Introduction

Derris indica (synonyms Pongamia pinnata, P. glabra and Cytisus pinnaus) is popularly known as Karanj or Karanja in Hindi and as Yi-Nam in Thai, and is a mangrove plant belonging to the Leguminosae family [1]. It is a medium-sized, fast-growing tree which is widely distributed in Southeast Asia and the Pacific Islands. In Thailand, it is widely found in the southern part [2]. The leaves of the plant consist of five or seven leaflets, the flowers are pinkish-white in color and the bark is thin gray to grayish-brown. It has been used in folk medicine for bronchitis, whooping cough, rheumatic arthritis, ulcers and diabetes [3]. Chemical investigation of various parts of the plant has led to the isolation of flavonoids, chalcones and rotenoids [4]. In addition, the seeds contain about 27–39 % oil which may be used for tanning leather, for making soap, as a liniment to treat scabies, for curing rheumatism and as lighting oil [5]. Nowadays, the oil is used as fuel in diesel engines, showing a good thermal efficiency. It has been reported that the oil from the seed is composed of saturated and unsaturated fatty acids such as oleic (42.44 %), stearic (29.64 %) and palmitic (18.58 %) acids [6]. In this study, the purification of the crude hexane extract led to two new compounds together with fourteen known compounds. Their cytotoxicity against cholangiocarcinoma (CCA) M156 cell line and human hepatoma HepG2 cell line were evaluated.

Results and discussion

The fruits of D. indica, collected in Krabi Province in August 2012, were air-dried, pulverized and sequentially extracted with hexane, EtOAc and MeOH. The hexane extract was subjected to silica gel column chromatography and further purified by chromatographic methods to obtain 16 pure compounds (1–16) (Fig. 1). The structures of all compounds were determined through the analysis of spectroscopic data, 1D- and 2D-NMR, and also MS analysis. It was found that 1 and 2 were new compounds.

Fig. 1
figure 1

The structure of all compounds isolated from Derris indica

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Compound 1 was obtained as colourless needles, m.p. 174–176 °C. It was assigned the molecular formula C25H26O8 as determined from its pseudo-molecular ion peak at m/z 477.1528 [M + Na]+ in the HRESIMS, corresponding to 13 degrees of unsaturation. The IR spectrum showed absorption bands of carbonyl groups at 1752, 1734 and 1681 cm−1. In addition, the 13C-NMR spectrum also showed three signals at δ 188.7, 170.0 and 169.8 which indicated the presence of three carbonyl groups. The 1H-NMR spectrum displayed doublet of doublets signals at δ 5.39 (J = 13.2, 2.8 Hz), δ 2.96 (J = 16.4, 13.2 Hz) and δ 2.81 (J = 16.4, 2.8 Hz) which were assigned as H-2, H-3a and H-3b, respectively (Table 1). The coupling constant (J = 13.2 Hz) between H-2 and H-3a indicated the axial–axial orientation of these two protons and confirmed the α-position of the phenyl group. Five aromatic protons displayed signals at δ 7.28–7.37, indicating that this compound is a flavanone possessing an unsubstituted B ring. The methoxy group at δ 3.86 was connected to an aromatic carbon at δ 162.7 in the HMBC spectrum. A methine proton at δ 6.05 which was connected to carbon at δ 93.8 in the HMQC spectrum was assigned as H-6 by the HMBC correlations with C-5 (δ 162.7), C-7 (δ 159.9), C-8 (δ 99.9) and C-10 (δ 106.0) in the spectrum. The 1H-NMR spectrum displayed two doublet signals at δ 6.23 (d, J = 4.8, H-4″) and δ 5.14 (d, J = 4.8, H-3″) which connected to carbons at δ 61.3 and 71.3, respectively. The 13C-NMR and DEPT spectra displayed two acetyl groups at δ C 169.8/20.6, OAc-3″ and 170.0/20.6, OAc-4″. The HMBC spectrum showed correlations between H-4″ and carbons at δ 163.1 (C-9), 159.9 (C-7), 99.9 (C-8), 77.3 (C-2″) and 71.3 (C-3″) (Fig. 2). The correlations of H-3″ to C-2″ and two geminal methyl groups at δ 26.2 and 21.6 were seen in this spectrum. In addition, the HMBC experiment showed the correlations of two methyl groups at δ 1.44 and 1.38 with an oxygenated quaternary carbon at δ 77.3 (C-2″). The relative stereochemistry at C-3″ and C-4″ was suggested as cis from the J value (4.8 Hz), which was similar to pongamone B [4] and 3-methoxy-(3″,4″-dihydro-3″,4″-diacetoxy)-2″,2″-dimethylpyrano-(7,8:5″,6″)-flavone [2] which was isolated from D. indica. The NOESY experiment showed correlations between H-3″/H-4″, H-3″/H-7″ and H-4″/H-7″; in addition, these protons (H-3″ and H-4″) showed no NOESY correlation with H-8″. The comparison of the 1H-, 13C- and also 2D-NMR spectra of this compound and pongamone B suggested that both compounds should have the same substructure of a pyrano ring in which H-3″ and H-4″ have β-configuration. The CD spectrum of this compound was similar to pongamone B, which showed a positive value at 334 nm and a negative value at 282 nm [4]. Thus, the absolute stereochemistry at C-2 was assumed as 2S. From the results, the structure of 1 was elucidated as (2S)-5-methoxy-(3″,4″-dihydro-3″,4″-diacetoxy)-2″,2″-dimethylpyrano-(7,8:5″,6″)-flavanone, and it was named derrivanone (Fig. 1).

Table 1 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectral data of compounds 1 and 2 (δ in ppm)
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Fig. 2
figure 2

The key HMBC () and NOESY () correlations of compounds 1 and 2

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Compound 2 was obtained as a dark yellow solid, m.p. 110–112 °C. It was assigned the molecular formula C27H30O6 as determined from its pseudo-molecular ion peak at m/z 473.1939 [M + Na]+ in the HRESIMS, corresponding to 13 degrees of unsaturation. The IR spectrum showed absorption bands at 3451, 1640 and 1575 cm−1. The 1H-NMR spectrum showed two doublets signals at δ 7.78 (J = 15.6, H-3′) and 7.55 (J = 15.6, H-2′) indicating the trans geometry double bond (Table 1). In the HMQC experiment, these protons connected to carbons at δ C 143.3(C-3′) and 121.1 (C-2′), respectively, which indicated the presence of an α,β-unsaturated double bond. The correlations of H-3′ to C-5′ (δ 106.7), C-9′ (δ 125.4) and C-1′ (δ 179.3), and of H-2′ to C-4′ (δ 130.1) and C-1′ (δ 179.3) were seen in the HMBC spectrum. Two doublet signals at δ 7.07 (J = 7.2, H-9′) and δ 6.84 (J = 7.2, H-8′) were located on carbons at δ C 125.4 and 108.9, respectively. The correlations of H-9′ with C-7′ (δ 150.1), C-5′ (106.7) and C-8′ (108.9), and of H-8′ with C-6′ (δ 148.6) and C-4′ (δ 130.1) were seen in the HMBC spectrum. The methylenedioxy group showed signals at δ H 6.03 (s, 2H) and at δ C 101.8. The 13C-NMR spectrum displayed four down-field signals at δ 205.6, 197.7, 179.3 and 171.2, which were assigned as C-3, C-1, C-1′ and C-5, respectively. The methoxy proton (δ H 3.92/δ C 56.2) correlated to C-5 in the HMBC spectrum. The proton H-6 showed a singlet signal at δ 5.51 and correlated with C-1, C-4 and C-5 in the HMBC experiment. The 1H-NMR spectrum exhibited dimethylallyl groups at δ 2.64 (4H, d, J = 7.2, H-1″ and H-1″′), δ 4.85 (2H, t, J = 7.2, H-2″ and H-2″′) and δ 1.56 (12H, s, H-4″, H-4″′, H-5″ and H-5″′). The methylene groups at H-1″ and H-1″′ both correlated with C-1 (δ 197.7), C-2 (δ 61.8) and C-3 (δ 205.6), confirming that dimethylallyl groups connected at the C-2 position. The HMBC correlation between H-2′/C-1′ (δ 179.3) and the correlation between H-1″/C-3 (δ 205.6) and C-1 (δ 197.7) confirmed the enolic hydroxy at C-1′ and ketone group at C-1 and C-3. From the results, the structure of 2 was identified as 4-[1′-hydroxy-2′-ene-3′-(6′,7′-methylenedioxyphenyl)-propenylene]-5-methoxy-2,2-bisdimethylallyl-5-cyclohexene-1,3-diketone, and it was named derrischalcone (Fig. 1).

In addition, 14 known compounds, tunicatachalcone (3) [7], obovatachalcone (4) [8], glabrachromene (5) [9], ovalichalcone (6) [10], pongamol (7) [11], desmethoxykanugin (8) [12], karanjin (9) [13], lanceolatin B (10) [14], kanjone (11) [15], pongaglabrone (12) [16], 8-methoxyfurano(6,7:4″,5″)-flavone (13) [17], pongaflavone (14) [18], (2R,3R)-3-hydroxy-5-methoxy-2″,2″-dimethylpyrano[7,8:5″,6″]-flavanone (15) [19] and candidone (16) [20], were isolated and their structures elucidated by comparing their spectroscopic data with those reported in the literature.

Cytotoxicity of tested compounds [21]

The anticancer activity of all compounds (1–16) was tested against human CCA M156 cells and human hepatoma HepG2 cells. Almost all compounds were active toward both cancer cell types (Table 2). Compound 4 showed the most potent and highest anticancer efficacy against M156 and HepG2 cells. Most compounds were active against both cell types at micromolar concentrations. In addition, compounds 2, 3, 4, 7, 15 and 16 showed strong cytotoxicity against these two cell lines. Comparing the cytotoxicity between 2 and 3, it seems that 3 was more efficacious than 2. The results show convincingly that the methylenedioxy group appears to be detrimental for the activity. This effect was shown in the case of cytotoxicity against the HepG2 cell line. Compound 4 (IC50 = 2.6 ± 0.9) exhibited stronger activity than 5 (IC50 = 19.8 ± 7.4). Compound 14 showed cytotoxicity against M156 and HepG2 cell lines with IC50 values of 0.59 ± 0.08 and 0.86 ± 0.58, respectively, but the E max values of this compound were 34.4 ± 8.2 and 66.9 ± 8.5 %. In contrast, flavanone 15 exhibited high values of E max (94.6 ± 12.7 and 100 %) and showed IC50 values of 7.8 ± 3.5 and 11.2 ± 3.6 µg/mL, respectively. The cytotoxicity results show that flavone derivatives (8–14) exhibited weak activity while flavanone derivatives (15 and 16) showed good activity. These results show convincingly that flavanone may play an important role in cytotoxicity. It should be noted that compounds 1, 10 and 12 were active only against M156 cells but not HepG2 cells. On the other hand, compound 5 was active against HepG2 cells but not M156 cells.

Table 2 Cytotoxicity of a series of compounds against M156 and HepG2 cells
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Experimental

General experimental procedures

Melting points were determined on a Sanyo Gallenkamp (UK) melting point apparatus and are uncorrected. UV spectra were measured on an Agilent 8453 UV–Visible spectrophotometer (Germany). IR spectra were recorded as KBr disks or thin films, using a Perkin Elmer Spectrum One FT-IR spectrophotometer (UK). The NMR spectra were recorded on a Varian Mercury plus spectrometer (UK) operating at 400 MHz (1H) and at 100 MHz (13C). Mass spectra were determined on a Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer with a Z-spray ES source (Micromass, Manchester, UK). Optical rotation was obtained using a Jasco DIP-1000 digital polarimeter. Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 TLC aluminum sheets. Column chromatography was done with silica gel 0.063–0.200 mm or less than 0.063 mm. Preparative layer chromatography (PLC) was carried out on glass-supported silica gel plates using silica gel 60 PF254 for PLC. All solvents were routinely distilled prior to use.

Plant material

The fruits of Derris indica were collected in August 2012 from Krabi Province, Thailand. The plant was identified by Prof. Dr. Pranom Chantaranothai, Faculty of Science, Khon Kaen University. A botanically identified voucher specimen (KKU0042012) was deposited at the herbarium of the Department of Chemistry, Faculty of Science, Khon Kaen University, Thailand.

Extraction and isolation

Air-dried fruits (5.8 kg) of D. indica were ground into powder and then extracted successively at room temperature with hexane (2 × 12 L), EtOAc (2 × 12 L) and MeOH (2 × 12 L). The filtered samples were combined, and the solvents were evaporated in vacuo to yield crude hexane (336 g), EtOAc (150 g) and MeOH (220 g) extracts. The crude hexane extract (336 g) was subjected to silica gel flash column chromatography and subsequently eluted with a gradient system of three solvents (hexane, EtOAc and MeOH) by gradually increasing the polarity of the elution solvent system. On the basis of their TLC characteristics, the fractions which contained the same major compounds were combined to give nine fractions, F1–F9. The first fraction was identified as the natural oil (262.4 g, 4.52 % yield). Purification of F2 was carried out on silica gel and eluted with the three gradient systems above to give compound 4 (72.4 mg, 0.0012 %). The solid in fraction F5 was recrystallized from CH2Cl2:EtOAc to afford a colorless solid of 9 (5.02 g, 0.0866 %). The filtrate of F5 was evaporated to yield a viscous yellow oil, which was subjected to silica gel column chromatography using gradient elution of hexane:EtOAc mixtures to give six subfractions, F5.1–F5.6. Crystallization of subfraction F5.3 from EtOAc:CH2Cl2 afforded a white solid of 14 (147 mg, 0.0025 %). Subfraction F5.5 was rechromatographed on silica gel column chromatography and eluted with an isocratic system of 10 % EtOAc:hexane to give four subfractions, F5.5.1–F5.5.4. Subfraction F5.5.2 was purified by reversed-phase silica gel column chromatography using 25 % water:MeOH as eluting solvent to give 3 (121.6 mg, 0.0021 %). Crystallization of subfraction F5.5.3 using EtOAc:CH2Cl2 afforded a colorless solid of 8 (47.8 mg, 0.0008 %). The filtrate of F5.5.3 was purified by reverse-phase silica gel chromatography using 20 % water:MeOH as eluting solvent to give 2 (25.9 mg, 0.0004 %) and 15 (4.3 mg, 0.0013 %). Subfraction F5.6 was rechromatographed on reverse-phase silica gel column chromatography and eluted with an isocratic system of 40 % water:MeOH to give five subfractions, F5.6.1–F5.6.5. Crystallizations of subfractions F5.6.4 using EtOAc:CH2Cl2 afforded a colorless solid of 10 (24.4 mg, 0.0004 %). Purification of F8 by silica gel column chromatography and elution with hexane:EtOAc mixtures of increasing polarity gave six subfractions, F8.1–F8.6. Subfraction F8.1 was further purified by preparative thin-layer chromatography using 2 % EtOAc:hexane as an eluting solvent to give 7 (35.3 mg, 0.0006 %) and 5 (4.4 mg, 0.00008 %). Subfraction F8.2 was further purified by preparative thin-layer chromatography using 15 % EtOAc:hexane as eluting solvent to give 1 (26.8 mg, 0.0005 %). Subfraction F8.4 was rechromatographed on silica gel column chromatography and eluted with an isocratic system of 75 % CH2Cl2:hexane to give 11 (11.4 mg, 0.0002 %) and 12 (3.2 mg, 0.00006 %). Subfraction F8.5 was subjected to silica gel column chromatography with 10 % acetone:hexane to give seven subfractions, F8.5.1–F8.5.7. Subfraction F8.5.1 was further purified by silica gel column chromatography using 10 % EtOAc:hexane as eluting solvent to give 6 (40.9 mg, 0.0007 %) and 16 (90.6 mg, 0.0016 %). The colorless solid in subfraction F8.5.4 was recrystallized with EtOAc:CH2Cl2 to give 13 (43.0 mg, 0.0007 %).

Cytotoxicity; cell culture

The human CCA cell line KKU-M156, kindly provided by Prof. Banchob Sripa, Faculty of Medicine, Khon Kaen University, was routinely cultured in Ham’s F12, supplemented with 10 % fetal bovine serum, 12.5 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, pH 7.3), 100 U/ml penicillin G and 100 µg/ml gentamicin. Human hepatoma HepG2 cells, purchased from the American Type Culture Collection (ATCC HB 8065), were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10 % fetal bovine serum (1 %), MEM nonessential amino acids (Gibco), 12.5 mM HEPES, pH 7.3, 100 U/ml penicillin and 100 µg/ml gentamicin. Cultured cells were maintained at 37 °C in a humidified atmosphere of 5 % CO2. The cells were subcultured every 2–3 days before cultured cell confluence using 0.25 % trypsin–EDTA, and medium was changed after an overnight incubation.

KKU-M156 and HepG2 cells were seeded onto 96-well plates at a density of 7.5 × 103 and 1.5 × 104 cells/well, respectively. After an overnight incubation, cultured media were changed to serum-free media. Test compounds, dissolved in DMSO and diluted with medium to various concentrations, were added into cultured cells and incubated for 24 h. The cytotoxicity was assessed by the sulforhodamine B (SRB) assay as previously described [22]. In brief, cultured cells were fixed with 10 % trichloroacetic acid and stained with 0.4 % SRB. The protein-bound dye was solubilized with 10 mM Tris-base solution for determination of the absorbance at 540 nm with a microplate reader. The cytotoxicity was calculated as percent absorbance of controls. The IC50 value was calculated by a non-linear curve-fitting program.

Derrivanone (1)

Colorless solid; m.p. 174–176 °C; [α] 24D −240.0 (c 0.001, CHCl3); UV (CHCl3) λ max (log ε) 242 (4.04), 283 (4.25) nm; IR (KBr) ν max 2923, 1752, 1734, 1681, 1603, 1583, 1419, 1369, 1292, 1236, 1137, 1106, 1049, 884, 771 cm−1; 1H-NMR (CDCl3, 400 MHz) and 13C-NMR (CDCl3, 100 MHz) spectroscopic data, see Table 1; HRESIMS m/z 477.1528 [M + Na]+ (calcd. for C25H26O8Na 477.1525).

Derrischalcone (2)

Dark yellow solid; m.p. 110.0–112.0 °C; UV (CHCl3) λ max (log ε) 264 (4.15), 321 (4.20), 411 (4.66) nm; IR (KBr) ν max 3451, 2916, 1640, 1575, 1446, 1406, 1358, 1243, 1035 cm−1; 1H-NMR (CDCl3, 400 MHz) and 13C-NMR (CDCl3, 100 MHz) spectroscopic data, see Table 1; HRESIMS m/z 473.1939 [M + Na]+ (calcd. for C27H30O6Na 473.1940).