Sepicanin A- A new geranyl flavanone from Artocarpus sepicanus with activity against methicillin-resistant Staphylococcus aureus (MRSA) (2025)

Abstract

Bioassay–guided fractionation of the EtOH extract of Artocarpus sepicanus Diels leaves has led to the isolation of a new geranyl flavanone (1), along with the known compounds, afzelechin-3-O-α-L-rhamnopyranoside and β-sitosterol glucoside. The structure of the new compound was established by UV, IR, HRESIMS, 1D and 2D NMR experiments. Antimicrobial testing of the three compounds indicated that 1 displayed a significant selective antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with IC₅₀ and MIC values of 1.4 and 2.9 μM, respectively.

Keywords: Flavanone, Artocarpus sepicanus, antibacterial, Methicillin-resistant Staphylococcus aureus

Graphical Abstract

1. Introduction

The genus Artocarpus (Moraceae) consists of approximately 50 species of large evergreen trees (Kijjoa et al., 1996). Many prenylated stilbenes (Hakim et al., 2002; Likhitwitayawuid et al., 2001; Puntumchai et al., 2004; Su et al., 2002), benzofurans (Hakim et al., 2002a, 2002b; Soekamto et al., 2003), flavonoids (Hakim et al. 2002a, Kijjoa et al. 1996; Ko et al. 2005; Suhartati et al. 2001) and chalcones (Jayasinghe et al. 2004; Shimizu et al. 2000) have been reported from various Artocarpus species. Several prenylated stilbenes and flavonoids showed cytotoxic (Hakim at al. 2002, Kijjoa at al. 1996, Ko et al. 2005, Suhartati et al. 2001), anti-mycobacterial (Puntumchai et al., 2004), and anti-inflammatory (Su et al. 2002; Wei at al. 2005) activities. As part of our ongoing research to investigate the antimicrobial activity of higher plants (Galal et al. 2001; Radwan at al. 2008; Ross et al. 2005, 2008) the ethanol extract of Artocarpus sepicanus leaves was evaluated for antimicrobial activities. Because this species has not been evaluated phytochemically, we decided to undertake a biologically-guided fractionation, which led to the isolation of a new geranyl flavanone (1), with an IC₅₀ value of 1.4 μM against methicillin-resistant Staphylococcus aureus (MRSA), along with two known compounds, afzelechin-3-O-rhamnopyranoside and β-sitosterol glucoside.

2. Results and Discussions

Compound 1 was isolated as a yellow powder. The HRESIMS (positive ion mode) showed [M+H] ⁺ at m/z 425.1931 and [M+Na] ⁺ at m/z 447.1750 compatible with a molecular formula of C₂₅H₂₈O₆. The IR spectrum showed that 1 contained hydroxyl (ν_max 3336 cm⁻¹) and conjugated ketone (ν_max 1635 cm⁻¹) functional groups. The UV absorption bands at λ_max 213 and 290 nm were suggestive of a flavanone skeleton (Mabry et al. 1970). A bathochromic shift (36 nm) produced in the UV spectrum (methanol) after the addition of NaOAc indicated a free hydroxyl group at C-7, while its bathochromic shift on the addition of AlCl₃ and AlCl₃/HCl (20 nm) suggested the presence of an hydroxyl group at C-5 which was supported by the presence of a hydrogen bonded proton at δ 12.1 in ¹H NMR spectrum (Mabry et al. 1970). The ¹H NMR spectroscopic data of 1 (Table 1) displayed a 1,2,4-trisubstituted benzene ring B [δ 7.36 ( 1H, d, J=8.4 Hz, H-6′), δ 6.43 (1H, dd, J=8.4, 2.0 Hz, H-5′) and δ 6.49 (1H, d, J=2.0, H-3′)], with two phenol groups at C-2′ (δ_C 158.5) and C-4′ (δ_C 155.3) along with one proton singlet at δ_H 6.06 which was assigned to H-8 and correlated to δ_C 95.5 in the HMQC spectrum. The ¹³C NMR, DEPT and HMQC NMR spectroscopic data of 1 displayed 25 signals, including three methyl, four methylene, seven methine and eleven quaternary carbons. Two of these signals resonated at δ_C 74.5 and 41.9 ppm characteristic for C-2 and C-3 of the flavanone skeleton (Agrawal et al. 1989). The absolute configuration at C-2 was determined to be S on the basis of the strong negative cotton effect at 293 nm observed in the CD spectrum of 1 (Gaffield, W. 1970) (Supplemntary data). The ¹H NMR also showed the presence of three methyl singlets at δ_H 1.56, 1.62 and 1.65, three methylenes at δ_H 1.93, 2.06 and 3.27 in addition to two olefinic protons at δ 5.07 (t, H-6″), 5.26 (t, H-2″), attributed to a geranyl substituent (Radwan et al. 2008). The geranyl moiety was located at C-6 of ring A based on HMBC correlations of H-1″ at δ 3.27 with C-6 (δ 107.5), C-5 (δ_C 164.4), and C-7 (δ_C 160.6) (Figure 1). The ¹³C NMR data further established the hydroxylation at C-5 and C-7 of ring A and at C-2′ and C-4′ of ring B. Therefore, compound 1 was assigned as 2(S)-6-geranyl- 5, 7, 2′, 4′-tetrahydroxy-flavanone. In fact, the structure of compound 1 has been reported by Sakamoto et al. (1989) as a copying error form Ohmoto et al. (1986). The later reported the isolation of 2(S)-5′-geranyl-5, 7, 2′, 4′-tetrahydroxy-flavanone (Ohmoto et al. 1986) in which the geranyl is located at C-5′ and not at C-6. Based on the above evidence 1 is a new flavanone named Sepicanin A, and this explains the discrepancy in the literature (Ohmoto et al. 1986; Sakamoto et al. 1989).

Table 1.

¹H [400 MHz] and ¹³C NMR [100 MHz] data for compound 1 [Acetone- d₆]

Position	δ_H	δ_C
2	5.70dd (12.8, 2.8)	74.5
3	3.08 dd (16.8, 12.8)	41.9
	2.78 dd (16.8, 2.8)
4	---	197.0
5	---	164.4
6	---	107.5
7	---	160.6
8	6.06 s	95.5
9	---	162.1
10	---	102.2
1′	---	117.0
2′	---	158.5
3′	6.49 d (2.0)	102.6
4′	---	155.3
5′	6.43 dd(8.4, 2)	106.9
6′	7.36 d (8.4)	127.7
1″	3.27 d (6.8)	21.3
2″	5.26 t (6.8)	122.9
3″	---	133.9
4″	1.93 t (8.0)	39.6
5″	2.06 m	26.5
6″	5.07 t (6.8)	124.3
7″	---	130.6
8″	1.65	24.9
9″	1.62	16.8
10″	1.56 s	15.3

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Assignments confirmed by DEPT, gHMQC, gCOSY and gHMBC experiments

Fig. 1.

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Afzelechin-3-O-α-L-rhamnopyranoside and β-sitosterol glucoside were identified by comparing their spectroscopic data with literature values (Drewes et al. 1992; Koizumi et al. 1979).

The three isolated compounds were evaluated in vitro for antimicrobial activity against the fungi Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and the bacteria Escherichia coli, Pseudomonas aeruginosa, Mycobacterium intracellulare, and MRSA. Compound 1 showed a selective and significant antibacterial activity against MRSA with an IC₅₀ value of 1.4 μM and MIC value of 2.9 μM. While it did show weak antifungal activity against Candida albicans with IC₅₀ and MIC values of 15.3 and 47.1 μM, respectively, it was completely inactive against the other test organisms at test concentrations of 118 μM. Moreover, compound 1 was potently bactericidal having a minimum bactericidal concentration (MBC) of 2.9 μg/mL (Table 2). The remaining two compounds were inactive against all test microbes. Additionally, none of the compounds showed toxicity against Vero cells (Monkey kidney fibroblast) at concentrations up to 10.0 μg/mL.

Table 2.

In vitro Antimicrobial Activity of Compound 1 (all Values in μM)a–c

Sample	MRSA			C. albicans

	IC₅₀a	MICb	MBCc	IC₅₀a	MICb	MFCc

1	1.4	2.9	2.9	15.3	47.1	47.1
amphotericin B	nte	nte	nte	0.3	0.7	1.3
ciprofloxacin	0.4	0.8	nad	nte	nte	nte

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IC₅₀ = the test concentration that affords 50% growth.

MIC (minimum inhibitory concentration) = the lowest test concentration that allows no detectable growth.

MBC/MFC (minimum bactericidal or fungicidal concentration) = the lowest test concentration that kills the organism.

na = not active at the highest test concentration.

nt = not tested.

3. Experimental

3.1. General

Optical rotation was measured on an Autoplot IV automatic polarimeter. CD spectrum was recorded on a Jasco DIP-370 spectropolarimeter. ¹H, ¹³C, and 2D-NMR spectra were recorded using the residual solvent signal as an internal standard on a Varian AS 400 spectrometer. IR spectra were taken on a Bruker Tensor 27. UV spectra were recorded on a Hewlett-Packard 8435 spectrometer High resolution mass spectra were measured using a Bruker BioApex FT mass spectrometer. TLC was performed on sheets precoated with silica gel 60 F₂₅₄ (Merck, Germany) using CHCl₃/MeOH (8:2). Compounds were detected by spraying with 1% vanillin in H₂SO₄/EtOH (1:9), followed by heating.

3.2. Plant material

The plant material was collected and identified by Dr. Topul Rali in Papua New Guinea in 1998 and a voucher specimen is deposited at the School of Natural and Physical Sciences, University of Papua New Guinea.

3.3. Extraction, isolation and characterization

Air-dried leaves of A. sepicanus (2 kg) were extracted by maceration in 95% EtOH (4 days) with frequent sonication. The ethanol extract was concentrated under vacuum to yield a residue (24 g). Twenty grams of the residue were subjected to fractionation by vacuum liquid chromatography over a silica gel column (300 g, 160–200 mesh) eluting with n-hexane-EtOAc [90:10, 80:20, 60:40, 40:60, 20:80, 10:90, 100:0, (500 mL of each solvent mixture)] followed by EtOAc-MeOH [80:20, 60:40, 40:60, 20:80, (500 mL of each solvent mixture)] yielding 11 fractions (A–K). All the fractions and the total extract, were evaluated for antimicrobial activity. Fractions F and I showed selective antimicrobial activity against MRSA with IC₅₀ values of 6.00 and 20.0 μg/mL, respectively. Fraction F (5.31 g, eluted with n-hexane-EtOAc, 10:90) was chromatographed over a silica gel column (250 g, 4×70 cm) eluted with CH₂Cl₂-MeOH (100:10 - 80:20) to afford 46 subfrs. (F 1–46, 100 mL each). Subfractions F33–35 were combined (784 mg, IC₅₀ = 1.5 μg/mL) and subjected to a silica gel column (40 g, 1.5 × 50 cm) eluted with CH₂Cl₂-MeOH (90:10) to afford 1 (6.3 mg, IC₅₀ = 0.60 μg/mL). Fraction I (2.15 g, eluted with EtOAc-MeOH, 60:40) was fractionated on a Si gel column (100 g, 5 × 120 cm) and eluted with CH₂Cl₂-MeOH (85:15) to afford 6 subfractions (I 1–6). Subfractions I1–3 (307 mg) were combined and were crystallized from MeOH to give 3 (93 mg). Subfraction I 5 (298 mg) was applied to a silica gel column (14 g, 4 × 70 cm) with CH₂Cl₂-MeOH (90:10) as an eluant to give 2 (30 mg).

3.3. 2(S)-6-geranyl-5, 7, 2′, 4′-tetrahydroxy-flavanone (Sepicanin A) (1)

Pale yellow Powder; UV (MeOH): λ_max 213, 290 nm, UV (NaOAc): λ_max 213, 326 nm, UV (AlCl₃) 213, 310 nm, UV (AlCl₃/HCl) 213, 310 nm; IR ν_max (neat): 3336, 1635, 1602, 1292 and 1068 cm⁻¹; for ¹H and ¹³ C NMR spectroscopic data see Table 1; HRESIMS m/z 425.1931 [M+H]⁺ (Calc. for C₂₅H₂₉O_6, 425.1964), 447.1750 [M+Na] ⁺ (Calc. for C₂₅H₂₈O₆Na, 447.1784).

3.4. Antimicrobial assay

The antibacterial and antifungal activities of the isolated compounds were evaluated according to the previously reported method (Radwan et al 2007, 2008; Ross et al. 2008).

Supplementary Material

NIHMS114037-supplement-01.doc^{(208.5KB, doc)}

Acknowledgments

We are grateful to Dr. Bharathi Avula, Dr. Shabana Khan, and Ms. Marsha Wright, National Center for Natural Products Research for assistance with the HRESIMS, cytotoxicity and antimicrobial assays, respectively. The authors also acknowledge the United States Department of Agriculture, Agriculture Research Services Specific Cooperation Agreement No. 58-6408-7-012 and the NIH, NIAID, Division of AIDS, Grant NO. AI 27094 for partial support of this work.

Footnotes

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Supplementary Materials

NIHMS114037-supplement-01.doc^{(208.5KB, doc)}