HeLa S3 cells with IC₅₀ values of 2.52 and 6.39 µM, respectively. In addition, compounds 8 and 9 showed significant cytotoxic effects against KB cells with IC₅₀ values of 5.70 and 5.03 µM, respectively. These data suggest that the presence of a 3,4-dihydroxybenzoyl group at C-2 might improve the cytotoxicity of phloroglucinols.

Table 3 In vitro cytotoxicity of compounds 1 – 10 against five human cancer cell lines.

Compounds	IC50 (µM); 95% CI
	KB	HeLa S3	HT-29	MCF-7	Hep G2
NT = not tested; adoxorubicin was used as the positive control
1	5.93; 5.36 – 6.49	5.33; 5.15 – 5.50	7.89; 7.28 – 8.50	5.42; 5.00 – 5.84	12.03; 11.39 – 12.66
2	7.13; 6.06 – 8.20	6.24; 4.6#7 – 7.81	12.73; 6.58 – 18.87	6.72; 6.03 – 7.41	14.39; 13.98 – 14.79
3	9.37; 7.33 – 11.41	6.65; 5.80 – 7.49	14.13; 10.64 – 17.63	6.50; 6.30 – 6.70	14.12; 11.96 – 11.28
4	> 100	> 100	NT	NT	NT
5	> 100	> 100	NT	NT	NT
6	5.38; 5.10 – 5.65	5.22; 3.85 – 6.60	5.05; 4.77 – 5.33	5.90; 4.33 – 7.47	7.03; 6.20 – 7.86
7	6.39; 5.91 – 6.88	5.48; 5.45 – 5.51	6.29; 4.63 – 7.95	4.06; 2.90 – 5.22	11.46; 9.34 – 13.58
8	5.70; 4.79 – 6.61	16.09; 15.95 – 16.24	20.80; 20.18 – 21.42	18.13; 14.47 – 21.49	> 100
9	5.03; 4.92 – 5.15	11.05; 10.35 – 11.74	19.17; 18.19 – 20.14	18.29; 9.13 – 27.44	> 100
10	2.52; 2.42 – 2.63	6.39; 5.44 – 7.34	13.48; 9.45 – 17.51	12.12; 2.57 – 21.67	11.20; 9.40 – 13.01
Doxorubicina	0.02; 0.00 – 0.02	0.15; 0.11 – 0.19	0.59; 0.51 – 0.67	1.29; 1.25 – 1.34	1.00; 0.57 – 1.43

Material and Methods

General experimental procedures

UV-visible absorption spectra were recorded on a UV-2550 UV-vis spectrometer. IR spectra were measured on a Nicolet 6700 FT-IR spectrometer using KBr discs. Optical rotations were measured with a Jasco P-1010 polarimeter. NMR spectra were recorded on a Bruker 400 AVANCE spectrometer (400 MHz for ¹H and 100 MHz for ¹³C). The HRESIMS were obtained using a Bruker MICROTOF model mass spectrometer. ECD data were recorded on a JASCO J-815 spectropolarimeter. Silica gel 60 G, silica gel 70 – 230 mesh, silica gel RP-C18 40 – 63 µm, and Sephadex LH-20 (all Merck) were used for column chromatography.

Plant material

Branches of G. schomburgkiana were collected in January 2020 from Pho Si Suwan district, Sisaket province, Thailand (15°16′55″ N 104°01′40″ E). The plant material was identified by Dr. Suttira Sedlak, botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University. A voucher specimen (Khumkratok no. 92 – 08) was deposited at Mahasarakham University.

Extraction and isolation

The air-dried branches of G. schomburgkiana (10.0 kg) were ground and then macerated with CH₂Cl₂ over a period of 5 days at room temperature with 2 × 15 L. Removal of the solvent under reduced pressure provided the CH₂Cl₂ crude extract (120.0 g), which was further separated by column chromatography (45 × 10 cm, i. d.) over silica gel with a gradient of n-hexane-EtOAc (1 : 0, 8 : 2, 6 : 4, 4 : 6, 2 : 8, each 5 L) to give 12 fractions (A – L). Fraction C (1.5 g) was purified by a silica gel RP-C18 column (55 × 3 cm, i. d.) with H₂O-MeOH (2 : 8, 1 L) to afford compound 4 (6.5 mg). Fraction D (6.2 g) was applied to a Sephadex LH-20 column (75 × 5 cm, i. d.) with CH₂Cl₂-MeOH (8 : 2, 2 L) and further purified by a silica gel RP-C18 column (55 × 3 cm, i. d.) with H₂O-MeOH (2 : 8, 1 L) to obtain compounds 8 (8.5 mg), 9 (4.2 mg), and 10 (6.0 mg). Compounds 3 (10.8 mg), 6 (15.5 mg), and 7 (16.2 mg) were obtained from fraction E (10.5 g) by chromatography on a Sephadex LH-20 column (75 × 5 cm, i. d.) with CH₂Cl₂-MeOH (8 : 2, 2 L) followed by a silica gel RP-C18 column (55 × 3 cm, i. d.) with H₂O-MeOH (2 : 8, 1 L). Compound 2 (12.5 mg) was isolated from fraction F (3.0 g) using a silica gel RP-C18 column (55 × 3 cm, i. d.) with H₂O-MeOH (2 : 8, 1 L). Finally, Fraction G (3.5 g) was subjected to a Sephadex LH-20 column (75 × 5 cm, i. d.) using CH₂Cl₂-MeOH (8 : 2, 2 L) to provide compounds 1 (15.5 mg) and 5 (7.5 mg).

Garschomcinol A (1): yellow gum; [α]_D ²⁰ + 12.5 (c 0.20, MeOH); UV (MeOH) λ _max (log ε) 324 (0.2), 257 (0.5), and 242 (0.5) nm.; IR (KBr) ν _max 3425, 1720, 1665 cm⁻¹; ECD (c 0.05, MeOH) λ _max (Δε) 330 (+ 4.9), 250 (− 21.0), 220 (+ 25.2) nm.; ¹H and ¹³C NMR data, see [Table 1]; HRESIMS (positive ion mode) m/z 711.4233 [M + Na]⁺ (calcd. for C₄₃H₆₀O₇Na, 711.4237).

Garschomcinol B (2): yellow gum; [α]_D ²⁰ + 13.5 (c 0.28, MeOH); UV (MeOH) λ _max (log ε) 326 (0.3), 253 (0.4), and 240 (0.5) nm.; IR (KBr) ν _max 3428, 1728, 1645 cm⁻¹; ECD (c 0.05, MeOH) λ _max (Δε) 332 (+ 5.7), 252 (− 32.2), 221 (+ 38.0) nm.; ¹H and ¹³C NMR data, see [Table 1]; HRESIMS (positive ion mode) m/z 725.4382 [M + Na]⁺ (calcd. for C₄₄H₆₂O₇Na, 725.4393).

Garschomcinol C (3): yellow gum; [α]_D ²⁰ + 14.7 (c 0.34, MeOH); UV (MeOH) λ _max (log ε) 318 (0.1), 260 (0.3), and 238 (0.2) nm.; IR (KBr) ν _max 3423, 1727, 1647 cm⁻¹; ECD (c 0.05, MeOH) λ _max (Δε) 331 (+ 5.2), 250 (− 25.8), 220 (+ 30.3) nm.; ¹H and ¹³C NMR data, see [Table 1]; HRESIMS (positive ion mode) m/z 739.4524 [M + Na]⁺ (calcd. for C₄₅H₆₄O₇Na, 739.4550).

Garschomcinol D (4): yellow gum; [α]_D ²⁰ + 13.1 (c 0.30, MeOH); UV (MeOH) λ _max (log ε) 328 (0.3), 259 (0.4), and 246 (0.2) nm.; IR (KBr) ν _max 1719, 1653, 1642 cm⁻¹; ECD (c 0.05, MeOH) λ _max (Δε) 316 (− 18.7), 254 (− 39.7), 212 (+ 49.9) nm.; ¹H and ¹³C NMR data, see [Table 2]; HRESIMS (positive ion mode) m/z 573.3786 [M + K]⁺ (calcd. for C₃₆H₅₄O₃K, 573.3710).

Garschomcinol E (5): yellow gum; [α]_D ²⁰ + 12.5 (c 0.20, MeOH); UV (MeOH) λ _max (log ε) 318 (0.2), 245 (0.2), and 238 (0.1) nm.; IR (KBr) ν _max 1725, 1648, 1635 cm⁻¹; ECD (c 0.05, MeOH) λ _max (Δε) 316 (− 22.7), 253 (− 52.5), 208 (+ 72.2) nm.; ¹H and ¹³C NMR data, see [Table 2]; HRESIMS (positive ion mode) m/z 521.3233 [M + Na]⁺ (calcd. for C₃₁H₄₆O₅Na, 521.3243).

Cytotoxicity assay

The cytotoxicity of compounds 1 – 10 was evaluated using the MTT colorimetric method against KB, HeLa S3, HT-29, MCF-7, and Hep G2 cell lines as previously reported [19], with doxorubicin as the positive control. The cancer cells were cultured in 100 µL/well of MEM containing 10% fetal bovine serum and 1% streptomycin-penicillin, seeded in a 96-well plate (3000 cells/well), and preincubated in a 5% CO₂ incubator at 37 °C for 24 h. Various concentrations of the sample, DMSO as the negative control, and the positive control (10 µL/well) were added, and then incubated for 72 h under the above conditions. The supernatant was removed and 100 µL of MTT solution (0.5 mg/mL) were added into each well and further incubated for 3 h. The supernatant was decanted and DMSO (100 µL/well) was added to dissolve Formosan, which was measured at 550 nm by a microplate reader. The tests were performed in triplicate. The IC₅₀ value was calculated by curve fitting with SigmaPlot 10 (Systat Software Inc.) and the 95% confidence interval for the mean values was identified by using IBM SPSS Amos 19 (SPSS Inc.).

Contributorsʼ Statement

Design of the work: Kaennakam, Sutin; critical revision of the manuscript: Tip-pyang, Santi and Sukandar, Edwin Risky; activity test: Siripong, Pongpun and Rassamee, Kitiya.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors are grateful to the Department of Agro-Industrial, Food, and Environmental Technology, Faculty of Applied Science, King Mongkutʼs University of Technology North Bangkok (KMUTNB). We also thank Dr. Suttira Khumkratok, Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham, Thailand, for identification and deposition of the plant material.

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Publication History

Received: 16 January 2022

Accepted after revision: 02 May 2022

Accepted Manuscript online:
02 May 2022

Article published online:
28 July 2022