Phytochemical study of Gypsophila trichotoma wend. (Caryophyllaceae)

ARTIGO

Phytochemical study of Gypsophila trichotoma wend. (Caryophyllaceae)

Ilina N. Krasteva^I,*; Ivo S. Popov^I; Vessela I. Balabanova^I; Stefan D. Nikolov^I; Ivanka P. Pencheva^II

^IDepartment of Pharmacognosy, Faculty of Pharmacy, 2 Dunav St., 1000 Sofia, Bulgaria

^IIDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, 2 Dunav St., 1000 Sofia, Bulgaria

ABSTRACT

Phytochemical studies of ethyl acetate extracts from the roots and aerial parts of Gypsophila trichotoma revealed the presence of stigmast-7-en-3-ol, stigmasta-7,22-dien-3-ol (spinasterol), ergost-7-en-3-ol, stigmasta-3,5dien7-one, β-sitosterol, 22,23-dihydrospinasterone, vitexin, orientin, homoorientin and hyperoside. The structures of the compounds were determined by GC/MS and HPLC analyses.

Keywords:Gypsophila trichotoma; Caryophyllaceae; flavonoids.

INTRODUCTION

The plants of genus Gypsophila (Caryophyllaceae) are well known by their medicinal, decorative and industrial application. The species of Gypsophila are studied for saponins,¹ flavonoids² and sterols.³Gypsophila saponins have anticarcinogenic properties, including direct cytotoxicity, immune-modulating effects, and normalization of carcinogen induced cell proliferation.⁴ From Gypsophila species were also isolated Δ sterols, which showed high biological activity against hyperplasia of the prostate,⁵ an antimutagen activity,⁶ therapeutic potential to modulate the development and/or progression of diabetic nephropathy⁷ and an antitumor activity.⁸

Gypsophila trichotoma Wend. (Caryophyllaceae) is a perennial herb, located in Southeast Europe (from East Bulgaria to Southeast Russia). The plant is spread in the northeast region of Black sea coast.⁹ Previous studies of G. trichotoma have led to isolation of triterpene saponins.^10-14 This report describes the identification of the steroids and flavonoids in Gypsophila trichotoma Wend.

EXPERIMENTAL

Plant material

The plant material (roots and aerial parts) was collected in June 2003 in the northeast region of Black sea coast, locality "Zelenka", near village Balgarevo, Bulgaria. The voucher specimen (SO 103887) was deposited in Herbarium of Sofia University, Bulgaria.

Extraction and fractionation

The dried roots of G. trichotoma (840 g) were exhaustively extracted with 80% MeOH and evaporated. The extracts were concentrated and the aqueous residue was extracted with Cl₂CH₂, EtOAc and BuOH. The EtOAc extract (10 g) was purified with column chromatography over Sephadex LH-20 (MeOH) and flash chromatography over silica gel (CHCl₃-MeOH, 9:1) to give two main purified sterol subfractions, which were submitted to GC/MS analysis.

The dried aerial parts of G. trichotoma (10 g) were exhaustively extracted with 80% MeOH. The MeOH extract was suspended in water and then successively extracted with CHCl₃ and EtOAc. The EtOAc extract was subjected to column chromatography using Sephadex LH-20 eluted with MeOH to give 23 fractions, which were combined after TLC. Three main purified flavonoid subfractions were further subjected to HPLC.

Analyses of the sterols and flavonoids

GC/MS analysis

GC/MS analysis was performed on a Hewlett Packard 6890 + MS 5973 with a capillary column HP-5, 23 m × 0.2 mm, 0.5 mm film thickness. Helium was used as a carrier gas with a temperature programme 100-315 °C at 5 °C min^-1 and a 10-min hold at 315 °C. GC/MS research was based on the interpretation of the mass spectral fragmentation followed by comparisons of the obtained spectra with those of authentic samples. Computer searches in a HP Mass Spectral Library NIST98 were also applied.

HPLC analysis

HPLC analysis was performed on a Spherisorb C18 ODS, column 5 mm, 250 x 4.6 mm; mobile phases: MeOH:H₂O (60:40 v/v) (1), ACN:H₂O (70:30 v/v) (2) and ACN:H₂O (45:55 v/v) (3); flow-rate: 1 mL/min^-1; UV-detection at 254 nm. The chromatograms were compared with those obtained from authentic samples analysed on the same mobile phases. The reference solutions of vitexin, hyperoside, orientin, and homoorientin were prepared by dissolving an equivalent amount of each of them in a mobile phase to obtain concentration 0.00001 g/mL. The standards were obtained from Extrasynthese (Genay, France). Test solutions – the samples were added 1.0 ml from solvents methanol – water (60:40 v/v).

RESULTS AND DISCUSSION

The ethyl acetate extract of G. trichotoma roots was purified by Silica gel and Sephadex LH-20 column chromatography to give two main sterol fractions, which were further analysed by GC/MS. The investigation was based on the interpretation of the mass spectral fragmentation followed by comparisons of the obtained spectra with those of authentic samples.¹⁵ Peak identification was accomplished by a comparison of their mass spectra with those stored on the GC/MS database. Six compounds were identified as: stigmast-7-en-3-ol, stigmasta-7,22-dien-3-ol (spinasterol), ergost-7-en-3-ol, 24-ethylcholesta-3,5dien7-one, β-sitosterol and 22,23-dihydros-pinasterone (Table 1).

Chromatographic separation of the ethyl acetate extract from the aerial parts over Sephadex LH-20 afforded three purified flavonoid fractions, which were analysed by HPLC. The prescribed HPLC method for analysis of flavonoids is based on literature methods for these compounds with some modifying elements – changed mobile phases and column.^16-18 To achieve an assuredly results the efficacy of the separation were tested with reference substance in different phases. The optimised results are presented on Table 2. Mobile phase (1) is the most suitable for studying of fractions with vitexin. For fractions with vitexin and orientin the most suitable is mobile phase (2). The relative retention of compounds in mobile phase (1) is about 0.90 but in mobile phase (2) – 0.73. The conditions are very suitable for simultaneous determination of both compounds without preliminary separation. Using mobile phase (3) were identified vitexin and homoorientin. Four compounds were identified as hyperoside,¹⁹ vitexin,²⁰ orientin^9-20 and homoorientin²⁰ by comparison of the retention time of the compounds of the flavonoid fractions and standards (Figure 1).

CONCLUSIONS

Phytochemical investigation of G. trichotoma led to identification of six steroids and four flavonoids by GC/MS and HPLC analyses. This is the first report about the presence of these compounds in the roots and aerial parts of the plant.

Recebido em 14/6/07; aceito em 14/11/07; publicado na web 2/7/08

• 1. Acebez, B.; Bernabe, M.; Diaz-Lanza, A. M.; Bartolome, C.; J. Nat. Prod 1998, 61, 1557.
• 2. Darmograi, V. N.; Litvinenko, V. I.; Krivenchuk, P. E.; Khim. Prir. Soed. 1968, 4, 248.
• 3. Schmidt, J.; Boehme, Fr., Adam, G.; Z. Naturforsch, C: J. Biosci. 1996, 51, 897.
• 4. Dalsgaard, K.; Henry, M.; San Martin Gamboa, R. M.; Grande, H. J.; Kamstrup, S.; PCT Int. Appl. WO. 95 09, 1995.
• 5. Strobl, M.; PhD Thesis, Luwing-Maximilians Universität, München, Germany, 2004.
• 6. Villasenor, I. M.; Domingo, A. P.; Teratog. Carcinog. Mutagen 2000, 20, 99.
• 7. Jeong, S. I.; Kim, K. J.; Choi, M. K.; Keum, K. S.; Lee, S.; Ahn, S. H.; Back, S. H.; Song, J. H.; Ju, Y. S.; Choi, B. K.; Jung, K. Y.; Planta Med 2004, 70, 736.
• 8. Jeon, G. Ch.; Park, M. S.; Yoon, D. Y.; Shin, Ch. H.; Sin, H. S.; Um, S. J.; Exp. Mol. Med. 2005, 37, 111.
• 9. Valev, S. In Flora Republicae Popularis Bulgaricae III; Jordanov, D., ed.; Aedibus Acad. Sci. Bulgaricae: Sofia, 1966, p. 388-394.
• 10. Luchanskaya, V. N.; Kondratenko, E. S.; Abubakirov, N. K.; Khim. Prir. Soed. 1970, 6, 434.
• 11. Luchanskaya, V. N.; Kondratenko, E. S.; Abubakirov, N. K.; Khim. Prir. Soed. 1971, 7, 431.
• 12. Luchanskaya, V. N.; Kondratenko, E.S.; Abubakirov, N. K.; Khim. Prir. Soed. 1971, 7, 608.
• 13. Luchanskaya, V. N.; Kondratenko, E. S.; Abubakirov, N. K.; Khim. Prir. Soed. 1972, 8, 69.
• 14. Gevrenova, R.; Voutquenne-Nazabadioko Harakat, L. D.; Prost, E.; Henry, M.; Magn. Res. Chem. 2006, 44, 686.
• 15. Carlson, R.; PhD Thesis, Stanford University, USA, 1978.
• 16. George, G .D.; Stewart, J. T.; Anal. Lett. 1990, 23, 1417.
• 17. Graham, T. L.; Plant Physiol 1991, 95, 584.
• 18. Hasler, A.; Stricher, O.; Meier, B.; J. Chromatography 1990, 580, 232.
• 19. Abourashed, A.; Vanderplank, J. R.; Khan, I. A.; Pharm. Biol 2002, 40, 81.
• 20. Bramati, L.; Aquilano, F.; Pietta, P.; J. Agric. Food Chem 2003, 51, 7472.

Publication Dates

• Publication in this collection
  12 Sept 2008
• Date of issue
  2008

History

• Accepted
  14 Nov 2007
• Received
  14 June 2007