BIOLOGICALLY ACTIVE COMPOUNDS FROM THE RHIZOMES OF IRIS HUNGARICA

Species of Iris genus (Iridaceae) have a long history of traditional medicinal use in different countries as alternative aperient, tonic, cathartic, diuretic, gall bladder diseases, liver complaints, dropsy, purification of blood, venereal infections, fever, bilious infections and for a variety of heart diseases. The rhizomes of Iris are the rich source of the secondary metabolites, in which flavonoids predominate. The clinical studies of substances from irises gave positive results in the treatment of cancer, bacterial and viral infections. Continuing the search of new biologically active compounds from the plants of Iridaceae family for the first time three isoflavones that are new for this species – irigenin, iristectorigenin B and its glucoside iristectorin B have been isolated from the ethanolic extract of the rhizomes of Iris hungarica Waldst. et Kit., which is widespread in Ukraine. The structure of the compounds is described as 5,7,3’-trihydroxy-6,4’,5’-trimethoxyisoflavone, 5,7,4’-trihydroxy-6,3’-dimethoxyisoflavone and iristectorigenin B-7-O-β-D-glucoside, respectively. The compounds were obtained from the ethyl acetate fraction of the iris rhizomes by column chromatography on silica gel with sequential elution of the chloroform – ethanol solvent with different concentrations. The structure of the compounds has been determined by chemical and spectral methods and in comparison with the literature data.


ISSN 2308-8303
Plants of Iris genus (the family Iridaceae) are perennial herbaceous plants of 30-100 cm in height, with well-developed ensiform leaves and peduncles, which are at the top of many large flowers: white, purple, violet. Iris is the genus of 260-300 species, which are mainly distributed across the Northern Hemisphere [1][2].
Plants of Iris genus are the rich source of the secondary metabolites: flavonoids [8], isoflavonoides and their glycosides, xanthones, quinones, triterpenoids and stilbene glycosides [9][10][11]. The clinical studies of biologically active compounds of irises gave positive results in the treatment of cancer, bacterial and viral infections [12][13].
The aim of the work was to isolate and identify phenolic compounds from the rhizomes of I. hungarica. The EtOAc extract of the rhizomes of I. hungarica was subjected to repeated chromatography on columns of silica gel to obtain compounds 2, 6, 7. Compounds are soluble in ethanol, benzene, chloroform and are poorly soluble in water, diethyl ether and petroleum ether (Scheme).
The data of the chemical analysis, the spectral characteristics of compound 2 are identical with the literature data on the structure of 5,7,3'-trihydroxy-6,4',5'-trimethoxy isoflavone or irigenin. It was first isolated from the rhizomes of Iris hungarica [12].
The chromatographic analysis of compound 6 using the system of 15% acetic acid (R f 0.49) produced a spot with a blue fluorescence. The mass spectrum of 6 showed the molecular ion peak at m/z 330 (M + ) in agreement with the molecular formula C 17 H 14 O 7 . UV absorption 6 maxima at 272 and 341 nm (sh) suggested the presence of the isoflavone moiety. In addition, the proton resonance for isoflavone C-2 was located at δ 8.32 (1Н, s) ppm, it also confirmed the nature of the ring. The IR-spectrum 6 showed intense absorptions at 3752 сm -1 (ОН), 2960, 2836 сm -1 (ОСН 3  With NOESY spectrum 6 arrangements of the substituents at C-3' and C-4′ were refined. The analysis showed the presence of two cross-peaks demonstrating the nuclear resonance, and they were spatially close (Nuclear Overhauser effect observed at a distance of 0.03-0.4 nm between atoms). The interaction of proton H-2 with protons H-2' and H-6' was observed (Fig.). Proton H-2' gave a cross-peak with the protons of the methoxy group, thus, it was located in position 3'.
Acid hydrolysis of 7 with 10% H 2 SO 4 gave aglycone (iristectorigenin B) and D-glucose and which were identified by co-PC and co-TLC.
The 1 H NMR-spectrum 7 indicated the isoflavonoid nucleus by the appearance of the H-2 signal at δ 8.32 (1Н, s) ppm. It had the aromatic nuclei spin system being the same as that of compound 6. It also displayed signals at δ 7.15, 6.90 and 6.70 ppm to form the spin-spin interaction (H-5′, H-2′ and H-6′). The spectrum also showed signals for two methoxy groups at δ 3.75 (3Н, s, 3'-ОСН 3 ) and δ 3.70 (3Н, s, 6-ОСН 3 ) ppm and one characteristic proton signal for H-8 at δ 6.47 (1H, s) ppm. It also indicated the presence of two singlet signals of the hydroxyl groups at δ 13.10 (1Н, s, 5-ОН), 9.15 (1Н, s, 4'-ОН) ppm. The ¹H NMRspectrum unlike to the spectra of aglycone as iristectorigenin B further noted the presence of groups of signals corresponding to the presence of six protons confirming the glycoside monoside nature.
Irigenin, Iristectorigenin B and Iristectorin B were previously isolated from the rhizomes of I. dichitoma

Extraction and Isolation of Compounds
Air-dried rhizomes (2.5-3 mm) of I. hungarica (1.0 kg) were extracted with EtOH (70%, 5 L) in a percolator for 24 h. The extraction was repeated thrice under the same conditions. The aqueous EtOH extracts were combined, filtered, evaporated in a rotary evaporator to 0.5 L of the aqueous residue, and left for 1 day. The supernatant liquid was separated. The resulting extract was treated successively with CHCl 3 , EtOAc and n-BuOH. The resulting extracts were evaporated in vacuo. The qualitative composition of CHCl 3 , EtOAc and BuOH fractions was controlled by PC and TLC in the solvent system of n-butanol -acetic acid -water (4:1:2).
The EtOAc extract was evaporated by heating under vacuum to complete stripping of the solvent, subjected to CC (120×5 cm) on silica gel and eluted with