Isolation and Structure Elucidation of Two New Xanthones from Gentiana azurium Bunge (Fam. Gentianaceae)
O. Pureva, Kh. Oyuna, G. Odontuyaa, A. M. Tankhaevaa, G. G. Nikolaevaa, Khalid M. Khanb, Syed Tasadaque Ali Shahb, and Wolfgang Voelterc
a Institute of Chemistry and Chemical Technology, MAS, Ulaanbaatar-51, Mongolia
b HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi-75270, Pakistan
c Abteilung für Physikalische Biochemie, Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Straße 4, D-72076 Tübingen, Germany
Reprint requests to Prof. Dr. Dr. h.c. Wolfgang Voelter.
Fax: +49(0)-70 71-29 33 48. E-mail: wolfgang.voelter@uni-tuebingen.de Dedicated to Prof. Viqar Uddin Ahmad on the occasion of his 60thbirthday Z. Naturforsch.57 b, 331Ð334 (2002); received November 27, 2001
Gentiana azuriumBunge, Gentianaceae, Xanthones
Two new natural xanthones, 1,3,5-trihydroxy-8-methoxyxanthone (1) and 3,5-dihydroxy-8- methoxy-1-O-β-D-glucopyranosyl xanthone (2) were isolated along with two known xan- thones from Gentiana azurium Bunge (Fam. Gentianaceae). Their structures were deter- mined on the basis of their spectral data.
Introduction
The species of Fam. Gentianaceae are wide- spread in the world flora including more than 200 species, 21 of them are growing in Mongolia. The species of genus of Gentiana, e.g., Gentiana Bar- bata (Froel)Ma, Gentiana algida Pall, Gentiana macrophylla. Pall.,Gentiana decumbenswere suc- cessfully used in Mongolian traditional medicine for the treatment of a variety of diseases [1,3].
Quite a few chemical investigations on the species of Gentianaceae were carried out by several au- thors [1Ð6], as they are rich in biologically active principles, such as xanthones, flavonoids, secoiri- doides and alkaloids.
The Gentiana species are the main natural re- sources for these kinds of compounds as men- tioned above which are also of interest for their chemotaxonomy. In this paper, the structure eluci- dation of two new natural xanthones, isolated fromGentiana azuriumBunge is described.
Results and Discussion
1was obtained from a chloroform fraction ofG.
azurium Bunge as yellow needles, m.p. 259Ð 261∞C (MeOH), analyzed for C14H1006(M+= 274) by EIMS. It gave a positive response to the HgCl2
test, and its UVspectrum showed maxima atλ =
0932Ð0776/2002/0300Ð0331 $ 06.00 ”2002 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com D
202.4, 254.2, 278.8, 330.4 nm in MeOH, suggesting 1to be a xanthone. Its IR spectrum (KBr) showed bands assigned to carbonyl groups (1657 cmÐ1) and aromatic rings (1585, 1450 and 900Ð650 cmÐ
1). The MS of 1 gave a base peak at m/z 273.8 (100) and other fragment ions at m/z 258 (90), 230.8 (70), 82.8 (99.5), 84.8 (56.0), 150.7 (26.9), 57.0 (42.6), typical for tetrasubstituted xanthones [2,4]. In the UVspectrum, recorded with the shift reagent AlCl3, bathochromic shifts in all absorp- tion bands (UVand visible) were observed from 202.4 to 215.2, 254.2 to 258.6, 278.8 to 284 and 330.4 to 358.2 nm, and the latter of these shifts is characteristic for the presence of chelating hydrox- yls at the positions C-1 or C-8 of the molecule [4,6,7,8]. Additionally, a bathochromic shift ap- peared in the UVabsorption band from 278.8 to 291.0 nm, when recording the UVspectrum with sodium acetate, which is characteristic for the presence of moderately acidic hydroxyl groups at positions C-3 or C-6 [3,7,8].
The 1H NMR spectral data indicated the pres- ence of one methoxy group at δ = 3.90 (singlet), one chelated hydroxyl at 12.80 (singlet), one phe- nolic hydroxyl at 5.13, (singlet) and four aromatic protons with meta- and ortho-coupling constants at 6.68, 6.37 (d, 2H,J= 2.1 Hz) and 7.25, 6.71 ppm (d, 2H, J= 9.0 Hz). Thus, the 1H NMR-spectral
332 O. Purevet al.· Two New Xanthones fromGentiana azuriumBunge (Fam. Gentianaceae)
data support those of UVand IR. The 1H NMR spectral shifts and signal assignments are collected in Table 1.
The 13C NMR spectrum of 1 was recorded in CDCl3; signal assignments of 1, 1,3,5,8-tertrahy- droxyxanthone and 1,5,8-trihydroxy-3-methoxy- xanthone are collected in Table 2.
On the basis of these spectral data,1(number- ing is according to reference [7]) was identified to
Table 1.1H NMR spectrum (δ/ppm,J/Hz, DMSO-d6) and signal assignments of1in comparison to 1,3,5,8-tetrahy- droxyxanthone and 1,5,8-trihydroxy-3-methoxyxanthone.
Protons 1 1,3,5,8-Tetrahydroxy- 1,5,8-Trihydroxy-3-
xanthone [4,6] methoxyxanthone [4,6]
2-H 6.68 (d, 1H,J= 2.1) 6.19 (d, 1H,J= 2.5) 6.32 (d, 1H,J= 2.5) 4-H 6.37 (d, 1H,J= 2.1) 6.40 (d, 1H,J= 2.5) 6.52 (d, 1H,J= 2.5) 6-H 7.25 (d, 1H,J= 9.0) 6.58 (d, 1H,J= 9.0) 6.60 (d, 1H,J= 9.0) 7-H 6.71 (d, 1H,J= 9.0) 7.20 (d, 1H,J= 9.0) 7.22 (d, 1H,J= 9.0)
OCH3 3.90 (s, 3H, 8-OCH3) Ð 3.88 (s, 3H, 3-OCH3)
OH 12.80 (s, 1-OH) 8.30 (s, 1-OH) 8.90 (s, 1-OH)
OH Ð 8.10 (s, 8-OH) 8.10 (s, 8-OH)
OH 5.13 (s, 5-OH) 5.80 (s, 5-OH) Ð
Table 2.13C-chemical shifts (δ/ppm, CDCl3) and signal assignments of1, 1,3,5,8- tetrahydroxyxanthone and 1,5,8- trihydroxy-3-methoxyxanthone.
Carbon atom 1 1,3,5,8-Tetrahydroxy- 1,5,8-Trihydroxy-3- xanthone [3] methoxyxanthone [3]
1 161.9 162.1 161.7
2 97.4 98.4 97.0
3 166.0 166.3 160.7
4 92.9 94.3 92.6
4a 157.3 157.3 157.0
5 137.3 137.1 137.1
6 123.8 123.6 123.6
7 109.5 109.3 109.1
8 146.8 151.7 151.7
8a 107.5 107.3 107.1
9 183.6 183.5 Ð
9a 101.0 101.2 Ð
10a 143.2 143.2 Ð
OCH3 56.2 Ð 55.8
be 1,3,5-trihydroxy-8-methoxyxanthone, which is a new natural compound.
Compound2 was isolated from the chloroform fraction ofG. azuriumBunge as a yellow powder which analyzed for C20H20O11and gave a molecu- lar ion peak atm/z = 436 by FABMS. It gave a positive response (red colour) in the presence of MgCl2which is characteristic for a xanthone glu- coside [3,5]. The UVmaxima are found at 201.8,
O. Purevet al.· Two New Xanthones fromGentiana azuriumBunge (Fam. Gentianaceae) 333
253.0, 275.4 and 324.2 nm, suggesting 2 to be a xanthone. Neither bathochromic nor hypsoch- romic shifts were observed in the UVspectrum, recorded in the presence of AlCl3, indicating the absence of free hydroxyl groups at positions C-1 and C-8. The UVspectrum, recorded in ethanolic sodium acetate showed a pronounced bathoch- romic shift of the longest wavelength maximum (λ = 324.25359.2 nm), indicating a free 3-OH [5,6]. The compound also did not respond to the quinol test [5], suggestive for the presence of a free hydroxyl group at C-5. After acidic hydrolysis with 5% HCl, the sugar residue was determined by paper chromatography, in comparison with au- thentic samples, as D-glucose [5], which must be attached to positions C-1 or C-8 of the molecule.
TheRf-values on silica gel/TLC cards (0.2 mm; 10
¥20 cm, Merck, Darmstadt, Germany, chloroform/
methanol = 9:1) for 1 and the aglycone of 2 are identical. Also, there was no difference in their melting points. In the 13C NMR spectrum of 2
Table 3. Comparison of1H NMR data of 2with 5,8-dihydroxy-3-methoxy-1-O-β-D- glucopyranosyl xanthone (δ/ ppm,J/Hz, CDCl3).
Protons 2 5,8-Dihydroxy-3-methoxy-1-O-β-D-
glucopyranosyl xanthone [4]
H-2 6.37 (d, 1H,J= 2.1) 6.52 (d, 1H,J= 3.0)
H-4 6.57 (d, 1H,J= 2.1) 6.60 (d, 1H,J= 3.0)
H-6 7.10 (d, 1H,J= 8.5) 7.30 (d, 1H,J= 9.0)
H-7 7.24 (d, 1H,J= 8.5) 6.68 (d, 1H,J= 9.0)
OCH3 3.88 (s, 3H, 8-OCH3) Ð
OH Ð 12.95 (s, 1H, chelating OH, 8-OH)
OH 10.0 (s, 1H, 3-OH) Ð
OH 5.12 (s,1H, 5-OH) Ð
H-1⬘ 4.88 (d, 1H,J= 6.0, anomeric proton) 5.0 (m, 1H, anomeric proton) Sugar protons 3.48 (six glucosyl protons) 3.50 (six glucosyl protons)
(DMSO-d6) twenty carbon signals were detected having the following chemical shifts: δ 56.1 (OMe), 162.6 (C-1), 97.1 (C-2), 165.1 (C-3), 92.1 (C-4), 156.3 (C-4a) 144.9 (C-10a), 141.0 (C-5), 121.0 (C-6), 112.4 (C-7), 149.4 (C-8), 111.8 (C-8a), 182.0 (C-9), 103.1 (C-9a), 103.5 (C-1⬘), 73.5 (C-2⬘), 76.0 (C-3⬘), 69.7 (C-4⬘), 77.4 (C-5⬘), 60.8 ppm (C- 6⬘). In Table 3, the 1H NMR data of2 are com- pared with those of the reference compound 5,8- dihydroxy-3-methoxy-1-O-β-D-glucopyranosyl xanthone [6].
From the above spectral evidences, compound2 was identified as 3,5-dihydroxy-8-methoxy-1-O-β- D-glucopyranosyl xanthone which is a new natural xanthone (numbering of compounds are according to literature [7]).
Two known compounds, 1,7-dihydroxy-3,8-di- methoxyxanthone (3) and 1-hydroxy-3,7,8-tri- methoxy xanthone (4) were also isolated and iden- tified by theirRfvalues and by comparing their1H NMR and mass spectra [6].
334 O. Purevet al.· Two New Xanthones fromGentiana azuriumBunge (Fam. Gentianaceae)
Experimental Section
1H NMR, 13C NMR and DEPT spectra were recorded on Bruker AM 400 and AMX 500 NMR spectrometers using a UNIX data system at 400 and 500 MHz. The IR spectra were performed on a JASCO A-302 spectrophotometer. The UV spectra were measured on a Hitachi U-3200 instru- ment. The low resolution and high resolution EI- MS were recorded on a JMS HX 110 mass spec- trometer with the data system DA 5000.
Collection, extraction and isolation: The aerial parts of Gentiana azurium Bunge were collected in September 1998 from the Khubsgul district of Mongolia and identified by Dr. Sanchir. A voucher specimen (Gentiana azurium Bunge No.
223Ð03) has been deposited in the herbarium of the Botanical Institute of the Mongolian Academy of Sciences, Ulaanbaatar-51, Mongolia. The dried plant material (100 g) was extracted with metha- nol at room temperature and after concentration
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and dilution with water the resulting solutions were fractionated by chloroform, ethyl acetate and n-butanol. Eight grams of the chloroform fraction (G. azuriumBunge) were loaded to a silica gel 60 (0.063Ð0.200 mm, Merck, Darmstadt, Germany) column (160 g) and eluted with hexane-ethyl ace- tate (from 1:10 to 1:1) to afford the pure com- pounds 1 (10 mg) and 2 (25 mg), along with two known xanthones.
Hydrolysis of 3,5-dihydroxy-8-methoxy-1-O-β- D-glucopyranosyl xanthone(2):2(10 mg) was hy- drolyzed with 5% HCl, andβ-D-glucose identified by paper chromatography with n-butanol:HOAc:
H2O (4:1:2) and visualized by spraying withp-ani- sidine phthalate [9].
Acknowledgements
One of us (Dr. O. Purev) gratefully acknowl- edges the financial support of TWAS in Italy and Deutscher Akademischer Austauschdienst (DAAD, Bonn, Germany).