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New Constituents from the Rhizomes of Egyptian Iris germanica L.

Chemical investigation of the methanolic extract of the rhizomes of Iris germanica L. (Iridaceae) afforded two new compounds; irigenin S (7) and iriside A (12), together with ten known compounds: stigmasterol (1), α-irone (2), γ-irone (3), 3-hydroxy-5-methoxyacetophenone (4), irilone (5), irisolidone (6), irigenin (8), stigmasterol-3-O-β-D-glucopyranoside (9), irilone 4'-O-β-D-glucopyranoside (10) and iridin (11). Their structures were established by UV, IR, 1D (1H and 13C) and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to mass spectroscopic data and comparison with literature data. The methanolic extract was evaluated for its antimicrobial activity. Both the methanolic extract and the isolated flavonoids were tested for their anti-inflammatory activity.


Introduction
The genus Iris belongs to the family Iridaceae, which comprises over 300 species [1]. Iris species have an immense medicinal importance and are used in the treatment of cancer, inflammation,

Results and Discussion
Irigenin S (7) 3 3.70 s 59.9, CH 3 4' ----5'-OCH 3 3.79 s 55.8, CH 3 5' ----6-OCH 3 3.76 s 59.9, CH 3 6 ----7-OCH 3 3.88 s 55.9, CH 3 7 ---- The 13 C-NMR showed nineteen carbon signals, their multiplicities were determined through DEPT, as well as, direct correlations of protons to their respective carbons in the HMQC spectrum. The placement of the methoxy groups at C-4', C-6, C-5', and C-7 was supported by the HMBC spectrum that displayed correlations between 4'-OCH 3 and C-4'/δ C 136.1, 6-OCH 3 and C-6/δ C 132.1, 5'-OCH 3 and C-5'/δ C 152.2, as well as 7-OCH 3 and C-7/δ C 154.5 ( Figure 2). On the basis of the above evidences, the structure of 7 was elucidated as 3',5-dihydroxy-4',5',6,7-tetramethoxyisoflavone and considered as a new natural product, for which we propose the name irigenin S. Iriside A (12) was isolated as yellow oil. The molecular formula C 7 H 14 O 5 for 12 was confirmed from the HRFABMS molecular ion peak at m/z 179.0840 [M+H] + , which required one degree of unsaturation. Its IR spectrum displayed an absorption band at 3452 cm −1 , indicating the presence of a hydroperoxy group [36]. The 13 C-NMR and HMQC spectra of 12 showed signals for one methyl carbon (δ C 15.1, C-8), three methylenes, two of them which are oxygenated [δ C 63.7 (C-6) and 62.1 (C-7)], and three oxygen-bonded methane carbons at δ C 103.2, 70.9, and 86.9, ascribed to C-2, C-4, and C-5, respectively. The COSY spectrum displayed the presence of two spin systems. The first spin system consists of the triplet methyl signal at δ H 1.06 (J = 6.5 Hz, H-8) coupled with the methylene protons at δ H 3.61 and 3.29 indicating the presence of an ethoxy group, which further confirmed by the fragment ion peak at m/z 150.1503 [M-C 2 H 5 +H] + . The second spin system was assigned to a substituted pyran moiety, and confirmed by the fragment ion peak at m/z 84.0354 [C 5 H 8 O] + . This moiety was also validated by the correlations observed in the HMBC spectrum ( Figure 2). H-2 showed HMBC correlations with C-4 and C-6 and a four bond coupling to C-5. H-6 correlated with C-2 and C-4 and H-3 with C-5. In the 1 H-NMR spectrum, the two signals at δ H 4.58 and 4.95 indicated the presence of two hydroxy groups; one for the hydroperoxy group at C-5 and the other for C-4-OH, which were confirmed by the observed COSY and HMBC correlations. The downfield shift of C-5 (δ C 86.9) suggested the presence of a hydroperoxy functional group at C-5 and was confirmed by the fragment ion peaks at m/z 133.0905 [M-C 2 H 6 O+H] + and 117.0782 [M-C 2 H 6 O 2 +H] + [37]. The connectivity of the ethoxy group at C-2 of the pyran ring was established through the HMBC correlations of H-2 with C-7, H-7 with C-2 and C-3. On the basis of these findings the structure of 12 was unambiguously elucidated as 2-ethoxytetrahydro-5-hydroperoxy-(2R,4R,5S)-2H-pyran-4-ol, for which we propose the name iriside A. The relative stereochemistry of 12 was determined by comparing the 1 H-, 13 C-NMR chemical shifts, and the coupling constants values with literature [38,39], and the absolute stereochemistry was confirmed using the Mosher procedure [40].
The methanolic extract of Iris germanica (MIG) and compounds 5-8, 10, 11 were tested for their anti-inflammatory effects using the induced paw edema test. All the tested compounds as well as MIG exhibited potent anti-inflammatory effects; compound 7 showed the highest activity, which was almost similar to that of dexamethasone (Table 2). These results are in accordance with previous studies that attributed the anti-inflammatory activity of flavonoids to the C-2,3 double bond [41] and the presence of a methoxyl group at C-5 and C-7, and the pyran ring [42]. The activity of MIG may be due to the presence of different classes of terpenes and flavonoids. The MIG also showed potent antimicrobial activity against different bacterial and fungal strains (Table 3) at concentrations 250 and 500 µg per disc. It exhibited highest activity at a concentration of 250 µg per disc against S. aureus, S. marcescens, E. coli, C. albicans, and A. flavus. This observed anti-microbial effects of the extract might be due to the presence of phenolic constituents in the plant.

General Procedures
Optical rotation was measured on a Perkin-Elmer Model 341 LC polarimeter. Melting points were determined using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd., Essex, England). HRESI and FABMS were recorded on a LTQ Orbitrap and an API 2000 (ThermoFinnigan, Bremen, Germany) mass spectrometers, respectively. Low resolution mass spectra were determined using a Finnigan MAT TSQ-7000 mass spectrometer. UV spectra were recorded in absolute MeOH on a Shimadzu 1601 UV/VIS spectrophotometer. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). 1D and 2D NMR spectra (chemical shifts in ppm, coupling constants in Hz) were recorded on Bruker BioSpin GmbH 500 MHz Ultrashield spectrometer using standard Bruker software and DMSO-d 6 , CDCl 3 , and C 5 D 5 N as solvents, with TMS as the internal reference. Solvents were distilled prior spectroscopic measurements. Column chromatographic separations were performed on silica gel 60 (0.04-0.063 mm), RP-18 (0.04-0.063 mm, Merck), and Sephadex LH-20 (0.25-0.1 mm, Merck). TLC was performed on precoated TLC plates with silica gel 60 F 254 (layer thickness 0.2 mm, Merck). The solvent systems used for TLC analyses were CHCl 3 -MeOH (97:3, solvent system I), CHCl 3 -MeOH (90:10, solvent system II), and CHCl 3 -MeOH (85:15, solvent system III). The compounds were detected by UV absorption at λ max 255 and 366 nm followed by spraying with anisaldehyde/H 2 SO 4 reagent and heating at 110 °C for 1-2 min.

Plant Material
The

Spectral Data
Stigmasterol (1) [40] Four mg of compound 12 was dissolved in pyridine-d 5 (0.7 mL) and transferred to NMR tubes. The 1 H-NMR spectrum of 12 was measured prior to adding 5 μL of (R)-MTPA-Cl and (S)-MTPA-Cl reagent (Fluka, Germany), respectively. The tubes were shaken thoroughly and were allowed to stand at room temperature for 72 h. The reaction was monitored by 1 H-NMR spectroscopy after every 24 h.

Anti-Inflammatory Activity
Hind paw edema (skin edema) was induced by 4% formalin solution injected into the subplantar region of the left hind paw [42]. Adult male albino rats 100-120 g (purchased from the Animal House, Pharmacology Department, Faculty of Medicine, Assiut University). The inflamed animals were divided randomly into ten groups (six in each group), inflamed control group, inflamed treated with dexamethasone (at a dose of 10 mg/kg subcutaneously), six groups of inflamed animals were treated with the tested compounds individually (at a dose of 10 mg/kg subcutaneously), and two groups were treated with MIG at doses of 50 and 100 mg/kg subcutaneously (the plant extract was dissolved in sterile distilled water). The change in paw thickness in all tested animals was measured at 1, 2, 4, and 24 h after formalin solution injection. The data were expressed as mean ± S.D. using the Student t test (Table 2).

Antimicrobial Assay
The procedure was carried out as previously described [43]. The antibacterial and antifungal activities were evaluated using the agar plate diffusion assay. Susceptibility discs (5. Each plate was inoculated with a single organism and the test was run in duplicates. The plates were incubated at 37 °C and checked for inhibition zones after 24 h for bacteria and after 48 h for fungi. Chloroamphenicol and clotrimazole at concentration 250 µg per disc were used as positive reference standards for antibacterial and antifungal activities, respectively.

Conclusions
Two new compounds; irigenin S (7) and iriside A (12), were isolated from the rhizomes of I. germanica L. growing in Egypt along with ten known compounds. The methanolic extract (MIG) and the isolated flavonoids exhibited potent anti-inflammatory effects. Compound 7 showed activity similar to that of dexamethasone. The MIG showed highest anti-microbial effect against S. aureus, S. marcescens, E. coli, C. albicans, and A. flavus.