Antitubercular Cassane Furanoditerpenoids from the Roots of Caesalpinia pulcherrima

• Abstract
• Materials and Method
• Acknowledgements
• References

Abstract

Activity-guided fractionation of a root extract of Caesalpinia pulcherrima led to the isolation of two cassane-furanoditerpenoids, 6β-benzoyl-7β-hydroxyvouacapen-5α-ol (1) and 6β-cinnamoyl-7β-hydroxyvouacapen-5α-ol (2). Compound 2 showed strong antitubercular activity with a minimum inhibitory concentration (MIC) of 6.25 μg/mL, whereas the benzoyl analogue (1) was less active (MIC 25 μg/mL). Both compounds expressed moderate cytotoxic activity towards KB (human oral carcinonoid cancer), BC (human breast cancer) and NCl-H187 (small cell lung cancer) cell lines.

The incidence of tuberculosis has rapidly increased worldwide, particularly among patients afflicted with HIV infection. It is estimated that approximately one-third of the global population is infected with Mycobacterium tuberculosis and that seven to eight million new cases of tuberculosis occur each year [1], [2]. Development of new drugs for use against the emerging multidrug resistant strains of M. tuberculosis is therefore urgently needed. Caesalpinia pulcherrima belongs to the Fabaceae family, a tropical shrub grown widely in south and south-east Asia. In addition to its well known use as an ornamental plant, its parts have been utilized as a component in several traditional remedies in Thailand. The flowers and leaves are believed to be able to relieve fever via infusion, whereas the stem is abortifacient and emmenagogue [3], [4]. Furthermore, its root has been recognized in the northern Thai region to be one of the ingredients in a traditional recipe for treating patients with tuberculous symptoms [5]; indeed, we have found that the crude CH₂Cl₂ extract of C. pulcherrima exhibited relatively strong anti-tubercular activity (MIC 25 μg/mL). This has prompted us to investigate the active ingredients of this plant.

Fractionation of a CH₂Cl₂ extract of the roots of C. pulcherrima by column chromatography yielded two furanoditerpenoids, which were identified as the known 6β-benzoyl-7β-hydroxyvouacapen-5α-ol (1) and 6β-cinnamoyl-7β-hydroxyvouacapen-5α-ol (2) by comparison of their spectral data (¹H- and ¹³C-NMR) with those published [6], [7]. Compound 1 has been previously isolated from leaves of C. pulcherrima from the Philippines [6], whereas 2 was found only in the root of the same species in Sri Lanka [7]. The presence of benzoate (e. g., 1) and cinnamate (e. g., 2) ester within the same plant is extremely rare since these two acids are competitive substrates during enzymatic esterification. Interestingly, the optical rotation (positive) of 1 was opposite to the value (negative) published [6]. This corresponds to the difference in melting point range. The present study found that compound 2 exhibited strong antitubercular activity with a minimum inhibitory concentration (MIC) of 6.25 μg/mL, whereas the benzoyl analogue (1) was less active (MIC 25 μg/mL) (Table [1]). Both compounds had moderate cytotoxic activity towards KB, BC and NCl-H187 cell lines (Table [1]). These results supported the traditional use of C. pulcherrima as an ingredient in Thai folklore medicine for the treatment of tuberculous symptoms. Cassanefuranoditerpenoids were previously reported to possess antibacterial activity [6], antitumor activity against KB and P338 cell lines [7], anti-tumor and potential fertility regulating activities [8], selective activity against DNA repair-deficient yeast mutants [9], and antiviral activity [10]. This is the first report for anti-tubercular activity of cassane diterpenes. Although plants of the genus Caesalpinia were previously found to exhibit several biological activities including anticomplementary activity [11], immune modulating function [12], antiulcer action [13], [14], and anti-inflammatory activity [15], there has been no report on anti-tubercular activity of this genus.[]

Materials and Method

General experimental procedures. ¹H, ¹³C, DEPT (135), ¹H-¹H COSY, NOESY, HMQC (optimized for ¹ J _HC = 45 Hz) and HMBC (optimized for ⁿ J _HC = 8 Hz) experiments were carried out on a Bruker DRX 400, operating at 400 MHz for proton and 100 MHz for carbon. The ESITOF mass spectra were obtained from a Micromass LCT mass spectrometer. IR spectra were recorded on a Nicolet 510 FT-IR spectrometer and UV spectra on a HP8452A UV-Vis spectrophotometer. Optical rotations were measured on a JASCO DIP370 polarimeter.

Plant material. The plant sample was collected from Lumphun Province, Thailand, in June 2001. It was identified as Caesalpinia pulcherrima (L.) Swartz. by J. F. Maxwell, and a voucher specimen (# N-01) is deposited in CMU Herbarium, Department of Biology, Faculty of Science, Chiang Mai University, Thailand.

Extraction and isolation. Air-dried roots of C. pulcherrima (0.562 kg) was ground and macerated in CH₂Cl₂ (2 L) for 2 days. The CH₂Cl₂ extract was filtered and the solvent was evaporated to dryness (17.43 g). This extract was column chromatographed on a Sephadex LH20 (200 g, MeOH as eluent) to yield 20 fractions (40 mL each). Fraction 7 was partially purified by flash column chromatography (silica gel 80 g, eluent CH₂Cl₂/EtOAc ranging from 9.5 : 0.5 to 1 : 1 by increasing 5 % of EtOAc for every 100 mL) from which 66 fractions (15 mL each) were collected. Fractions 13 - 25 were combined and rechromatographed (silica gel 40 g, eluent hexane/CH₂Cl₂/EtOAc, 1 : 0.5 : 0.25, 1 : 0.75 : 0.25 and 1 : 1 : 0.25, 300 mL each) to yield 85 fractions (10 mL each). Crystallization of the combined fractions 20 - 23 from CHCl₃/MeOH afforded compound 1 (226 mg) whereas fractions 4 - 6 were combined and rechromatographed [silica gel 110 g, eluent hexane/CH₂Cl₂/EtOAc, 1 : 0.25 : 0.25 (500 mL) and 1 : 0.5 : 0.25 (540 mL)] to give 52 fractions (20 mL each). Recrystallization of the solid material (CHCl₃/MeOH) obtained from fractions 6 - 9 yielded 2 (147 mg).

6β-Benzoyl-7β-hydroxyvouacapen-5α-ol (1): Colorless needles, m. p. 193 - 195 °C (CHCl₃/MeOH) (Lit. [6] 116 - 118 °C from petroleum ether), R_f 0.29 (CH₂Cl₂) and 0.75 (hexane/EtOAc 1 : 0.5), [α]_D ³⁰: + 18.4 (c 0.977, CHCl₃), ESITOF-MS: m/z = 439.2 ([M + H]⁺, 100 %).

6β-Cinnamoyl-7β-hydroxyvouacapen-5α-ol (2): Colorless needles, m. p. 213 - 215 °C (CHCl₃/MeOH) (Lit. [7] 218 - 221 °C from CHCl₃), R_f 0.24 (CH₂Cl₂) and 0.73 (hexane/EtOAc 1 : 0.5), [α]_D ³⁰: + 53.4 (c 1.1.04, CHCl₃), ESITOF-MS: m/z = 465.5 ([M + H]⁺, 100 %).

Biological activities. The antitubercular activity was assessed against Mycobacterium tuberculosis H37Ra using the microplate Alamar blue assay (MABA) [16]. The standard drugs, isoniazide and kanamycin sulfate, were employed as references. The cytotoxicity assay against the BC (human breast cancer cells), KB (human epidermoid carcinoma of the mouth) and NCI-H87 (lung cancer cells) was performed employing the colorimetric method [17]; the reference substance was ellipticine.

Original copies of NMR spectra of compounds 1 and 2 are obtainable from the author of correspondence.

Acknowledgements

We thank the Biodiversity Research and Training Program (BRT) for financial support. Bioassay Research Facility of BIOTEC is gratefully acknowledged for biological activity tests.

References

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  Thieme ConnectPubMedSearch in Google Scholar
• 15 Hikino H, Taguchi T, Fujimura H, Hiramatsu Y. Anti-inflammatory principles of Caesalpinia sappan wood and of Haematoxylon campechianum wood.  Planta Med. 1977;  31 214-20
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar

Dr. Pakawan Nongkunsarn

Department of Chemistry

Faculty of Science

Chiang Mai University

Chiang Mai 50200

Thailand

Fax: +66-5389-2277

Email: pakawan@science.cmu.ac.th

References

• 1 Small P M, Schecter G F, Goodman P C, Sande M A, Chaisson R E, Hopewell P C. Treatment of tuberculosis in patients with advanced human immunodeficiency virus infection.  New Engl J Med. 1991;  324 289-94
  PubMedSearch in Google Scholar
• 2 Bradford W Z, Martin J N, Reingold A L, Schecter G F, Hopewell P C, Small P M. The changing epidemiology of acquired drug-resistant tuberculosis in San Francisco, USA.  Lancet. 1996;  348 928-31
  CrossrefPubMedSearch in Google Scholar
• 3 Lotschert W, Beese G. Collins Guide to Tropical plants. London; Collins 1983: p. 45
  Search in Google Scholar
• 4 Jain S K, DeFilipps R A. Medicinal Plants of India. Reference Publications Michigan; 1991: pp. 210-1
  Search in Google Scholar
• 5 Wutthithammaweach W. Encyclopedia of herbs: compilation of Thai pharmaceuticals. 1st edn Bangkok; Odion store 1997: p. 488
  Search in Google Scholar
• 6 Ragasa C Y, Hofilena J G, Rideout J A. New furanoid diterpenes from Caesalpinia pulcherrima .  J Nat Prod. 2002;  65 1107-10
  CrossrefPubMedSearch in Google Scholar
• 7 Mcpherson D D, Che C T, Cordell G A, Soejarto D D, Pezzuto J M, Fong H HS. Diterpenoids from Caesalpinia pulcherrima .  Phytochemistry. 1986;  25 167-70
  PubMedSearch in Google Scholar
• 8 Che C T, McPherson D D, Cordell G A, Fong H HS. Pulcherralpin, a new diterpene ester from Caesalpinia pulcherrima .  J Nat Prod. 1986;  49 561-9
  CrossrefPubMedSearch in Google Scholar
• 9 Patil A D, Freyer A J, Webb R L, Zuber G, Reichwein R, Bean M F. et al . Pulcherrimins A - D, novel diterpene dibenzoates from Caesalpinia pulcherrima with selective activity against DNA repair-defiant yeast mutants.  Tetrahedron. 1997;  53 1593-7
  CrossrefPubMedSearch in Google Scholar
• 10 Jiang R W, Ma S C, He Z D, Huang X S, But P P, Wang H. et al . Molecular structures and antiviral activities of naturally occurring and modified cassane furanoditerpenoids and friedelane triterpenoids from Caesalpinia minax .  Bioorg Med Chem. 2002;  10 2161-70
  PubMedSearch in Google Scholar
• 11 Oh S R, Kim D S, Lee I S, Jung K Y, Lee J J, Lee H K. Anticomplementary activity of constituents from the heartwood of Caesalpinia sappan .  Planta Med. 1998;  64 456-8
  PubMedSearch in Google Scholar
• 12 Choi S Y, Yang K M, Jeon S D, Kim J H, Khil L Y, Chang T S. et al . Brazilin modulates immune function mainly by augmenting T cell activity in halothane administered mice.  Planta Med. 1997;  63 405-8
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Bacchi E M, Sertie J A, Villa N, Katz H. Antiulcer action and toxicity of Styrax camporum and Caesalpinia ferrea .  Planta Med. 1995;  61 204-7
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Bacchi E M, Sertie J A. Antiulcer action of Styrax camporum and Caesalpinia ferrea in rats.  Planta Med. 1994;  60 118-20
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Hikino H, Taguchi T, Fujimura H, Hiramatsu Y. Anti-inflammatory principles of Caesalpinia sappan wood and of Haematoxylon campechianum wood.  Planta Med. 1977;  31 214-20
  PubMedSearch in Google Scholar
• 16 Collins L, Franzblau S G. Microplate Alamar Blue Assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium .  Antimicrobial Agents and Chemotherapy. 1997;  41 1004-9
  PubMedSearch in Google Scholar
• 17 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  Journal of National Cancer Institute. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar

Dr. Pakawan Nongkunsarn

Department of Chemistry

Faculty of Science

Chiang Mai University

Chiang Mai 50200

Thailand

Fax: +66-5389-2277

Email: pakawan@science.cmu.ac.th