Cucumol A: a cytotoxic triterpenoid from Cucumis melo seeds

ABSTRACT

Phytochemical investigation of the MeOH extract of Cucumis melo L. var. reticulates, Cucurbitaceae, seeds led to the isolation of a new triterpenoid: cucumol A (27-hydroxy taraxerol-3β-ol), along with three known compounds: α-spinasterol and D:B-friedoolean-5-ene-3-β-ol. Their structures were established by extensive 1D (¹H, ¹³C, and DEPT) and 2D (¹H–¹H COSY, HMQC, and HMBC) NMR, as well as IR and HRESIMS spectral analyses. Compound 3 displayed cytotoxic activity against L5178Y and Hela cancer cell lines with ED₅₀ of 1.30 and 5.40 µg/ml, respectively compared to paclitaxel (0.07 and 0.92 µg/ml, respectively).

Keywords:
Cucumis melo; Cucurbitaceae; Triterpenoid; Cucumol A; Cytotoxic activity

Introduction

Cucumis melo L. (cantaloupe) belonging to Cucurbitaceae family, is cultivated in temperate, subtropical, and tropical regions worldwide (Yasar et al., 2006; Ibrahim, 2010). Its fruits are consumed in the summer period because the pulp of the fruit is very refreshing, high nutritional, and sweet with a pleasant aroma, which may be used as an appetizer, a dessert or as salad (Melo et al., 2000; Baghaei et al., 2008). In Chinese folk medicine, the seeds of C. melo are used as digestive, febrifuge, antitussive, demulcent, and vermifuge (Duke and Ayensu, 1985; De Marino et al., 2009). Their extract can be used as an anti-diabetic and is useful in chronic eczema (Lal and Lata, 1980; Teotia and Ramakrishna, 1984). Seed kernel is commonly used in renal disorders such as kidney and bladder stones, ulcers in the urinary tract and stomach, painful and burning micturation, jaundice, vitiligo, ascites, suppression of urine, chronic fevers, inflammation of the liver and kidney, and in general debility in the Indian traditional medicine (Nayar and Singh, 1998; Baitar, 2003; Gill et al., 2011; Milind and Kulwant, 2011; Ibrahim, 2014; Ullah et al., 2015). The fruit's pulp possesses diuretic and anthelmintic properties (Ullah et al., 2015). Its lotion is employed for acute and chronic eczema. Roots are emetic agents. The fruits are used as a first aid treatment for burns and abrasions. Peduncle is used to manage anasarca and indigestion (Milind and Kulwant, 2011). C. melo revealed a wide range of biological activities such as antioxidant, analgesic, anti-inflammatory, and antimicrobial (Vouldoukis et al., 2004; Mariod and Matthaus, 2008; Gill et al., 2011; Ibrahim, 2014). Previous phytochemical studies on C. melo L. var. reticulates seeds resulted in the isolation of chromone derivatives, triterpene, and sterols (Ibrahim, 2010; Ibrahim, 2014, Ibrahim and Mohamed, 2015a, b). In the present work, investigation of the MeOH extract of C. melo L. var. reticulates seeds afforded a new triterpenoid: cucumol A (27-hydroxy taraxerol-3β-o1) (3), along with α-spinasterol (1) and D:B-friedoolean-5-ene-3-β-ol (2). The new compound was evaluated for its cytotoxic activity against L5178Y, PC12, and Hela cancer cell lines.

Materials and methods

General experimental procedures

Melting point was carried out using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). Optical rotation was recorded on a Perkin-Elmer Model 341 LC Polarimeter (Perkin-Elmer, Waltham, MA, USA). EIMS was recorded on JEOL JMS-SX/SX 102A mass spectrometer (Joel, Peabody, MA, USA). HRESIMS spectrum was recorded on a LTQ Orbitrap (Thermo Finnigan, Bremen, Germany). 1D and 2D NMR spectra were recorded on a Bruker DRX400 NMR spectrometer using standard Bruker software and C₅D₅N and CDCl₃ as solvents, with TMS as the internal reference (Bruker, Rheinstetten, Germany). Column chromatographic separations were performed on silica gel 60 (0.04–0.063 mm, Merck, Darmstadt, Germany). TLC was performed on precoated TLC plates with silica gel 60 F₂₅₄ (layer thickness 0.2 mm, Merck, Darmstadt, Germany). The chromatograms were developed using the following solvent systems: hexane:EtOAc (95:5, S₁) and hexane:EtOAc (90:10, S₂). The compounds were detected by spraying with p-anisaldehyde/H₂SO₄ reagent and heating at 110 ºC for 1–2 min.

Plant material

Seeds of Cucumis melo L. var. reticulates, Curcubitaceae, were obtained from the cultivated plants El-Galaa Village, Samalout, Minia, Egypt. The plant material was identified and authenticated (voucher specimen 2014-5) by Prof. Dr. Mohamed A. Farghali, Professor of Horticulture (Vegetable Crops), Faculty of Agriculture, Assiut University.

Extraction and isolation

Fruits were cut and seeds were removed from stringy piths. The seeds were rubbed by hand and washed quickly with tap water, then transferred to a colander and dried at room temperature. Dried seeds (200 g) were triturated in a ball mill and screened through a mesh of 0.5 mm diameter. The triturated seeds (175 g) were packed in a Soxhlet apparatus and defatted using hexane (3 × 1 l), then extracted with MeOH several times (4 × 1 l). The MeOH extract was evaporated and concentrated under reduced pressure to afford a dark brown residue (9.3 g). The latter was subjected to VLC (vacuum liquid chromatography) using hexane:EtOAc and EtOAc:MeOH gradients to afford five fractions: CA-I to CA-V; CA-I (2.6 g, hexane:EtOAc 75:25), CA-II (1.3 g, hexane:EtOAc 50:50), CA-III (2.1 g, hexane:EtOAc 25:75), CA-IV (0.7 g, EtOAc 100%), and CA-V (1.2 g, MeOH 100%). Fraction CA-I (2.6 g) was subjected to silica gel column chromatography (120 g × 50 × 2 cm) using hexane:EtOAc gradient to afford four subfractions CA-IA:CA-ID. Silica gel column chromatography (80 g × 50 × 2 cm) of subfraction CA-IB (0.55 g) using hexane:EtOAc as an eluent gave compound 1 (15 mg, white crystalline needles). Subfraction CA-IC (0.67 g) was chromatographed over silica gel column (90 g × 50 × 2 cm) using hexane:EtOAc gradient to yield compound 2 (10 mg, white crystals). Subfraction CA-ID was subjected to silica gel column using hexane:EtOAc (95:5–80:20) to afford compound 3 (7.5 mg, white needles).

Spectral data

Cucumol A (3): White needles (7.5 mg); mp 203–204 ºC; [α]_D ²² + 36.1 (C = 1.0, CHCl₃); UV (MeOH) λ_max (log ε): 258 (4.25) nm; IR (KBr) γ_max: 3435, 2982, 1610, 1070 cm⁻¹; NMR data (C₅D₅N, 400 MHz and 100 MHz), see Table 1: HRESIMS m/z 443.38840 (calcd for C₃₀H₅₁O₂ [M+H(proton)]⁺, 443.38836).

Table 1
NMR spectral data of compound 3 (C₅D₅N, 400 and 100 MHz).

No.	δH [mult., J (Hz)]	δC (mult.)	HMBC
1	1.51 m 1.47 m	37.7 (CH2)	–
2	2.05 m 1.83 m	28.0 (CH2)	3
3	3.42 dd (9.2, 6.6)	78.1 (CH)	5, 24
4	–	39.2 (C)	–
5	0.81 dd (9.6, 1.3)	55.9 (CH)	1, 4, 6, 24
6	1.53 m 1.34 m	19.1 (CH2)	–
7	1.92 m 1.89 m	41.6 (CH2)	14
8	–	39.4 (C)	–
9	1.42 m	49.5 (CH)	–
10	–	38.7 (C)	–
11	1.64 m 1.41 m	17.8 (CH2)	–
12	1.31 m 1.27 m	33.2 (CH2)	–
13	–	41.1 (C)	–
14	–	158.6 (C)	–
15	5.62 dd (10.6, 4.3)	116.8 (CH)	8, 13, 17
16	2.68 dd (13.8, 10.6) 1.86 dd (13.8, 4.3)	31.7 (CH2)	14, 15, 17, 18, 27, 15
17	–	41.1 (C)	–
18	0.70 dd (10.8, 4.8)	45.5 (CH)	14, 27
19	1.50 m 1.03 m	36.8 (CH2)	–
20	–	38.7 (C)
21	–	28.4 (CH2)	–
22	1.62 m 1.46 m	33.8 (CH2)	–
23	1.21 s	28.6 (CH3)	3, 4, 5, 24
24	1.05 s	16.4 (CH3)	3, 4, 5, 23, 25
25	0.92 s	15.6 (CH3)	1, 5, 9, 24
26	1.08 s	26.2 (CH3)	7, 9, 14
27	3.59 d (11.8) 3.44 d (11.8)	64.5 (CH2)	12, 14, 16, 18, 16
28	1.02 s	33.7 (CH3)	18, 19, 21
29	0.98 s	30.1 (CH3)	19, 21, 30
30	1.09 s	21.9 (CH3)	21, 22, 29
OH	5.96 brs	–	–
OH	5.75 brs	–	–

Cytotoxicity assay

The cytotoxic activity of compound 3 was examined towards mouse lymphoma (L5178Y), rat brain (PC12), and human cervix (Hela) cancer cell lines using MTT assay as described earlier (Mohamed, 2014; Mohamed et al., 2013). Exponentially growing cells were harvested, counted, and diluted appropriately. Of the cell suspension, 50 µl containing 3750 cells were pipetted into 96-well microtiter plates. Subsequently, 50 µl of a solution of the tested sample was added to each well. The test plates were incubated at 37 ºC with 5% CO₂ for 72 h. A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was prepared at 5 mg/ml in phosphate buffered saline (PBS; 1.5 mM KH₂PO₄, 6.5 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl; pH 7.4) and from this solution, 20 µl was pipetted into each well. The yellow MTT penetrates the healthy living cells and in the presence of mitochondrial dehydrogenases, MTT is transformed to its blue formazan complex. After an incubation period of 4 h at 37 ºC in a humidified incubator with 5% CO₂, the medium was centrifuged (15 min, 20 ºC, 210 × g) with 200 µl DMSO, the cells were lysed to liberate the formed formazan product. Cell viability was evaluated by measurement of the absorbance 520 nm using a scanning microtiter-well spectrophotometer. Compound concentrations that produce 50% cell growth inhibition (ED₅₀) were calculated from curves constructed by plotting cell survival (%) versus drug concentration (µg/ml). All experiments were carried out in triplicates and repeated three times. As negative controls, media with 0.1% (v/v) EtOH were included in all experiments. The paclitaxel was used as a positive control (Ibrahim et al., 2015c).

Results and discussion

Compound 3 was obtained as white needles. It gave a positive Liebermann-Burchard's test, indicating its triterpenoidal nature (Ibrahim et al., 2012; Sayed et al., 2007; Reinhold, 1935). Its HRESIMS spectrum showed a quasi-molecular ion peak at m/z 443.3884 [M+H]⁺, suggesting a molecular formula C₃₀H₅₀O₂, which implied six degrees of unsaturation. The IR spectrum showed absorption bands at 3435 and 1070 cm⁻¹ characteristic for the presence of a hydroxyl group (Silverstein and Webster, 1998; Al Musayeib et al., 2014). This was confirmed by the appearance of two exchangeable protons signals at δ_H 5.96 (1H, brs) and 5.75 (1H, brs) in ¹H NMR spectrum. ¹³C NMR and DEPT spectra of 3 revealed the presence of resonances for thirty carbons: seven methyls, eleven methylenes one of them is oxygen-bonded at δ_C 64.5 (C-27), five methines, and seven quaternary carbons. The ¹H and ¹³C NMR spectra exhibited seven methyl groups signals at δ_H 1.21 (H-23)/δ_C 28.6 (C-23), 1.05 (H-24)/16.4 (C-24), 0.92 (H-25)/15.6 (C-25), 1.08 (H-26)/26.2 (C-26), 1.02 (H-28)/33.7 (C-28), 0.98 (H₃-29)/30.1 (C-29), and 1.09 (H-30)/21.9 (C-30), suggesting a pentacyclic triterpenoidal nature of 3 (Mahato and Kundu, 1994; Laphookhieo et al., 2004; Al Muqarrabun et al., 2014). Moreover, signals for tri-substituted double bond at δ_H 5.62 (dd, J = 10.6, 4.3 Hz, H-15)/δ_C 116.8 (C-15) and 158.6 (C-14) were observed. Its position at C₁₄–C₁₅ was established by the ³ J HMBC cross peaks of H-7, H-16, and H-18 to C-14 and H-15 to C-8, C-13, and C-17. The Z geometry of the double bond was assigned based on the coupling constant value J_15,16ax = 10.6 and J_15,16eq 4.3 Hz (Ibrahim, 2014). The ¹H NMR spectrum revealed signals for an oxymethine at δ_H 3.42 (1H, dd, J = 9.2, 6.6 Hz, H-3) and oxymethylene at δ_H 3.59 and 3.44 (each d, J = 11.8 Hz, H-27), which correlated with the carbon signals resonating at δ_C 78.1 (C-3) and 64.5 (C-27), respectively in HMQC spectrum. The HMBC cross peaks of H-2, H-23, and H-24 to C-3, H-16 and H-18 to C-27, and H-27 to C-12, C-14, and C-18 established the positions of the oxymethine and oxyemthylene moieties at C-3 and C-27, respectively. Three methine proton signals at δ_H 0.81 (dd, J = 9.6, 1.3 Hz, H-5), 1.42 (m, H-9), and 0.70 (dd, J = 10.8, 4.8 Hz, H-18), which correlated with the carbon signals resonating at δ_C 55.9 (C-5), 49.5 (C-9), and 45.5 (C-18), respectively in HMQC spectrum were observed. Their assignment was established based on the observed correlation in the ¹H–¹H COSY and HMBC spectra (Fig. 1). On the basis of the above spectra evidence and by comparison with literature (Mahato and Kundu, 1994; Laphookhieo et al., 2004), the structure of compound 3 was established as 27-hydroxy taraxerol-3β-o1 and named cucumol A.

Fig. 1
Some key ¹H–¹H COSY and HMBC correlations of compound 3.

The known compounds were identified by analysis of the spectroscopic data (¹H, ¹³C NMR, COSY, and HMQC) and comparison of their data with those in the literature to be: α-spinasterol (2) (Ibrahim, 2014) and D:B-friedoolean-5-ene-3-β-ol (3) (Ibrahim, 2014).

The cytotoxic effect of compound 3 was tested towards L5178Y, PC12, and Hela cancer cell lines. Compound 3 was found to display cytotoxic activity towards L5178Y and Hela cancer cell lines with ED₅₀ values of 1.30 and 5.40 µg/ml, respectively compared to paclitaxel (0.07 and 0.92 µg/ml, respectively). While, it was inactive against PC12 cancer cell line.

Conclusion

A new triterpenoid: cucumol A (3) and three known compounds were isolated from C. melo seeds afforded. Their structures were established by different spectroscopic analyses. Compound 3 showed cytotoxic activity towards L5178Y and Hela cells.

• Ethical disclosures. Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.
  Confidentiality of data. The authors declare that no patient data appear in this article.
  Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Acknowledgments

We would like to express our deep thanks to Dr. Volker Brecht for acquiring NMR and MS spectroscopic data. We are grateful to Prof. Dr. W. E. G. Müller (Institute für Physiologische Chemie, Dues bergweg 6, D-55099 Mainz, Germany) for carrying out cytotoxicity assay.

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Publication Dates

• Publication in this collection
  Nov-Dec 2016

History

• Received
  31 Jan 2016
• Accepted
  2 Mar 2016