A New Prenylated Isoflavonoid From Limonium leptophyllum

Abstract

Phytochemical investigation of methanolic extract of Limonium leptophyllum (Plumbaginaceae), led to the isolation of 1 new isoflavonoid with a rare 5-membered dihydrofuran ring (1, leptoisoflavone A) and 8 known compounds. The known isolated compounds were identified as euchrenone b₉ (2), auriculasin (3), kaempferol (4), avicularoside (5), myricetin-3-arabinoside (6), trans-N-feruloyltyramine (7), trans-N-caffeoyltyramine (8), and β-sitosterol (9). The crude methanolic extract exhibited moderate activity toward endocannabinoid receptors. Auriculasin (3) showed activity toward cannabinoid receptor type 1 (86.7% displacement with IC₅₀ 8.92 μM).

The genus Limonium (Plumbaginaceae family) is known as halophytes and consists of 300 species worldwide, of which 18 are found in Kazakhstan. ¹ Species in this genus are also known as statice, caspia, sea-lavender, or marsh-rosemary. The genus is widely distributed in Asia, Africa, Australia, Europe, and North America. More recently, several species of the Limonium genus are used to treat arthritis and fever. ² This genus possesses anti-inflammatory, antibacterial, ^3,4 antiviral, ⁵ and cytotoxic properties. ³ Previous phytochemical and biological studies on the Limonium genus have shown the presence of different classes of compounds with various biological activities. ^6–14 In continuation of our previous work and search for novel plant-derived biological agents, ^6,7,15–17 further investigations on the phytochemical constituents of Limonium leptophyllum (Schrenk) O. Kuntze were carried out. Herein, we are reporting the isolation and characterization of 1 new and 8 known compounds from L. leptophyllum.

Compound 1 was obtained as a yellow solid. The high-resolution mass spectrometric (MS) data showed a peak corresponding to the molecular formula C₂₅H₂₂O₅, based on the [M + H]⁺ ion signal at m/z 403.1532 (calc. 403.1545). The ¹³C nuclear magnetic resonance (NMR) data of 1 (Table 1) showed resonances of 25 carbon atoms, which were classified by distortionless enhancement by polarization transfer 135 and heteronuclear single quantum coherence (HSQC) experiments as 3 methyls, 2 methylenes, 8 methines, and 12 quaternary carbons. The ¹H and ¹³C NMR spectra (Table 1) showed resonances of 3 methyls [δ _H/δ _C 1.48/28.3, 1.52/28.5, and 1.92/19.9 (C-5′′′, 4′′′, and 5′′)], methylene [δ _H/δ _C 3.16 (dd, J = 9.0, 14.0 Hz), 3.03 (dd, J = 4.5, 14.0 Hz)/23.4 (C-1′′)], an oxymethine proton [δ _H/δ _C 4.45 (dd, J = 4.5, 9.0 Hz)/86.6 (C-2′′)], olefinic methylene [δ _H/δ _C 5.01 (s)/113.1 (C-4′′)], a pair of olefinic protons [δ _H/δ _C 5.61 (d, J = 10.0 Hz)/127.3, 6.71 (d, J = 10.0 Hz)/115.0, (C-2′′′, 1′′′)], a set of A₂B₂/ortho coupled protons of 1,4-disubstituted aromatic ring [δ _H/δ _C 6.90 (d, J = 8.5 Hz)/115.7 (C-3′, 5′), and 7.39 (d, J = 8.5 Hz)/130.5 (C-2′, 6′)], and an oxygenated aromatic proton [δ _H/δ _C 7.90 (s)/152.7 (C-2)]. Furthermore, the ¹³C NMR showed characteristic resonances attributed to an isoflavonoid skeleton of the conjugated oxo group [δ _C 181.2 (C-4)], 5 nonprotonated aromatic carbons [δ _C 101.1, 105.6, 109.6, 123.7, and 127.3 (C-8, 4a, 6, 1′, and 3)], 4 oxygenated aromatic carbons [δ _C 159.9, 157.5, 156.1, and 151.1 (C-5, 7, 4′, and 8a)], nonprotonated olefinic carbon [δ _C 144.4 (C-3′′)], and nonprotonated oxygenated carbon [δ _C 78.8 (C-3′′′)]. The assignments of ¹H and ¹³C NMR spectroscopic data of 1 (Table 1) were based on HSQC, heteronuclear multiple bond correlation (HMBC) correlations, and ¹H-¹H correlated spectroscopy (COSY) couplings (Figure 1). The HMBC spectrum of compound 1 showed the following key ² J and ³ J correlations (Figure 1): H-4′′′ and H-5′′′ exhibited correlation with C-2′′′, C-3′′′, and with each other, which confirmed the attachment of methyls to C-3′′′. The methyl (H-5′′) in the isoprenyl moiety showed correlations with C-2′′, 3′′, and 4′′. Compound 1 showed similarities with the known compound 2 with observed variation at C-2′′ (δ _C 86.6 for compound 1 whereas δ _C 76.1 for compound 2), which was similar to δ _C 89.5, observed in lachnoisoflavones A, ¹⁸ which suggested the cyclization in compound 1. It was also confirmed by comparing the oxo carbon (C-4) value of compound 1 (δ _C 181.2) with lachnoisoflavone A (182.9 ppm). Correlated spectroscopy spectrum showed the following correlations (Figure 1): δ _H 7.39 (H-2′, 6′) with δ _H 6.90 (H-3′, 5′), δ _H 6.71 (H-1′′′) with δ _H 5.61 (H-2′′′), 4.45 (H-2′′) with δ _H 3.16 (H-1′′a) and δ _H 3.03(H-1′′b). Based on spectral data, compound 1 is identified to be a rare new natural pentacyclic isoflavonoid including a rare 5-membered dihydrofuran ring between C-5 and C-6. Finally, compound 1 was elucidated as 10-(4-hydroxyphenyl)-5,5-dimethyl-2-(prop-1-en-2-yl)-2,3-dihydro-5H,11H-furo[2,3 f]pyrano[2,3 h]chromen-11-one and named as leptoisoflavone A.

Table 1

¹H and ¹³C NMR Data for Compounds 1 and 2 in CDCl₃ (δ _Cand δ _H in parts per minute; J in Hertz).

Position	Leptoisoflavone A (1)		Euchrenone b9 (2)
	1H NMR a	13C NMR b	1H NMR a	13C NMR b
2	7.90 (s)	152.7 (D)	7.87 (s)	152.58 (D)
3	-	127.3 (S)	-	123.05 (S)
4	-	181.2 (S)	-	181.10 (S)
4a	-	105.6 (S)	-	105.7 (S)
5	-	159.9 (S)	-	160.09 (S)
6	-	109.6 (S)	-	110.03 (S)
7	-	157.5 (S)	-	157.44 (S)
8	-	101.1 (S)	-	100.99 (S)
8a	-	151.1 (S)	-	150.8 (S)
1′	-	123.7 (S)	-	123.63 (S)
2′	7.39 (d, 8.5)	130.5 (D)	7.37 (d, 8.0)	130.39 (D)
3′	6.90 (d, 8.5)	115.7 (D)	6.85 (d, 8.0)	115.70 (D)
4′	-	156.1 (S)	-	156.16 (S)
5′	6.90 (d, 8.5)	115.7 (D)	6.85 (d, 8.0)	115.70 (D)
6′	7.39 (d, 8.5)	130.5 (D)	7.37 (d, 8.0)	130.39 (D)
1′′	3.16 (dd, 9.0, 14.0)	23.4 (T)	2.90 (dd, 9.2, 14.0)	29.20 (T)
	3.03 (dd, 4.5, 14.0)		3.03 (dd, 3.6, 14.0)
2′′	4.45 (dd, 4.5, 9.0)	86.6 (D)	4.30 (dd, 3.6, 9.2)	76.14 (D)
3′′	-	144.4 (S)	-	147.57 (S)
4′′	5.01 (s)	113.1 (T)	4.85 (s), 5.03 (s)	110.40 (T)
5′′	1.92 (s)	19.9 (Q)	1.88 (s)	18.39 (Q)
1′′′	6.71 (d, 10.0)	115.0 (D)	5.60 (d, 10.0)	127.22 (D)
2′′′	5.61 (d, 10.0)	127.3 (D)	6.71 (d, 10.0)	114.99 (D)
3′′′	-	78.8 (S)	-	78.60 (S)
4′′′	1.52 (s)	28.5 (Q)	1.51 (s)	28.57 (Q)
5′′′	1.48 (s)	28.3 (Q)	1.45 (s)	28.37 (Q)

NMR, nuclearmagnetic resonance.

Recorded at ^a500 MHz, ^b125MHz.

Figure 1

Key HMBC and COSY correlations of compound 1.

The known isolated compounds (Figure 2) were identified as euchrenone b₉ (2), ¹⁹ auriculasin (3), ²⁰ kaempferol (4), avicularoside (5), ²¹ myricetin-3-arabinoside (6), ²² trans-N-feruloyltyramine (7), trans-N-caffeoyltyramine (8), ²³ and β-sitosterol (9).

Figure 2

Compounds isolated from L. leptophyllum.

Crude methanolic extract of aerial parts of L. leptophyllum exhibited moderate activity toward endocannabinoid receptors (cannabinoid receptor type 1 [CB1] and CB2). Auriculasin (3) showed activity toward CB1 receptor (86.7% displacement with IC₅₀ 8.92 µM). ¹⁵

Experimental

General

Bruker model AMX 500 NMR and 400 NMR spectrometers operating on a standard pulse system were used to acquire ¹H and ¹³C NMR and 2D spectra. The instruments ran at 500 and 400 MHz for ¹H whereas they ran at 125 and 100 MHz for ¹³C. CDCl₃, dimethyl sulfoxide-d ₆, and acetone-d ₆ were used as NMR solvents, and trimethyl silane was used as an internal standard. Electrospray ionizationmass spectrometric (ESI-MS) data were recorded on Thermo Orbitrap Fusion (Thermo Scientific). Samples were analyzed in the positive mode of ionization and were directly infused at 3 µL/min. Mass was analyzed in Orbitrap (mass error on the instrument <2 ppm). Electrospray ionization-MS data were obtained on a Micromass Q-Tof micromass spectrometer. Fourier transform mass spectrometry-ESI was analyzed on Thermo Orbitrap Fusion (Thermo Scientific). The sample was analyzed in the positive mode of ionization. Thin-layer chromatography was performed on precoated silica gel GF254 plates and column chromatography was performed on silica gel (200-300 mesh) and Sorbadex-LH20 (Sorbent Technologies, Atlanta, GA, USA) with detection provided by UV light (254 and 366 nm) and by spraying with 1% vanillin-H₂SO₄ reagent followed by heating for 5 to 10 minutes (105°C). Quantitative high-performance liquid chromatography (HPLC) was conducted using an Agilent 1100 HPLC system equipped with a degasser (G1379A), quaternary pump (G13311A), auto sampler (G1313A), column oven (G1316A), and UV-diode array detector (G1315B) controlled by Chemstation software. The analysis was carried out on RP-C18 columns (150 × 4.6 mm; particle size 5 µm; Luna) and (250 × 10.0 mm; particle size 10 µm; Luna) with column oven temperature set at 25°C and using the isocratic system of eluent water (A) and acetonitrile (B) for the separation of the target compounds. Acetonitrile and water solvents were of HPLC grade, where acetic acid was added as a modifier to achieve a final concentration of 0.1% in each solvent. The flow rates of the solvent were 1.0 mL/min for the analytical injections whereas 5 mL/min for the semipreparative ones and the injection volumes were 5.0 and 50 µL for the analytical and semipreparatives, respectively. All the analysis was carried out at wavelengths of 254 and 280 nm with a run time of 40 minutes.

Plant Material

Limonium leptophyllum (Schrenk) O. Kuntze aerial parts were collected in August 2016, from Kyzylorda region, Kazakhstan. The plant material was authenticated by Akhtaeva Nursulu and the specimen voucher (No.0916.) was stored at the department of biology and biotechnology of al-Farabi Kazakh National University.

Extraction and Isolation

Limonium leptophyllum aerial parts (830 g) were extracted using MeOH (3 L × 3 times); the extract was thereafter filtered and concentrated on a rotary evaporator at 40°C yielding 90.3 g of crude methanolic extract. This extract (50 g) was fractionated over HP 20 gel (200 g) column using H₂O-methanol (1:9), methanol, acetone, and dichloromethane (DCM) (each 750 mL). Each fraction was evaporated to dryness to give 16.47 g fraction A (H₂O:methanol 1:9), 7.87 g fraction B (methanol), 11.25 g fraction C (acetone), and 1.17 g fraction D (DCM).

Fractions C and D were combined and the mixture (E, 12 g) was loaded onto a normal phase silica gel vacuum liquid column (210 g) and eluted using hexane-acetone gradient to yield 12 fractions (E1-E12) and continued with methanol-DCM gradient to yield 10 more fractions (E13-E22). Fraction E5 was identified as β-sitosterol (9, 20 mg). Fraction E13 was identified as kaempferol (4, 10 mg). Fractions E5-E8 were combined together and purified on HPLC using water-acetonitrile gradient 60:40 to 20:80 over a period of 20 minutes, to yield euchrenone b₉ (2, 2.5 mg, RT 13.1 min) and compound 1 (2.1 mg, RT 14.6 min). Fractions E11 and 12 were combined (497 mg) and purified over silica gel column to afford auriculasin (3, 25 mg). Combined fractions E20-22 (5 g) were loaded on silica gel and purified using methanol-DCM gradient system to afford avicularoside (5, 10 mg), myricetin-3-arabinoside (6, 15 mg), trans-N-feruloyltyramine (7, 5 mg), and trans-N-caffeoyltyramine (8, 8 mg).

Leptoisoflavone a (1)

Yellow solid.

[α]_D ^22.5: –3.125 (c 0.064, MeOH).

¹H and ¹³C NMR: Table 1.

High-resolution ESI-MS: m/z 403.1532 [M + H]⁺ (calc. C₂₅H₂₂O₅ + H, 403.1545).

Cannabinoid and Opioid Receptor Assays

The affinity of the total extracts, fractions, and isolated compounds toward cannabinoid and opioid receptors was analyzed according to the published method. ²⁴ The crude methanolic extract exhibited moderate activity toward endocannabinoid receptors. Compound 3 showed activity toward CB1 receptor.