A pair of new enantiomers of xanthones from the stems and leaves of Cratoxylum cochinchinense

Abstract

Background

The simple and caged xanthones from Clusiaceae showed significant antineoplastic activity. This study aims to identify structural diverse xanthones and search for novel antitumor natural products from this family plants.

Methods

The structures of new compounds 1a and 1b were elucidated mainly through comprehensive NMR and MS spectroscopic data, and their absolute configurations were determined by the comparison of the experimental and calculated electronic circular dichroism.

Results

A pair of new xanthone enantiomers, (+)- and (−)-cracochinxanthone A (1a and 1b), along with thirty known analogues (2–31), were isolated from extracts of the stems and leaves of C. cochinchinense. Preliminary biological assay of some isolates against HL-60, PC-3, and MDA-MB-231 cancer cell lines.

Conclusion

Some isolated xanthones exhibited high sensitivity against three human malignant cell lines and the structure–activity relationship study showed that the prenyl and geranyl units may play an important role in antitumor activity.

Electronic supplementary material

The online version of this article (10.1186/s13020-019-0235-z) contains supplementary material, which is available to authorized users.

Background

Cratoxylum cochinchinense Blume (Clusiaceae) is a deciduous shrub tree growing abundantly in southeast Asian countries [1]. The leaves, stems, barks, roots and latex of C. cochinchinense have been used as traditional Chinese medicine for the treatment of various diseases such as jaundice, edema, cough, itch, fever, diarrhea, hoarseness, diuretic, flu, colic, ulcer and dental problems and so on [2–4]. In addition, the young leaves have been used as an herbal substitute for tea and the immature fruit as a spice for cooking [5]. The simple and caged xanthones with significant antineoplastic activity have been reported from previous phytochemical investigations [6–12]. Aiming to identify structural diverse xanthones and search for novel antitumor natural products from the Clusiaceae [13–18], we continued our studies on the petroleum ether-soluble and dichloromethane-soluble portions of the stems and leaves of C. cochinchinense which exhibited moderate cytotoxicity against human myeloid leukemia (HL-60), human prostate cancer (PC-3) and human breast carcinoma (MDA-MB-231) cell lines with IC₅₀ values of 7.59, 21.49, 19.63 and 7.86, 32.48, 30.40 μg/ml, respectively. A pair of new enantiomers of xanthones, (+)- and (−)-cracochinxanthone A (1a and 1b), as well as thirty known analogues (2–31) were obtained (Fig. 1). In the present paper, the isolation and structure elucidation of new enantiomers of 1a and 1b, as well as the biological evaluation of some selected xanthones are presented.

Fig. 1.

Chemical structures of xanthones 1–31

Materials and methods

Information of experimental design and resources

The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study (Additional file 1).

General experimental procedures

¹H NMR, ¹³C NMR, HSQC, and HMBC were recorded on the Bruker-ARX-400 and Bruker-AV-600 NMR with tetramethylsilane (TMS) as internal standard. HRESIMS spectra were measured on a Bruker micrOTOF-Q mass spectrometer. Optical rotations were measured by the JASCO P-2000 polarimeter. UV spectra were recorded on a Shimadzu UV-2201 spectrometer. ECD spectra were measured on the BioLogic MOS 450 AF/CD at room temperature. Multimode Reader were used by a Varioskan Flash. The semi-preparative HPLC was a Shimadzu SPD-20A series equipped with an YMC C₁₈ column (250 × 20 mm, 5 μm, 2 mL/min). Chiral HPLC was a CHIRALPAK IB (250 × 4.6 mm) from Daicel Chiral Technologies Co., Ltd., China. Column chromatography (CC) was conducted on silica gel (100–200 and 200–300 mesh) and preparative and analytical TLC was performed on precoated GF254 plates (Qingdao Haiyang Chemical Co., Ltd., China), octadecyl silane (ODS) (50 µm, YMC Co., Ltd., Kyoto, Japan) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). All the organic solvents were purchased from Yuwang and Laibo Chemicals Industries, Ltd., China.

Plant material

Stems and leaves of Cratoxylum cochinchinense were collected in December 2016, at Mengla County, Xishuangbanna Autonomous Prefecture, People’s Republic of China, and were identified by Zhi Na (Kunming Institute of Botany, Chinese Academy of Sciences). The voucher specimen (HNMJY-2016) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China.

Extraction and isolation

The smashed leaves and stems of C. cochinchinense (10 kg) were macerated with 80% aqueous acetone at room temperature (3 × 80 L, 3 days each time). The combined extracts was suspended in water, and successively partitioned to produce petroleum ether (PE), dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), n-butyl alcohol (n-BuOH) and water (H₂O) fractions. The CH₂Cl₂ extract (140 g) was fractionated on a silica gel CC and eluted with a PE/EtOAc gradient (100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, 100:50, 0:100) to give ten fractions (Fr. A–J). Fraction D was purified by ODS CC with a stepwise gradient elution using MeOH/H₂O to afford 23 (154.8 mg), 29 (8.2 mg), 22 (5.8 mg), 26 (8.7 mg) and yield two subfractions D3 and D4. Fr. D3 was subsequently refined over Sephadex LH-20 (MeOH), followed by semi-preparative HPLC using 77% MeOH in H₂O as a mobile phase to get 19 (15.8 mg, t_R = 63.5 min) and 1 (3.2 mg, t_R = 93.3 min). Then, 1 was separated by chiral HPLC eluting with n-hexane: isopropanol (90:10) to yield 1a (0.92 mg, t_R = 15.4 min) and 1b (1.1 mg, t_R = 18.4 min). Fr. D4 was also chromatographed on Sephadex LH-20 (MeOH) and semi-preparative HPLC (64% MeOH in H₂O) to produce 6 (4.5 mg, t_R = 38.4 min) and 9 (6.5 mg, t_R = 40.3 min). Fr. F was fractionated by ODS CC (MeOH/H₂O) to give 27 (7.5 mg), 18 (5.3 mg), 20 (7.9 mg) and three major subfractions F2, F5 and F8. Fr. F2 was successively partitioned by a Sephadex LH-20 column (MeOH) to provide the key subfraction F2.2. Fr. F2.2 was further processed via semi-preparative HPLC using 56% aqueous MeOH as the mobile phase to afford 3 (10.2 mg, t_R = 28.0 min) and 4 (7.8 mg, t_R = 32.2 min). Fr. F5 was recrystallized with methanol to yield 19 (50.1 mg). Fr. F8 was loaded onto semi-preparative HPLC using 82% aqueous MeOH to gain 24 (6.9 mg, t_R = 87.5 min), 25 (10.2 mg, t_R = 92.5 min) and 21 (15.2 mg, t_R = 121.5 min). Fr. H was subjected to ODS CC, which afford 15 (6.9 mg) through further recrystallization and subfractions H3 and H5. Fr. H3 and Fr. H5 were applied to Sephadex LH-20 column and eluted with MeOH to obtain 7 (5.4 mg) and 8 (7.5 mg), respectively. Fr. I was subjected to ODS CC to furnish 13 (5.2 mg), 14 (2.8 mg), and subfraction I4. Fr. I4 followed by Sephadex LH-20 CC to afford 28 (11.6 mg). Fr. J was rechromatographed over silica gel CC, affording 12 (4.6 mg), 11 (3.2 mg) and 10 (13.4 mg).

The PE extract (69 g) was chromatographed on a silica gel CC and eluted stepwise with a PE/EtOAc gradient system (100:1, 100:3, 100:7, 100:15, 100:50, 100:100, 0:100) to afford the major fractions A′-G′. Fr. C′ was subjected to separation over ODS CC to yield 30 (8.8 mg) and subfraction C′8. Fr. C′8 was further purified over a silica gel CC and followed by semi-preparative HPLC with 90% aqueous MeOH as mobile phase under isocratic condition to furnish 31 (10.7 mg, t_R = 25.5 min). Fr. D′ was separated via ODS CC to provide 2 (15.5 mg), which was crystallized from the 65% MeOH/H₂O solution, and to give subfraction D′5. Fr. D′5 was chromatographed over Sephadex LH-20 eluting with MeOH to give 17 (6.7 mg). Fr. E′ was initially subjected to ODS CC to yield subfraction E′3 and E′8. Fr. E′3 was further purified by semi-preparative HPLC eluted with 60% MeOH/H₂O to give 16 (9.3 mg, t_R = 35.8 min). Fr. E′8 was again subjected to ODS CC to obtain 5 (3.1 mg).

Cracochinxanthone A (1): yellow needle crystal; UV (MeOH) λ_max (log ε) 319 (3.86), 268 (4.23), 235 (4.21) nm; ¹H, ¹³C NMR and HMBC data see Table 1; HRESIMS m/z 379.1541 [M + H]⁺ (calcd for C₂₃H₂₃O₅, 379.1540).

Table 1.

¹H (600 MHz), ¹³C NMR (150 MHz) and HMBC data for compound 1 in DMSO-d₆

Position	1H-NMR (mult, J in Hz)	13C–NMR	HMBC (1H → 13C)
1		158.0
2		108.0
3		163.5
4		106.3
5	7.46 (1H, d, J = 9.0 Hz)	118.6	C-7, 8a, 10a
6	7.28 (1H, dd, J = 9.0, 3.0 Hz)	124.4	C-8, 10a
7		153.8
8	7.40 (1H, d, J = 3.0 Hz)	107.9	C-6, 9, 10a
9		179.9
4a		152.9
8a		120.1
9a		101.5
10a		149.0
1′	2.95 (1H, dd, J = 14.7, 3.1 Hz) 2.82 (1H, dd, J = 14.7, 8.0 Hz)	28.9	C-1, 2, 3, 2′, 3′
2′	4.24 (1H, dd, J = 8.0, 2.3 Hz)	74.8	C-2, 1′, 3′
3′		147.1
4′	4.89 and 4.75 (each 1H, s)	110.0	C-2′, 3′, 5′
5′	1.76 (3H, s)	18.1	C-2′, 3′, 4′
1″	3.43 (2H, d, J = 7.2 Hz)	21.6	C-3, 4, 4a, 2″, 3″
2″	5.19 (1H, t, J = 7.2 Hz)	122.5	C-1″, 4″, 5″
3″		130.6
4″	1.82 (3H, s)	17.8	C-2″, 3″, 5″
5″	1.62 (3H, s)	25.6	C-2″, 3″
1-OH	13.35 (1H, s)		C-1, 2, 9a
7-OH	9.95 (1H, s)		C-6, 7, 8

(+) Cracochinxanthone A (1a). Yellow needles; ${\left[\alpha \right]}_{\text{D}}^{25}$ + 10.0 (c 0.06 MeOH); ECD (MeOH 0.58) λ_max (Δε) 241 (+ 3.65), 270 (− 4.22), 317 (− 1.91) nm.

(−) Cracochinxanthone A (1b). Yellow needles; ${\left[\alpha \right]}_{\text{D}}^{25}$ − 11.3 (c 0.07 MeOH); ECD (MeOH 0.70) λ_max (Δε) 242 (− 4.06), 273 (+ 3.72), 316 (+ 1.45) nm.

Anticancer assay in vitro

The antiproliferative activities of some selected compounds against the HL-60, PC-3, and MDA-MB-231 cancer cell lines were evaluated. 5-Fluorouracil (5-FU) was used as a positive control. Detailed methodology for the cell growth inhibition test has been described in a previous report [19]. The IC₅₀ values were calculated by SPSS 16.0 software and results were repeated three times that were expressed as mean ± SD.

Results

Cracochinxanthone A (1) was obtained as a yellow needle, and its molecular formula was determined as C₂₃H₂₂O₅ with 13° of unsaturation from the HRESIMS data of [M + H]⁺ ion at m/z 379.1541 (calcd for C₂₃H₂₃O₅, 379.1540). The UV bands observed at λ_max 319, 268 and 235 nm suggested a xanthone skeleton [13]. The ¹H NMR data showed signals for a hydrogen bond hydroxy proton at δ_H 13.35 (1H, s, OH-1), a free phenolic hydroxy proton at δ_H 9.95 (1H, s, OH-7), a set of ABX coupling system aromatic protons at δ_H 7.40 (1H, d, J = 3.0 Hz, H-8), 7.46 (1H, d, J = 9.0 Hz, H-5) and 7.28 (1H, dd, J = 9.0, 3.0 Hz, H-6), along with the typical signals of a 3-methylbut-2-enyl (prenyl) moiety at δ_H 3.43 (2H, d, J = 7.2 Hz, H-1″), 5.19 (1H, t, J = 7.2 Hz, H-2″), 1.82 (3H, s, CH₃-4″) and 1.62 (3H, s, CH₃-5″). The remaining proton signals were assigned to a dihydrofuran ring with an isopropenyl group at δ_H 2.95 (1H, dd, J = 14.7, 3.1 Hz, Ha-1′), 2.82 (1H, dd, J = 14.7, 8.0 Hz, Hb-1′), 4.24 (1H, dd, J = 8.0, 2.3 Hz, H-2′), 4.89 and 4.75 (each 1H, s, Ha-4′, Hb-4′), 1.76 (3H, s, CH₃-5′), and the corresponding carbon signals at δ_C 147.1 (C-3′), 110.0 (C-4′), 74.8 (C-2′), 28.9 (C-1′) and 18.1 (C-5′) were assigned through HSQC correlations [20]. The ¹³C NMR displayed 23 carbon resonances including one conjugated carbonyl carbon, sixteen aromatic/olefinic carbons, three methyl, two methylene and one oxygenated methine (Table 1). The dihydrofuran ring with an isopropenyl group was fused with xanthone skeleton at position C-2 and C-3, based on the HMBC correlations from Ha-1′ (δ_H 2.95) and Hb-1′ (2.82) to C-1 (δ_C 158.0), C-2 (108.0) and C-3 (163.5), as well as from H-2′ (δ_H 4.24) to C-2. The cross peaks between H-1″ (δ_H 3.43) and C-3, C-4a (δ_C 152.9) and C-4 (106.3) confirmed the location of the prenyl group at C-4. The correlations of H-5 (δ_H 7.46) with C-7 (δ_C 153.8), C-8a (120.1) and C-10a (149.0), H-8 (δ_H 7.40) with C-6 (124.4), C-9 (δ_C 179.9) and C-10a, H-6 (δ_H 7.28) with C-8 (δ_C 107.9) and C-10a, and OH-7 (δ 9.95) with C-6, C-7 and C-8 indicated that the free phenolic hydroxy located at C-7 (Fig. 2). Based on these results, the structure of 1 was assigned to a new compound, namely cracochinxanthone A.

Fig. 2.

Key HMBC correlations of 1

Cracochinxanthone A might be a racemic mixture due to the smooth ECD curve as well as close to zero optical rotation. Subsequent chiral HPLC separation of 1 gave the corresponding enantiomers 1a and 1b possessing the opposite ECD curves. Their experimental ECD spectra matched well with the calculated ones for R and S, respectively, thus, explicitly assigning the absolute configurations of 1a and 1b (Fig. 3). And the optical rotations of 1a and 1b were + 10.0 (c 0.06 MeOH) and − 11.3 (c 0.07 MeOH), respectively. Therefore, the structures of 1a and 1b were named as (+) and (−)-cracochinxanthone A.

Fig. 3.

Experimental and calculated ECD spectra of 1a and 1b

By comparison with those data from the literatures, the known analogues were identified as cochinchinoxanthone (2) [21], 1,4,7-trihydroxy-8-methoxyxanthone (3) [22], gentisein (4) [23], 1,6-dihydroxy-2,5,8-trimethoxyxanthone (5) [23], 1,7-dihydroxyxanthone (6) [24], 1,7-dihydroxy-4-methoxyxanthone (7) [25], 1,7-dihydroxy-3,6-dimethoxyxanthone (8) [26], 1,7-dihydroxy-8-methoxyxanthone (9) [27], 1,5,6-trihydroxy-7-methoxyxanthone (10) [28], 1,4,7-trihydroxyxanthone (11) [29], 1,5,6-trihydroxy-3,7-dimethoxyxanthone (12) [30], 1,3,5,6-tetrahydroxyxanthone (13) [31], 1,3,6,7-tetrahydroxyxanthone (14) [32], 1,3,6-trihydroxy-7-methoxyxanthone (15) [29], cratoxanthone C (16) [33], 1,2,4-trimethoxy-3,8-dimethoxyxanthone (17) [34], 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (18) [35], dulcisxanthone B (19) [20], cudratricusxanthone E (20) [36], γ-mangostin (21) [37], 1,3,7-trihydroxy-2,4-diisoprenylxanthone (22) [38], cochinchinone A (23) [33], cochinchinone B (24) [33], pruniflorone Q (25) [39], 1,3,5-trihydroxy-6′,6′-dimethyl-2H-pyrano(2′,3:6,7)xanthone (26) [30], pruniflorone N (27) [40], xanthone V₁ (28) [41], osajaxanthone (29) [42], cochinchinone I (30) [43], 1,7-dihydroxy-4-(3,7-dimethylocta-2,6-dienyl)-5′-(1-hydroxy-1-methylethyl)-4′,5′-dihydrofuro[2′,3′:3,2]-xanthone (31) [44].

Discussions

The antiproliferative activities of some xanthones were evaluated against HL-60, PC-3, and MDA-MB-231 cancer cell lines (Table 2). The isolates 1a, 1b, 3, 4, 6, 10, 12, 19–25, 27 and 28 displayed antiproliferative effect against HL-60 cells with IC₅₀ values ranging from 1.00 to 19.78 μM, especially 23 bearing one prenyl and one geranyl groups with an IC₅₀ value of 1.0 μM and 27 possessing a pyran ring with 1-hydroxy-4,4-dimethyl with an IC₅₀ value of 1.89 μM. Compounds 19–25, 27 and 28 exhibited potent inhibitory activity against PC-3 cells with IC₅₀ values ranging from 11.77 to 27.11 μM and compounds 1b, 19–25 and 28 displayed significant cytotoxicity against MDA-MB-231 cells with IC₅₀ values ranging from 7.94 to 18.46 μM, respectively. It is worth to mention that compounds 23–25 possessing one prenyl and one geranyl and 19–22 with two prenyl groups showed high sensitivity against three human cancer cell lines than others without prenyl unit.

Table 2.

Cytotoxicities of selected compounds (IC₅₀ μM)

Compounds	HL-60	PC-3	MDA-MB-231
1a	12.08 ± 0.84	> 50	> 50
1b	19.24 ± 1.51	> 50	18.46 ± 1.65
3	19.78 ± 2.09	> 50	> 50
4	18.00 ± 1.04	> 50	> 50
6	15.56 ± 0.51	> 50	> 50
9	> 50	> 50	> 50
10	10.43 ± 0.31	> 50	> 50
11	> 50	> 50	> 50
12	10.77 ± 0.13	> 50	> 50
19	2.62 ± 0.74	21.87 ± 1.94	7.94 ± 0.94
20	4.50 ± 0.17	11.77 ± 0.19	11.97 ± 0.65
21	3.07 ± 0.16	27.11 ± 1.49	13.30 ± 1.09
22	9.64 ± 0.34	20.60 ± 1.64	14.59 ± 1.26
23	1.00 ± 0.21	11.95 ± 1.36	9.40 ± 1.28
24	6.18 ± 0.31	14.99 ± 1.28	15.96 ± 0.46
25	4.47 ± 0.14	14.57 ± 1.27	11.55 ± 1.25
27	1.89 ± 0.54	22.94 ± 1.97	> 50
28	4.52 ± 0.97	20.72 ± 2.04	16.37 ± 1.32
5-FU a	2.20 ± 0.08	25.98 ± 1.08	38.69 ± 2.84

IC₅₀ values expressed as mean ± standard deviation, n = 3

^aPositive control

Conclusions

A pair of new racemic mixture of xanthones, (+)-cracochinxanthone A (1a) and (−)-cracochinxanthone A (1b), along with 30 known analogues (2–31) were isolated from the stems and leaves of C. cochinchinense. The antiproliferative activities of some selected compounds against human HL-60, PC-3, and MDA-MB-231 cancer cell lines were screened by the trypan blue and MTT methods. The polyprenylated or geranylated xanthones exhibited potent cytotoxicity against three human malignant cell lines, which could be further developed as potential lead compounds in the design for the treatment of cancer.

Abbreviations

HL-60

human myeloid leukemia cell line

PC-3

human prostate cancer cell line

MDA-MB-231

human breast carcinoma cell line

ODS

octadecyl silane

CC

column chromatography

TMS

tetramethylsilane

PE

petroleum ether

CH₂Cl₂

dichloromethane

EtOAc

ethyl acetate

n-BuOH

n-butyl alcohol

H₂O

water

ECD

electronic circular dichroic spectroscopy

HPLC

high performance liquid chromatography

IC₅₀

the concentration of drug required to inhibit cell growth by 50% compared with untreated control