Cucumin S, a new phenylethyl chromone from Cucumis melo var. reticulatus seeds

ABSTRACT

A new phenylethyl chromenone, cucumin S [(R)-5,7-dihydroxy-2-[1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)ethyl]chromone] (1), along with five known compounds: 5,7-dihydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (2), 5,7-dihydroxy-2-[2-(3,4-dihydroxyphenyl)ethyl]chromone (3), luteolin (4), quercetin (5), and 7-glucosyloxy-5-hydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (6) were isolated from the EtOAc fraction of Cucumis melo var. reticulatus Ser., Cucurbitaceae, seeds. Their structures were determined by spectroscopic means (1D and 2D NMR), as well as HRESIMS, optical rotation measurement, and comparison with literature data. The isolated compounds 1–6 were assessed for their antioxidant activity using DPPH assay. Compounds 3, 4, and 5 showed potent activities compared to propyl gallate at concentration 100 µM.

Keywords:
Cucumis melo ; Cucurbitaceae; Chromone; Flavonoid; Antioxidant

Introduction

Family Cucurbitaceae includes pumpkins, squash, musk melons, watermelons, cucumbers, and grounds, which are known as vine crops. Cucurbit fruits are significant source of dietary fibers, beta-carotene (pro-vitamin A), minerals (potassium), and vitamin C (Fleshman et al., 2011; Adams and Richardson, 1981). Naturally, they are low in sodium, fat, and cholesterol (Lester, 1997). Cucumis melo var. reticulatusSer., Cucurbitaceae (netted muskmelon or cantaloupe) is one of the most cultivated cucurbits. It is an orange-fleshed, sweet, and aromatic melon that is highly popular for its nutritive and medicinal properties. It is a worm-season crop grows in all tropical and subtropical regions of the world. C. melo has potential in the treatment of pain, inflammation, cough, dysuria, diabetes, liver diseases, and cardiovascular disorders (Ibrahim, 2010, 2014). It exhibited antioxidant, antimicrobial, and anti-inflammatory activities (Ibrahim, 2010, 2014; Vouldoukis et al., 2004). Previous studies on C. melo L. led to isolation of hydrocarbons (Velcheva and Donchev, 1997), peptides (Ribeiro et al., 2007), fatty acids, volatile sesquiterpenes (Lewinsohn et al., 2008; Portnoy et al., 2008), phenylethyl chromone derivatives, triterpenes, sterols, and one triacylglyceride (Ibrahim, 2010, 2014). In the present work, we reported the isolation and structural characterization of a new phenylethyl chromone derivative: (R)-5,7-dihydroxy-2-[1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)ethyl]chromone (1), along with five known compounds from the EtOAc fraction of C. melo seeds. The antioxidant activity of compounds 1–6 was evaluated using DPPH assay.

Materials and methods

General experimental procedures

Melting points were 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. Shimadzu 1601 UV/VIS and Shimadzu Infrared-400 (Shimadzu, Kyoto, Japan) spectrophotometers were used to measure the Ultraviolet (UV) and Infrared (IR) spectra, respectively. HRESIMS spectra were measured with a Micromass Qtof 2 mass spectrometer (ThermoFinnigan, Bremen, Germany). Bruker Avance DRX 500 and 700 (Bruker BioSpin, Billerica, MA, USA) were used to record NMR spectra. Chromatographic separation was achieved using RP₁₈ (0.04–0.063 mm), silica gel 60 (0.04–0.063 mm), and Sephadex LH-20 (0.25–0.1 mm) (Merck, Darmstadt, Germany). TLC analysis was performed on pre-coated silica gel 60 F₂₅₄TLC plates (0.2 mm, Merck, Darmstadt, Germany). The compounds were detected by UV absorption at λ_max 255 and 366 nm followed by spraying with p-anisaldehyde/H₂SO₄ reagent and heating at 110 °C for 1–2 min. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and propyl gallate were purchased from Sigma Chemical Co. (Taufkirchen, Germany).

Plant material

Seeds of Cucumis melo var. reticulatus Ser., Cucurbitaceae, were obtained from cultivated plants at Mankabad, Assiut, Egypt. Plant material was identified and authenticated (voucher specimen 2013-5) by Prof. Dr. Mohamed A. Farghali, Professor of Horticulture (Vegetable Crops), Faculty of Agriculture, Assiut University.

Extraction and isolation

The dried seeds (263 g) were defatted in a Soxhlet apparatus using n-hexane (3 × 1 l), then extracted with MeOH (4 × 2 l). The MeOH extract was concentrated under reduced pressure to afford a dark brown residue (18.9 g). The latter was subjected to silica gel vacuum liquid chromatography (VLC) using n-hexane and EtOAc to afford 12.6 and 2.9 g, respectively. The EtOAc fraction was subjected to silica gel VLC using n-hexane:EtOAc gradient to give five subfractions: C-1 (0.93 g, n-hexane:EtOAc 8:2), C-2 (0.41 g, n-hexane:EtOAc 6:4), C-3 (0.48 g, n-hexane:EtOAc 4:6), C-4 (0.24 g, n-hexane:EtOAc 2:8), and C-5 (0.51 g, EtOAc). Subfraction C-2 (0.41 g) was chromatographed over silica gel column (80 g × 50 × 3 cm) using n-hexane:EtOAc gradient to obtain 1 (4.9 mg, yellow crystals). Silica gel column chromatography (70 g × 50 × 3 cm) of subfraction C-3 (0.48 g) using n-hexane:EtOAc gradient afforded 2 (15 mg, yellow crystals) and 3 (9 mg, yellow crystals). Subfraction C-4 (0.24 g) was chromatographed over silica gel column (50 g × 30 × 2 cm) using n-hexane:EtOAc gradient to yield 4 (6.2 mg, yellow powder) and 5 (11 mg, yellow powder). Compound 6 (8.1 mg, yellow powder) was isolated from subfraction C-5 (0.51 g) through silica gel column (50 g × 50 × 2 cm) using CHCl₃:MeOH gradient elution.

Antioxidant activity

The antioxidant activity of compounds 1–6 was evaluated using 2,2′-diphenylpicrylhydrazyl (DPPH) assay as previously outlined (Mohamed et al., 2013,2014). The decrease in the absorption of each compound (100 µM) in DPPH solution was monitored at 517 nm using a spectrophotometer. The absorbance of DPPH in MeOH (with or without compounds) was measured after 2 min. The antioxidant activity of each compound was measured in relation to propyl gallate (a standard antioxidant). Determinations were performed in triplicate. The % activity was calculated by the following equation:

Spectral data

Cucumin S (1). Yellow crystals, mp 115–116 °C; [α]²⁵_D + 51.6 (c 0.5, CHCl₃), + 49.7 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 255 (3.89), 352 (3.15) nm; IR (KBr) ν_max 3445, 2978, 1655, 1620, 1584, 1065 cm⁻¹; NMR data (CD₃OD, 500 and 125 MHz), see Table 1; HRESIMS m/z 345.0976 [M+H]⁺ (calc. for C₁₈H₁₇O₇, 345.0974).

Table 1
NMR spectral data of compound 1(CD₃OD).

No.	δH [mult., J (Hz)]a	δC (mult.)a	HMBCb
2	-	168.6 (C)	-
3	6.47 s	110.6 (CH)	2, 4, 10, 8'
4	-	181.7 (C)	-
5	-	161.5 (C)	-
6	6.06 d (2.5)	98.2 (CH)	5, 7, 8, 10
7	-	164.2 (C)	-
8	6.24 d (2.5)	95.8 (CH)	7, 9, 10
9	-	158.6 (C)	-
10	-	102.3 (C)	-
1'	-	129.6 (C)	-
2'	7.28 d (2.5)	115.6 (CH)	1', 4', 6', 7'
3'	-	149.0 (C)	-
4'	-	145.9 (C)	-
5'	6.78 d (7.5)	117.8 (CH)	1', 3', 4', 6'
6'	7.23 dd (7.5, 2.5)	119.8 (CH)	1', 4', 7'
7'	3.26 dd (14.5, 6.0)	40.6 (CH2)	2, 1', 2', 6', 8'
	2.73 dd (14.5, 3.5)
8'	5.91 dd (6.0, 3.5)	85.8 (CH)	2, 3, 1'
3'-OCH3	3.87 s	57.3 (CH3)	3'

• ^a
  Measured at 500 and 125 MHz.
• ^b
  Measured at 700 MHz.

Results and discussion

Compound 1 was obtained as yellow crystals. Its molecular formula C₁₈H₁₆O₇ was determined from the HRESIMS (positive ion-mode): m/z 345.0976 [M+H]⁺ (calc. for C₁₈H₁₇O₇, 345.0974). The UV absorption maxima at 255 and 352 nm and the IR absorption bands at 1655, 1620, and 1584 cm⁻¹ suggested the presence of a chromone moiety in 1 (Ibrahim, 2010, 2014). The IR spectrum showed a band at 3445 cm⁻¹ characteristic for hydroxyl groups. The ¹H NMR spectrum (Table 1) showed signals for an olefinic proton at δ_H 6.47 (1H, s, H-3) and two meta-coupled protons for 1,2,3,5-tetrasubstituted phenyl moiety at δ_H 6.24 (1H, d, J = 2.5 Hz, H-8) and 6.06 (1H, d, J = 2.5 Hz, H-6). They correlated to the carbon signals at δ_C 110.6, 95.8, and 98.2, respectively in the HSQC spectrum. Furthermore, the ¹H and ¹³C NMR spectra displayed diastereotopic methylene protons at δ_H 3.26 (dd, J= 14.5, 6.0 Hz, H-7′a) and 2.73 (dd, J = 14.5, 3.5 Hz, H-7′b)/δ_C 40.6 (C-7′) and an oxymethine at δ_H5.91 (dd, J = 6.0, 3.5 Hz, H-8′)/δ_C 85.8 (C-8′), indicating the presence of an AMX spin system. It was confirmed by the observed ¹H-¹H COSY and HMBC correlations (Fig. 1). The oxymethine was located at C-8′ based on the HMBC cross peaks of H-8′ to C-2, C-3, and C-1′ and absence of cross peaks with C-2′ and C-6′. Also, the presence of a trisubstituted phenylethyl moiety was evident by the signals at δ_H 7.23 (dd, J = 7.5, 2.5 Hz, H-6′)/δ_C 119.8 (C-6′), 7.28 (d, J = 2.5 Hz, H-2′)/115.6 (C-2′), and 6.78 (d, J = 7.5 Hz, H-5′)/117.8 (C-5′). This moiety was confirmed by the ¹H-¹H COSY and HMBC cross peaks. Moreover, the singlet signal at δ_H 3.87, correlating with the carbon signal at δ_C 57.3 in the HSQC experiment was assigned to a methoxyl group. The ¹³C NMR and HSQC spectra of 1 revealed the presence of 18 carbons: one methoxyl, one methylene, seven methines, and nine quaternary carbons including carbonyl carbon at δ_C 181.7 (C-4). The connectivity of the phenylethyl moiety at C-2 of the chromone ring was secured through the HMBC correlations of H-7′ to C-2 and H-8′ to C-2 and C-3 (Fig. 1). The HMBC cross peaks of H-8 to C-7 (δ_C 164.2), C-9 (δ_C 158.6), and C-10 (δ_C 102.3), H-6 to C-5 (δ_C 161.5), C-7 (δ_C 164.2), C-8 (δ_C 95.8), and C-10 (δ_C 102.3), H-5′ to C-3′ (δ_C 149.0), and H-2′ and H-6′ to C-4′ (δ_C 145.9) supported the presence of hydroxyl groups at C-5, C-7, and C-4′. The HMBC correlation of the methoxy group at δ_H 3.87 to the carbon at δ_C 149.0 established its attachment at C-3′. Due to the scarcity of the compound, the relative configuration at C-8′ was assigned to be R-form based on the comparison ¹H chemical shift and coupling constant of H-8′ as well as optical rotation of 1 with those of series of analogous compounds (Bernhardt et al., 2000; Kim et al., 2010; Park et al., 2010; Ibrahim et al., 2014). On the basis of these findings, 1 was identified as (R)-5,7-dihydroxy-2-[1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)ethyl]chromone and named cucumin S.

Fig. 1
Some important HMBC correlations of compound 1.

The known compounds were identified by analysis of the spectroscopic data (¹H and ¹³C NMR) and comparison of their data with those in the literature, in addition to co- chromatography with authentic samples to be: 5,7-dihydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (2) (Ibrahim, 2010; Ibrahim and Mohamed, 2014), 5,7-dihydroxy-2-[2-(3,4-dihydroxyphenyl)ethyl]chromone (3) (Ibrahim, 2010; Ibrahim and Mohamed, 2014), luteolin (4) (Harborne, 1988; Mohamed, 2008), quercetin (5) (Harborne, 1988; Mohamed, 2008), and 7-glucosyloxy-5-hydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (6) (Ibrahim, 2010).

DPPH radical is widely used as a model system to investigate the scavenging activity of several natural compounds. DPPH is scavenged by the antioxidants through the donation of proton forming the reduced DPPH which can be quantified by its decrease of absorbance.

Compounds 1–6 showed a concentration dependent scavenging activity by quenching DPPH radical. They exhibited maximum inhibition of DPPH free radical that ranged from 91.24 to 56.07% (Table 2). Their antioxidant effects were related to the number of free hydroxyl groups especially that located at C-3′ and C-4′ in their structures. Compounds 3, 4, and 5 showed potent activities compared to propyl gallate at the same concentration. However, absence or blocking of the hydroxyl group by a methyl or glucose moiety leads to a decrease in the activity as in 1, 2, and 6 (Dugas et al., 2000; Al-Musayeib et al., 2014).

Table 2
The DPPH radical scavenging activity results.

Sample*	Inhibition%
PG	97.39 ± 1.8
1	63.12 ± 1.6
2	67.09 ± 1.8
3	86.20 ± 0.9
4	87.16 ± 0.7
5	91.24 ± 0.8
6	56.07 ± 1.2

• ^*
  Conc. (100 µM); Each value represents the mean ± S.D., n = 3.

References

• Adams, C.F., Richardson, M., 1981. Nutritive value of foods. USDA Home and Garden Bul. 72. Government Printing Office, Washington DC, USA, pp. A2-A35.
• Al-Musayeib, N.M., Mohamed, G.A., Ibrahim, S.R.M., Ross, S.A., 2014. New thiophene and flavonoid from Tagetes minuta leaves growing in Saudi Arabia. Molecules 19, 2819-2828.
• Bernhardt, M., Shaker, K.H., Elgamal, M.H.A., Seifert, K., 2000. The new bishomoflavone ononin and its glucoside from Ononis vaginalis Z. Naturforsch. C 55, 516-519.
• Dugas, A.J.J., Castañeda-Acosta, J., Bonin, G.C., Price, K.L., Fischer, N.H., Winston, G.W., 2000. Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure–activity relationships. J. Nat. Prod. 63, 327-331.
• Fleshman, M.K., Lester, G.E., Riedl, K.M., Kopec, R.E., Narayanasamy, S., Curley Jr., R.W., Schwartz, S.J., Harrison, E.H., 2011. Carotene and novel apocarotenoid concentrations in orange-fleshed Cucumis melo melons: determinations of β-carotene bioaccessibility and bioavailability. J. Agric. Food Chem. 59, 4448-4454.
• Harborne, J.B., 1988. The Flavonoids. Advances in Research Since 1980. Chapman & Hall, New York.
• Ibrahim, S.R.M., Fouad, M.A., Abdel-Lateff, A., Okino, T., Mohamed, G.A., 2014. Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica Nat. Prod. Res. 28, 1765-1771.
• Ibrahim, S.R.M., 2010. New 2-(2-phenylethyl)chromone derivatives from the seeds of Cucumis melo L var. reticulatus Nat. Prod. Commun. 5, 403-406.
• Ibrahim, S.R.M., 2014. New chromone and triglyceride from Cucumis melo seeds. Nat. Prod. Commun. 9, 205-208.
• Ibrahim, S.R.M., Mohamed, G.A., 2014. Natural occurring 2-(2-phenylethyl) chromones, structure elucidation and biological activities. Nat. Prod. Res., http://dx.doi.org/10.1080/14786419.2014.991323.
  » http://dx.doi.org/10.1080/14786419.2014.991323
• Kim, J.M., Ko, R.K., Jung, D.S., Kim, S.S., Lee, N.H., 2010. Tyrosinase inhibitory constituents from the stems of Maackia fauriei Phytother. Res. 24, 70-75.
• Lester, G., 1997. Melon (Cucumis melo L.) fruit nutritional quality and health functionality. HortTechnology 7, 222-227.
• Lewinsohn, E., Portnoy, V., Benyamini, Y., Bar, E., Harel-Beza, R., Gepstein, S., Giovannoni, J.J., Schaffer, A.A., Burger, Y., Tadmor, Y., Katzir, N., 2008. Sesquiterpene biosynthesis in melon (Cucumis melo L.) rinds. In: Pitrat, M. (Ed.), Cucurbitaceae Proceedings of the IXth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae. INRA, Avignon, France, pp. 249–255.
• Mohamed, A.G., 2008. Alliuocide G, a new flavonoid with potent α-amylase inhibitory activity from Allium cepa L. ARKIVOC xi, 202-209.
• Mohamed, G.A., Ibrahim, S.R.M., Ross, S.A., 2013. New ceramides and isoflavone from the Egyptian Iris germanica L. rhizomes. Phytochem. Lett. 6, 340-344.
• Mohamed, G.A., Ibrahim, S.R.M., Al-Musayeib, N.M., Ross, S.A., 2014. New anti-inflammatory flavonoids from Cadaba glandulosa Forssk. Arch. Pharm. Res. 37, 459-466.
• Park, D., Kim, H.J., Jung, S.Y., Yook, C., Jin, C., Lee, Y.S., 2010. A new diarylheptanoid glycoside from the stem bark of Alnus hirsuta and protective effects of diarylheptanoid derivatives in human HepG2 cells. Chem. Pharm. Bull. 58, 238-241.
• Portnoy, V., Benyamini, Y., Bar, E., Harel-Beza, R., Gepstein, S., Giovannoni, J.J., Schaffer, A.A., Burger, Y., Tadmor, Y., Lewinsohn, E., Katzir, N., 2008. The molecular and biochemical basis for varietal variation in sesquiterpene content in melon (Cucumis melo L.) rinds. Plant Mol. Biol. 66, 647-661.
• Ribeiro, S.F.F., Agizzio, A.P., Machado, O.L.T., Neves-Ferreira, A.G.C., Oliveira, M.A., Fernandes, K.V.S., Carvalho, A.O., Perales, J., Gomes, V.M.A., 2007. A new peptide of melon seeds which shows sequence homology with vicilin: partial characterization and antifungal activity. Sci. Hortic. 111, 399-405.
• Velcheva, M.P., Donchev, C., 1997. Isoprenoid hydrocarbons from the fruit of extant plants. Phytochemistry 45, 637-639.
• Vouldoukis, I., Lacan, D., Kamate, C., Coste, P., Calenda, A., Mazier, D., Conti, M., Dugas, B., 2004. Antioxidant and anti-inflammatory properties of Cucumis melo LC. Extract rich in superoxide dismutase activity. J. Ethnopharmacol. 94, 67-75.

Publication Dates

• Publication in this collection
  Oct 2015

History

• Received
  29 May 2015
• Accepted
  30 June 2015