Two New Abietane Diterpenes From the Stems of Clerodendrum trichotomum Thunb

Abstract

Two new abietane diterpenoids (4S, 5S, 10S)-12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-triene-19-oic acid β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (1) and (3S, 5S, 10S, 15S)-3β-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol (2), along with 5 known terpene glycosides (3–7) were isolated from the n-butanol extract of the stems of Clerodendrum trichotomum Thunb. The structures of the new compounds were determined by analysis of spectroscopic data, chemical correlations, and electronic circular dichroism calculations. Besides, all compounds were evaluated for cytotoxic activities against cultured K562, MCF-7, A549, and HepG2 cell lines. None of them showed good antitumor activities.

Keywords

abietane diterpenoids terpene glycosides structure identification

Introduction

Clerodendrum trichotomum Thunb., genus Clerodendrum family Verbenaceae, also called “chou wu tong” in Chinese owing to its bad smell,^1,2 has been used as a folk medicine for the treatment of rheumatic arthritis, hypertension, and migraine.³ The major constituents reported include phenylpropanoids, terpenoids, flavonoids and steroids.^4–7 In earlier work,^{5, 7} the chemical constituents of the ethyl acetate fraction from the stems of C trichotomum have been studied systematically. However, relatively few papers have been devoted to the n-butanol extract so far.

Abietane diterpenoids are characteristic constituents of the genus Clerodendrum, which exhibit remarkable antitumor,⁸ anti-inflammatory,⁹ and insecticidal activities.¹⁰ This paper focused on the study of the chemical constituents from the n-butanol extract of C trichotomum stems, and 2 new abietane diterpenoids, (4S, 5S, 10S)-12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-triene-19-oic acid β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (1), and (3S, 5S, 10S, 15S)-3β-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol (2), along with 5 known terpene glycosides (3–7) were isolated and characterized (Figure 1, 1–7). Compounds 1–7 were evaluated for cytotoxicity against several cancer cell lines, and all of them were inactive.

Figure 1.

Structures of compounds 1 to 11.

Results and Discussion

The n-butanol part of 85% ethanol extract from the stems of C trichotomum Thunb. was subjected to repeated column chromatography (CC) to yield 7 compounds 1–7. Compounds 3–7 were identified as szemaoenoid A (3),¹¹ leucasinoside (4),¹² melittoside (5),¹³ 8-epiloganic acid (6),¹⁴ and trijugaoside A (7)¹⁵ by comparison of their spectroscopic data with those previously reported in the literature. Compound 4 has been named as 12-O-β-D-glucopyranosyl-11, 16-dihydroxyabieta-8, 11, 13-triene,¹² but it should be 11,16-dihydroxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-19-oic acid (an unfortunate typing/printing error deleted the main suffix). A few closely related abietane diterpene glycosides (Figure 1, 8–11) have been previously reported: 12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-trien-19-oic acid β-D-glucopyranosyl ester (8, unfortunately named as 12,19-di-O-D-glucopyranosyl-11-hydroxyabieta-8,11,13-trien-19-one¹⁶ or 19-O-β-D-carboxyglucopyranosyl-12-O-β-D-glucopyranosyl-11,16-dihydroxyabieta-8,11,13-triene¹⁷), 3,12-bis(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol¹⁷ (9, correct name is 3-(β-D-glucopyranosyl)oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol), 12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-3β,11,16-triol (10, 12-O-β-D-glucopyranosyl-3,11,16-trihydroxyabieta-8,11,13-triene¹⁷), and ajugoside A¹⁷ (11, ie 12,19-bis-β-D-glucopyranosyloxyabieta-8,11,13-triene-11,16-diol).

Compound 1 was obtained as a yellow solid, which had the molecular formula C₃₈H₅₈O₁₉ based on the negative high resolution electrospray ionization mass spectrometry (HR-ESI-MS) ion at m/z 817.3496 [M-H]⁻; calcd for 817.3486 (Supplemental Figure S1), indicating 10 degrees of unsaturation. The infrared (IR) spectrum of 1 showed absorption (3392, 2930, 2873, and 1726 cm⁻¹) bands assignable to hydroxy, phenyl and carbonyl groups (Supplemental Figure S3).

As shown in Supplemental Figure S5, there were 2 doublet methyl and 2 singlet methyl proton resonances at δ_H 1.09 (3H, d, J = 6.6 Hz, H-16), 1.11 (3H, d, J = 6.6 Hz, H-17), 1.30 (3H, s, H-18), and 1.20 (3H, s, H-20) in the ¹H-nuclear magnetic resonance (NMR) spectrum. In addition, 3 doublet anomeric proton resonances at δ_H 4.30 (1H, d, J = 7.8 Hz, H-1′′′), 4.36 (1H, d, J = 7.8 Hz, H-1′), and 5.41 (1H, d, J = 7.8 Hz, H-1′′) showed the presence of 3 sugar units as a glycoside. Gas chromatography (GC) analysis (Supplemental Figure S4) showed that the sugar, after acid hydrolysis of 1, had the same retention time (t_R = 28.97 min) as the derivative of authentic D-glucose, which indicated that 1 had β-D-glucopyranose moieties. Besides, a singlet resonance at δ_H 6.39 (1H, s, H-14) was a phenylic proton signal; the others were proton signals of diterpene and sugar fragments. The ¹³C-NMR spectrum (Supplemental Figure S6) showed 38 carbon signals, including 1 oxycarbonyl signal at δ_C 178.4 (C-19) and 6 aromatic carbon signals at δ_C 135.4 (C-8), 133.0 (C-9), 149.3 (C-11), 142.7 (C-12), 140.9 (C-13), and 118.0 (C-14); the anomeric carbon signals of 3 glucopyranosyl moiety resonances were at δ_C 108.1 (C-1′), 95.7 (C-1′′), and 104.9 (C-1′′′). The other resonances belonged to diterpene and sugar moieties.

The ¹H-¹H homonuclear chemical-shift correlated spectroscopy (COSY) spectrum (Supplemental Figure S9) of 1 showed that correlations of H-1a (δ_H 1.08, m, 1H), H-2b (δ_H 1.99, m, 1H), and H-3a (δ_H 2.24, m, 1H) indicated the fragment C-1/C-2/C-3 in ring A, and correlations of H-5 (δ_H 1.47, m, 1H), H-6b (δ_H 1.85, m, 1H), and H-7 (δ_H 2.70, m, 2H) corresponded to fragment C-5/C-6/C-7 in ring B. The key correlations are shown with blue bold lines in Figure 2. These characteristic signals implied that 1 was an abietane diterpenoid. Further examination of the ¹H detected heteronuclear multiple quantum coherence (HMQC) and ¹H detected heteronuclear multiple bond correlation (HMBC) spectroscopic data (Supplemental Figures S7 and S8) of compound 1 indicated that it was structurally similar to 8 [12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-triene-19-oic acid β-D-glucopyranosyl ester].¹⁶ The differences in the NMR spectra were a group of carbon signals at δ_C 104.9, 78.0, 78.0, 75.3, 71.2, and 70.0, which were resonances of 1 additional sugar unit. This observation was verified by the HMBC correlations from δ_H 4.30 (1H, d, J = 7.8 Hz, H-1′′′) to δ_c 70.0 (C-6″). The nuclear overhauser effect spectroscopy (NOESY) correlations (Supplemental Figure S10) of H-5/H-18, H-1a/H-5, and H-3a/H-5 suggested that these protons were co-facial, pointing out their α-orientation, whereas the correlation of H-20/H-6b indicated that these protons were β-oriented (Figure 3). All ¹H and ¹³C NMR data of compound 1 were assigned (Table 1). The absolute configuration of 1 was assigned as 4S, 5S, and 10S by comparing the computational and experimental electronic circular dichroism (ECD) (Figure 4). Thus, compound 1 was established as (4S, 5S, 10S)-12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-triene-19-oic acid β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.

Figure 2.

Key ¹H-¹H COSY, HMBC correlations for compound 1.

Figure 3.

Key NOESY correlations for compound 1.

Figure 4.

Experimental and calculated electronic circular dichroism (ECD) spectra of compound 1 (in MeOH).

Table 1.

NMR Spectroscopic Data of Compound 1 [¹H (600 MHz) and ¹³C (150 MHz)] and 2 [¹H (400 MHz) and ¹³C (100 MHz)] in CD₃OD.

Position		1		2
Position		δ_H (ppm, J in Hz)	δ_C (ppm)	δ_H (ppm, J in Hz)	δ_C (ppm)
1	Ha	1.08, m 3.27, m	37.4	1.36, m 3.35, m	36.2
1	Hb	1.08, m 3.27, m	37.4	1.36, m 3.35, m	36.2
2	Ha	1.44, m 1.99, m	21.0	2.00, m 1.81, m	28.2
2	Hb	1.44, m 1.99, m	21.0	2.00, m 1.81, m	28.2
3	Ha	2.24, m 1.09, m	39.0	3.25, m	90.7
3	Hb	2.24, m 1.09, m	39.0	3.25, m	90.7
4			45.6		40.9
5		1.47, m	57.3	1.21, d (12.0)	54.6
6	Ha	2.14, m 1.85, m	22.5	1.81, m 1.57, m	20.1
6	Hb	2.14, m 1.85, m	22.5	1.81, m 1.57, m	20.1
7		2.70, m	34.9	2.75, m	33.9
8			135.4		135.2
9			133.0		134.7
10			41.3		40.3
11			149.3		149.2
12			142.7		143.3
13			140.9		136.5
14		6.39, s	118.0	6.36, s	118.3
15		3.63, m	26.7	3.65, m	35.0
16	Ha	1.09, d (6.6)	24.3	3.62, m 3.43, m	69.2
16	Hb	1.09, d (6.6)	24.3	3.62, m 3.43, m	69.2
17		1.11, d (6.6)	24.4	1.13, d (7.2)	18.5
18		1.30, s	29.7	1.12, s	28.9
19			178.4	0.95, s	17.4
20		1.20, s	18.2	1.30, s	20.0
1′		4.36, d (7.8)	108.1	4.36, d (7.6)	106.9
2′		3.49, m	75.6	3.45, m	75.8
3′			78.4		78.2
4′		3.30, m	71.6	3.30, m	71.7
5′			78.1		77.9
6′	Ha	3.78, d (2.4) 3.74, d (4.8)	62.3	4.09, dd (12.0, 1.6) 3.83, m	69.8
6′	Hb	3.78, d (2.4) 3.74, d (4.8)	62.3	4.09, dd (12.0, 1.6) 3.83, m	69.8
1′′		5.41, d (7.8)	95.7	4.46, d (8.4)	104.9
2′′		3.42, m	74.3	3.21, m	75.2
3′′			78.3		77.1
4′′		3.39, m	71.2	3.30, m	71.7
5′′			78.2		77.7
6′′	Ha	4.08, dd (12.0, 2.4) 3.72, d (4.8)	70.0	3.89, m 3.71, m	62.8
6′′	Hb	4.08, dd (12.0, 2.4) 3.72, d (4.8)	70.0	3.89, m 3.71, m	62.8
1‴		4.30, d (7.8)	104.9	4.44, d (7.6)	107.9
2‴		3.18, m	75.3	3.49, m	75.7
3‴			78.0		79.0
4‴		3.45, m	71.2	3.44, m	71.4
5‴			78.0		68.0
6‴	Ha	3.80, d (2.4) 3.63, m	62.4	3.88, m 3.68, m	63.1
6‴	Hb	3.80, d (2.4) 3.63, m	62.4	3.88, m 3.68, m	63.1

Compound 2 was obtained as a yellow solid. Its negative HR-ESI-MS data (Supplemental Figure S11) showed an [M-H]⁻ ion at m/z 819.3646 (calcd for 819.3645), which is in accordance with the molecular formula C₃₈H₆₀O₁₉, indicating 9 degrees of unsaturation. The presence of hydroxy (3392 cm⁻¹) and phenyl (2932 and 2876 cm⁻¹) groups was confirmed by its IR spectrum (Supplemental Figure S12).

In the ¹H-NMR spectrum (Supplemental Figure S15), 1 doublet methyl and 3 singlet methyl proton resonances were displayed at δ_H 0.95 (3H, s, H-19), 1.12 (3H, s, H-18), 1.30 (3H, s, H-20), and 1.13 (3H, d, J = 7.2 Hz, H-17). In addition, 3 doublet anomeric proton resonances at δ_H 4.36 (1H, d, J = 7.6 Hz, H-1′), 4.46 (1H, d, J = 8.4 Hz, H-1′′), and 4.44 (1H, d, J = 7.6 Hz, H-1‴) showed the presence of 3 sugar units as a glycoside. The GC analysis spectrum (Supplemental Figure S14) showed that the sugar derivative in the acid hydrolysate of 2 had the same retention time t_R = 28.97 min as the derivative of D-glucose, which indicated that 2 had β-D-glucopyranosyl moieties. A singlet proton at δ_H 6.36 (1H, s, H-14) was the proton signal on a benzene ring, and the others were diterpene hydrogen and sugar proton signals. The ¹³C-NMR spectrum (Supplemental Figure S16) showed 38 carbon signals, including 6 aromatic carbon signals at δ_C 135.2 (C-8), 134.7 (C-9), 149.2 (C-11), 143.3 (C-12), 136.5 (C-13), and 118.3 (C-14); the anomeric carbon signals of 3 glucopyranose moieties were at δ_C 106.9 (C-1′), 104.9 (C-1′′), and 107.9 (C-1′′′), an obvious methylol resonance was at δ_C 69.2 (C-16), and the others were of diterpene and sugar moieties.

In addition, the ¹H-¹H COSY spectrum (Supplemental Figure S19) of compound 2 showed that correlations of H-1a (δ_H 1.36, m, 1H), H-2b (δ_H 1.81, m, 1H), and H-3 (δ_H 3.25, m, 1H) indicated the fragment C-1/C-2/C-3 in ring A; correlations of H-5 (δ_H 1.21, d, J = 12.0 Hz, 1H), H-6b (δ_H 1.57, m, 1H), and H-7 (δ_H 2.75, m, 2H), corresponded to fragment C-5/C-6/C-7 in ring B. These correlations are shown with blue bold lines in Figure 5. Based on the 1D NMR spectrum, compound 2 was an abietane diterpenoid. Examination of the HMQC and HMBC spectra (Supplemental Figures S17 and S18) of 2 indicated that it was structurally similar to 9 [3-(β-D-glucopyranosyl)oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol].¹⁷ The differences in the NMR spectra were a group of carbon signals (δ_C 104.7，77.7，77.1，75.2，71.7，and 69.8) in 2. This observation was correlated by the HMBC correlations from δ_H 4.46 (1H, d, J = 8.4 Hz, H-1′′) to δ_C 69.8 (C-6′). NOESY correlations (Supplemental Figure S20) of H-6a/H-18, H-3/H-1a, and H-3/H-5 revealed their co-facial relationship, and they were assigned arbitrarily as α-oriented, whereas those of H-2b/H-20 and H-6b/H-19 implied these protons were β-oriented (Figure 6). All the ¹H and ¹³C NMR data of compound 2 were assigned (Table 1). The absolute configuration of 2 was elucidated as 3S, 5S, 10S, and 15S by comparison of the calculated ECD data with the experimental data (Figure 7). Based on the above evidence, compound 2 was established as (3S, 5S, 10S, 15S)-3β-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol.

Figure 5.

Key ¹H-¹H COSY, HMBC correlations for compound 2.

Figure 6.

Key NOESY correlations for compound 2.

Figure 7.

Experimental and calculated electronic circular dichroism (ECD) spectra of compound 2 (in MeOH).

The antitumor activities of compounds 1–7 were evaluated against 4 cell lines, K562 human leukemia, MCF-7 human breast cancer, A549 human lung cancer, and HepG2 human hepatoma, using the MTT (methyl thiazolyl tetrazolium) assay. However, all the compounds were inactive (IC₅₀ > 50 µM).

Experimental

General

HR-ESI-MS were measured on a Thermo Fisher Q Exactive-Plus mass spectrometer (Thermo Fisher Scientific), 1D and 2D NMR spectra on a BRUKER 600 NEO NMR spectrometer (Bruker Co. Ltd) and JEOL ECS 400 NMR spectrometer (Jeol), and UV spectra on an UV-2700 spectrometer (Shimadzu Co. Ltd). Optical rotation values were measured on a Bellingham-Stanley ADP 440 + polarimeter (Bellingham-Stanley Co. Ltd), and IR spectra on an IR Tracer-100 spectrophotometer (Shimadzu Co. Ltd). A JASCO J-715 spectrometer (Jasco) was used to record the ECD. GC analysis was carried out on a Shimadzu-2010 Plus gas chromatograph (Shimadzu Co. Ltd), using a ZB-5 capillary column (30 m × 0.25 mm i.d. × 0.25 μm), an flame ionization detector at 280 °C, and a column initial temperature of 200 °C. CC was performed with a macroporous resin (D101, Tianjin Haiguang Chemical Co. Ltd), silica gel (200–300 mesh and 300–400 mesh, Qingdao Haiyang chemical Co. Ltd), Toyopearl HW-40C (Tocho corporation), Toyopearl HW-40F (Tocho corporation), and Sephadex LH-20 (Pharmacia Biotech). For thin-layer chromatography, silica gel GF 254 plates were used (Qingdao Haiyang Chemical Co. Ltd).

Plant Material

C trichotomum Thunb. was collected from Nanjing, Jiangsu Province, and identified by Yao Gan researcher, Jiangsu Institute of Botany Chinese Academy of Sciences. The voucher specimens are preserved in the Engineering Research Center for the Development and Application of Ethnic Medicine and Traditional Chinese Medicine (Ministry of Education), Guizhou Medical University.

Extraction and Isolation

The stems of C trichotomum (15 kg) were extracted with 85% ethanol under reflux, and the extract was partitioned with petroleum, ethyl acetate, and n-butanol, sequentially. Three parts were obtained: the petroleum part (50 g), the EtOAc part (70 g), and the n-butanol part (470 g). The n-butanol part was dissolved in water and separated through a D101 macroporous resin, following elution in proper order by water, ethanol–water (30%, 60%, 95%, v/v).

The 60% ethanol eluted fraction was separated into 8 fractions (fr.1∼8) by octadecylsilyl silica gel (ODS) (MeOH/H₂O 0:100→100:0) CC. Fr.2 was fractionated by silica gel CC eluting with EtOAc–MeOH (30:1 to 5:1) to yield compound 5 (23 mg). Fr.5 was chromatographed on a Sephadex LH-20 column (MeOH–Water, 1:1) to obtain 5 sub-fractions (Fr.5.1∼5.5). Fr.5.2 was further separated by ODS (MeOH/H₂O 10:90→70:30), silica gel (EtOAc–MeOH 30:1 to 5:1), Sephadex LH-20 (MeOH–Water, 1:1), and HW-40F CC (MeOH) to obtain compounds 1 (21 mg), 2 (25 mg), 3 (51 mg), and 7 (33 mg). Fr.7 was separated repeatedly by ODS (MeOH/H₂O 10:90→50:50) and HW-40F (MeOH) CC to obtain compound 4 (3 mg). Compound 6 (3 mg) was purified from the 30% ethanol eluted fraction by repeated ODS (MeOH/H₂O 10:90→50:50) and Sephadex LH-20 (MeOH–Water, 1:1) CC.

(4S, 5S, 10S)-12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-triene-19-oic acid β-D-glucopyranosyl-(1→6)- β -D-glucopyranosyl ester (1)

Yellow solid; mp 182 to 185 °C; [α] $\underset{D}{25}$ + 9.3 (c 0.29, MeOH), UV (MeOH) λ_max (log ε) 273 (3.56) nm; IR(KBr) ν_max 3392, 2930, 2873, 1726, 1630, 1423, 1382, 1331, 1229, 1072, 1033, and 896 cm⁻¹; HR-ESI-MS m/z 817.3496 [M-H]⁻ (calcd for [C₃₈H₅₈O₁₉-H]⁻, 817.3486); ECD (MeOH, c = 0.20) nm (Δε): 204 ( + 2.36), 212(−1.20), 221 ( + 11.10), 236 (−10.23); ¹H and ¹³C NMR: Table 1.

(3S, 5S, 10S, 15S)-3β-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol (2)

Yellow solid; mp 174 to 176 °C; [α] $\underset{D}{25}$ + 26.1 (c 0.27, MeOH), UV (MeOH) λ_max(log ε) 280 (3.37) nm; IR(KBr) ν_max 3392, 2932, 2876, 1640, 1425, 1370, 1330, 1227, 1073, 1037, 937, and 905 cm⁻¹; HR-ESI-MS m/z 819.3646 [M-H]⁻ (calcd for [C₃₈H₆₀O₁₉-H]⁻, 819.3645); ECD (MeOH, c = 0.22) nm (Δε): 237(−19.67), 278 (−1.31); ¹H and ¹³C NMR Table 1.

Acid Hydrolysis and Sugar Identification

Compounds 1 and 2 (each 0.5 mg) were hydrolyzed with 2 mol/L HCl (2.0 mL) at 95 °Cfor 3 h, respectively. After cooling, the reaction mixture was partitioned between water and ethyl acetate, 3 times. The water extract was dried under reduced pressure in a rotary evaporator to obtain the test sample. Separately, the test sample, D-glucose and L-glucose (each 0.5 mg) were dissolved in pyridine (0.4 mL) and reacted with L-cysteine methyl ester hydrochloride (1.0 mg), followed by heating at 60 °C for 1 h. Then, N-trimethylsilylimidazole (0.15 mL) was added to the mixture and reacted at 60 °C for another 1 h. After that, the reaction solution was blown to dryness by nitrogen. The reaction residue was dissolved in water (1.0 mL), and extracted with n-hexane (0.5 mL) 3 times. The n-hexane extract was subjected to GC analysis using an HP-5 column. Acid hydrolysis derivatives of compounds 1 and 2 (t_R = 28.97 min) were compared with D-glucose (t_R = 28.97 min) and L-glucose (t_R = 29.31 min) derivatives.

ECD Calculations

Conformational analysis of all the plausible stereoisomers of compounds 1 and 2 were performed by using a random search algorithm, the MMFF94s force field, and MMFF94 charge in Sybyl X 2.1 software. All the conformers obtained were screened based on the energy of optimized structures at the B3LYP/6- 31 + G(d) level in an energy window of 3 kcal/mol in Gaussian 16 software. The ECD data of all the selected conformers were calculated with the TD/B3LYP/6-311 + G(2d, p) methods in the gas phase, respectively. The overall simulated CD curves were weighted by Boltzmann distributions of each conformer (with a half-bandwidth of 0.3 eV) and compared to the experimental ones in SpecDis 1.7 software.

Cytotoxicity Assay

Antiproliferative activities were evaluated as IC₅₀ against the K562 human leukemia, MCF-7 human breast cancer, A549 human lung cancer, and HepG2 human hepatoma cell lines, using the methyl thiazolyl tetrazolium (MTT) assay. The cell lines were cultured in humidified incubators (37 °C and 5% CO₂). They were maintained in RPMI1640 medium (Sigma), supplemented with 10% fetal bovine serum (HyClone), 100 Unit/mL of penicillin G (Sigma), and 100 mg/mL of streptomycin (Sigma). The cells were seeded in a 96-well plate and cultured overnight, then treated with compounds 1–7 at 5 concentrations (0.15, 1.5, 5, 15, and 50 μM) for 24 h. Then, 20 μL of MTT solution (5 mg/mL) was added to each well. Following an additional 4 h incubation, 150 μL of dimethylsulfoxide (DMSO) solution was added to each well to dissolve the formazan crystals. Absorbance was determined at 490 nm by a Bio-Rad Model 680 multi-well scanning spectrophotometer (Bio-Rad). The concentrations required to inhibit growth by 50% (IC₅₀ values) were calculated from survival curves.

Conclusions

Two new abietane diterpenoids, (4S, 5S, 10S)-12-(β-D-glucopyranosyl)oxy-11-hydroxyabieta-8,11,13-triene-19-oic acid β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (1) and (3S, 5S, 10S, 15S)-3β-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]oxy-12-(β-D-glucopyranosyl)oxyabieta-8,11,13-triene-11,16-diol (2), together with five known terpene glycosides, named szemaoenoid A (3), leucasinoside (4), melittoside (5), 8-epiloganic acid (6), and trijugaoside A (7), were isolated from the n-butanol extract of C trichotomum stems. The new structures were determined by extensive analysis of HR-ESI-MS, NMR spectra, chemical correlations, and ECD calculations. All these isolated compounds were evaluated for in vitro cytotoxicity against the K562, MCF-7, A549, and HepG2 tumor cell lines, but their IC₅₀ values were all over 50 μM.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221125053 - Supplemental material for Two New Abietane Diterpenes From the Stems of Clerodendrum trichotomum Thunb

Supplemental material, sj-docx-1-npx-10.1177_1934578X221125053 for Two New Abietane Diterpenes From the Stems of Clerodendrum trichotomum Thunb by Lin-Zhen Li, Yu Zhang, Liang Chen, Yin-Zhi Cen and Yang-Li Tu, Xiao-Sheng Yang, Yong-Jun Li in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China, Science and Technology Cooperation Projects of Guizhou Province (grant numbers 81860689, and 20157359).

Supplemental Material

Supplemental material for this article is available online.

ORCID iD

Lin-Zhen Li

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