Fitoterapia 95 (2014) 258–265

Contents lists available at ScienceDirect

Fitoterapia

j ourna l homepage: www.e lsev ie r .com/ locate / f i to te

Anti-ulcer xanthones from the roots ofHypericum oblongifolium Wall

Mumtaz Ali a,⁎, Abdul Latif a,⁎, Khair Zaman b, Mohammad Arfan c, Derek Maitland d,Habib Ahmad e, Manzoor Ahmad a

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

a r t i c l e i n f o

⁎ Corresponding authors. Tel.: +92 345 8747744.E-mail addresses: mumtazphd@gmail.com (M. Ali)

(A. Latif).

http://dx.doi.org/10.1016/j.fitote.2014.03.0140367-326X/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

Article history:Received 25 January 2014Accepted in revised form 20 March 2014Available online 29 March 2014

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-2-methoxyxanthone (5), 3,5-dihydroxy-1-methoxyxanthone (6), 3-acetylbetulinic acid (7), 10H-1,3-dioxolo[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 structuresof the new compounds 1, 2 and 3were deduced by spectroscopic techniques [ESI MS, 1H NMR, 13CNMR, and 2D NMR (HMQC, HMBC, COSY and NOESY)]. The entire series of compounds wereevaluated for anti-ulcer activity.

© 2014 Elsevier B.V. All rights reserved.

Keywords:Hypericum oblongifolium WallXanthonesHypericorin CHypericorin D2D-NMRAnti-ulcer

1. Introduction

Hypericum oblongifolium Wall., which belongs to thefamily Guttiferae, is an erect evergreen shrub, which growsto a height of 6–12 m, that is commonly found in theKhasia Hills in India at an altitude of 5000–6000 ft and inthe Himalayas [1]. In Chinese traditional herbal medicineH. oblongifolium has been used for the treatment of hepatitis,bacterial diseases nasal hemorrhage, and as a remedy for dogbites and bee stings [2]. In various parts of the world, theplants of genus Hypericum have been used in traditionalmedicines as a sedative, an antiseptic, and an antispasmodic,as well as for the treatment of external wounds and gastriculcers [3].

, dralatif@uom.edu.pk

Plants of the genusHypericum are a rich source of xanthones;many of which exhibit a broad spectrum of activities. Thexanthones and their derivatives, isolated from different speciesof Hypericum, exhibit potent anti-tumor, anti-fungal, cytotoxic[4], anti-microbial, anti-ulcer, anti-depressant, inhibition of lipidperoxidase [5], anti-inflammatory, anti-septic, anxiolytic, di-uretic, digestive, expectorant, and vermifugal [3] activities andhave received attention for the anti-viral action of hypericin andpseudohypericin on lipid enveloped and non-enveloped DNAand RNA viruses [6,7]. The most common compounds isolatedfrom 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 through-out the animal and plant kingdom. Many microorganisms usethis enzyme to provide a source of nitrogen for growth, and italso plays an important role in plant nitrogen metabolismduring the germination process [13,14]. The presence of urease

259M. Ali et al. / Fitoterapia 95 (2014) 258–265

activity in soils is exploited widely in agriculture. Unfor-tunately, excessive levels of soil urease can degrade urea infertilizers too rapidly and result in phytopathic effects and lossof volatilized ammonia [15]. On the other hand in human andveterinary medicine, urease is a virulent factor in certain humanand animal pathogens, which participate in the development ofkidney stones, pyelonephritis, peptic ulcers, and other diseasestates [16]. The obvious remedy for treating bacterial infectionwith anti-microbials has often proven futile [17], and only a fewcombination regimes have reached clinical practice. Thus theneed for alternative or novel treatments is paramount. Thediscovery of potent and safe urease inhibitors is a veryimportant area of pharmaceutical research due to the involve-ment of ureases in different pathological conditions.

In a continuation of our study on the genusHypericum, hereinwe report the isolation and structure elucidation of threenew xanthones, hypericorin C, {(2R,3R)-rel-2-[(acetyloxy)methyl]-3-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-5-methoxy-7H-1,4-dioxino[2,3-c]xanthen-7-one}(1), hypericorinD, {(2R,3R)-rel-2-[hydroxymethyl]-3-(2,3,4-trihydroxy-5-methoxyphenyl)-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 fromHypericum; namely 2,3-dimethoxyxanthone (4), 3,4-dihydroxy-2-methoxyxanthone (5), 3,5-dihydroxy-1-methoxy-xanthone (6), and 3-acetylbetulinic acid (7) (Fig. 1). Alsoisolated for the first time from H. oblongifolium were 10H-1,3-dioxolo[4,5-b]xanthen-10-one (8) [18], 3-hydroxy-2-methoxyxanthone (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, asthe solvent. IR spectra were recorded on a Nicolet 205 andImpact 410 FT-IR spectrometers, using KBr windows withacetone as solvent against an air background. 1H, 13C, and 2DNMR spectra were recorded on a JEOL ECA-600 FT NMRspectrometer fitted with an X-H auto-tune 5 mm probe.Chemical shifts (δ) are expressed in ppm relative totetramethylsilane (TMS) and coupling constants are givenin Hz. Mass spectra (ESI in either positive- or negative-ionmode) were measured on a Micromass Quattro Ultima(Triple Quad). TLC was performed on pre-coated silica gelF-254 plates (Plastic plates; F254; Macherey Germany); thevisualization was done at 254 nm and by spraying with cericsulphate reagent. Column silica gel (Silica gel-60, 70-230 mesh;Material Harvest UK) and flash silica gel 230-400 mesh wereused for column chromatography. Melting points were deter-mined 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 BunerDistrict, Khyber Pakhtunkhwa, Pakistan. A voucher speci-men (HUH-002) was retained for verification purposes in

the Department of Botany, Hazara University, KhyberPakhtunkhwa, Pakistan.

2.3. Extraction and isolation

The air-dried, powdered roots (4 kg) were exhaustivelysuccessively extracted with n-hexane, ethyl acetate andmethanol (3 × 25 L, each for 3 days) at room temperature.The extracts were concentrated in a rotary evaporator anddried under vacuum to yield gummy residues. The ethylacetate fraction (70 g) was subjected to column chromatog-raphy over silica gel eluting with n-hexane–ethyl acetate andethyl acetate–methanol in increasing order of polarity toafford 180 fractions, which were grouped according to thesimilarity 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 com-pound 7. Fraction 5 was also subjected to column chroma-tography. Elution with n-hexane: chloroform in increasingorder of polarity starting with a 80:20 mixture yielded threesub-fractions (5.1–5.3), which were further purified bypreparative TLC using chloroform as eluent to give 4 (4 mg)and 8 (3 mg). Fraction 11 was also subjected to columnchromatography and elution with n-hexane:chloroform inincreasing order of polarity (started at 1:1) gave fivesub-fractions (11.1–11.5). Further purification by preparativeTLC using methanol:chloroform (5:95) as eluent gave 9(3 mg) and 10 (4 mg). Fraction 12 was also subjected topreparative TLC using methanol:chloroform (5:95) as eluentand yielded pure 11 (20 mg). Fraction 17 was subjected tofurther column chromatography. Elution with n-hexane:chlo-roform (80:20) through to pure chloroform and then metha-nol:chloroform (1:99) afforded 1 (15 mg). Compound 5 wasobtained from fraction 18 by preparative TLC using methanol:chloroform (7:93) as eluent. Fraction 19 was also subjected tocolumn 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]D20 =+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/z479.0 (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 NMRspectral 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]D20 =+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/z467.0 (consistentwith 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 NMRspectral data (150 MHz, DMSO-d6): given in Table 1.

3,4-Dihydroxy-5-methoxyxanthone (3): Yellow amor-phous 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)+:

Table 11H and 13C NMR Spectral data for compounds 1–3 (acetone-d6, 600 MHz).

Compound 1 2 3

C. no. 13C NMR(δ) 1HNMR(δ) couplingconstants JHH (Hz)

13C NMR(δ) 1HNMR(δ) couplingconstants JHH (Hz)

13CNMR(δ)

1HNMR(δ) couplingconstants JHH (Hz)

1 97.4 7.28, s 97.0 7.16, s 114.6 7.27, d(J = 9.1)

1a 114.8 – 114.4 – 117.5 –

2 147.8 – 146.4 – 124.3 7.39, d(J = 9.1)

3 140.5 – 140.1 – 147.3 –

4 132.2 – 133.0 – 151.1 –

4a 141.3 – 141.8 – 145.3 –

5 118.0 7.61, dd(J = 8.4, 1.0)

118.6 7.66, d (J = 8.4) 146.8 –

5a 156.7 – 155.8 – 146.1 –

6 134.4 7.80, td(J = 8.4,1.7)

135.4 7.81, t(J = 8.4)

120.2 7.25, dd(J = 7.6, 1.5)

7 124.0 7.45, td(J = 7.9, 1.0)

124.8 7.46, t(J = 7.5)

124.3 7.19, t(J = 7.8)

8 126.1 8.23, dd(J = 7.9,1.3)

126.4 8.17, d(J = 7.5)

116.8 7.75, dd(J = 7.6, 1.5)

8a 121.1 – 121.2 – 123.8 –

9 174.9 – 175.3 – 176.7 –

5/ 77.0 5.1,d (J = 7.8) 77.2 5.05, d(J = 7.8)

– –

6/ 75.5 4.64, m 78.2 4.42, td (m) – –

CH2O 62.5 4.18 (Ha-7/), dd(J = 12.0,4.4)4.36 (Hb-7/), dd(J = 12.0,2.8)

60.4 3.68 (Ha-7/), dd(J = 12.0, 4.6)3.38 (Hb-7/), dd(J = 12.0, 2.7)

– –

CH2COCH3 169.8 – – – – –

OCH2COCH3 19.7 2.03, s – – – –

1// 126.8 – 126.4 – – –

2// 111.4 7.16, d (J = 1.9) 137.8 – – –

3// 147.3 – 133.5 – – –

4// 147.0 – 136.8 – – –

5// 115.2 6.9, d(J = 8.3)

148.5 – – –

6// 121.8 7.02, dd(J = 8.3, 1.9)

106.2 6.7, s – –

MeO-2 55.6 3.90, s 56.3 3.8, s – –

MeO-4// 55.8 3.86, s – – – –

MeO-5// – – 56.7 3.7, s – –

MeO-5 – – – – 62.1 3.83, s

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

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 NMRspectral data (150 MHz, CDCl3): given in Table 2.

3,4-Dihydroxy-2-methoxyxanthone (5): Yellowish amor-phous powder; Rf = 0.54; methanol:chloroform (3:97); mp245–250 °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) and13C NMR spectral data (150 MHz, (CD3)2O): given in Table 2.

3, 5-Dihydroxy-1-methoxyxanthone (6): White amor-phous powder; Rf = 0.45; Methanol:Chloroform (2:98); mp320–325 °C [lit., mp [22], 354–55 °C]; IR νmax(KBr) cm−1

3455 (OH), 2959, 1658 (C_O), 1604, 1457, 1275, 1143 and

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);1H (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]; IRνmax(KBr) cm−1 2946, 1732, 1696, 1452, 1369, 1244, 1105and 1024; ESI (M − 1)− m/z 497.0 (consistent withC32H50O4); 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

Table 21H and 13C NMR spectral data for xanthones 4–5 (acetone-d6, 600 MHz) and 6 (methanol-d3, 600 MHz).

C. no. 4 5 6

13C (δ) 1HNMR(δ) couplingconstants JHH (Hz)

13C (δ) 1HNMR(δ) couplingconstants JHH (Hz)

13C (δ) 1HNMR(δ) couplingconstants JHH (Hz)

1 105.5 7.68, s 96.29 7.22, s 162.1 –

1a 115.0 – 113.8 – 103.4 –

2 146.8 – 145.8 – 97.5 6.22, dd (J = 2.3)3 152.5 – 142.5 – 162.3 –

4 97.5 6.9, s 141.0 – 96.6 6.36, d (J = 2.3)4a 155.5 – 133.8 – 160.1 –

5 117.7 7.46, d (J = 8.4) 117.9 7.55, dd (J = 8.3, 1.8) 146.2 –

5a 156.2 – 156.0 – 144.6 –

6 134.1 7.71, t (J = 8.4) 134.1 7.76, dt (J = 8.3, 1.8) 118.9 7.10, m7 123.8 7.36, t (J = 7.5) 123.7 7.40, dt (J = 8.3, 1.2) 122.8 7.10, m8 126.8 8.36, d (J = 7.5) 126.1 8.22, dd (J = 8.3, 1.8) 115.2 7.58, dd (J = 7.0,2.3)8a 121.6 – 121.4 – 123.4 –

9 176.2 – 175.1 – 175.2 –

CH3O-1 – – – – 61.1 3.88, sCH3O-2 56.6 4.0, s 55.7 3.93, s – –

CH3 O-3 56.4 4.05, s – – – –

261M. Ali et al. / Fitoterapia 95 (2014) 258–265

(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 (jackbean urease) solution and 55 μL of buffers containing100 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 ammoniaproduction using the indophenol method as described byweather burn [24]. Briefly, 45 μL of each phenolic reagent (1%w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μLof alkaline reagent (0.5% w/v NaOH and 0.1% active chlorideNaOCl) were added to each well. The increasing absorbanceat 630 nm was measured after 50 min, using a microplatereader (Molecular Device, USA). All reactions were performedin triplicate in a final volume of 200 μL. The results (change inabsorbance per min) were processed by using SoftMax Prosoftware (Molecular Device, USA). The entire assays wereperformed at pH 6.8. Percentage inhibitions were calculat-ed 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 floweringperiod (September 2011) from Buner district, KhyberPakhtunkhwa 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-methoxyxanthoneand 1,3,7-trihydroxyxanthone. In our present study, we herein

report the isolation of eleven compounds (1–11) from theEtOAc soluble fraction of H. oblongifolium.

The molecular formula of Compound 1 (Fig. 1), a whiteamorphous powder, was determined as C26H22O9 by acombination 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 exhibitedcharacteristic absorptions of a xanthone nucleus at 248, 308and 346 nm [27]. The IR spectrum showed absorptions at3416, 1742, 1643 and 1608 cm−1 indicating the presence ofhydroxyl and ester groups, conjugated carbonyl and aromaticdouble bonds, respectively [27].

The NMR data (Table 1) of 1 suggested the presence of axanthone and phenylpropanoid moieties [27,28]. In the 1HNMR spectrum (Table 1), signals of aromatic protonsbelonging to rings A and C of the xanthone skeletonresonated 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 phenyl-propanoid 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′) and4.36 (1H, dd, J = 12.0, 2.8 Hz, Hb-7′). The deshielded doubletat δH 5.1 suggested a O-benzylic methylene proton with atran-configuration (J = 7.8 Hz). A singlet at δH 2.03 wasattributed to the methyl group of the 6′-acetoxymethylenylsub-unit. Furthermore, two singlet peaks, each of threeprotons intensity at δH 3.86 and 3.90 were assigned tomethoxy groups (ring C and E). From the data mentioned, itwas deduced that 1 consists of a xanthone nucleus connectedto a phenyl group through 1,4-dioxane ring [29,30], whichwas further supported by the presence of a significant EI-MSpeak atm/z 222 due to retro-Diels-Alder fragmentation in thedioxane ring. The 13C NMR spectra {broad band decoupledand DEPT} (Table 1) of 1 showed twenty six carbon signals,

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

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

comprising of three methyl, one methylene, ten methine, andtwelve quaternary carbons. HMBC correlations (Fig. 2) wereused to decide the location of the 1,4-dioxane ring, twomethoxy groups and a hydroxymethylene group. Correlationpeaks 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-3and 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′′ (δC121.8) and C-6′′ (δC 111.4) showed that the trisubstitutedbenzene ring (ring E) is linked to 1,4-dioxane at C-5′ andC-1′′. The positions of hydroxymethylene and methoxygroups were fixed to their respective carbon atoms for HMBCcorrelations of H-7′ to C-6′ and methoxy protons (δH 3.86) toC-4′′, respectively. COSY cross peaks betweenH-5/H-6, H-6/H-7,H-7/H-8 and H-2′′/H-3′′/H-6′′ and NOE difference correla-tions between H-1 (δH 7.28) and the methoxy (MeO-2)signal at δH 3.90 further supported the structure of 1, which wasdetermined as (2R,3R)-rel-3-(3-hydroxy-4-methoxyphenyl)-5-

methoxy-7-oxo-2,3-dihydro-7H[1,4]dioxino[2,3,c]xanthen-2yl acetate (Hypericorin C) (1).

The molecular formula, C24H20O10, of compound 2 (Fig. 1),also a white amorphous powder, was determined via acombination of NMR spectroscopy and electrospray negativeion mass spectrometry. The latter gave a molecular ion peak[M − 1]+ at m/z 467. The UV spectrum showed the presenceof a xanthone giving absorption bands at 250, 302 and387 nm [27]. The IR spectrum showed absorptions at 3384 br,1639 and 1599 cm−1 indicating the presence of OH, conju-gated carbonyl and aromatic ring, respectively [27].

A comparison of the NMR data (Table 1) of 2with that of 1indicated that the compounds were broadly similar instructure with a few differences in the phenylpropanoidpart. The first difference is the absence of the acetyl group in2, which is attached to C-6′ in 1, and second is the differencein number and pattern of substituents on the phenyl ring(ring E). The single proton and an aromatic methoxy of thepentasubstituted aromatic ring (ring E) were assigned

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

263M. Ali et al. / Fitoterapia 95 (2014) 258–265

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 NMRspectrum, the deshielded signals at δC 137.8, 133.5, and136.8 were assigned to the hydroxyl bearing carbons (C-2′′,C-3′′and C-4′′, respectively) of ring E. The positions of amethoxy group and aromatic proton in ring E were decided onthe basis of HMBC NMR correlations (Fig. 2). Thus compound2 was identified as (2R,3R)-rel-2,3-dihydro-5-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl)-2-(hydroxylmethyl)-7H-1,4-dioxino[2,3-c]xanthen-7-one (Hypericorin D) (2).

Compound 3 (Fig. 1), a pale yellow amorphous solid, wasassigned a molecular formula of C14H10O5 on the basis ofNMR data and electrospray positive mass spectrometry,which gave a parent peak [M + 1]+ at 259. The UV spectrumof 3 also showed absorption peaks for the presence ofxanthone at 240, 258 and 376 nm. IR absorptions at 3437,1622 and 1595 cm−1 indicated the presence of OH, conju-gated carbonyl and aromatic ring, respectively.

The NMR spectrum of 3 (Table 1) also exhibitedcharacteristic peaks of xanthone functionality [27]. In the 1HNMR 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 assignedto 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 methoxygroup positioned at C-5 on the basis of an HMBC correlation(Fig. 2) and was also validated through the 2D-NOESYexperiment where a cross peak was observed betweenMeO-5 and H-6. The 13C and DEPT NMR spectra (Table 1) of3 showed fourteen carbon signals, including one methoxy, sixmethine, and seven quaternary carbons. In the HMBC spectrumH-1 proton (δH 7.27) showed 2J and 3J correlationwith C-1a (δC117.5), 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

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

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

(δC 147.3) and C-4 (δC 151.1) in the HMBC spectrum, thusconfirming the positions of two hydroxyls at C-3 and C-4.Thus, compound 3 was identified as 3,4-dihydroxy-5-methoxyxanthone.

A SciFinder search confirmed that xanthones 1–3 have notbeen reported before, either as natural products or as synthet-ically prepared. Four other compounds, 2,3-dimethoxyxanthone(4) [21], 3,4-dihydroxy-2-methoxyxanthone (5) [18,28], 3,5-dihydroxy-1-methoxyxanthone (6) [22] and 3-acetoxybetulinicacid (7) [29] have been reported before but here they arepresented with supplementary data (13C and 2D NMR), whichhas not been reported previously. Three new source xanthoneswere identified as 10H-[1,3]dioxolo[4,5-b]xanthen-10-one (8)[18], 3-hydroxy-2-methoxyxanthone (9) [19] and 3,4,5-trihydroxyxanthone (10) [20] along with a well-knowntriterpenoid betulinic acid (11) by comparing their spec-troscopic data with published values.

The enzyme inhibitory activities of the isolated com-pounds (1–11) were evaluated for urease inhibition activitiesby using a contemporary assay [25]. Table 3 summarizes the

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

Compound % inhibition at 1000 μg/ml IC50 (μM) ± SEM

1 52.22 483.60 ± 5.22 60.12 289.80 ± 0.53 76.12 92.60 ± 0.414 52.20 257.50 ± 5.25 38.42 Inactive6 50.20 270.50 ± 6.47 45.50 Inactive8 35.56 Inactive9 21.45 Inactive10 90.32 85.50 ± 0.9411 21.76 InactiveThiourea 98.86 21.01 ± 0.51

IC50 values and percent inhibition against a positive control(thiourea). Only compounds 3 and 10 were found to showgood activity with IC50 values of 92.60 ± 0.41 and 85.50 ±0.94 μM, respectively. Compounds 2, 4 and 6 possessedmoderate activity against urease with IC50 values of289.80 ± 0.5, 257.50 ± 5.2 and 270.50 ± 6.4 μM, respec-tively. A very weak activity was shown by compound 1having IC50 value of 483.60 ± 5.2 μM. The activities of 3 and10 can be attributed to their capabilities to bind to the centralmetal atom (Ni) of the enzyme [30]. The greater activity of 10as compared to 3 may be attributed to the presence of anadditional hydroxyl group in 10. From these results it can beconcluded that the plants of genus Hypericum are goodsources of novel xanthones with potent urease inhibitoryeffects.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgment

The financial support of the Higher Education Commissionof Pakistan under indigenous fellowship PhD programme andIRSIP is gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.fitote.2014.03.014.

References

[1] Chopra RN, Chopra IC, Verma BS. Supplement to glossary of Indianmedicinal plants; 1998.

[2] Ferheen S, Ahmed E,Malik A, AfzaN, LodhiMA, ChoudharyMI. Hyperinols Aand B, chymotrypsin inhibiting triterpenes from Hypericum oblongifolium.Chem Pharm Bull 2006;54:1088–90.

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

[3] Cakir OA, Mavi A, Yildirim A, Duru ME, Harmandar M, Kazaz C. Isolationand characterization of antioxidant phenolic compounds from theaerial parts of Hypericum hyssopifolium L. by activity-guided fraction-ation. J Ethnopharmacol 2003;87:73–83.

[4] Iinuma M, Tosa H, Tanaka T, Yonemori S. Two xanthones from root barkof Calophyllum inophyllum. Phytochemistry 1994;35:527–32.

[5] Peres V, Nagem TJ. Trioxygenated naturally occurring xanthones. Phyto-chemistry 1997;44:191–214.

[6] Sommer H, Harrer G. Placebo-controlled double blind study examiningthe effectiveness of an Hypericum preparation in 105 mildly depressedpatients. J Geriatr Psychiatry Neurol 1994;7:S9–S11.

[7] Wagner H, Bladt S. Pharmaceutical quality of Hypericum extracts.J Geriatr Psychiatry Neurol 1994;7:S65–8.

[8] Bennett GJ, Lee HH. Xanthones from Guttiferae. Phytochemistry1989;28:967–98.

[9] Rocha L, Marston A, Potterat O, Kaplan MAC, Stoeckli Evans H. Antibacterialphloroglucinols and flavonoids from Hypericum brasiliense. Phytochemistry1995;40:1447–52.

[10] Jayasuriya H, Clark AM, McChesney JD. New antimicrobial filicinic acidderivatives from Hypericum drummondii. J Nat Prod 1991;54:1314–20.

[11] Ferraz AB, Bordignon SA, Staats C, Schripsema J, von Poser GL.Benzopyrans from Hypericum polyanthemum. Phytochemistry2001;57:1227–30.

[12] Ana PaulaMB, Alexandre BFF, Daniela VA, Sérgio ALB, Jan S, Raquel B, et al.Benzophenones from Hypericum carinatum. J Nat Prod 2005;68:784–6.

[13] Mobley HLT, Hausinger RP. Microbial ureases: significance, regulation,and molecular characterization. Microbiol Rev 1989;53:85–108.

[14] Zonia LE, Stebbins NE, Polacco JC. Essential role of urease ingermination of nitrogen-limited Arabidopsis thaliana seeds. Plant Physiol1995;107:1097–103.

[15] Mulvaney RL, Bremner JM. In: Paul EA, Ladd JN, editors. Soil biochemistry.New York: Marcel Dekker, Inc.; 1981. p. 153–96.

[16] Mobley HLT, Island MD, Hausinger RP. Molecular biology of microbialureases. Microbiol Rev 1995;59:451–80.

[17] Bayerdorffer E, Ottenjhan R. The role of antibiotics in the Campylobacterpylori associated peptic ulcer disease (suppl. 142). Scand J Gastroenterol1988;23:93–100.

[18] Cardona ML, Fernandez MI, Pedro JR, Serrano A. Xanthones fromHypericum reflexum. Phytochemistry 1990;29:3003–6.

[19] Nielsen H, Arends P. Xanthone constituents of Hypericum androsaemum.J Nat Prod 1979;42:303–6.

[20] Patanaik M. Synthesis of new trioxygenated xanthones of Tovomitaexcelsa. Acta Chem Scand B 1987;41:210–2.

[21] Gunatilaka AAL, Balasubramaniam S, Kumar V. 2,3-Dimethoxyxanthonefrom Hypericum mysorense. Phytochemistry 1979;18:182–3.

[22] Ghosal S, Chauhan RBPS, Biswas K, Chaudhuri RK. New 1,3,5-trioxygenatedxanthones in Canscora decussata. Phytochemistry 1976;15:1041–3.

[23] Urban M, Sarek J, Klinot J, Korinkova G, Hajduch M. Synthesis of a-secoderivatives of betulinic acid with cytotoxic activity. J Nat Prod2004;67:1100–5.

[24] Weatherburn MW. Phenol hypochlorite reaction for determination ofammonia. Anal Chem 1967;39:971–4.

[25] Khan KM, Iqbal S, Lodhi MA, Maharvi GM, Zia-Ullah, Choudhary MI,et al. Biscoumarin: new class of urease inhibitors; economical synthesisand activity. Bioorg Med Chem 2004;12:1963–8.

[26] Ali M, Arfan M, Ahmad M, Singh K, Anis I, Ahmad H, et al. Anti-inflammatory xanthones from the twigs of Hypericum oblongifoliumWall. Planta Med 2011;77:2013–8.

[27] Wu C-C, Yen M-H, Yang S-H, Lin C-N. Phloroglucinols withantioxidant activity and xanthonolignoids from the heartwood ofHypericum geminiflorum. J Nat Prod 2008;71:1027–31.

[28] Pinto MM De M, Mesquita AAL, Gottlieb OR. Xanthonolignoids fromKielmeyera coriacea. Phytochemistry 1987;26:2045–8.

[29] Otsuka H, Fujioka S, Komiya T, Goto M, Hiramatsu Y, Fujimura H. Studieson anti-inflammatory agents. V. A new anti-inflammatory constituent ofPyracantha crenulata roem. Chem Pharm Bull 1981;29:3099–104.

[30] Tanaka T, Kawase M, Tani S. Urease inhibitory activity of simple α,β-unsaturated ketones. Life Sci 2003;73:2985–90.