Isolation of Mangiferin from Bombax malabaricum and Structure Revision of Shamimin

• Abstract
• Materials and Methods
• Acknowledgments
• References

Abstract

Repeated column chromatography of the n-BuOH fraction of the 70 % EtOH of the dried leaves of Bombax malabaricum led to the isolation of mangiferin, a xanthone. Mangiferin was identified by UV, ¹H- and ¹³C-NMR spectroscopy and electrospray mass spectrometry. It was found to be identical to shamimin, a compound for which originally a flavonol structure was proposed, and the structure of which has to be revised.

Bombax is the common name for members of the Bombaceae, a family including many commercially important members, e. g., the baobab, the balsa, the kapok and several species of the genera Bombax and Ceriba whose seed fibres are used as filling materials. Bombax malabaricum DC. (syn. B. ceiba L. and Salmalia malabaricum DC.) is also known as silk-cotton tree. It is widely cultivated in Pakistan, India, China and Australia. B. malabaricum was introduced into Egypt several decades ago as an ornamental plant and shade tree [1], [2], [3]. All parts of the plant have medicinal significance, e. g., leaves are reported to have application in skin eruptions and they are used to reduce blood sugar levels; roots and flowers are regarded as having diuretic, laxative and restorative properties. The gum is useful in acute dysentery. Phytochemical investigations of different parts of this plant resulted in the isolation of naphthol and naphthoquinone derivatives, anthocyanins, shamimin (a flavonoid C-glycoside), and lupeol [4], [5], [6].

Compound 1 was isolated from the n-BuOH fraction of the 70 % alcoholic extract of the leaves of Bombax malabaricum by column chromatography on polyamide, followed by column chromatography on Sephadex LH-20. Compound 1 was obtained as an amorphous yellow powder. The ¹³C-NMR spectrum of compound 1 showed an obvious similarity with 1,3,6,7-tetrahydroxyxanthone; however, one of the singlets in the ¹H-NMR spectrum and a CH signal in the ¹³C-NMR spectrum were missing [7]. An additional quaternary carbon atom was observed at δ = 107.58, which was attributed to C-2. Remaining signals in the ¹H- and ¹³C-NMR spectra were in agreement with a C-glycoside. Indeed, the ¹H- and ¹³C-NMR spectral data were in complete agreement with those reported for mangiferin or 2-β-D-glucosyl-1,3,6,7-tetrahydroxyxanthone (listed without any assignment) [8]. This structural proposal was confirmed by positive ion electrospray mass spectrometry, showing a molecular ion at m/z = 423 ([M + H]⁺). The NMR data were also in complete agreement with those reported for shamimin or 6-β-D-glucosyl-3,5,7,2′,4′,5′-hexahydroxyflavone reported before from Bombax ceiba (Fig. [1]) [6]. However, the mass spectral data reported for shamimin, i. e., a signal at m/z = 421 in negative FAB-MS, rather point towards a compound with a molecular weight of 422, such as mangiferin, whereas the putative structure of shamimin requires a molecular weight of 480. On the other hand, the EI mass spectrum reported for shamimin is in good agreement with the fragmentation pattern published before for mangiferin [9]. In addition, the ¹³C-NMR assignments reported for shamimin contain two overlapping signals, C-2 and C-3′, at δ = 150.81, and C-3 and C-5′ at δ = 143.70 and, as a consequence, the ¹H- and ¹³C-NMR spectral data could just as well be assigned to mangiferin. The same authors have reported a ¹H- and ¹³C-NMR study of some acetylated derivatives of shamimin [10]. Also for these derivatives, double assignments are always needed to allow complete ¹³C assignments. In addition, heptaacetylmangiferin has a relative molecular mass of 716, and several EI-mass spectra of acetylated shamimin derivatives reported in the same study show a signal at m/z = 716. Therefore, the NMR and MS data reported for acetyl derivatives of shamimin cannot support its structure. Also the optical rotation reported for shamimin {[α]_D ²⁴: + 30.55° (c 0.11, DMSO)} is in good agreement with the [α]_D value of mangiferin (+ 32°). Finally, a direct comparison on TLC, and comparison of the UV, ¹H- and ¹³C-NMR spectra of our compound isolated from Bombax malabaricum, a sample of shamimin obtained from Faizi et al., and a commercially available sample of mangiferin, revealed that the three compounds were identical. The UV spectra of all three samples were identical to those reported before for mangiferin [8], showing a characteristic xanthone spectrum presenting four maxima [11], whereas for a flavonol with a free hydroxyl group in position 3 such as shamimin rather two bands (band I, 350 - 385 nm; band II, 250 -280 nm) are expected [12]. Therefore, we conclude that the structure reported for shamimin [6] is not correct and has to be revised, and that in fact it is identical to mangiferin. Consequently, it should be noted that the hypotensive and hypoglycaemic activities reported for shamimin [5] apply in fact to mangiferin.

Materials and Methods

General experimental procedures: Column chromatography was carried out on polyamide 6 S (Riedel-de Haën, Germany) and Sephadex LH-20 (Pharmacia). TLC was carried out on silica gel 60 F-254 plates (Merck); solvent system: EtOAc-HOAc-HCOOH-H₂O (30/0.8/1.2/8) v/v; detection: Neu’s spray reagent (1 % diphenylboric acid ethanolamine complex), UV detection at 254 and 366 nm.

Electrospray ionisation (ESI) mass spectra were recorded on an Autospec TOF MS (Micromass) of EBE time-of-flight design equipped with an electrospray ionisation (ESI) source and microchannel plate (MCP) detector. The ESI source was operated at 4 kV and a cone voltage of + 40 V. ¹H- and ¹³C-NMR spectra were recorded in DMSO-d ₆ on a Bruker DRX-400 instrument. The chemical shifts were reported in δ values (ppm) with TMS as the internal standard. UV spectra were recorded in MeOH using a Kontron Uvikon 931 instrument.

Plant material: Leaves of Bombax malabaricum DC. were collected at Giza Street, Area of Imbaba, Giza, Egypt in May, 2000. The plant was kindly identified by Prof. Dr. K. H. Al-Batanony, Professor of Taxonomy, Faculty of Science, Cairo University to whom the authors are greatly indebted. A voucher specimen (Bombaceae/Bombax malabaricum/05-2000) is kept at the Herbarium of the NRC, Dokki, Cairo, Egypt.

A reference sample of mangiferin was obtained from Extrasynthèse, France. A sample of shamimin was obtained from Prof. S. Faizi, University of Karachi, Pakistan [6].

Extraction and isolation: 500 g of the dried powdered leaves were defatted with petroleum ether (40 - 60 °C). After drying, the residue was extracted with 70 % EtOH (4 × 1000 mL). The alcohol extract was evaporated under reduced pressure and kept in the refrigerator overnight. After filtration the supernatant was successively extracted with CHCl₃, EtOAc and n-BuOH (5 × 300 mL each). The n-BuOH fraction (4 g) was subjected to column chromatography on polyamide (60 × 3 cm) and eluted first with H₂O (150 mL), and then with a gradient of H₂O and MeOH with an increasing amount of MeOH, in steps of 10 % (elution volume each time 200 mL). Fractions eluted with 60 % MeOH were collected and concentrated. Repeated column chromatography on Sephadex LH-20 (Pharmacia) (60 × 1.5 cm) using MeOH as eluent (3 × 200 mL) gave compound 1 (98 mg).

Mangiferin (2-β- D -glucosyl-1,3,6,7-tetrahydroxyxanthone) (1). UV (MeOH): λ_max (log ε) = 243 (4.56), 259 (4.61), 318 (4.26), 368 nm (4.22) [8]. ESI-MS (positive ion mode): m/z = 423 ([M + H]⁺). ¹H-NMR (400 MHz, DMSO-d ₆): Table [1]. ¹³C-NMR (100 MHz, DMSO-d ₆): Table [1].

Acknowledgments

Dr. M. Claeys (Department of Pharmaceutical Sciences, University of Antwerp) is kindly acknowledged for recording mass spectra. Prof. S. Faizi (University of Karachi) is kindly acknowledged for sending a sample of shamimin.

References

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Dr. A. A. Shahat

Department of Pharmaceutical Sciences

University of Antwerp

Universiteitsplein 1

2610 Antwerp

Belgium

Fax: +32 3 820 27 09

Email: shahat@uia.ua.ac.be

References

• 1 Chanda Y R. The Wealth of India, Raw Material. Vol. IX New Delhi; CSIR Hillside Rood 1962: pp. 175-83
  Search in Google Scholar
• 2 Bircher W H. Gardens of Hesperides. Cairo; The Anglo-Egyptian Book Shop 1960
  Search in Google Scholar
• 3 Nadkarni K M. The Indian Materia Medica. Bombay; Popular Parkashan 1976: pp. 207-9
  Search in Google Scholar
• 4 MacDonald Hocking G. A Dictionary of Natural Products. Medford; Plexus Publishing, Inc 1997: p. 119
  Search in Google Scholar
• 5 Saleem R, Ahmad M, Hussain S, Qazi A M, Ahmad S I, Qazi M H, Ali M, Faizi S, Akhtar S, Hussain S N. Hypotensive, hypoglycaemic and toxicological studies on the flavonol C-glycoside shamimin from Bombax ceiba .  Planta Med. 1999;  65 331-4
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Faizi S, Ali M. Shamimin: A new flavonol C-glycoside from leaves of Bombax ceiba .  Planta Med. 1999;  65 383-5
  PubMedSearch in Google Scholar
• 7 Hano Y, Matsumoto Y, Shinohara K, Sun J -Y, Nomura T. Structures of four new isoprenylated xanthones, cudraxanthones L, M, N, and O from Cudrania tricuspidata .  Planta Med. 1991;  57 172-5
  PubMedSearch in Google Scholar
• 8 Wada H, Shimizu Y, Tanaka N, Cambie R C, Braggins J E. Chemical and chemotaxonomical studies of ferns. LXXXVII. Constituents of Trichomanes reniforme .  Chem Pharm Bull. 1995;  43 461-5
  PubMedSearch in Google Scholar
• 9 Aritomi M, Komori T, Kawasai T. Massenspektren von Isomangiferin und Mangiferin sowie verwandter peracetylierter C-Glucoside.  Liebigs Ann Chem. 1970;  734 91-101
  PubMedSearch in Google Scholar
• 10 Faizi S, Muhammad  A. ¹H and ¹³C NMR of acetyl derivatives of shamimin, a flavonol C-glycoside.  Magn Reson Chem. 2000;  38 701-3
  CrossrefPubMedSearch in Google Scholar
• 11 Hostettmann K, Hostettmann M. Xanthones. In: Dey PM, Harborne JB, editors Methods in Plant Biochemistry Vol. 1 Plant Phenolics. London; Academic Press 1989: pp. 493-508
  Search in Google Scholar
• 12 Markham K R. Techniques of Flavonoid Identification. London; Academic Press 1982: pp. 36-51
  Search in Google Scholar

Dr. A. A. Shahat

Department of Pharmaceutical Sciences

University of Antwerp

Universiteitsplein 1

2610 Antwerp

Belgium

Fax: +32 3 820 27 09

Email: shahat@uia.ua.ac.be