Anti-ulcer xanthones from the roots of Hypericum oblongifolium Wall

Fitoterapia 95 (2014) 258–265

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Anti-ulcer xanthones from the roots of Hypericum oblongifolium Wall Mumtaz Ali a,⁎, Abdul Latif a,⁎, Khair Zaman b, Mohammad Arfan c, Derek Maitland d, Habib Ahmad e, Manzoor Ahmad a a b c d e

Department of Chemistry, University of Malakand, Chakdara, Dir (L), Pakistan Department of Chemistry, Abdul Wali Khan University, Mardan, Pakistan Institute of Chemical Sciences, University of Peshawar, Peshawar 25120, Pakistan Chemical and Forensic Sciences, University of Bradford, BD7 1DP, UK Department of Genetics, Hazara University, Mansehra, Pakistan

a r t i c l e

i n f o

Article history: Received 25 January 2014 Accepted in revised form 20 March 2014 Available online 29 March 2014 Keywords: Hypericum oblongifolium Wall Xanthones Hypericorin C Hypericorin D 2D-NMR Anti-ulcer

a b s t r a c t Three new xanthones, hypericorin C (1), hypericorin D (2) and 3,4-dihydroxy-5-methoxyxanthone (3), along with eight known compounds; 2,3-dimethoxyxanthone (4), 3,4-dihydroxy-2methoxyxanthone (5), 3,5-dihydroxy-1-methoxyxanthone (6), 3-acetylbetulinic acid (7), 10H-1,3dioxolo[4,5-b]xanthen-10-one (8), 3-hydroxy-2-methoxyxanthone (9), 3,4,5-trihydroxyxanthone (10) and betulinic acid (11) were isolated from the roots of Hypericum oblongifolium. The structures of the new compounds 1, 2 and 3 were deduced by spectroscopic techniques [ESI MS, 1H NMR, 13C NMR, and 2D NMR (HMQC, HMBC, COSY and NOESY)]. The entire series of compounds were evaluated for anti-ulcer activity. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Hypericum oblongifolium Wall., which belongs to the family Guttiferae, is an erect evergreen shrub, which grows to a height of 6–12 m, that is commonly found in the Khasia Hills in India at an altitude of 5000–6000 ft and in the Himalayas [1]. In Chinese traditional herbal medicine H. oblongifolium has been used for the treatment of hepatitis, bacterial diseases nasal hemorrhage, and as a remedy for dog bites and bee stings [2]. In various parts of the world, the plants of genus Hypericum have been used in traditional medicines as a sedative, an antiseptic, and an antispasmodic, as well as for the treatment of external wounds and gastric ulcers [3]. ⁎ Corresponding authors. Tel.: +92 345 8747744. E-mail addresses: [email protected] (M. Ali), [email protected] (A. Latif).

http://dx.doi.org/10.1016/j.fitote.2014.03.014 0367-326X/© 2014 Elsevier B.V. All rights reserved.

Plants of the genus Hypericum are a rich source of xanthones; many of which exhibit a broad spectrum of activities. The xanthones and their derivatives, isolated from different species of Hypericum, exhibit potent anti-tumor, anti-fungal, cytotoxic [4], anti-microbial, anti-ulcer, anti-depressant, inhibition of lipid peroxidase [5], anti-inflammatory, anti-septic, anxiolytic, diuretic, digestive, expectorant, and vermifugal [3] activities and have received attention for the anti-viral action of hypericin and pseudohypericin on lipid enveloped and non-enveloped DNA and RNA viruses [6,7]. The most common compounds isolated from plants of this genus are xanthones [8], flavonoids [9], phloroglucinol, licinic acid derivatives [10], benzopyrans [11] and benzophenones [12]. Urease (urea amidohydrolase, EC: 3.5.1.5) occurs throughout the animal and plant kingdom. Many microorganisms use this enzyme to provide a source of nitrogen for growth, and it also plays an important role in plant nitrogen metabolism during the germination process [13,14]. The presence of urease

M. Ali et al. / Fitoterapia 95 (2014) 258–265

activity in soils is exploited widely in agriculture. Unfortunately, excessive levels of soil urease can degrade urea in fertilizers too rapidly and result in phytopathic effects and loss of volatilized ammonia [15]. On the other hand in human and veterinary medicine, urease is a virulent factor in certain human and animal pathogens, which participate in the development of kidney stones, pyelonephritis, peptic ulcers, and other disease states [16]. The obvious remedy for treating bacterial infection with anti-microbials has often proven futile [17], and only a few combination regimes have reached clinical practice. Thus the need for alternative or novel treatments is paramount. The discovery of potent and safe urease inhibitors is a very important area of pharmaceutical research due to the involvement of ureases in different pathological conditions. In a continuation of our study on the genus Hypericum, herein we report the isolation and structure elucidation of three new xanthones, hypericorin C, {(2R,3R)-rel-2-[(acetyloxy) methyl]-3-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-5methoxy-7H-1,4-dioxino[2,3-c]xanthen-7-one}(1), hypericorin D, {(2R,3R)-rel-2-[hydroxymethyl]-3-(2,3,4-trihydroxy-5methoxyphenyl)-2,3-dihydro-5-methoxy-7H-1,4-dioxino[2,3-c] xanthen-7-one} (2) and 3,4-dihydroxy-5-methoxyxanthone (3), along with four compounds previously isolated from Hypericum; namely 2,3-dimethoxyxanthone (4), 3,4dihydroxy-2-methoxyxanthone (5), 3,5-dihydroxy-1-methoxyxanthone (6), and 3-acetylbetulinic acid (7) (Fig. 1). Also isolated for the first time from H. oblongifolium were 10H1,3-dioxolo[4,5-b]xanthen-10-one (8) [18], 3-hydroxy-2methoxyxanthone (9) [19], 3,4,5-trihydroxyxanthone (10) [20] and betulinic acid (11) (Fig. 3). 2. Experimental 2.1. General UV spectra were obtained on Optima SP3000 plus (Japan) UV–Visible spectrometer using chloroform, or methanol, as the solvent. IR spectra were recorded on a Nicolet 205 and Impact 410 FT-IR spectrometers, using KBr windows with acetone as solvent against an air background. 1H, 13C, and 2D NMR spectra were recorded on a JEOL ECA-600 FT NMR spectrometer fitted with an X-H auto-tune 5 mm probe. Chemical shifts (δ) are expressed in ppm relative to tetramethylsilane (TMS) and coupling constants are given in Hz. Mass spectra (ESI in either positive- or negative-ion mode) were measured on a Micromass Quattro Ultima (Triple Quad). TLC was performed on pre-coated silica gel F-254 plates (Plastic plates; F254; Macherey Germany); the visualization was done at 254 nm and by spraying with ceric sulphate reagent. Column silica gel (Silica gel-60, 70-230 mesh; Material Harvest UK) and flash silica gel 230-400 mesh were used for column chromatography. Melting points were determined on a Gallenkamp apparatus and are uncorrected. 2.2. Plant material H. oblongifolium Wall, which was authenticated by Dr. Habib Ahmad, Dean Faculty of Science, Hazara University, was collected at flowering period in June from Buner District, Khyber Pakhtunkhwa, Pakistan. A voucher specimen (HUH-002) was retained for verification purposes in

259

the Department of Botany, Hazara University, Khyber Pakhtunkhwa, Pakistan. 2.3. Extraction and isolation The air-dried, powdered roots (4 kg) were exhaustively successively extracted with n-hexane, ethyl acetate and methanol (3 × 25 L, each for 3 days) at room temperature. The extracts were concentrated in a rotary evaporator and dried under vacuum to yield gummy residues. The ethyl acetate fraction (70 g) was subjected to column chromatography over silica gel eluting with n-hexane–ethyl acetate and ethyl acetate–methanol in increasing order of polarity to afford 180 fractions, which were grouped according to the similarity on TLC profiles to give 21 major fractions (1–21). Fraction 4 was purified through column chromatography (n-hexane:chloroform; 1:1) to yield 20 mg of pure compound 7. Fraction 5 was also subjected to column chromatography. Elution with n-hexane: chloroform in increasing order of polarity starting with a 80:20 mixture yielded three sub-fractions (5.1–5.3), which were further purified by preparative TLC using chloroform as eluent to give 4 (4 mg) and 8 (3 mg). Fraction 11 was also subjected to column chromatography and elution with n-hexane:chloroform in increasing order of polarity (started at 1:1) gave five sub-fractions (11.1–11.5). Further purification by preparative TLC using methanol:chloroform (5:95) as eluent gave 9 (3 mg) and 10 (4 mg). Fraction 12 was also subjected to preparative TLC using methanol:chloroform (5:95) as eluent and yielded pure 11 (20 mg). Fraction 17 was subjected to further column chromatography. Elution with n-hexane:chloroform (80:20) through to pure chloroform and then methanol:chloroform (1:99) afforded 1 (15 mg). Compound 5 was obtained from fraction 18 by preparative TLC using methanol: chloroform (7:93) as eluent. Fraction 19 was also subjected to column chromatography. Elution with n-hexane:chloroform (80:20 through to pure chloroform and then methanol: chloroform 1:99) gave 2 (17 mg). Finally compound 6 (6 mg) was purified from fraction 20 by preparative TLC (methanol: chloroform 7:93). Hypericorin C (1): Whitish amorphous powder; Rf = 0.6; methanol:chloroform (1:99); mp 230–232 °C; [α]20 D =+0.33° (0.01 acetone); IR νmax(KBr) cm−1 3416, 2941, 1742, 1642, 1608, 1485, 1343, 1228, 1140 and 1089; ESI [M + 1]+ m/z 479.0 (consistent with C26H24O9); HR-ESIMS (+): ([M + H]+ m/z 479.1359; calcd 479.1337); UV λmax(MeOH) nm (log ε): 248 (4.34), 308 (3.83), 346 (3. 82). 1H (600 MHz) and 13C NMR spectral data (150 MHz, (CD3)2CO): given in Table 1. Hypericorin D (2): White amorphous powder; Rf = 0.4; methanol:chloroform (1:99); mp 250–254 °C; [α]20 D = +0.58° (0.01 acetone); IR νmax(KBr) cm−1 3384, 2940, 1704, 1639, 1599, 1464, 1325, 1285, 1138 and 1089; ESI (M − 1)− m/z 467.0 (consistent with C24H20O10); HR-ESIMS (+): ([M + H]+ m/z 467.1359; calcd 467.1337)UV λmax(MeOH) nm (log ε): 250 (4.5), 302 (4.38), 387 (3.71); 1H (600 MHz) and 13C NMR spectral data (150 MHz, DMSO-d6): given in Table 1. 3,4-Dihydroxy-5-methoxyxanthone (3): Yellow amorphous solid; Rf = 0.35; chloroform:hexane (8:2); mp 230– 235 °C; UV λmax(MeOH) nm (log ε): 240 (4.32), 258 (4.37), 269 (4.45), 376 (3.58); IR νmax(KBr): 3437, 2900, 1622, 1585, 1470, 1455, 1345, 1310, 1245, 1215 cm−1; ESI (M + H)+:

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Table 1 1 H and 13C NMR Spectral data for compounds 1–3 (acetone-d6, 600 MHz). Compound

1

C. no.

13

1

2

3

1

H NMR(δ) coupling constants JHH (Hz)

13

1

H NMR(δ) coupling constants JHH (Hz)

13

C NMR(δ)

1 H NMR(δ) coupling constants JHH (Hz)

97.4

7.28, s

97.0

7.16, s

114.6

1a 2

114.8 147.8

– –

114.4 146.4

– –

117.5 124.3

3 4 4a 5

140.5 132.2 141.3 118.0

140.1 133.0 141.8 118.6

– – – 7.66, d (J = 8.4)

147.3 151.1 145.3 146.8

5a 6

156.7 134.4

155.8 135.4

124.0

8

126.1

8a 9 5/

121.1 174.9 77.0

6/ CH2O

75.5 62.5

CH2COCH3 OCH2COCH3 1// 2// 3// 4// 5//

169.8 19.7 126.8 111.4 147.3 147.0 115.2

– – 126.4 137.8 133.5 136.8 148.5

– 7.81, t (J = 8.4) 7.46, t (J = 7.5) 8.17, d (J = 7.5) – – 5.05, d (J = 7.8) 4.42, td (m) 3.68 (Ha-7/), dd (J = 12.0, 4.6) 3.38 (Hb-7/), dd (J = 12.0, 2.7) – – – – – – –

146.1 120.2

7

– – – 7.61, dd (J = 8.4, 1.0) – 7.80, td (J = 8.4,1.7) 7.45, td (J = 7.9, 1.0) 8.23, dd (J = 7.9,1.3) – – 5.1,d (J = 7.8)

7.27, d (J = 9.1) – 7.39, d (J = 9.1) – – – –

6//

121.8

106.2

MeO-2 MeO-4// MeO-5// MeO-5

55.6 55.8 – –

56.3 – 56.7 –

C NMR(δ)

4.64, m 4.18 (Ha-7/), dd (J = 12.0,4.4) 4.36 (Hb-7/), dd (J = 12.0,2.8) – 2.03, s – 7.16, d (J = 1.9) – – 6.9, d (J = 8.3) 7.02, dd (J = 8.3, 1.9) 3.90, s 3.86, s – –

C NMR(δ)

124.8 126.4 121.2 175.3 77.2 78.2 60.4

m/z 259.0 (consistent with C14H10O5); 1H NMR (600 MHz) and 13C NMR, (150 MHz, (CD3)2CO): given in Table 1. 2,3-Dimethoxyxanthone (4): White crystalline solid; Rf = 0.39; Chloroform (100%); mp 145–150 °C [lit., mp [21], 154–55 °C]; IR νmax(KBr) cm−1 1657 (C_O), 1590, 1444, 1315, 1281, 1138 and 1089; ESI (M + 1)+ m/z 257.0 (consistent with C15H12O4); UV λmax(MeOH) nm (log ε): 242 (3.5), 272 (3.38), 307 (2.71); 1H (600 MHz) and 13C NMR spectral data (150 MHz, CDCl3): given in Table 2. 3,4-Dihydroxy-2-methoxyxanthone (5): Yellowish amorphous powder; Rf = 0.54; methanol:chloroform (3:97); mp 245–250 °C [lit., mp [18], 243–245 °C]; IR νmax(KBr) 3339, 3240, 2930, 1726, 1604, 1466 and 1273 cm−1; ESI (M + 1)+ m/z 259.0 (consistent with C14H10O5); UV λmax(MeOH) nm (log ε): 232 (4.5), 270 (3.78), 367 (3.01); 1H (600 MHz) and 13 C NMR spectral data (150 MHz, (CD3)2O): given in Table 2. 3, 5-Dihydroxy-1-methoxyxanthone (6): White amorphous powder; Rf = 0.45; Methanol:Chloroform (2:98); mp 320–325 °C [lit., mp [22], 354–55 °C]; IR νmax(KBr) cm−1 3455 (OH), 2959, 1658 (C_O), 1604, 1457, 1275, 1143 and

123.8 176.7 –

– 7.25, dd (J = 7.6, 1.5) 7.19, t (J = 7.8) 7.75, dd (J = 7.6, 1.5) – – –

– –

– –

– – – – – – –

– – – – – – –

6.7, s

–

–

3.8, s – 3.7, s –

– – – 62.1

– – – 3.83, s

124.3 116.8

1084; ESI (M − H)− m/z 257.0 (consistent with C14H10O5); UV λmax(MeOH) nm (log ε): 242 (4.1), 278 (3.8), 307 (2.91); 1 H (600 MHz) and 13C NMR spectral data (150 MHz, CD3OD): given in Table 2. 3-Acetylbetulinic acid (7): White needles; Rf = 0.49; chloroform:hexane (1:1); mp 180–182 °C [lit., mp [23]; IR νmax(KBr) cm−1 2946, 1732, 1696, 1452, 1369, 1244, 1105 and 1024; ESI (M − 1)− m/z 497.0 (consistent with C32H50O4); UV λmax(MeOH) nm (log ε): 240 (5.1), 269 (4.31); 1H NMR (600 MHz, CDCl3): δH 4.72 (1H, d, J = 1.6, H-30a), 4.59 (1H, brs, H-30b), 3.0 (1H,td J = 10.4,5.5, H-19), 2.3–2.70 (5H, m, H2-21, H2-22, H-1a), 2.27 (1 H, td, J = 13.8, 3.4 Hz, H-16), 2.16, 2.03(3H, s, CH3CO), (1 H, dt, J = 11.9, 3.4 Hz, H-12), 1.93 (2 H, m, H-1), 1.67 (1H, m, H-18), 1.67 (3H, brs, H-28), 1.6 8(1H, m, H-5), 1.56 (1H, m, H-9), 1.42 (2H, m,H-15), 1.40 (4H, m, H-11,12), 1.27-1.48 (4H, m, H-6,7), 1.27 (1H, m, H-1b), 0.91-0.95 (6H, brs, Me-24, 26), 0.83 (3H, brs, Me-27), 0.82 (3H, brs, Me-23), 0.81 (3H, brs, Me-25); 13C NMR (150 MHz, CDCl3): δC 34.2 (C-1), 23.8 (C-2), 81.0 (C-3), 37.2 (C-4), 50.4 (C-5), 18.2 (C-6), 37.1

M. Ali et al. / Fitoterapia 95 (2014) 258–265

261

Table 2 1 H and 13C NMR spectral data for xanthones 4–5 (acetone-d6, 600 MHz) and 6 (methanol-d3, 600 MHz). C. no.

4

5 1

H NMR(δ) coupling constants JHH (Hz)

13

105.5 115.0 146.8 152.5 97.5 155.5 117.7 156.2 134.1 123.8 126.8 121.6 176.2 – 56.6 56.4

7.68, s – – – 6.9, s – 7.46, d (J = 8.4) – 7.71, t (J = 8.4) 7.36, t (J = 7.5) 8.36, d (J = 7.5) – – – 4.0, s 4.05, s

96.29 113.8 145.8 142.5 141.0 133.8 117.9 156.0 134.1 123.7 126.1 121.4 175.1 – 55.7 –

C (δ)

1 1a 2 3 4 4a 5 5a 6 7 8 8a 9 CH3O-1 CH3O-2 CH3 O-3

6

13

C (δ)

(C-7), 40.7 (C-8), 55.5 (C-9), 37.9 (C-10), 20.9 (C-11), 25.5 (C-12), 38.5 (C-13), 42.25 (C-14), 29.8 (C-15), 32.2 (C-16), 56.5 (C-17), 49.0 (C-18), 47.3 (C-19), 150.5 (C-20), 30.67 (C-21), 38.4 (C-22), 28.0 (C-23), 16.3 (C-24), 16.5 (C-25), 14.4 (C-26), 16.1 (C-27), 182.4 (C-28), 19.4 (C-29), 109.9 (C-30), 171.2 (CH3COO), 21.4 (CH3COO). 2.4. Urease inhibition assay Reaction mixtures comprising 25 μL of the enzyme (jack bean urease) solution and 55 μL of buffers containing 100 mM urea were incubated with 5 μL of test compounds (0.5 mM concentration) at 30 °C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described by weather burn [24]. Briefly, 45 μL of each phenolic reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μL of alkaline reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μL. The results (change in absorbance per min) were processed by using SoftMax Pro software (Molecular Device, USA). The entire assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula 100 − (ODtestwell / ODcontrol) × 100. Thiourea was used as the standard inhibitor of urease [24,25]. 3. Results and discussion H. oblongifolium was collected during the flowering period (September 2011) from Buner district, Khyber Pakhtunkhwa province of Pakistan. A previous study [26] reported the isolation from this plant of hypericorin A, hypericorin B, kielcorin, 4-hydroxy-2,3-dimethoxyxanthone, 3,4,5-trihydroxyxanthone, 1,3-dihydroxy-5-methoxyxanthone and 1,3,7-trihydroxyxanthone. In our present study, we herein

1

H NMR(δ) coupling constants JHH (Hz)

13

7.22, s – – – – – 7.55, dd (J = 8.3, 1.8) – 7.76, dt (J = 8.3, 1.8) 7.40, dt (J = 8.3, 1.2) 8.22, dd (J = 8.3, 1.8) – – – 3.93, s –

162.1 103.4 97.5 162.3 96.6 160.1 146.2 144.6 118.9 122.8 115.2 123.4 175.2 61.1 – –

C (δ)

1 H NMR(δ) coupling constants JHH (Hz)

– – 6.22, dd (J = 2.3) – 6.36, d (J = 2.3) – – – 7.10, m 7.10, m 7.58, dd (J = 7.0,2.3) – – 3.88, s – –

report the isolation of eleven compounds (1–11) from the EtOAc soluble fraction of H. oblongifolium. The molecular formula of Compound 1 (Fig. 1), a white amorphous powder, was determined as C26H22O9 by a combination of electrospray ionization mass spectrometry (ESI-MS) and NMR spectroscopy. ESI-MS (positive mode) produced molecular ion peaks [M + 1]+ at 479 and [M + Na]+ at 501. The ultraviolet (UV) spectrum exhibited characteristic absorptions of a xanthone nucleus at 248, 308 and 346 nm [27]. The IR spectrum showed absorptions at 3416, 1742, 1643 and 1608 cm−1 indicating the presence of hydroxyl and ester groups, conjugated carbonyl and aromatic double bonds, respectively [27]. The NMR data (Table 1) of 1 suggested the presence of a xanthone and phenylpropanoid moieties [27,28]. In the 1H NMR spectrum (Table 1), signals of aromatic protons belonging to rings A and C of the xanthone skeleton resonated at δH 7.61 (1H, dd, J = 8.4, 1.0 Hz, H-5), 7.80 (1H, td, J = 8.4, 1.7 Hz, H-6), 7.45 (1H, td, J = 7.9, 1.0 Hz, H-7), 8.23 (1H, dd, J = 7.9, 1.3 Hz, H-8), and 7.28 (1H, s, H-1). Similarly, aromatic protons (ring E) of the phenylpropanoid moiety appeared at δH 7.02 (1H, dd, J = 8.3, 1.9 Hz, H-2′′), 6.90 (1H, d, J = 8.3 Hz, H-3′′) and 7.16 (1H, d, J = 1.9 Hz, H-6′′), whereas aliphatic protons of CH(O)CH(O) CH2O linkage resonated at δH 5.1 (1H, d, J = 7.8 Hz, H-5′), 4.64 (1H, m, H-6′), 4.18 (1H, dd, J = 12.0, 4.4 Hz, Ha-7′) and 4.36 (1H, dd, J = 12.0, 2.8 Hz, Hb-7′). The deshielded doublet at δH 5.1 suggested a O-benzylic methylene proton with a tran-configuration (J = 7.8 Hz). A singlet at δH 2.03 was attributed to the methyl group of the 6′-acetoxymethylenyl sub-unit. Furthermore, two singlet peaks, each of three protons intensity at δH 3.86 and 3.90 were assigned to methoxy groups (ring C and E). From the data mentioned, it was deduced that 1 consists of a xanthone nucleus connected to a phenyl group through 1,4-dioxane ring [29,30], which was further supported by the presence of a significant EI-MS peak at m/z 222 due to retro-Diels-Alder fragmentation in the dioxane ring. The 13C NMR spectra {broad band decoupled and DEPT} (Table 1) of 1 showed twenty six carbon signals,

262

M. Ali et al. / Fitoterapia 95 (2014) 258–265

Fig. 1. Chemical structures of compounds 1–7.

comprising of three methyl, one methylene, ten methine, and twelve quaternary carbons. HMBC correlations (Fig. 2) were used to decide the location of the 1,4-dioxane ring, two methoxy groups and a hydroxymethylene group. Correlation peaks of H-1 (δH 7.28) to C-4a (δC 141.3), C-3 (δC 140.5) and C-9 (δC 174.9) and methoxy protons (δH 3.90) to C-2 (δC 141.3) clearly indicated that 1,4-dioxane ring is connected to C-3 and C-4 with a methoxy carbon attached to C-2. Similarly, correlations of H-5′ (δH 5.1) to C-1′′ (δC 126.8), C-2′′ (δC 121.8) and C-6′′ (δC 111.4) showed that the trisubstituted benzene ring (ring E) is linked to 1,4-dioxane at C-5′ and C-1′′. The positions of hydroxymethylene and methoxy groups were fixed to their respective carbon atoms for HMBC correlations of H-7′ to C-6′ and methoxy protons (δH 3.86) to C-4′′, respectively. COSY cross peaks between H-5/H-6, H-6/H-7, H-7/H-8 and H-2′′/H-3′′/H-6′′ and NOE difference correlations between H-1 (δH 7.28) and the methoxy (MeO-2) signal at δH 3.90 further supported the structure of 1, which was determined as (2R,3R)-rel-3-(3-hydroxy-4-methoxyphenyl)-5-

methoxy-7-oxo-2,3-dihydro-7H[1,4]dioxino[2,3,c]xanthen2yl acetate (Hypericorin C) (1). The molecular formula, C24H20O10, of compound 2 (Fig. 1), also a white amorphous powder, was determined via a combination of NMR spectroscopy and electrospray negative ion mass spectrometry. The latter gave a molecular ion peak [M − 1]+ at m/z 467. The UV spectrum showed the presence of a xanthone giving absorption bands at 250, 302 and 387 nm [27]. The IR spectrum showed absorptions at 3384 br, 1639 and 1599 cm−1 indicating the presence of OH, conjugated carbonyl and aromatic ring, respectively [27]. A comparison of the NMR data (Table 1) of 2 with that of 1 indicated that the compounds were broadly similar in structure with a few differences in the phenylpropanoid part. The first difference is the absence of the acetyl group in 2, which is attached to C-6′ in 1, and second is the difference in number and pattern of substituents on the phenyl ring (ring E). The single proton and an aromatic methoxy of the pentasubstituted aromatic ring (ring E) were assigned

M. Ali et al. / Fitoterapia 95 (2014) 258–265

263

Fig. 2. Selected HMBC and NOESY correlations of compounds 1–7.

chemical shift values of δH 6.70 (1H, s, H-′′) and 3.70 (3H, s, 5′′-OCH3) in the 1H NMR spectrum of 2. In the 13C NMR spectrum, the deshielded signals at δC 137.8, 133.5, and 136.8 were assigned to the hydroxyl bearing carbons (C-2′′, C-3′′and C-4′′, respectively) of ring E. The positions of a methoxy group and aromatic proton in ring E were decided on the basis of HMBC NMR correlations (Fig. 2). Thus compound 2 was identified as (2R,3R)-rel-2,3-dihydro-5-hydroxy-3-(4hydroxy-3,5-dimethoxyphenyl)-2-(hydroxylmethyl)-7H-1,4dioxino[2,3-c]xanthen-7-one (Hypericorin D) (2). Compound 3 (Fig. 1), a pale yellow amorphous solid, was assigned a molecular formula of C14H10O5 on the basis of NMR data and electrospray positive mass spectrometry, which gave a parent peak [M + 1]+ at 259. The UV spectrum of 3 also showed absorption peaks for the presence of xanthone at 240, 258 and 376 nm. IR absorptions at 3437, 1622 and 1595 cm−1 indicated the presence of OH, conjugated carbonyl and aromatic ring, respectively.

The NMR spectrum of 3 (Table 1) also exhibited characteristic peaks of xanthone functionality [27]. In the 1H NMR spectrum of 3, five signals observed at δH 7.75 (dd, J = 7.6, 1.5 Hz), 7.39 (d, J = 9.1 Hz), 7.27 (d, J = 9.1 Hz), 7.25 (dd, J = 7.6, 1.5 Hz) and 7.19 (t, J = 7.8 Hz) were assigned to the H-8, H-2, H-1, H-6 and H-7 aromatic protons, respectively. A singlet at δH 3.83 was assigned to a methoxy group positioned at C-5 on the basis of an HMBC correlation (Fig. 2) and was also validated through the 2D-NOESY experiment where a cross peak was observed between MeO-5 and H-6. The 13C and DEPT NMR spectra (Table 1) of 3 showed fourteen carbon signals, including one methoxy, six methine, and seven quaternary carbons. In the HMBC spectrum H-1 proton (δH 7.27) showed 2J and 3J correlation with C-1a (δC 117.5), C-4a (δC 145.3), C-3 (δC 147.3) and C-9 (δC 176.7). Similarly, H-8 (δH 7.75) showed 2J and 3J correlation with C-7 (δC 124.3), C-5 (δC 146.8), C-5a (δC 146.1) and C-9 (δC 176.7), whereas, H-2 showed its correlation with C-1a (δC 117.5), C-3

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Fig. 3. Chemical structures of compounds 8–11.

(δC 147.3) and C-4 (δC 151.1) in the HMBC spectrum, thus confirming the positions of two hydroxyls at C-3 and C-4. Thus, compound 3 was identified as 3,4-dihydroxy-5methoxyxanthone. A SciFinder search confirmed that xanthones 1–3 have not been reported before, either as natural products or as synthetically prepared. Four other compounds, 2,3-dimethoxyxanthone (4) [21], 3,4-dihydroxy-2-methoxyxanthone (5) [18,28], 3,5dihydroxy-1-methoxyxanthone (6) [22] and 3-acetoxybetulinic acid (7) [29] have been reported before but here they are presented with supplementary data (13C and 2D NMR), which has not been reported previously. Three new source xanthones were identified as 10H-[1,3]dioxolo[4,5-b]xanthen-10-one (8) [18], 3-hydroxy-2-methoxyxanthone (9) [19] and 3,4,5trihydroxyxanthone (10) [20] along with a well-known triterpenoid betulinic acid (11) by comparing their spectroscopic data with published values. The enzyme inhibitory activities of the isolated compounds (1–11) were evaluated for urease inhibition activities by using a contemporary assay [25]. Table 3 summarizes the

IC50 values and percent inhibition against a positive control (thiourea). Only compounds 3 and 10 were found to show good activity with IC50 values of 92.60 ± 0.41 and 85.50 ± 0.94 μM, respectively. Compounds 2, 4 and 6 possessed moderate activity against urease with IC50 values of 289.80 ± 0.5, 257.50 ± 5.2 and 270.50 ± 6.4 μM, respectively. A very weak activity was shown by compound 1 having IC50 value of 483.60 ± 5.2 μM. The activities of 3 and 10 can be attributed to their capabilities to bind to the central metal atom (Ni) of the enzyme [30]. The greater activity of 10 as compared to 3 may be attributed to the presence of an additional hydroxyl group in 10. From these results it can be concluded that the plants of genus Hypericum are good sources of novel xanthones with potent urease inhibitory effects. Conflict of interest The authors declare no conflict of interest. Acknowledgment

Table 3 The IC50 values and percent inhibition of urease to compounds. Compound

% inhibition at 1000 μg/ml

IC50 (μM) ± SEM

1 2 3 4 5 6 7 8 9 10 11 Thiourea

52.22 60.12 76.12 52.20 38.42 50.20 45.50 35.56 21.45 90.32 21.76 98.86

483.60 ± 5.2 289.80 ± 0.5 92.60 ± 0.41 257.50 ± 5.2 Inactive 270.50 ± 6.4 Inactive Inactive Inactive 85.50 ± 0.94 Inactive 21.01 ± 0.51

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