Antitubercular Constituents from the Roots of Engelhardia roxburghiana

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolates
• Antituberculosis activity assay
• Results and Discussion
• Acknowledgements
• References

Abstract

Three new compounds, engelhardione, (-)-5-hydroxy-4-methoxy-1-tetralone, and 3-methoxycarbonyl-1,5-dihydroxyanthraquinone, together with twelve known compounds have been isolated from the roots of Engelhardia roxburghiana. The structures of these new compounds were determined through spectral analyses. Engelhardione, 3-methoxyjuglone, and (-)-4-hydroxy-1-tetralone showed antitubercular activities with MIC values = 3.125, 3.125, and 6.25 μg/mL against Mycobacterium tuberculosis 90 - 221 387, respectively, and with MIC values = 0.2, 0.2, and 4.0 μg/mL against M. tuberculosis H37Rv, individually.

Introduction

Tuberculosis and the incidence of multidrug-resistant TB are ever increasing world health problems. Searches for new structural types of effective drugs against this infectious disease have also become important. Recently, about 200 species of Formosan plants were screened for antitubercular activity and Engelhardia roxburghiana Wall. [E. chrysolepis Hance; E. formosana (Hay.) Hayata; E. spicata Bl. var. formosana Hayata] (Juglandaceae) was shown to be an active species. E. roxburgiana is a deciduous tree growing in India, Indochina, China, and Taiwan [1]. Leaves of this plant have been used as a sweet tea to prevent obesity and to treat abdominal pain and fever in China [2]. The bark and the leaves are also used as fish poison [3]. Past studies have revealed dihydroflavonol glycosides and flavonol glycosides to be the major constituents of leaves and stem bark of this species [4], [5], [6]. Examination of the root has led to the isolation of three new (1 - 3) and twelve known compounds. This paper describes the structural elucidation of 1 - 3 and the antitubercular activity of the isolates.

Materials and Methods

General experimental procedures

All melting points were determined on a Yanaco micro-melting point apparatus and are uncorrected. IR spectra (KBr or neat) were taken on a Perkin-Elmer system 2000 FT-IR spectrometer. UV spectra were obtained on a Jasco UV-240 spectrophotometer in EtOH or MeOH. EI-mass spectra were recorded on a Micromass TRIO-2000 spectrometer. HR-mass spectra were recorded on a Finnigan/Thermo Quest NAT mass spectrometer. NMR spectra, including COSY, NOESY, DEPT, HETCOR, HMQC and HMBC experiments, were recorded on Varian Unity 400 and Bruker AV-500 spectrometers at 400 or 500 MHz (¹H) and at 100 or 125 MHz (¹³C), chemical shifts being given in ppm (δ) with TMS as internal standard. Silica gel (60 - 230, 230 - 400 mesh) (Merck) was used for CC, and silica gel 60 F-254 (Merck) for TLC and preparative TLC. Optical rotations were measured using a Jasco P-1020 polarimeter in CHCl₃, MeOH or EtOH.

Plant material

The root of E. roxburghiana Wall. was collected from Lai-I, Pingtung County, Taiwan, in September 2001 and identified by Dr. I. S. Chen. A voucher specimen (Chen 6043) was deposited in the herbarium of the School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.

Extraction and isolation

Dried roots (11.4 kg) were extracted with cold MeOH, and the extract concentrated under reduced pressure. The MeOH extract (2.05 kg), when partitioned between H₂O-CHCl₃ (1 : 1), afforded a CHCl₃-soluble fraction (fr. A, 50.5 g). Fr. A (50.5 g) was chromatographed on silica gel (1.53 kg), eluting with n-hexane, gradually increasing the polarity with EtOAc and MeOH to give 14 frs: fr. A1 (1800 mL, n-hexane), fr. A2 (600 mL, n-hexane-EtOAc, 98 : 2), fr. A3 (7200 mL, n-hexane-EtOAc, 95 : 5), fr. A4 (4200 mL, n-hexane-EtOAc, 90 : 10), frs. A5 - A7 (each 2000 mL, n-hexane-EtOAc, 80 : 20), fr. A8 and A9 (each 4200 mL, n-hexane-EtOAc, 50 : 50), fr. A10 (6000 mL, n-hexane-EtOAc, 50 : 50), fr. A11 (6000 mL, n-hexane-EtOAc, 30 : 70), fr. A12 (6000 mL, EtOAc), fr. A13 (1800 mL, EtOAc-MeOH, 50 : 50), fr. A14 (1800 mL, MeOH). Fr. A6 (1.85 g) was washed with MeOH to yield 9 (834 mg). The washings (652 mg) of fr. A6 were chromatographed on silica gel (20 g) eluting with a gradient of n-hexane-EtOAc to give 11 frs (each 100 mL, fr. A6 - 1 - fr. A6 - 11). Fr. A6 - 2 (7.5 mg) was further purified by preparative TLC (n-hexane-CH₂Cl₂, 1 : 1) to obtain 3 (1.0 mg). Fr. A6 - 4 (265 mg) was further purified by preparative TLC (n-hexane-CH₂Cl₂-EtOAc, 3 : 6:1) to yield 12 (16.3 mg). Fr. A8 (2.55 g) was chromatographed on silica gel (76 g) eluting with CH₂Cl₂-acetone step gradients to give 6 frs (each 150 mL, fr. A8 - 1 - fr. A8 - 6). Part (150 mg) of fr. A8 - 5 was further purified by preparative TLC (n-hexane-EtOAc, 19 : 1) to obtain 15 (7.4 mg). Fr. A9 (2.08 g) was chromatographed on silica gel (60 g) eluting with n-hexane-EtOAc (1 : 1) to give 7 frs (each 200 mL, fr. A9 - 1 - fr. A9 - 7). Fr. C9 - 2 (1.52 g) was chromatographed on silica gel (46 g) eluting with CH₂Cl₂-EtOAc step gradients to give 12 frs (each 100 mL, fr. A9 - 2-1 - fr. A9 - 2-12). Fr. A9 - 2-2 (10.5 mg) was further purified by preparative TLC (n-hexane-EtOAc, 4 : 1) to yield 7 (2.5 mg). Fr. A9 - 2-5 (168 mg) was chromatographed on silica gel (10 g) eluting with n-hexane-acetone step gradients to give 13 frs (each 100 mL, fr. A9 - 2-5 - 1 - fr. A9 - 2-5 - 13). Fr. C9 - 2-5 - 4 (39.7 mg) was further purified by preparative RP-18 TLC (CH₃CN) to yield 14 (2.9 mg). Fr. C9 - 2-5 - 6 (10.2 mg) was purified with preparative TLC (n-hexane-acetone, 1 : 1) to yield 13 (3.8 mg). Fr. C9 - 2-5 - 9 (26.8 mg) was purified with preparative RP-18 TLC (CH₃N-H₂O, 1 : 1) to yield 2 (4.4 mg). Fr. A9 - 2-8 (181 mg) was chromatographed on silica gel (10 g) eluting with CH₂Cl₂-EtOAc step gradients to give 9 frs (each 100 mL, fr. A9 - 2-8 - 1 - fr. A9 - 2-8 - 9). Fr. C9 - 2 - 8 - 7 (16 mg) was purified with preparative RP-18 TLC (MeOH-H₂O, 9 : 1) to yield 10 (0.7 mg). Fr. A9 - 2-9 (432.6 mg) was chromatographed on silica gel (13 g) eluting with n-hexane-acetone step gradients to give 7 frs (each 100 mL, fr. A9 - 2-9 - 1 - fr. A9 - 2-9 - 7). Fr. A9 - 2 - 9 - 4 (200 mg) was purified by preparative TLC (CH₂Cl₂-EtOAc, 10 : 1) to yield 1 (34.1 mg). Fr. A9 - 2 - 11 (32.1 mg) was washed with MeOH to yield 11 (10.4 mg). Fr. C9 - 5 (24.3 mg) was purified by preparative TLC (n-hexane-EtOAc, 3 : 1) to obtain 8 (1.2 mg). Fr. A10 (4.96 g) was chromatographed on silica gel (150 g) eluting with CH₂Cl₂-EtOAc step gradients to give 10 frs (each 600 mL, fr. A10 - 1 - fr. A10 - 10). Part (145 mg) of fr. A10 - 5 was purified by preparative TLC (CH₂Cl₂-EtOAc, 19 : 1) to yield 4 (47.1 mg). Part (152 mg) of fr. A10 - 7 was purified by preparative TLC (CH₂Cl₂-EtOAc, 19 : 1) to yield 5 (8.7 mg). Part (180 mg) of fr. A11 was purified by preparative TLC (CH₂Cl₂-EtOAc, 4 : 1) to yield 6 (4.4 mg).

Isolates

Engelhardione (1): Colorless needles from CH₂Cl₂-MeOH, m. p. 73 - 75 °C. UV (EtOH): λ_max (log ε) = 281 (3.68); (KOH): 288 nm (3.69); IR: ν_max = 3380 (OH), 1697 cm^-1 (C = O); EI-MS: m/z (rel. int.) = 312 (M⁺, 23), 120 (12), 107 (14), 85 (60), 83 (100), 47 (18), 44 (29). HR-EI-MS: found: 312.1367 [M]⁺; calcd. for C₁₉H₂₀O₄ : 312.1372; ¹H-NMR (CDCl₃, 400 MHz): δ = 1.56 (2H, m, H-5), 1.67 (2H, m, H-6), 1.86 (2H, m, H-4), 2.33 (2H, m, H-2), 2.69 (2H, m, H-7), 2.85 (2H, m, H-1), 5.57 (1H, d, J = 2.0 Hz, H-2′), 5.64 (2H, br s, OH-4′, 4′′, exchangeable with D₂O), 6.65 (1H, dd, J = 8.0, 2.0 Hz, H-6′), 6.82 (1H, dd, J = 8.2, 2.0 Hz, H-6′), 6.84 (1H, d, J = 8.0 Hz, H-5′), 6.89 (1H, d, J = 8.2 Hz, H-5′′), 6.95 (1H, d, J = 2.0 Hz, H-2′′); ¹³C-NMR (CDCl₃, 100 MHz): δ = 19.0 (C-5), 27.2 (C-6), 27.3 (C-1), 35.6 (C-7), 41.1 (C-2), 46.4 (C-4), 112.6 (C-2′), 115.6 (C-5′), 117.9 (C-2′′), 122.8 (C-6′), 122.9 (C-6′′), 123.4 (C-5′′), 134.0 (C-1′), 140.6 (C-1′′), 140.7 (C-4′′), 142.9 (C-4′), 146.8 (C-3′′), 148.8 (C-3′), 210.3 (C-3).

(-)-5-Hydroxy-4-methoxy-1-tetralone (2): Colorless needles from CH₂Cl₂-MeOH, m. p. 110 - 112 °C; [α]_D ²⁶: -27.8°(c 0.088, EtOH); UV (EtOH): λ_max (log ε) = 222 (3.25), 258 (2.59), 315 nm (2.09); (KOH): 245 (3.00), 371 nm (2.05); IR: ν_max = 3271 (OH), 1679 cm^-1 (C = O); EI-MS: m/z (rel. int.) = 192 (M⁺, 25), 160 (100), 132 (24), 131 (60), 103 (9), 77 (9). HR-EI-MS: found: 192.0784 [M]⁺; calcd for C₁₁H₁₂O₃ : 192.0782; ¹H-NMR (acetone-d ₆, 500 MHz): δ = 2.17 (1H, m, H-3a), 2.40 (1H, m, H-3b), 2.46 (1H, m, H-2b), 2.85 (1H, m, H-2a), 3.42 (3H, s, OCH₃ - 4), 4.92 (1H, dd, J = 4.0, 3.0 Hz, H-4), 7.14 (1H, dd, J = 8.0, 1.0 Hz, H-6), 7.29 (1H, t, J = 8.0 Hz, H-7), 7.46 (1H, dd, J = 8.0, 1.0 Hz, H-8), 8.91 (1H, br s, OH-5, exchangeable with D₂O); ¹³C-NMR (acetone-d ₆, 125 MHz): δ = 27.8 (C-3), 33.8 (C-2), 56.8 (OCH₃), 70.8 (C-4), 118.7 (C-8), 121.6 (C-6), 129.8 (C-4a), 130.0 (C-7), 134.3 (C-1a), 156.5 (C-5), 197.7 (C-1).

3-Methoxycarbonyl-1,5-dihydroxyanthraquinone (3): Yellow needles from CH₂Cl₂-MeOH; m. p. 216 - 218 °C; UV (EtOH): λ_max (log ε) = 230 (4.25), 290 (sh) (3.71), 417 nm (3.34); (KOH): 399 (3.11), 427 (3.11), 500 nm (3.41); IR: ν_max = 3000 (OH), 1726 (C = O); EI-MS: m/z (rel. int.) = 298 (M⁺, 43), 267 (26), 239 (16), 167 (58), 149 (100), 83 (31), 71 (46), 57 (89), 44 (70). HR-EI-MS: found: 298.0473 [M]⁺; calcd for C₁₆H₁₀O₆ : 298.0469; ¹H-NMR (CDCl₃, 400 MHz): δ = 4.01 (3H, s, OCH₃), 7.37 (1H, dd, J = 8.0, 1.2 Hz, H-6), 7.73 (1H, t, J = 8.0 Hz, H-7), 7.89 (1H, dd, J = 8.0, 1.2 Hz, H-8), 7.97 (1H, d, J = 1.6 Hz, H-2), 8.64 (1H, d, J = 1.6 Hz, H-4), 12.58 (1H, s, exchangeable with D₂O, OH-1), 12.59 (1H, s, exchangeable with D₂O, OH-5).

(-)-Regiolone (4): [α]_D ²⁶: -11.3° (c 0.20, EtOH) {lit. [α]_D: -3.3° [7]}.

(-)-4-Hydroxy-1-tetralone (5): [α]_D ²⁶: -41.5° (c 0.17, CHCl₃) {lit. [α]_D: -35.5° [8]}.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)-3-heptanone (6): [α]_D ²⁶: + 1.3° (c 0.06, EtOH) {lit. [α]_D: + 1.05° [9]}.

β-Sitosterol (9): [α]_D ²⁵: -38.2° (c 0.15, CHCl₃) {lit. [α]_D: -36.1° [10]}.

Oleanolic acid (11): [α]_D ²⁶: + 75.1° (c 0.21, MeOH) {lit. [α]_D: + 72.8° [11]}.

3-epi-Betulinic acid acetate (12): [α]_D ²⁶: -36.5° (c 0.18, EtOH) {lit. [α]_D: -21° [12]}.

Antituberculosis activity assay

The antituberculosis activity of each test compound was evaluated and compared with the minimal inhibitory concentration (MICs) using the clinical susceptible isolate of Mycobacterium tuberculosis (90 - 221 387). The agar dilution method with Middlebrook 7H10 agar was used to determine the MICs as described in the tentative standard (M24-T2) of the National Committee of Clinical Laboratory Standards (NCCLS), USA. [13]. Briefly, each test compound was added to Middlebrook 7H10 agar medium supplemented with OADC (oleic acid-albumin-dextrose-catalase) at 50 - 56 °C in serial dilutions to yield a final concentration of 100 to 0.2 μg/mL. 10 mL of each concentration of antimycobacterial agent-containing medium as well as drug-free control medium were dispensed into plastic quadrant Petri dishes. The inoculum of the test isolate of M. tuberculosis was prepared by diluting the initial inoculum in Middlebrook 7H9 broth until a turbidity was reduced that is the equivalent of McFarland no.1 standard. Final suspensions were performed by adding Middlebrook 7H9 and preparing 10 ^{- 2} dilutions of the standardized suspensions. After solidification of the Middlebrook 7H10 medium, 33 μL portions of the dilutions were placed on each quadrant of the agar plates, and the agar plates were incubated at 35 - 37 °C with 10 % CO₂ for 3 weeks. The MIC of each test compound was determined to be the lowest concentration that inhibited macroscopic growth. The test strain included M. tuberculosis H37Rv (ATCC 27*294) in this study for quality control testing. Also, the test compound included ethambutol (Sigma, St. Louis, Missouri, USA) in this experiment for the comparison of in vitro activity against M. tuberculosis.

Results and Discussion

Engelhardione (1) was isolated as colorless needles. The EI-MS afforded the molecular ion [M]⁺ at m/z = 312, implying a molecular formula of C₁₉H₂₀O₄, which was confirmed by the HR-EI-MS. The UV absorptions at 281 nm and a bathochromic shift upon addition of KOH suggested the presence of a phenolic benzenoid moiety. The IR spectrum revealed a hydroxy absorption at 3380 cm^-1 and a ketone group at 1697 cm^-1, the latter was supported by a resonance signal in the ¹³C-NMR spectrum at δ = 210.3. The ¹H-NMR spectrum of 1 indicated two trisubstituted benzene rings, two hydroxy groups, and a 3-heptanonyl group. Due to its DBE of 10, compound 1 was suggested to be a diarylheptanoid with a 13-member ring. The correlations of 12 methylene protons of the 3-heptanonyl moiety were easily established by the COSY spectrum. Correlations between H-7 (δ = 2.69) and H-2′′ (δ = 6.95), H-6′′ (δ = 6.82); H-1 (δ = 2.85) and H-2′ (δ = 5.57), H-6′ (δ = 6.65) were found in the NOESY spectrum. The C-3′ signal at δ = 148.8 and C-3′′ at δ = 146.8 in the ¹³C-NMR spectrum and the correlations between H-2′′ (δ = 6.95) and C-3′′ (δ = 146.8), H-2′ (δ = 5.57) and C-3′ (δ = 148.8) found in the HMBC spectrum could support two phenyl moieties connected by an ether linkage and two hydroxy groups that were located at C-4′′ and C-4′, respectively. According to the above data, the structure of engelhardione was elucidated as 1, which was further confirmed by the COSY, NOESY (Fig. [1]), HETCOR, and HMBC experiments (Fig. [1]). Cyclic diarylheptanoids have been reported in Betulaceae and Myricaceae of Amentiferae [14]. The occurrence of 1 with a 14-membered ring of a diarylheptanoid with an O-ether linkage from Juglandaceae in this study adds meaningful evidence in the chemotaxonomy of Amentiferae.

(-)-5-Hydroxy-4-methoxy-1-tetralone (2) was isolated as colorless needles. Its molecular formula, C₁₁H₁₂O₃, was determined by EI-MS ([M]⁺, m/z = 192) and HR-EI-MS. The UV absorptions at 222, 258, and 315 nm were similar to those of (-)-regiolone (4) [7], also isolated in this study and the exhibition of a bathochromic shift in alkaline solution suggested the presence of a phenolic 1-tetralone nucleus. The IR spectrum showed absorption bands for a hydroxy group at 3271 cm^-1 and a conjugated ketone group at 1679 cm^-1, along with a resonance signal in the ¹³C-NMR spectrum at δ = 197.7. The ¹H-NMR spectrum of 2 showed three mutually-coupling aromatic protons at δ = 7.14 (1H, dd, J = 8.0, 1.0 Hz, H-6), 7.29 (1H, t, J = 8.0 Hz, H-7), and 7.46 (1H, dd, J = 8.0, 1.0 Hz, H-8), a hydroxy group at δ = 8.91 (1H, br s, OH-5), an oxymethine proton at δ = 4.92 (1H, dd, J = 4.0, 3.0 Hz, H-4), four non-equivalent methylene protons at δ = 2.85 and 2.46 (each 1H, m, H-2), δ = 2.40 and 2.17 (each 1H, m, H-3). The assignments of H-4, H-3, and H-2 were supported by the COSY spectrum. Thus, a methoxy group signal at δ = 3.42 was reasonably assigned to C-4. According to the above data, the structure of 2 was elucidated as 5-hydroxy-4-methoxy-1-tetralone, which was further confirmed by ¹³C-NMR, DEPT, NOESY, HMQC, and HMBC (Fig. [2]). Compound 2 showed a levorotatory optical activity with [α]_D ²⁶: -27.8° (c 0.088, EtOH) as in the cases of (-)-regiolone (4) [7] {[α]_D ²⁶: -11.3° (c 0.2, EtOH)} and the relative configuration of C-4 in 2 has to be S [7].

3-Methoxycarbonyl-1,5-dihydroxyanthraquinone (3) was obtained as yellow needles. Its molecular formula of C₁₆H₁₀O₆ was determined by EI-MS ([M]⁺, m/z = 298) and HR-EI-MS. The UV absorption maxima at 230 (4.25), 290 (sh) (3.71) and 417 (3.34) nm and the exhibition of a bathochromic shift in alkaline solution suggested the presence of a phenolic anthraquinone nucleus [15]. The presence of a conjugated C = O group was revealed by IR absorption at 1726 cm^-1. The fragmentation at m/z = 239 in the EI-MS indicated the presence of a dihydroxyanthraquinone skeleton. The ¹H-NMR spectrum of 3 showed signals of three mutually coupling aromatic protons at δ = 7.89 (1H, dd, J = 8.0, 1.2 Hz, H-8), 7.73 (1H, t, J = 8.0 Hz, H-7), 7.37 (1H, dd, J = 8.0, 1.2 Hz, H-6), two aromatic protons with meta-coupling at δ = 8.64 (1H, d, J = 1.6 Hz, H-4), 7.97 (1H, d, J = 1.6 Hz, H-2). Moreover, the signals at δ = 12.58, 12.59 (each 1H, s, exchangeable with D₂O, OH-1 and OH-5) were due to two hydrogen-bonded hydroxy groups. Thus, a methyl signal of methyl ester group at δ = 4.01 (3H, s) was located at the C-3 position. Comparing with ziganein (with 1,5-dihydroxys) (δ = 12.94 and 13.02), chrysophanol (with 1,8-dihydroxys) (δ = 11.98 and 12.09), and rhein methyl ether (with 1,8-dihydroxys) (δ = 11.97 and 12.03) [16], the chemical shifts of the two hydroxy groups [δ = 12.58 and 12.59] of 3 are larger than ca. δ = 12.10 and this supports that compound 3 should be a 3-methoxycarbonyl-1,5-dihydroxyanthraquinone.

The known compounds, (-)-regiolone (4) [7], (-)-4-hydroxy-1-tetralone (5) [8], 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)-3-heptanone (6) [9], 2-methoxyjuglone (7) [17], 3-methoxyjuglone (8) [18], β-sitosterol (9) [10], muurolan-3-en-9β-ol-2-one (10) [19], oleanolic acid (11) [11], 3-epi-betulinic acid acetate (12) [12], linoleic acid [20], oleic acid [8], methylparaben [21], were readily identified by comparison of physical and spectroscopic data ([α]_D, UV, IR, ¹H-NMR and mass spectrometry) with an authentic sample or literature data.

Fractionation of the active CHCl₃ fraction led to the isolation of engelhardione (1), 3-methoxyjuglone (8), and (-)-4-hydroxy-1-tetralone (5) as active constituents with potent in vitro activity against M. tuberculosis 90 - 221 387, showing MIC values of 3.125, 3.125, and 6.25 μg/mL, respectively. When these three test compounds were tested against the standard strain of M. tuberculosis H37Rv, the MICs obtained were 0.2, 0.2, and 4.0 μg/mL, individually. However, ethambutol against the test strains of M. tuberculosis 90 - 221 387 and M. tuberculosis H37Rv showed an in vitro activity with the MICs of 6.25 μg/mL and 3.13 μg/mL, respectively.

Acknowledgements

This work was kindly supported by grant from the National Science Council of the Republic of China.

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Prof. Dr. I. S. Chen

Graduate Institute of Natural Products

College of Pharmacy

Kaohsiung Medical University

Kaohsiung

Taiwan

Republic of China

Fax: +886-7-3210683

Email: m635013@kmu.edu.tw

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Prof. Dr. I. S. Chen

Graduate Institute of Natural Products

College of Pharmacy

Kaohsiung Medical University

Kaohsiung

Taiwan

Republic of China

Fax: +886-7-3210683

Email: m635013@kmu.edu.tw