Two new pterocarpans and a new pyrone derivative with cytotoxic activities from Ptycholobium contortum (N.E.Br.) Brummitt (Leguminosae): revised NMR assignment of mundulea lactone
© The Author(s) 2016
Received: 19 June 2016
Accepted: 28 September 2016
Published: 5 October 2016
Ptycholobium is a genus related to Tephrosia which comprises only three species. Compared to Tephrosia, which has been phytochemically and pharmacologically studied, Ptycholobium species have only few or no reports on their chemical constituents. Moreover, no studies on the cytotoxic activities of its secondary metabolites have been previously documented.
From the non polar fractions of the roots bark of Ptycholobium contortum (syn Tephrosia contorta), two new pterocarpans: seputhecarpan C 1 and seputhecarpan D 2 and a new pyrone derivative, ptycholopyrone A 3 were isolated. Alongside, five known compounds identified as 3-α,α-dimethylallyl-4-methoxy-6-styryl-α-pyrone or mundulea lactone 4, glyasperin F 5, seputhecarpan A 6, seputheisoflavone 7 and 5-O-methyl-myo-inositol or sequoyitol 8 were also obtained. Their structures were established by the mean means of spectroscopic data in conjunction to those reported in literature. The NMR assignment of the major compound mundulea lactone 4 is revised in this paper. In addition, the cytotoxicity of the isolated metabolites was evaluated on two lung cancer cell lines A549 and SPC212. 8 was not active while compounds 1, 2, 4–7 displayed antiproliferative effects against the two carcinoma cell lines with IC50 values below 75 µM. IC50 values below 10 µM were obtained for 4, 6 and 7 on SPC212 cells.
Based on the obtained results, Ptycholobium contortum turns to be a rich source of phenolic metabolites among them some bearing prenyl moieties. This study reports for the first time the isolation of pyrone derivatives 3 and 4 from Ptycholobium genus. The cytotoxicity observed for the isolate is also reported for the first time and shows that 4, 6 and 7 could be chemically explored in order to develop a hit candidate against lung cancer.
KeywordsCytotoxic activities Ptycholobium contortum Ptycholopyrone A Seputhecarpan C Seputhecarpan D
There is a considerable burden due to lung cancer which is the most common cause of death from the cancer diseases worldwide. Approximately 20 % (1.59 million deaths, 19.4 % of the total) of cancer death are victims of lung cancer . This estimation is continuously constant since several decades and 1.8 million new cases were diagnosed in 2012 (12.9 % of the total, 58 % of which occurred in the less developed regions). The disease remains also prominent in men (1.2 million, 16.7 % of the total) with the highest estimated age-standardized incidence rates in Central and Eastern Europe (0.054 %) and Eastern Asia (0.050 %) . The use of medicinal plants as an alternative or complementary solution remains a partial healthcare solution since the plant kingdom represents one of the sources of hit compounds and drugs candidates against cancer. Chemical constituents of Tephrosia species (a related genus of Ptycholobium) and their biological benefit (cytotoxic activities) are well known . Recently, we reported on two pterocarpans and one isoflavanone together with their antimicrobial, α-glucosidase and antioxidant properties from the polar fractions of the root bark of P. contortum. This work is up to date the only on this genus . This work is the only report on this genus up to date . We herein report the isolation and the structure elucidation of two new pterocarpans, a new pyrone derivative along with the cytotoxic activities of the isolated compounds.
Results and discussion
The crude extract of P. contortum roots was partitioned with n-hexane, chloroform, ethyl acetate and n-butanol. Purification of the hexane and ethyl acetate fractions by successive column chromatography afforded eight compounds among them three new (1–3).
1H- and 13C-NMR Data (300 and 75 MHz, resp) of 1 in (D6)acetonea and 2 in CDCl 3 a . δ in ppm, J in Hertz
7.02 (s, 1H)
7.29 (s, 1H)
6.41 (s, 1H)
6.40 (s, 1H)
4.11 (t, J = 10.3, 1H)
3.62 (m, 1H)
4.37 (ddd, J = 10.3; 3.4; 2.0, 1H)
4.02 (dd, J = 5.4; 2.1, 1H)
3.51 (m, 1H),
3.62 (m, 1H)
6.99 (d, J = 8.2, 1H)
7.25 (d, J = 8.4, 1H)
6.41 (dd, J = 8.2; 2.5, 1H)
6.47 (dd, J = 8.4; 2.7, 1H)
4.02 (s, 1H)
6.40 (d, J = 2.5 Hz, 1H)
6.31 (brs, 1H)
5.02 (brs, 1H)
5.55 (d, J = 6.0, 1H)
3.42 (dd, J = 15.1; 9.3, 1H)
3.12 (dd, J = 15.1; 7.6, 1H)
6.18 (dd, J = 18.0; 10.1, 1H)
5.37 (t, J = 9.3, 1H)
4.99 (m, 2H)
1.44 (s, 3H)
5.22 (m, 1H)
1.44 (s, 3H)
5.22 (m, 1H)
4.29 (brs, 1H)
3.78 (s, 3H),
3.77 (s, 3H)
1H- and 13C-NMR Data (300 and 75 MHz, resp) of 3 in MeOD a and 4 in CDCl 3 a , δ in ppm, J in Hertz
6.14 (s, 1H)
6.56 (s, 1H)
6.63 (d, J = 15.2, 1H),
6.88 (d, J = 15.0, 1H)
7.53 (d, J = 15.2, 1H,)
7.44 (d, J = 15.0, 1H)
7.51 (m, 1H)
7.60 (m, 1H)
7.39 (m, 3H)
7.38 (m, 3H)
6.23 (dd, J = 17.4; 10.5, 1H)
6.18 (dd, J = 17.4; 10.5, 1H)
4.98 (dd, J = 17.4; 1.2, 1H)
4.92 (dd; J = 10.5; 1.2, 1H)
4.87 (m, 2H)
1.54 (s, 6H)
1.49 (s, 6H)
4.71 (d, J = 6.6, 2H)
5.50 (t, J = 1.2, 1H)
1.83 (s, 3H)
1.80 (s, 3H)
Compound 4 was isolated as a yellow crystal, mp: 104.3–106.2 °C as the major constituent of the plant. Its molecular formula C19H20O3 was deduced from the analysis of HR-ESI–MS in which the pseudo-molecular ion [M+H]+ was observed at m/z 297.1514. NMR data of 4 (see Additional file 4; Table 2) were closely comparable to those of mundulea lactone 4 previously isolated from Mundulea suberosa by Dutta . The structure was revised by Lalitha et al.  and the full NMR data were reported by Venkata et al. . The 13C chemical shifts of 1′a and 6a were correctly assigned in the previous report. However, the 1H chemical shifts of H-1′a and H-6a were wrongly assigned at δ 6.55 (d, J = 16 Hz) and 7.50 (d, J = 16 Hz) respectively. The analysis of the HMQC spectra of 4 revealed correlations between the proton at δ 7.53 (current H-1′a) and the carbon at δ 135.4 and between the proton at δ 6.63 (current H-6a) and the carbon at δ 118.7. This can be justified by the fact that H-1′a is highly deshielded by the conjugation with pyrone ring; therefore, its 1H chemical shift should be higher than the one of H-6a. Additionally, the 13C chemical shifts of the aromatic oxymethines C-4 and C-6 and the carbonyl of the lactone C-2 were assigned as δ 157.7, 162.6 and 166.6 respectively . We herein revise the above NMR assignment of 4. Correlations were observed on the HMBC spectrum (Additional file 4) of 4 from the trio H-1′a, H-6a and H-5 to C-6 at δ 157.7. Based on this information, the chemical shift of C-6 was unequivocally assigned at δ 157.7. Furthermore, HMBC correlation was observed between the hydrogen atoms of the methoxyl at δ 3.87 and C-4 at δ 166.5 and no correlation was observed with the carbon at δ 162.6 suggesting that the chemical shift of C-4 and C-2 were respectively δ 166.5 and 162.6. Based on these data, the NMR assignment of mundulea lactone 4 was revised accordingly (Fig. 1; Table 2).
Cytotoxicity of compounds and doxorubicin towards lung carcinoma cells
Cell lines and IC50 values (µM)
73.49 ± 8.64
63.47 ± 5.99
26.39 ± 1.27
12.99 ± 0.95
11.39 ± 1.52
9.02 ± 0.07
13.19 ± 1.55
16.38 ± 1.89
46.70 ± 3.63
9.35 ± 0.98
38.68 ± 3.65
0.59 ± 0.16
1.01 ± 0.20
0.07 ± 0.00
NMR spectra were recorded on Bruker DMX Avance 300 and 600 instruments equipped with an auto-tune probe and using the automation mode aided by the Bruker program, Icon-NMR using Acetone-d6, CDCl3 and CD3OD as solvents and internal standards. HR EISMS spectra were determined on a microTOF-Q 98 spectrometer. Infra-Red spectra were recorded as KBr disk. For column chromatography, silica gel 60 particles size 0.04–0.063 mm (Merck) or Sephadex LH-20 (Sigma) were used. Analytical and Preparative TLC were performed respectively using silica gel 60 PF254 + 366 (Merck) and silica gel 60-F254 precoated aluminum sheets (Merck). The plates were visualized using UV (254 and 366 nm) and revealed by spraying with vanillin-sulphuric acid.
The roots of P. contortum were collected around Maun, Ngamiland District in North-Western Botswana and were botanically authenticated by Joseph Madome of the Okavango Research Institute (ORI) Herbarium. Voucher specimen (No KM-1-Maun-2013; KM-2-Maun-2014) were deposited at the University of Botswana Herbarium and at ORI Herbarium, respectively.
Extraction and isolation
Dried and powdered stem bark of P. contortum (1255 g) were extracted twice at room temperature with 4L of CH2Cl2–MeOH (1:1) for 48 h. The solvent was evaporated under reduced pressure to give 20.53 g of crude extract. The residue was extracted with 2 L of MeOH at room temperature for 24 h to give 7.39 g of crude extract. The two extracts were combined on the basis of their TLC profile to give 27.92 g of crude extract. This extract was defatted with n-hexane to give 4.33 g of n-hexane fraction. The residue was suspended in H2O and partitioned between CHCl3 (300 mL × 3), AcOEt (300 mL × 3) and n-butanol (300 mL × 3) to give 8.05 g of CHCl3; 12.41 g of AcOEt and 1.52 g of n-BuOH fractions. The chloroform fraction was subjected to silica gel column chromatography (40–63 μm, 4.5 × 50 cm) using n-hexane-AcOEt gradients as eluents. 83 fractions of 300 ml each were collected and combined on the basis of their TLC profile to give 9 sub-fractions (F 1 –F 9 ) as follows F 1 [(1–10), n-hexane-AcOEt 5 %, 0.80 g], 2 [(11–19), n-hexane-AcOEt 7.5 % 1.20 g], 3 [(20–27), n-hexane-AcOEt 10 %, 1.01 g], 4 [(28–49), n-hexane-AcOEt 15 %, 1.03 g], 5 [(50–55), n-hexane-AcOEt 20 % 0.60 g], 6 [(55–68), n-hexane-AcOEt 25 %, 1.05 g], 7 [(69–75), n-hexane-AcOEt 30 %, 0.50 g] 8 [(76–80), AE, 0.75 g] and 9 [(81–83), MeOH, 0.30 g] Purification of F 1 by a preparative TLC plate afforded 3 [UV (+), Rf = 0.70 at Hex-AE 10 %, 2.1 mg], a yellow compound, The yellow precipitate in F 2 was washed with Hex-AE 2.5 % followed by a filtration to yield 4 [UV (+); Rf = 0.33 at Hex-AE 10 %, 640.0 mg]. F 3 –F 4 were subjected to silica gel column chromatography (40–63 μm, 4.5 × 50 cm) using n-hexane-AcOEt gradients as eluents. F 3 afforded 6 [UV (+); Rf = 0.30 at Hex-AE 20 %, 12.3 mg] while 1 [UV (-); Rf = 0.50 at Hex-AE 20 %, 28.5 mg] and 7 [UV (+); Rf = 0.40 at Hex-AE 25 %, 32.0 mg] were isolated from F 4 respectively as yellowish and brownish powders. F 5 was purified using Sephadex LH-20 with CHCl3–MeOH (7:3) as eluent to afford 2 [UV (+); Rf = 0.35 at Hex-AE 20 %, 26.7 mg] as a red oil and 5 [UV (+); Rf = 0.30 at Hex-AE 20 %, 10.2 mg] as a white powder. Precipitate in F 8 was washed twice with a mixture of Hexane–ethyl acetate (1:3) and compound 8 was obtained as a white powder. The n-hexane fraction (3.76 g) was absorbed on a silica gel and chromatographed on a silica gel column using a mixture of hexane–ethyl acetate of increasing polarity as eluent. From this fraction, compound 4 (45.7 mg) was also re-isolated.
Seputhecarpan C (1). Brownish crystals. M.p. 108.5–109.9 °C. UV (acetone) λmax nm (log ε): 345 (3.73), 320 (3.67). IR KBr ν (cm−1): 3308, 1618, 1496, 963, 814. CD (c 5.0 × 10 −3, MeOH): ([θ230] −44,925, [θ300] +10,135), [θ475] + 3885. 1H-and 13C-NMR: see Table 1. HR-ESI–MS: 353.1353 ([M+H] +, C21H21O5 +; calc. 353.1389), 375.1178 ([M + Na] +, C21H20O5Na+; calc. 375.1208).
Seputhecarpan D (2). Yellowish oil. UV (acetone) λmax nm (log ε): 340 (4.35), 320 (3.38), 324 (4.40). IR KBr ν (cm−1): 3395, 1610, 1490, 1215, 1150, 1080, 965, 902, 836. 1H-and 13C-NMR: see Table 1. HR-ESI–MS: 361.1047 ([M + Na] +, C21H22O4Na+; calc. 361.1416),
Ptycholopyrone A (=4-(3-methylbut-2-enyloxy)-3-(2-methylbut-3-en-2-yl)-6-styryl-2H-pyran-2-one ; 3). Yellow oil. IR KBr ν (cm−1): 2956, 1686, 1524, 1348, 1024, 909, 685. 1H-and 13C-NMR: see Table 2. HR-ESI–MS: 351.1940 ([M+H] +, C23H27O3 +; calc. 351.1960), 701.3817 ([2 M+H]+, C 46 H 53 O 6 + ; calc. 701.3842).
Mundulea lactone (=4-methoxy-3-(2-methylbut-3-en-2-yl)-6-styryl-2H-pyran-2-one ; 4). Yellow crystals. M.p. 104.3-106.2 °C. IR KBr ν (cm−1): 2959, 1686, 1523, 1348, 1080, 909, 685. 1H-and 13C-NMR: see Table 2. HR-ESI–MS: 297.1514 ([M+H] +, C19H21O3 +; calc. 297.1491), 593.2901 ([2 M+H] +, C38H41O6 +; calc. 593.2903).
Cell lines and culture
Two lung cancer cell lines were used in this study. They include the human non-small cell lung cancer (NSCLC) cell line A549, obtained from Institute for Fermentation, Osaka (IFO, Japan) and the human mesothelioma cell line, SPC212 provided by Doc. Dr. Asuman Demiroğlu Zergeroğlu, Department of Molecular Biology and Genetic, Gebze Technical University, Turkey. The cells were maintained as a monolayer in DMEM (Sigma-aldrich, Munich, Germany) medium supplemented with 10 % fetal calf serum and 1 % penicillin (100 U/mL)-streptomycin (100 μg/mL) in a humidified 5 % CO2 atmosphere at 37 °C.
Neutral red uptake assay
The cytotoxicity of compounds and doxorubicin (purchased from Sigma Chemical Co., St. Louis, MO, USA) used as standard anticancer drug was performed by neutral red assay as previously described . This method is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. The procedure is cheaper and more sensitive than other cytotoxicity tests . Compounds were added in the culture medium so that dimethylsulfoxide (DMSO) used prior for dilution did not exceed 0.1 % final concentration. The viability was evaluated based on a comparison with untreated cells. IC50 values represent the sample’s concentrations required to inhibit 50 % of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel [16, 17].
This work reports the chemical investigation of the non polar fractions of Ptycholobium contortum from which two new pterocarpans and a new pyrone derivative were isolated. The interesting cytotoxic activities obtained with mundulea lactone 4 seputhecarpan A 6 and seputheisoflavone 7 (IC50 values below 10 µM) gives evidence that the genus Ptycholobium is a rich source of prenylated flavonoids and pyrone derivatives with potent cytotoxic activities. These results open a way for the study of the two others species of this genus P. plicatum and P. biflorum on which no phytochemical nor pharmacological studies have been carried out so far.
DN, FK and BTN have been involved in the isolation of compounds; DN, GWF, FN and GK acquisition of data (NMR, UV, IR, MS, CD) of the compounds; DN, GWF, LPS and BTN were involved in the structural elucidation of compounds; VK, OK, HS and KAM performed the cytotoxic assays; DN, GWF, LPS, BTN and VK drafted the manuscript. All authors read and approved the final manuscript.
DN and GWF are grateful to the Network of Analytical and Bioassay Services in Africa (NABSA) for 2 months financial support (Travel grant and maintenance allowance) at the University of Botswana. VK and HS are thankful to Türkiye Bilimsel Ve Teknolojik Araştirma Kurumu (Tubitak) for 6 months travel grant (to VK) and to Anadolu University, Eskisehir, Turkey for the funding grant 1507F563 (to VK and HS). The traditional healers, Mr. and Mrs. Seputhe are also acknowledged for providing the plant material.
The authors declare that they have no competing interests.
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- WHO 2012: Lung cancer estimated incidence, mortality and prevalence worldwide in 2012. http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx?cancer=lung. Accessed 13 Apr 2016
- Saad T, Muhammad AS, Muhammad A (2013) A review on the phytochemistry and pharmacology of genus Tephrosia. Phytopharmacol 4:598–637Google Scholar
- Fotso GW, Maher FA, Ngnintedo D, Ango PY, Kapche DGFW, Ngameni B, Ngwenya B, Yeboah SO, Ngadjui BT, Andrae-Marobela K (2015) Three new isoflavonoids with antioxidant properties from Ptycholobium contortum (N.E.Br.) Brummitt (Leguminosae). Phytochem Lett 14:254–259View ArticleGoogle Scholar
- Babu UV, Bhandari SPS, Garg HS (1998) Barbacarpan, a pterocarpan from Crotalaria barbata. Phytochemistry 48:1457–1459View ArticleGoogle Scholar
- Schüffler A, Sterner O, Anke H: Cytotoxic α-pyrones from Xylaria hypoxylon. Z. Naturforsch C 2007, 62 c: 169-172Google Scholar
- Lalitha VR, Srimannarayana G, Subba NVR (1966) Structure of mundulea lactone isolated from the roots of Mandelea suberosa. Cur Sci India 16:410Google Scholar
- Dutta N (1959) Constitution of munetone, the principal crystalline product of the root bark of Mundulea suberosa. J India Chem Soc C 36:165Google Scholar
- Venkata ER, Sridhar P, Rajendra YP (1997) Two prenylated flavanones from Mundulea suberosa. Phytochemistry 46:1271–1274View ArticleGoogle Scholar
- Zeng L, Fukai T, Nomura T, Zhang R-Y, Lou Z-C (1992) Five new isoprenoid-substituted flavonoids glyasperins F, G, H, I and J from the roots of Glycyrrhiza aspera. Heterocycles 34:1813–1828View ArticleGoogle Scholar
- Sultana N, Hartley TG, Waterman PG (1999) Two novel prenylated flavones from the aerial parts of Melicope micrococca. Phytochemistry 5:1249–1253View ArticleGoogle Scholar
- Boik J (2001) Natural compounds in cancer therapy. Oregon Medical Press, Minnesota USAGoogle Scholar
- Brahemi G, Kona FR, Fiasella A, Buac D, Soukupova J, Brancale A, Burger AM, Westwell AD (2010) Exploring the structural requirements for inhibition of the ubiquitin E3 ligase breast cancer associated protein 2 (BCA2) as a treatment for breast cancer. J Med Chem 53:2757–2765View ArticleGoogle Scholar
- Kuete V, Efferth T (2015) African flora has the potential to fight multidrug resistance of cancer. Biomed Res Int. doi:https://doi.org/10.1155/2015/914813 Google Scholar
- Borenfreund E, Puerner J (1984) A simple quantitative procedure using mono-layer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Cult Methods 9:7–9View ArticleGoogle Scholar
- Repetto G, Del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3:1125–1131View ArticleGoogle Scholar
- Kuete V, Wabo HK, Eyong KO, Feussi MT, Wiench B, Krusche B, Tane P, Folefoc GN, Efferth T (2011) Anticancer activities of six selected natural compounds of some Cameroonian medicinal plants. PLoS One 6:e21762View ArticleGoogle Scholar
- Kuete V, Sandjo LP, Wiench B, Efferth T (2013) Cytotoxicity and modes of action of four Cameroonian dietary spices ethno-medically used to treat cancers: Echinops giganteus, Xylopia aethiopica, Imperata cylindrica and Piper capense. J Ethnopharmacol 149:245–253View ArticleGoogle Scholar