Benzopyrans, Biphenyls and Xanthones from the Root of Garcinia linii and their Activity against Mycobacterium tuberculosis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A new benzopyran, (S)-3-hydroxygarcibenzopyran, and three new biphenyls, garcibiphenyl C, garcibiphenyl D, garcibiphenyl E, together with thirteen known compounds have been additionally isolated from the root of Garcinia linii. The structures of these new compounds were determined through spectral analyses. Among the isolates, 1,7-dihydroxy-3-methoxyxanthone and 1,5-dihydroxy-3-methoxyxanthone showed antitubercular activities with MICs of 3.1 ± 0.5 and 6.3 ± 1.0 μg/mL against Mycobacterium tuberculosis 90 - 221 387 in vitro, respectively.

Garcinia linii C. E. Chang (Guttiferae) is a small endemic evergreen tree which grows on Lanyu Island and Green Island of Taiwan [1]. The plants of the genus Garcinia are rich in xanthonoids, depsidones, and benzophenones, for some of which cytotoxic activity has been demonstrated [2], [3], [4], [5]. In a preliminary screening, the methanolic extract of the root of this species showed cytotoxic and antitubercular activities in vitro. In a previous study [6], we have reported three new xanthones, linixanthones A - C, two new biphenyls, garcibiphenyl A, garcibiphenyl B, and a new benzopyran, garcibenzopyran, together with thirteen known compounds, including several cytotoxic agents [6] from the root of Garcinia linii. Continuing investigation of the minor constituents and the antitubercular principles of the root of this plant led to the isolation and characterization of a new benzopyran, (S)-3-hydroxygarcibenzopyran (1), three new biphenyls, garcibiphenyl C (2), garcibiphenyl D (3), garcibiphenyl E (4), and thirteen known compounds. This paper describes the structural elucidation of 1 - 4 and the antitubercular activities of the isolates.[]

(S)-3-Hydroxygarcibenzopyran (1) was isolated as colorless amorphous powder. The molecular formula of 1 was established as C₁₈H₂₀O₄ by EI-MS and HR-EI-MS ([M]⁺, m/z = 300.1359). The UV absorptions at 210, 227 (sh), and 268 nm were similar to those of garcibenzopyran [6] and the exhibition of a bathochromic shift in alkaline solution also suggested the presence of a phenolic biphenyl nucleus. The presence of hydroxy groups in the molecule was revealed by a band at 3377 cm^-1 in the IR spectrum, which was confirmed by the signal at δ = 2.35 (br s, D₂O exchangeable) in the ¹H-NMR spectrum. The ¹H-NMR spectrum of 1 was similar to that of garcibenzopyran [6] except that H-3 [δ = 1.77 (2H, t, J = 7.1 Hz)] in garcibenzopyran was replaced in 1 by an OH-3 (δ = 2.35) and H-3 [δ = 3.83 (1H, t, J = 5.0 Hz)]. The stereochemistry of the OH group at C-3 was determined as the 3S-configuration based on the chemical shifts of the hydrogens, Me-2α (δ = 1.33), Me-2β (δ = 1.39), H-3α [δ = 3.83 (1H, t, J = 5.0 Hz)], H-4α [δ = 2.92 (1H, dd, J = 17.6, 5.0 Hz)], H-4β [δ = 2.73 (1H, dd, J = 17.6, 5.0 Hz)], and the carbons, Me-2α (δ = 24.5), Me-2β (δ = 22.3), C-2 (δ = 76.7), C-3 (δ = 69.4), C-4 (δ = 26.3), and [α]_D ²⁴: -10.8° which are in agreement with those of the similar compound, (S)-3,5-dihydroxy- 2,2-dimethyl-7-(2-phenylethenyl)benzopyran {[α]_D ²⁰: -12°} [7]. This was also supported by the marked NOESY correlations (see Fig. 1 in Supporting Information) which were observed between H-3α and Me-2α, H-4α. On the basis of the above evidence, 1 is a new structure determined to be (S)-7-(4-hydroxyphenyl)-5-methoxy-2,2-dimethylchroman-3-ol, named (S)-3-hydroxygarcibenzopyran; this was further confirmed by the NOESY experiments (see Fig. 1 in Supporting Information). The assignments of ¹³C-NMR resonances were confirmed by the HSQC and HMBC techniques (see Fig. 1 in Supporting Information), which also supported the structure of 1.

Garcibiphenyl C (2) was isolated as a colorless oil. The HR-EI-MS gave an [M]⁺ ion at m/z = 246.0893 (calcd. 246.0892), consistent with a molecular formula of C₁₄H₁₄O₄. The UV absorptions at 206 and 264 nm were similar to those of garcibiphenyl A [6], and the exhibition of a bathochromic shift in alkaline solution suggested the presence of a phenolic biphenyl skeleton. The presence of hydroxy groups in the molecule was revealed by the band at 3353 (br) cm^-1 in the IR spectrum, which was confirmed by the signals at δ = 4.81 (br s, D₂O exchangeable, OH-4′) and δ = 5.50 (br s, D₂O exchangeable, OH-4) in the ¹H-NMR spectrum. The ¹H-NMR spectrum of 2 was similar to that of aucuparin [6] except that in 2 a 4′-hydroxyphenyl group [δ = 4.81 (1H, br s, OH-4′), 7.42 (2H, d, J = 8.8 Hz, H-2′ and 6′), 6.89 (2H, d, J = 8.8 Hz, H-3′ and 5′)] replaced the phenyl group [δ = 7.32 - 7.54 (5H, m, H-2′ - H-6′)] in aucuparin. According to the above data, 2 is a new structure determined to be 3,5-dimethoxy-[1,1′-biphenyl]-4,4′-diol, named garcibiphenyl C. This was further confirmed by the NOESY experiments (see Fig. 2 in Supporting Information).

Garcibiphenyl D (3) was isolated as a colorless amorphous powder. The EI-MS afforded the molecular ion [M]⁺ at m/z = 300, implying a molecular formula of C₁₈H₂₀O₄, which was confirmed by the HR-EI-MS. The UV absorptions at 208, 228 (sh), and 268 nm were similar to those of garcibiphenyl C (2), and the exhibition of a bathochromic shift in alkaline solution suggested the presence of a phenolic biphenyl nucleus. The presence of hydroxy groups in the molecule was revealed by the band at 3352 cm^-1 in the IR spectrum, which was confirmed by the signal at δ = 7.96, 4.95, and 2.42 (each br s, D₂O exchangeable, OH-3, 4′, and 2′′, respectively) in the ¹H-NMR spectrum. The ¹H-NMR spectrum of 3 was similar to that of garcibiphenyl A [6] except that the H-4 in garcibiphenyl A at δ = 6.37 (1H, t, J = 1.9 Hz) was replaced in 3 by a 2-hydroxy-3-methyl-3-butenyl group [δ = 1.87 (3H, s, H-5′′), 2.42 (1H, br s, OH-2′′), 2.80 (1H, dd, J = 14.5, 8.8 Hz, H-1′′), 3.17 (1H, dd, J = 14.5, 2.0 Hz, H-1′′), 4.34 (1H, d, J = 8.8 Hz, H-2′′), 4.88, 5.02 (each 1H, s, H₂ - 4′′)]. The stereochemistry at the C-2′′ position of 3 follows from correlation of optical activity {[α]_D ²⁴: + 8.8° (c 0.15, CHCl₃)} with that of 7-hydroxy-6-(2-(R)-hydroxy-3-methylbut-3-enyl)coumarin {[α]_D ²⁴: + 8° (c 0.2, CHCl₃)} [8]. The formation of 3 may be the result of the cleavage at the pyran ring O-C bond of 1 in the biogenetic process. According to the above data, 3 is a new structure determined to be (R)-5-methoxy-4-(2′′-hydroxy-3′′-methylbut-3′′-enyl)-[1,1′-biphenyl]-3,4′-diol, named garcibiphenyl D; this was further confirmed by the NOESY experiments (see Fig. 3 in Supporting Information). The assignment of ¹³C-NMR resonances was confirmed by HSQC and HMBC techniques (see Fig. 3 in Supporting Information), which also supported the structure of 3.

Garcibiphenyl E (4) was isolated as a colorless oil. The molecular formula of 4 was established as C₁₈H₂₀O₄ by EI-MS ([M]⁺, m/z = 300) and HR-EI-MS. The UV absorptions at 209, 227 (sh), and 268 nm were similar to those of garcibiphenyl D (3), and the observation of a bathochromic shift in alkaline solution also suggested the presence of a phenolic biphenyl nucleus. The presence of hydroxy groups in the molecule was revealed by the band at 3354 cm^-1 in the IR spectrum, which was confirmed by the signal at δ = 4.90 (br s, D₂O exchangeable, OH-4′) in the ¹H-NMR spectrum. The ¹H-NMR spectrum of 4 was similar to that of garcibiphenyl D (3) except that the 2-hydroxy-3-methyl-3-butenyl group in 3 at C-4 was replaced in 4 by a 4-hydroxy-3-methyl-2-butenyl group [δ = 1.78 (3H, d, J = 1.4 Hz, H-5′′), 3.50 (2H, d, J = 8.0 Hz, H-1′′), 4.32 (2H, s, H-4′′), 5.45 (1H, td, J = 8.0, 1.4 Hz, H-2′′)]. On the basis of the above results, 4 is a new structure determined to be 5-methoxy-4-(4′′-hydroxy-3′′-methylbut-2′′-enyl)-[1,1′-biphenyl]-3,4′-diol, named garcibiphenyl E; this was further confirmed by the NOESY experiments (see Fig. 4 in Supporting Information). The assignment of ¹³C-NMR resonances was confirmed by HSQC and HMBC techniques (see Fig. 4 in Supporting Information), which also supported the structure of 4.

The known isolates, including two xanthones, 1,5-dihydroxy-6-methoxyxanthone (5) [9] and 1,7-dihydroxy-3-methoxyxanthone (6) [10], a benzaldehyde, syringaldehyde (7) [11], a triterpenoid, squalene (8) [12], α-tocospiro B (9) [13], a benzoquinone, α-tocopherylquinone (10) [14], seven steroids, 6β-hydroxystigmast-4-en-3-one (11) [15], a mixture of β-sitosterol (12) and stigmasterol (13) [16], a mixture of β-sitostenone (14) and stigmasta-4,22-dien-3-one (15) [16], a mixture of 6α-hydroxystigmast-4-en-3-one (16) and 6α-hydroxystigmasta-4,22-dien-3-one (17) [17] were readily identified by comparison of physical and spectroscopic data (UV, IR, ¹H-NMR, and mass spectrometry data) with corresponding authentic samples or literature values.

The antitubercular effects of the isolates from the roots of Formosan G. linii were tested in vitro against Mycobacterium tuberculosis 90 - 221 387. The antitubercular activity data are shown in Table [3]. The clinically used antitubercular agent, ethambutol, was used as the positive control. From the results of our antitubercular tests, the following conclusions can be drawn regarding these isolates: (a) Among the analogues (5, 6, 22, and 23), 6 and 22 (with 3-methoxy) exhibited more potent antitubercular activities than 5 (with 6-methoxy) and 23 (with 5-methoxy) against Mycobacterium tuberculosis 90 - 221 387. This revealed that 3-methoxy group probably plays an important role in the antitubercular activity. (b) Compounds 6 and 22 are the most effective among the isolates, with MICs of 3.1 ± 0.5 and 6.3 ± 1.0 μg/mL against Mycobacterium tuberculosis 90 - 221 387 in vitro, respectively. Especially, 6 exhibited more potent antitubercular activities than ethambutol (MICs value = 6.2 ± 1.3 μg/mL) against Mycobacterium tuberculosis 90 - 221 387 in vitro. (c) The biphenyls 1, 3, 4, and 24, and the chromenoxanthones 18, 19, and 20 exhibited less antitubercular activities than 6 and 22 against Mycobacterium tuberculosis 90 - 221 387 in vitro.

Materials and Methods

Extraction and isolation: The roots of G. linii were collected from Lanyu Island, Taiwan, in August 2002 and identified by Dr. I. S. Chen. A voucher specimen (Chen 6107) was deposited in the herbarium of the School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. The dried root (10.3 kg) was extracted with cold MeOH, and the extract concentrated under reduced pressure. The MeOH extract (1.56 kg), when partitioned between H₂O-CHCl₃ (1 : 1), afforded a CHCl₃-soluble fraction (fr. A, 21 g). Fr. A (21 g) was chromatographed on silica gel (660 g), eluting with CH₂Cl₂, gradually increasing the polarity with MeOH to give 15 frs: fr. A1 (1200 mL, CH₂Cl₂), fr. A2 - A5 (each 1200 mL, CH₂Cl₂-MeOH, 50 : 1), fr. A6 - A7 (each 1200 mL, CH₂Cl₂-MeOH, 30 : 1), fr. A8 (2400 mL, CH₂Cl₂-MeOH, 20 : 1), fr. A9 - A11 (each 1200 mL, CH₂Cl₂-MeOH, 10 : 1), fr. A12 - A13 (each 1200 mL, CH₂Cl₂-MeOH, 5 : 1), fr. A14 (1200 mL, CH₂Cl₂-MeOH, 1 : 1), fr. A15 (3600 mL, MeOH). Fr. A5 (1.5 g) was chromatographed on silica gel (54 g) eluting with CH₂Cl₂-acetone (8 : 1) to give 8 frs (each 500 mL, fr. A5 - 1 - fr. A5 - 8). Fr. A5 - 5 (153 mg) was further purified by preparative TLC (CHCl₃-MeOH, 10 : 1) to yield 2 (4.3 mg) (R_f = 0.43) and 4 (4.3 mg) (R_f = 0.48). Fr. A6 (2.2 g) was chromatographed on silica gel (72 g) eluting with CH₂Cl₂-acetone (6 : 1) to give 10 frs (each 500 mL, fr. A6 - 1 - fr. A6 - 10). Fr. A6 - 5 (254 mg) was further purified by preparative TLC (CHCl₃-MeOH, 6 : 1) to yield 3 (4.5 mg) (R_f = 0.59). Fr. A6 - 6 (211 mg) was further purified by preparative TLC (CHCl₃-MeOH, 5 : 1) to yield 1 (1.4 mg) (R_f = 0.64).

Isolation of known compounds 5 - 17 : see Supporting Information.

(S)-3-Hydroxygarcibenzopyran (1): Colorless amorphous powder; [α]_D ²⁴: -10.8° (c 0.17, CHCl₃); UV (MeOH): λ_max (log ε) = 210 (4.38), 227 (sh, 4.18), 268 (4.10); (MeOH + KOH): 210 (4.42), 282 nm (4.06); IR: ν_max = 3377 (br, OH), 1612, 1574, 1490, 1453 cm^-1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 300 (M⁺, 18), 285 (22), 282 (16), 269 (22), 229 (51), 147 (47), 128 (31), 115 (30), 97 (46), 84 (59), 73 (71); HR-EI-MS: C₁₈H₂₀O₄, found: 300.1359 [M]⁺, calcd: 300.1356; ¹H-NMR: see Table [1]; ¹³C-NMR: see Table [2].

Garcibiphenyl C (2): Colorless oil; UV (MeOH): λ_max (log ε) = 206 (4.66), 264 (4.11); (MeOH + KOH): 207 (4.85), 281 nm (4.08); IR: ν_max = 3353 (br, OH), 1607, 1500, 1463 cm^-1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 246 (M⁺, 32), 231 (100), 216 (47), 215 (28), 153 (23), 138 (16), 89 (31); HR-EI-MS: C₁₄H₁₄O₄, found: 246.0893 [M]⁺, calcd: 246.0892; ¹H-NMR: see Table [1].

Garcibiphenyl D (3): Colorless amorphous powder; [α]_D ²⁴: + 37.5° (c 0.15, CHCl₃); UV (MeOH): λ_max (log ε) = 208 (4.40), 228 (sh, 4.10), 268 (4.10); (MeOH + KOH): 208 (4.47), 283 nm (4.04); IR: ν_max = 3352 (br, OH), 1613, 1569, 1502, 1453 cm^-1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 300 (M⁺, 6), 230 (41), 229 (100), 199 (38), 184 (11), 171 (18), 157 (13), 128 (17); HR-EI-MS: C₁₈H₂₀O₄, found: 300.1354 [M]⁺, calcd: 300.1356; ¹H-NMR: see Table [1]; ¹³C-NMR: see Table [2].

Garcibiphenyl E (4): Colorless oil; UV (MeOH): λ_max (log ε) = 209 (4.37), 227 (sh, 4.16), 268 (4.08); (MeOH + KOH): 207 (4.49), 282 nm (4.04); IR: ν_max = 3354 (br, OH), 1610, 1501, 1454 cm^-1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 300 (M⁺, 18), 282 (37), 267 (100), 252 (13), 229 (28), 216 (45), 197 (15), 115 (11); HR-EI-MS: C₁₈H₂₀O₄, found: 300.1357 [M]⁺, calcd: 300.1356; ¹H-NMR: see Table [1]; ¹³C-NMR: see Table [2].

Compounds 18 - 24: refer to our previous paper [6].

Antitubercular activity assay: Antitubercular activity of each test compound was evaluated and compared with the minimal inhibitory concentration (MICs) using the strain of Mycobacterium tuberculosis 90 - 221 387. Middlebrook 7H10 agar was used to determine the MICs as recommended by the proportion method [18]. Briefly, each test compound was added to Middlebrook 7H10 agar supplemented with OADC (oleic acid-albumin-dextrose-catalase) at 50 - 56 °C by a serial dilutions to yield a final concentration of 100 to 0.8 μg/mL. 10 mL of each concentration of test compound-containing medium were dispensed into plastic quadrant petri dishes. The inoculum of test isolate of M. tuberculosis was prepared by diluting the initial inoculum in Middlebrook 7H9 broth until the turbidity was reduced to that of an equivalent of McFarland no. 1 standard. Final suspensions were performed by adding Middlebrook 7H9 broth and preparing 10 ^{- 2} dilutions of the standardized suspensions. After solidification 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 °C with 10 % CO₂ for 2 weeks.

Acknowledgements

This work was supported by a grant from the National Science Council of the Republic of China (NSC 91-2320-B-127-006).

References

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Dr. J. J. Chen

Department of Pharmacy

Tajen University

Pingtung 907

Taiwan

Republic of China

Phone: +886-8-762-4002 ext. 332

Fax: +886-8-762-5308

Email: jjchen@mail.tajen.edu.tw

References

• 1 Robson N KB. Guttiferae in the Flora of Taiwan. Vol 2, 2nd Edition. Taipei; Editorial Committee of the Flora of Taiwan 1996: p 694-714
  Search in Google Scholar
• 2 Asano J, Chiba K, Tada M, Yoshii T. Cytotoxic xanthones from Garcinia hanburyi .  Phytochemistry. 1996;  41 815-20
  CrossrefPubMedSearch in Google Scholar
• 3 Xu Y J, Yip S C, Kosela S, Fitri E, Hana M, Goh S H. et al . Novel cytotoxic, polyprenylated heptacyclic xanthonoids from Indonesian Garcinia gaudichaudii (Guttiferae).  Org Lett. 2000;  2 3945-8
  CrossrefPubMedSearch in Google Scholar
• 4 Xu Y J, Chiang P Y, Lai Y H, Vittal J J, Wu X H, Tan B KH. et al . Cytotoxic prenylated depsidones from Garcinia parvifolia .  J Nat Prod. 2000;  63 1361-3
  CrossrefPubMedSearch in Google Scholar
• 5 Ito C, Itoigawa M, Miyamoto Y, Onoda S, Rao K S, Mukainaka T. et al . Polyprenylated benzophenones from Garcinia assigu and their potential cancer chemopreventive activities.  J Nat Prod. 2003;  66 206-9
  CrossrefPubMedSearch in Google Scholar
• 6 Chen J J, Chen I S, Duh C Y. Cytotoxic xanthones and biphenyls from the root of Garcinia linii .  Planta Med. 2004;  70 1195-200.
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Li Y, Hu Y, Xie Z, Chen X. Enantioselective total synthesis of chiricanine B.  Tetrahedron Asymmetry. 2003;  14 2355-60
  CrossrefPubMedSearch in Google Scholar
• 8 Ahsan M, Gray A I, Leach G, Waterman P G. Novel angular pyranocoumarins from Boronia lanceolata .  Phytochemistry. 1994;  36 777-80
  CrossrefPubMedSearch in Google Scholar
• 9 Patnaik M, Sharma V K, Parmar V S, Boll P M. Synthesis of new trioxygenated xanthones of Tovomita excelsa .  Acta Chem Scand B. 1987;  41 210-2
  PubMedSearch in Google Scholar
• 10 Ikeya Y, Sugama K, Okada M, Mitsuhashi H. Two xanthones from Polygala tenuifolia .  Phytochemistry. 1991;  30 2061-5
  CrossrefPubMedSearch in Google Scholar
• 11 Gutierrez A B, Herz W. Bisabolones and other constituents of Mikania shushunensis .  Phytochemistry. 1988;  27 3871-4
  CrossrefPubMedSearch in Google Scholar
• 12 Deiana M, Corongiu F P, Dessi M A, Scano P, Casu M, Lai A. NMR determination of site-specific deuterium distribution (SNIF-NMR) in squalene from different sources.  Magn Reson Chem. 2001;  39 29-32
  CrossrefPubMedSearch in Google Scholar
• 13 Chiang Y M, Kuo Y H. Two novel α-tocopheroids from the aerial roots of Ficus microcarpa .  Tetrahedron Lett. 2003;  27 5125-8
  PubMedSearch in Google Scholar
• 14 Tsai I L, Jeng Y F, Duh C Y, Chen I S. Cytotoxic constituents from the leaves of Litsea akoensis .  Chin Pharm J. 2001;  53 291-301
  PubMedSearch in Google Scholar
• 15 Achenbach H, Loewel M. Constituents of Isolona maitlandii .  Phytochemistry. 1995;  40 967-74
  CrossrefPubMedSearch in Google Scholar
• 16 Chen J J, Huang Y C, Chen Y C, Huang Y T, Wang S W, Peng C Y. et al . Cytotoxic amides from Piper sintenense .  Planta Med. 2002;  68 980-5
  Thieme ConnectPubMedSearch in Google Scholar
• 17 Chen J J, Chang Y L, Teng C M, Chen I S. Anti-platelet aggregation alkaloids and lignans from Hernandia nymphaeifolia .  Planta Med. 2000;  66 251-6
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Inderlied C B, Nash K A. Antibiotics in laboratory medicine. 4th Edition. Philadelphia; Lippincott Williams & Wilkins 1996: p 127-75
  Search in Google Scholar

Dr. J. J. Chen

Department of Pharmacy

Tajen University

Pingtung 907

Taiwan

Republic of China

Phone: +886-8-762-4002 ext. 332

Fax: +886-8-762-5308

Email: jjchen@mail.tajen.edu.tw

• Supplementary Material