A new alpinumisoflavone derivative from Genista pichisermolliana

Phytochemistry Letters 4 (2011) 342–344

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A new alpinumisoflavone derivative from Genista pichisermolliana Cecilia Noccioli a, Letizia Meini a, Maria Cecilia Loi b, Donatella Potenza c, Luisa Pistelli a,* a

Dipartimento di Chimica Scienze Farmaceutiche-sede Chimica Biorganica e Biofarmacia, Universita` di Pisa, via Bonanno 33, 56126 Pisa, Italy Dipartimento di Scienze Botaniche, Universita` di Cagliari, V.le S. Ignazio da Laconi 13, 09100 Cagliari, Italy c Dipartimento di Chimica Organica e Industriale, Universita` di Milano, Via Venezian 21, 20133 Milano, Italy b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 February 2011 Received in revised form 6 July 2011 Accepted 8 July 2011 Available online 23 July 2011

The aerial parts of Genista pichisermolliana Valsecchi (Fabaceae), an endemic plant of Sardinia, were extracted in Soxhlet apparatus and purified by several chromatographic methods. The new compound alpinumisoflavone 40 -O-glucopyranoside (6) was isolated together with nineteen flavonoids, p-coumaric methylester and D-pinitol, while no alkaloids were detected. All the chemical structures were elucidated by spectroscopic analysis. Since flavonoids represent the main constituents of this plant, the total flavonoid content was determined according to the Italian Pharmacopoeia IX Ed. method. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Genista pichisermolliana Genisteae Fabaceae Flavonoids Alpinumisoflavone Flavonoid content

1. Introduction The genus Genista L. (Fabaceae) includes about 100 species of shrubs or small trees in the Mediterranean area and in Western Asia and more than 30 of these species are endemic in Sardinia, Sicily, in the North Africa and in the Iberian peninsula. Genista pichisermollina Valsecchi is an endemic shrub that grows on the sunny sloping grounds of the eastern and central Sardinian rises. Flowering occurs between May and June (Valsecchi, 1993). G. pichisermolliana is not used in folk medicine, but other species of the same genus, G. anglica, G. germanica, are used as medicinal plants as diuretics and against gout and kidney stones (Guarrera and Leporatti, 2007; Adams et al., 2009). Flavonoids are characteristic secondary metabolites of the Fabaceae family and are especially abundant in the tribes Genisteae. They are chemotaxonomic markers of the genus Genista together with quinolizidine alkaloids (Harborne, 1994). In our previous phytochemical studies on the Genisteae tribe, we isolated flavonoids from five species growing in the Mediterranean area (Pistelli et al., 1998, 2000, 2003, 2004; Giachi et al., 2002; Lucchesini et al., 2010), as well as alkaloids from G. ephedroides (Pistelli et al., 2001). In the present paper the isolation and the structure elucidation of a novel compound from G. pichisermolliana is described (Fig. 1).

* Corresponding author. Tel.: +39 0502219700; fax: +39 0502219660. E-mail address: [email protected] (L. Pistelli).

Nineteen known flavonoids, p-coumaric methylester and D-pinitol were also detected. 2. Results and discussion Compound 6, a yellowish amorphous powder, obtained from AcOEt extract after repeated chromatographies (see Section 3), gave yellow fluorescence at 366 nm on TLC after spraying with NTS-PEG reagent. The FT-ICR mass spectrum of compound 6 showed a molecular ion peak at 521.14195 m/z [M+Na]+ (calc. [M]+ 498.47864), consistent with a molecular formula of C26H26O10. Analysis of the 1H and 13C NMR spectra (Table 1) of 6 together with the HMQC, COSY and NOESY spectral data revealed an isoflavonoidic skeleton, as shown by the singlet at d 8.13 (H-2) in the proton spectrum and by the signal at d 181.0 (C-4) in the carbon spectrum. The singlet at d 6.37, corresponding to the signal at 95.9 ppm in the 13 C spectrum, was assigned to H-8, suggesting a 5,6,7-trisubstitution on the A ring. Two doublets at d 7.50 and 7.17 (J = 8.3 Hz, H-20 /H-60 and H-30 /H-50 , respectively) in the 1H NMR spectrum indicated a 40 hydroxylated pattern for the B ring (AA’BB’ system). These protons showed a downfield shift, suggesting the presence of a Oglycosylation in 40 . This hypothesis was confirmed by the signals of the anomeric proton at d 4.98 correlated to the characteristic Oglycosidic anomeric carbon at d 101.8 and by five signals between 60 and 80 ppm. The interpretation of 1H–1H COSY and 1H–1H NOESY spectra revealed the presence of a b-glucopyranosyl unit in a 1C4chair. The conformation and the position of the glucose moiety was confirmed by the NOE correlations of H-1000 with H-3000 and H-5000 , and

1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.07.005

C. Noccioli et al. / Phytochemistry Letters 4 (2011) 342–344

O

O

H3C H3C

OH

O

O

OH

HO

OH CH2OH

O

H O

Position

dH (J in Hz)

dC

NOESY

HMBC

8.13 – – – – – 6.37 – – – 7.50 7.17 – 7.17 7.50 6.70 5.73 – 1.47 1.47 4.98 3.50 3.49 3.43 3.46 3.72 3.92

154.4 123.7 181.0 159.7 105.4 156.8 95.5 157.8 106.3 125.3 130.3 117.7 157.4 117.7 130.3 114.9 129.7 78.1 27.5 27.5 101.8 75.0 78.2 71.4 78.1 62.6 –

H-20 ,60 – – – – – –CH3 – – – – 100 0 – 100 0 – H-200 H-100 – H-8 H-8 H-30 ,50 ,300 0 ,500 0 – – – –

C-4,9,10 ,20 ,60 – – – – – C-6,10 – – – C-10 ,30 ,50 C-10 ,40 – C-10 ,40 C-10 ,30 ,50 C-5,7,300 ,400 ,500 C-6,300 ,400 ,500 – – – – – – 600 0 – 400 0

s

br s

d (8.3) d (8.8) d d d d

(8.8) (8.3) (10.3) (10.3)

s s d (6.3) dd (9.4, 6.3) m m m dd (12.5, 2.5) dd (12.5, 5.7)

with H-30 and H-50 , respectively (Fig. 2) and by comparison with literature data (Watanabe et al., 1993). In the 1H NMR spectrum the presence of a g,g-dimethylpyran system was evidenced by two doublets at d 6.70 and 5.73 (J = 10.3 Hz, characteristic of two vinyl protons) and a singlet at d 1.47 integrating for six protons and it was positioned on C-6 and C-7 of the A ring, by the comparison with the literature data (Martinez Olivares et al., 1982). These data were confirmed on the basis of the HMBC correlations reported in Table 1 and Fig. 3. So the new compound 6 was identified as 40 -Oglucopyranosyl-40 ,5-dihydroxy-200 ,200 -dimethylpyran-[500 ,600 ;6,7]isoflavone, also named alpinumisoflavone 40 -O-b-glucopyranoside.

H3C H3C

H O

H

O

H H

H H

OH

O

O

H H

HO H

OH O

OH CH2OH

H

H H

H H

OH

O

O

H H

Fig. 1. The new isoflavone alpinumisoflavone 4 -O-glucopyranoside (6).

2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 100 0 200 0 300 0 400 0 500 0 600 0 Ha 600 0 Hb

H

O

0

Table 1 1 H NMR, 13C NMR, NOESY and HMBC spectral data of compound 6 (CD3OD, d ppm).

343

HO H

OH O

OH CH2OH

H H

Fig. 3. Significant HMQC correlations of alpinumisoflavone 40 -O-glucopyranoside (6).

The analysis of flavonoidic content yielded nineteen known compounds. They were identified and sorted as: eight isoflavones: daidzein (1), genistein (2), genistein 7-O-b-glucopyranoside (3), biochanin A 7-O-b-glucopyranoside (4) (Agrawal, 1989), genistein 40 ,7-di-O-b-glucopyranoside (5) (Watanabe et al., 1993), genistein 8-C-b-glucopyranoside (7) (Kinjo et al., 1987), orobol 8-C-bglucopyranoside (8) (Nunes et al., 1989), 30 -O-methylorobol 8-C-bglucopyranoside (9) (Adinarayana and Rajasekhara, 1972); five flavonols: rutin (10) (Agrawal, 1989), quercetin 3-O-robinobioside (11) (Brasseur and Angeuot, 1986), isorhamnetin 3-O-b-glucopyranoside (12) (Alaniya and Shalashvili, 1981), isorhamnetin 3-O-bgalattopyranoside (13) (Agrawal, 1989), dell’isorhamnetin 3-Orobinobioside (14) (Yasukawa et al., 1989); two flavones: apigenin (15) and luteolin 7-O-b-glucopyranoside (16) (Agrawal, 1989); the flavanone eriodictiol (17) (Agrawal, 1989); the dihydroflavonol aromadendrin 7-O-b-glucopyranoside (18) (Nørbæk et al., 1999); two pterocarpans: maackiain (19) (Matsuura et al., 1994) and 4methoxymaackiain (20) (Ma´ximo and Lourenc¸o, 1998). The structures of all these constituents were confirmed by comparison with authentic samples previously isolated in our laboratory or published data. The results of the present phytochemical study are in agreement with the flavonoidic patterns in the tribe Genisteae reported by Harborne (1969), namely high concentration of isoflavones, absence of leucoanthocyanidins and regular occurrence of glycoflavones and flavonols. Among the Italian Genista species (Harborne, 1994; Luczkiewicz et al., 2004; Giachi et al., 2002; Pistelli et al., 2000, 1998), G. pichisermolliana showed a considerable presence of flavonol glycosides, while the two pterocarpans and all the isorhamnetin glycosides were not already detected in the genus Genista; the presence of D-pinitol is considered a further chemotaxonomic marker in the Leguminosae family (Plouvier, 1962). Since flavonoids represented the main constituents of the analysed plant, the total flavonoid content in the extracts was determined by the Italian Pharmacopoeia IX Ed. Method; the results showed similar amounts of flavonoids among the three extracts, with percentages of 1.9%, 2.4% and 1.5% in the chloroformic, ethyl acetate and butanolic extracts, respectively. In the present analysis no quinolizidine alkaloids were detected in the extracts of G. pichisermolliana, since no characteristic colours were evidenced on TLC after spraying with iodoplatinic reagent; however the distribution of these compounds in the genus Genista is not constant. 3. Experimental 3.1. General experimental procedures

H

Fig. 2. Significant NOE correlations observed for alpinumisoflavone 40 -Oglucopyranoside (6).

Silica gel 60 (230–410 mesh and 63–230 mesh, Merck Kieselgel) were used for flash and column chromatography, respectively; Sephadex LH-20 (Pharmacia Fine Chemicals) for gel filtration.

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Analytical TLC were performed on silica gel (Merck Kieselgel 60 F254) and visualized under UV light at 256 and 366 nm and/or sprayed with NTS-PEG. 1H and 13C NMR spectra (samples solved in CD3OD or DMSO-d6) were registered on a Bruker AC-200 (200.13 MHz for proton and 50.33 MHz for carbon); two-dimensional NMR experiments (COSY, TOCSY, NOESY, HSQC and HMQC) were performed on a Bruker Advance 400 NMR spectrometer. The FT-ICR MS high resolution spectra were performed on a APEX II spectrometer & Xmass software (Bruker Daltonics).

prep. TLC in the same conditions of fraction B12, afforded genistein 8-C-b-glucopyranoside (7, 4 mg) and quercetin 3-O-robinbioside (11, 8 mg). A total of 16 mg of 1, 38 mg of 2, 49 mg of 3, 14 mg of 4, 15 mg of 5, 8 mg of 6, 19 mg of 7, 19 mg of 8, 2 mg of 9, 5 mg of 10, 8 mg of 11, 6 mg of 12, 15 mg of 13, 36 mg of 14, 5 mg of 15, 19 mg of 16, 4 mg of 17, 7 mg of 18, 17 mg of 19, 23 mg of 20, 11 mg of 21 and 446 mg of 22 were isolated from the titled plant and identified by comparison with authentic samples previously isolated in our laboratory or published data.

3.2. Plant materials The aerial parts of G. pichisermolliana were collected at Fonni (Nuoro, Sardinia island) in July 2005; the plant material was authenticated by Dr. M.C. Loi and voucher specimens (290/b) are deposited at Herbarium CAG (Department of Botanical Science, Cagliari, Italy). 3.3. Extraction and isolation Dried and powdered aerial parts (434 g) were extracted in Soxhlet apparatus with petroleum ether (E, 5.4 g), chloroform (C, 13.3 g) and methanol (M, 31.8 g) in sequence. The chloroformic extract, submitted to gel filtration on Sephadex LH-20 eluted with MeOH–CHCl3 (9:1), afforded to thirteen fractions (C1–C13); fractions C8 and C9, put together and flash chromatographated eluting with CHCl3–EtOAc (99:1), furnished methoxymaackiain (20, 21 mg) and daidzein (1, 16 mg). Maackiain (19, 17 mg), genistein (2, 26 mg) and p-coumaric methylester (21, 11 mg) were obtained from fractions C10 and C11 after flash chromatography on silica gel (CHCl3–EtOAc 99:1). Fraction C13, submitted to prep. TLC on silica gel (CHCl3–MeOH 85:15), afforded to eriodictiol (17, 4 mg). The methanolic extract was partitioned first with EtOAc and then with n-BuOH to obtain residues A (8.7 g) and B (8.0 g), respectively. The A residue was fractionated by gel filtration on Sephadex LH-20 column (eluting with MeOH) to yield seventeen fractions (A1–A17). Fractions A9 and A10, put together and submitted to MPLC in MeOH–H2O 1:1 afforded to aromadendrin 7O-b-glucopyranoside (18, 7 mg), genistein 8-C-b-glucopyranoside (7, 7 mg), isorhamnetin 3-O-b-glucopyranoside (12, 6 mg), isorhamnetin 3-O-robinobioside (14, 27 mg) and alpinumisoflavone 40 -O-b-glucopyranoside (6, 8 mg). Genistein 7-O-b-glucopyranoside (3, 37 mg) and luteolin 7-O-b-glucopyranoside (16, 19 mg) were isolated as precipitate from fraction A12, which, after filtration, was submitted to MPLC in MeOH–H2O 4:6, affording to fourteen subfractions (A12.1–A12.14). Orobol 8-C-b-glucopyranoside (8, 19 mg) and isorhamnetin 3-O-b-galattopyranoside (13, 15 mg) were separated from subfraction A12.2 and A12.9, respectively after repeated chromatography. Fraction 15, submitted to Lobar eluting with MeOH–H2O 6:4, furnished apigenin (15, 5 mg) and genistein (2, 12 mg). The n-BuOH residue was subjected to gel filtration on Sephadex LH-20 column, eluting with MeOH–H2O (8:2), to obtain thirteen fractions (B1–B13). Fraction B5 showed a precipitate identified as D-pinitol (22, 446 mg). Fraction B9 was purified by flash chromatography in CHCl3–MeOH–H2O 7:3:0.3 to afford biochanin A 7-O-b-glucopyranoside (4, 14 mg) and genistein 7,40 -O-bdiglucopyranoside (5, 15 mg). 30 -methylorobol 8-C-b-glucopyranoside (9, 2 mg), isorhamnetin 3-O-robinobioside (14, 9 mg) and rutin (10, 5 mg) were obtained as pure compounds after prep. TLC in CHCl3–MeOH–H2O 7:3:0.3, while fraction B13, submitted to

References Adams, M., Berset, C., Kessler, M., Hamburger, M., 2009. Medicinal herbs for the treatment of rheumatic disorders. A survey of European herbals from the 16th and 17th century. J. Ethnopharmacol. 121 (3), 343–359. Adinarayana, D., Rajasekhara, R.J., 1972. Isoflavonoid glycosides of Dalbergia paniculata: the constitutions of dalpanitin and dalpatin. Tetrahedron 28, 5377– 5384. Agrawal, P.K., 1989. Carbon-13 NMR of Flavonoids. Elsevier, Amsterdam. Alaniya, M.D., Shalashvili, K.G., 1981. Flavonoids from Bupleurum exaltatum. Khim. Prir. Soedin. 6, 800–801. Brasseur, T., Angeuot, L., 1986. Flavonol glycosides from leaves of Strychnos variabilis. Phytochemistry 25, 563–564. Giachi, I., Manunta, A., Morelli, I., Pistelli, L., 2002. Flavonoids and isoflavonoids from Genista morisii. Biochem. Syst. Ecol. 30, 801–803. Guarrera, P.M., Leporatti, M.L., 2007. Ethnobotanical remarks on Central and Southern Italy. J. Ethnobiol. Ethnomed. 3, 23. Harborne, J.B., 1969. Chemosystematics of the Leguminosae. Flavonoid and isoflavonoid patterns in the tribe Genisteae. Phytochemistry 8 (8), 1449–1456. Harborne, J.B., 1994. Plants and their constituents. In: Phytochemical Dictionary of the Leguminosae, Chapmann & Hall, London, pp. 313–330. Istituto Poligrafico Zecca dello Stato, Roma. Kinjo, J., Kurusawa, J., Baba, J., Takeshita, T., Yamasaki, M., Nohara, T., 1987. Studies on the constituents of Pueraria lobata. III. Isoflavonoids and related compounds in the roots and the voluble stems. Chem. Pharm. Bull. 35 (12), 4846–4850. Lucchesini, M., Bertoli, A., Mensuali Sodi, A., Cappelli, E., Noccioli, C., Luciardi, L., Pistelli, L., 2010. Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production. Cent. Eur. J. Biol. 5 (1), 111–120. Luczkiewicz, M., Glod, D., Baczek, T., Bucinski, A., 2004. LC-DAD UV and LC-MS for the analysis of isoflavones and flavones from in vitro and in vivo biomass of Genista tinctoria L. Chromatographia 60, 179–185. Martinez Olivares, E., Lwande, W., Delle Monache, F., Marini Bettolo, G.B., 1982. Tephrosieae. Part 12. A pyranoisoflavone from seeds of Milletia thonningii. Phytochemistry 21, 1763–1765. Matsuura, N., Nakai, R., Iinuma, M., Tanaka, T., Inoue, K., 1994. A prenylated flavanone from roots of Maackia amurensis subsp. Buergeri. Phytochemistry 36 (1), 255–256. Ma´ximo, P., Lourenc¸o, A., 1998. A pterocarpan from Ulex parviflorus. Phytochemistry 48, 359–362. Nørbæk, R., Nielsen, J.K., Kondo, T., 1999. Flavonoids from flowers of two Crocus chrysanthus-biflorus cultivars: eye-catcher and Spring Pearl (Iridaceae). Phytochemistry 51, 1139–1146. Nunes, D.S., Haag, A., Bestmann, H.J., 1989. Components from the stem bark of Dalbergia monetaria L. Three new isoflavone C-glucosides. Liebigs. Ann. Chem. 4, 331–335. Pistelli, L., Bader, A., Santucci, M., Noccioli, C., 2004. Alcaloidi chinolizidinici e flavonoidi da Cytisus aeolicus. Italus Hortus 11 (4), 179–181. Pistelli, L., Bertoli, A., Giachi, I., Manuta, A., 1998. Flavonoids from Genista ephedroides. J. Nat. Prod. 61, 1404–1406. Pistelli, L., Bertoli, A., Giachi, I., Morelli, I., Rubiolo, P., Bicchi, C., 2001. Quinolizidine alkaloids from Genista ephedroides. Biochem. Syst. Ecol. 29, 137–141. Pistelli, L., Fiumi, C., Morelli, I., Giachi, I., 2003. Flavonoids from Calicotome villosa. Fitoterapia 74, 417–419. Pistelli, L., Giachi, I., Potenza, D., Morelli, I., 2000. A New Isoflavone from Genista corsica. J. Nat. Prod. 63, 504–506. Plouvier, V., 1962. The cyclitols in some botanical groups; L-inositol of the composites and D-pinitol of the legumes. C. R. Acad. France 255, 1770–1772. Valsecchi, F., 1993. Il genere Genista L. in Italia. Le specie delle sezioni Erinacoides Spach, Ephedrospartum Spach, Aureospartum sect. nova. Webbia 48, 779–824. Watanabe, K., Kinjo, J., Nohara, T., 1993. Leguminous plants. XXXIX. Three new isoflavonoid glycosides from Lupinus luteus and L. polyphyllus  arboreus. Chem. Pharm. Bull. 41 (2), 394–396. Yasukawa, K., Sekine, H., Takido, M., 1989. Two flavonol glycosides from Lysimachia fortunei. Phytochemistry 28 (8), 2215–2216.