Acylated flavonol glycosides from Epimedium elatum, a plant endemic to the Western Himalayas

Fitoterapia 83 (2012) 665–670

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Acylated flavonol glycosides from Epimedium elatum, a plant endemic to the Western Himalayas Mudasir A. Tantry a,⁎, Javid A. Dar b, Ahmed Idris c, Seema Akbar d, Abdul S. Shawl e a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS 38677, United States b Department of Urology Research Laboratories, University of Pittsburgh Medical Center Pittsburgh, PA 15232, United States c Department of Medicinal Chemistry, School of Pharmacy, The University of Mississippi, MS 38677, United States d Drug Standardisation Research Unit, Regional Research Institute of Unani Medicine, Naseem Bagh, Srinagar 190006, Kashmir, India e Natural Product Chemistry Division, Indian Institute of Integrative Medicine, Srinagar 190005, Kashmir, India

a r t i c l e

i n f o

Article history: Received 17 December 2011 Accepted in revised form 4 February 2012 Available online 18 February 2012 Keywords: Epimedium elatum Acylated flavonol glycosides Elatoside A Elatoside B Antimicrobial PPAR-γ

a b s t r a c t Herba Epimedii is a well-known Botanical preparation used over long time in traditional Chinese medicine. The extracts and chemical constituents from Epimedium species are aphrodisiac as well as to treat many ailments. Chemical investigation of lonely species growing in Kashmir Himalaya Epimedium elatum was undertaken to evaluate its chemical profile. Two unusual substituted acylated flavonol glycosides named Elatoside A (1) and Elatoside B (2) have been isolated from the ethanolic extract of E. elatum along with 23 previously known ones (3–25). All isolates were evaluated for antimicrobial and PPAR-γ ligand binding activity, and some of them appeared to be modestly active. Published by Elsevier B.V.

1. Introduction Epimedium (Berberidaceae), also known as Barrenwort is a genus of about 52 species of herbaceous plants [1]. Some of the species in this genus have a long history of use in traditional Chinese medicine (TCM) and are believed to be an aphrodisiac and to nourish the kidney. Extracts of the aerial parts of this genus are used in famous botanical preparations that have been used as a tonic, an aphrodisiac and an antirheumatic in China, Japan, and Korea for more than 2000 years. This preparation has shown in vivo and in vitro activity against sexual dysfunction, osteoporosis, cardiovascular diseases, menstrual irregularity, asthma, chronic nephrites and immunoregulation [2–4]. In the last few decades, the use of Epimedium plants in traditional Chinese medicine has led to rapid increase in the information available on the active components of Epimedium, there chemical compounds and the pharmacological activities ⁎ Corresponding author. Tel.: + 1 662 607 9594; fax: + 1 662 915 1708. E-mail address: [email protected] (M.A. Tantry). 0367-326X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.fitote.2012.02.003

of these compounds. More than 260 compounds have been identified from different species of Epimedium [5], among them prenyl flavonoids are the major constituents and are also important chemotaxonomic markers. In vivo or in vitro experimental and clinical practice has demonstrated that Epimedium and its active compounds posses wide reaching pharmacological actions. In the process of bioprospecting Epimedium elatum, a native and only one species growing in Western Himalaya (Kashmir), we tried to investigate the chemical profile of this particular species for its chemotaxonomic markers. Ethanolic extract of whole plant led to isolation of two new acylated flavonol glycosides Elatoside A (1) and Elatoside B (2) along with 23 known ones, which were identified by comparison of their physical and spectral data with those of reported compounds such as anhydroicaritin (3) [6], 8isoprenylkaempferol (4) [7], breviflavone B (5) [8], luteolin (6) [9], hyperoside (7) [10], epimedokoreanin B (8) [11], daidzein (9) [12], β-sitosterol glucoside (10) [13], γ-sitosterol glucoside (11) [13], quercetin (12) [14], desmethylanhydroicaritin (13) [15], epimedoside A (14) [17], epimedoside B (15) [18], apigenin (16) [19], icariin (17) [20], epimedin B

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(18) [21], epimedin C (19) [22], icariside II (20) [23], icaritin (21) [10], ikarisoside A (22) [25], isoliquiritigenin (23) [10], caohuoside F (24) [27] and diphylloside A (25) [9]. The compounds were characterized by NMR including extensive 2D NMR techniques. All compounds were evaluated for antimicrobial and PPAR-γ activities. 2. Experimental 2.1. General NMR spectra were recorded on a Bruker 400 and 600 NMR spectrometer instrument using TMS as internal standard. Chemical shifts were reported in δ units and coupling constants (J) in Hz. ESIMS and HRESIMS were obtained on Agilent Series 1100 SL mass spectrometer. IR spectra were recorded using KBr pellet on a Bruker Tensor 27 FT-IR spectrometer. Optical rotations were measured on a Rudolph Research AutoPol IV polarimeter. Melting points were determined on a Meltemp melting point apparatus and are uncorrected. Column chromatography was performed by using silica gel (40 μm mesh, JT Baker), ODS silica gel and reversed-phase RP-C18, C-8 silica (Polarbond; JT Baker). Medium Pressure Liquid Chromatography was performed on Biotage Inc. Horizon MPLC System. TLC analysis was carried out on a silica gel 60 F254 plates (Merck) and spots on TLC plates were observed under UV light (254/365 nm). Spraying reagents p-anisaldehyde-H2SO4 (Sigma-Aldrich), FeCl3 solution, 10% H2SO4 in ethanol and water, followed by heating were used for the detection of spots. All HPLC experiments were carried out on a Shimadzu SIL-10A with an auto injector, SCL-10A System with Activation GoldPak 100 5 μm ODS 250 × 10 mm or Waters C18 300 × 8 mm 5 μm semipreparative columns. All solvents used for extractions and chromatographic separations were of analytical grade with the exception of HPLC grade solvents used for HPLC separations. 2.2. Plant material The root and aerial parts of E. elatum collected from Naranag Ganderbal (Kashmir Valley) and identified by the taxonomist at Centre for Biodiversity and Taxonomy Biodiversity (CBT), University of Kashmir, Srinagar India. A voucher specimen (No.: EE-WP-09-108; dated 27/07/2009) of the plant was deposited in the same center. 2.3. Extraction and isolation The dried whole plant material of E. elatum (1.0 kg) was extracted with EtOH (20 L, 45 °C, 2 h). The EtOH extract (62 g) was chromatographed on a silica gel eluted with CHCl3–MeOH gradients (19:1; 9:1; 2:1), and finally with MeOH alone, to give four fractions (I–IV). Since the activity was found to be concentrated in fraction I (15 g), the fraction was subjected to silica gel column chromatography eluted with CHCl3–MeOH (99:1), and finally with MeOH alone, to give eight fractions (I-A–H). Fraction I-B (8 g) was separated by silica gel column chromatography using hexane-Me2CO (3:1) to afford eight fractions (I-B1–8). Fraction I-B2 (1.3 g) was subjected to silica gel column chromatography eluted

with hexane–EtOAc (10:1) to give seven fractions (I-B2a– g). Fraction IB2c (44.7 mg) was subjected to preparative HPLC (flow rate, 0.8 mL/min) using MeOH–H2O (20:1) to give 9 (7.3 mg). Fraction I-B2e (274 mg) was subjected to preparative HPLC using MeOH–H2O (4:1) to give 13 (14.1 mg). Fraction I-B4 (3.5 g) was separated by ODS silica gel column chromatography eluted with MeCN–H2O (2:1) to give five fractions (I-B4a–e). Fraction I-B4a (1.02 g) was subjected to ODS silica gel column chromatography eluted with MeCN–H2O (2:1) to give nine fractions (I-B4a1–9). Compound 15 (5.8 mg) was obtained from fraction I-B4a1 (92.8 mg) by subjecting it to preparative HPLC using MeCN– H2O (2:1). Fraction IB4a4 (83.7 mg) was subjected to preparative HPLC using MeCN–H2O (2:1) to give 8 (4.9 mg) and 6 (8.6 mg). Similarly, purification of fraction I-B4a5 (124 mg) yielded 10 (30.2 mg), and fraction I-B4c (70 mg) gave 11 (5.4 mg), 14 (17.4 mg), 16 (2.5 mg) and 7 (18.5 mg). Fraction IB6 (1.8 g) was separated by ODS silica gel column chromatography eluted with MeCN–H2O (3:1) to give eight fractions (I-B6a–h). Fraction I-B6b (215 mg) was subjected to preparative HPLC using MeCN–H2O (2:1) to give 4 (16.3 mg), 5 (12.4 mg) and 22 (24.7 mg). Similarly, purification of fraction I-B6d (201 mg) yielded 21 (11.4 mg) and 19 (37.1 mg). Fraction I-C (13.2 g) was subjected to silica gel column chromatography eluted with CHCl3–MeOH (99:1), and finally with MeOH alone, to give seven fractions (I-C1–7). Fraction I-C5 (1.5 g) was subjected to ODS silica gel column chromatography eluted with MeOH–H2O (5:1) and MeCN–H2O (1:1) to give three fractions (I-C5a–c). Fraction I-C5a (458 mg) was chromatographed on ODS silica gel eluted with MeOH–H2O (5:2) to give a mixture, which was then separated by preparative HPLC using MeOH–H2O (5:2) to yield 17 (42.5 mg), 18 (28.1 mg) and 20 (13.1 mg). Compound 1 (4.5 mg) was obtained from fraction I-C5c (350 mg) by subjecting it to ODS silica gel column chromatography eluted with MeOH– H2O (5:1). Fraction I-D (11.3 g) was subjected to an ODS silica gel column eluted with MeOH–H2O (4:1; 7:3) and MeCN–H2O (1:1), a silica gel column with hexane–Me2CO (2:1), and to preparative HPLC using MeCN–H2O (1:1) to afford 2 (3.2 mg). Fraction I-E (10.7 g) was subjected to ODS silica gel column chromatography using MeCN–H2O (3:2) to afford four fractions (I-E1–4). Fraction I-E1 (756 mg) was separated by ODS silica gel column chromatography eluted with MeCN–H2O (2:3) to give seven fractions (I-E1a–g). Purification of fractions I-E1a (77.7 mg), I-E1c (55.4 mg) and IE1d (171 mg) by preparative HPLC using MeOH–H2O (7:3) led to the isolation of compounds 3 (18.0 mg), 12 (17.3 mg), 23 (13.7 mg) and 7, respectively. Fraction I-E1f (84.7 mg) was subjected to preparative HPLC (flow rate, 0.6 mL/min) with MeOH–H2O (8:2) to give 24 (10.7 mg). Fraction I-E2 (2.9 g) was separated by silica gel column chromatography eluted with CHCl3–EtOAc (10:1) to give nine fractions (I-E2a–i). Purification of fraction I-E2f (261 mg) by preparative HPLC (flow rate, 0.7 mL/min) with MeCN–H2O (3:2) to give 25 (28.8 mg). 2.3.1. 8-(3,3-Dimethylallyl)-3′,5,7-trihydroxy-4′, 5′-dimethoxyflavonol 3-[O-5-O-Acetyl-β-D-xylopyranosyl-(1→ 2)-α-L-rhamnopyranoside] 7-(β-D-glucopyranoside) (1): [α]D25 = −77 (0.011, MeOH); UV (MeOH): 270 (4.72), 314 (4.35), 352 (4.28); IR

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(KBr): 3352, 2921, 1738, 1650, 1596, 1440, 1370, 1221, 1175, 1076, 1045; HR-ESIMS: m/z 935.2789 [M+ Na]+ (Calcd. for C41H52O23Na); 1H and 13C NMR: (see Table 1).

2.3.2. 8-(3,3-Dimethylallyl)-3′,5′,5, 7-tetrahydroxy-4′-methoxyflavonol 3-[O-5-O-Acetyl-β- D -xylopyranosyl-(1 → 2)-α-Lrhamnopyranoside] 7-(β-D-glucopyranoside) (2): [α]D25 =−70 (0.010, MeOH); UV (MeOH): 271 (4.70), 315 (4.36), 351 (4.25); IR (KBr): 3420, 2928, 1736, 1648, 1590, 1438, 1368, 1220, 1170, 1070, 1045; HR-ESIMS: m/z 899.2819 [M+H]+ (Calcd. for C40H50O23H); 1H and 13C NMR: (see Table 1).

Table 1 1 H (500 MHz, C5D5N, δH, J/Hz) and compounds 1 and 2.

13

C-NMR (125 MHz, C5D5N, δC) data of

1

2 δH

δC

δH

Position

δC

C (2) C (3) C (4) C (5) CH (6) C (7) C (8) C (9) C (10) CH2 (11) CH (12) C (13) CH3 (14) CH3 (15) C (1′) CH (2′) C (3′) C (4′) C (5′) CH (6′) \OCH3 (C-4′) \OCH3 (C-5′)

157.2 133.4 179.1 158.7 98.8 160.1 106.4 152.8 104.9 21.4 123.0 132.2 24.1 18.1 120.3 109.2 151.4 135.6 155.7 130.6 55.2 56.1

7-O-Glc CH (1″) CH (2″) CH (3″) CH (4″) CH (5″) CH2 (6″)

100.6 73.3 76.6 70.3 76.6 60.6

5.00 (d, J = 6.5) 3.35 (m) 3.29 (m) 3.15 (m) 3.42 (m) 3.40, 3.68 (2 m)

100.5 73.4 76.6 70.2 76.5 60.5

4.99 (d, J = 6.4) 3.36 (m) 3.29 (m) 3.14 (m) 3.39 (m) 3.40, 3.67 (2 m)

3-O-Rha CH (1‴) CH (2‴) CH (3‴) CH (4‴) CH (5‴) CH3 (6‴)

101.9 69.9 77.2 71.2 69.9 17.2

5.15 3.15 3.40 3.03 3.29 0.72

(br, s) (m) (m) (m) (m) (d, J = 6.0)

101.7 69.8 77.2 71.3 69.9 17.2

5.17 3.13 3.49 3.04 3.29 0.74

(br, s) (m) (m) (m) (m) (d, J = 6.0)

2‴-O-Xyl CH (1⁗) CH (2⁗) CH (3⁗) CH (4⁗) CH (5⁗) C (6⁗) CH3 (7⁗)

104.1 73.4 75.9 71.5 90.2 170.2 21.0

5.03 3.49 3.73 3.96 5.45

(s, J = 7.1) (m) (m) (m) (s, J = 6.9)

104.1 73.5 75.8 71.5 90.2 170.2 20.2

5.02 3.49 3.75 3.96 5.41

(s, J = 7.1) (m) (m) (m) (s, J = 6.9)

12.4 (s, OH) 6.62 9 (s)

3.35, 3.40 5.17 (t, J = 6.0) 1.68 (s) 1.59 (s) 6.97

6.99 3.78 (s) 3.82 (s)

1.98 (s)

157.1 133.5 178.8 157.7 98.6 160.1 105.4 153.8 105.9 20.8 123.2 132.7 24.5 18.6 121.3 110.2 152.4 131.7 153.2 130.5 55.5

12.0 (s, OH) 6.69 (s)

3.34, 3.39 5.15 (t, J = 6.0) 1.65 (s) 1.58 (s) 6.99

6.96 3.78 (s)

2.01 (s)

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2.4. Antimicrobial activity 2.4.1. Microbial strains A total of six micro-organisms belonging to one Gram (+) bacterial species (Staphylococcus aureus) and five Gram (−) bateria (Escherichia coli, Salmonella typhi, Shigella dysenteriae, Klebsiella pneumoniae, and Pseudomonas aeruginosa) were clinically isolated from patients. They were maintained on agar slants at 4 °C. 2.4.2. Antimicrobial assays Antimicrobial activity was evaluated using the agar diffusion method, according to the NCCLS (2002) protocol with slight modifications. Briefly, sterile cylinders of 6 mm were used to make wells inside Muellar-Hinton agar plates. The plates were inoculated with 2 × 10 − 4 L of the test of the test microorganisms equivalent to 5 × 10 5 CFU/mL. All the compounds were filled with 15 × 10 − 5 L of solution of each test compound, the positive control drug (gentamicin) and the negative control DMSO, and allowed to diffuse for 45 min at 4 °C. The plates were incubated at 37 °C for 24 h. The sensitivity was recorded by measuring clear zone of growth inhibition around the wells (mm diameter), each set was tested in triplicate. 2.5. PPAR-γ ligand binding activity PPAR-γ ligand-binding activity was carried out using a GAL4-PPAR-γ chimera assay system . CV-1 monkey kidney cells from the American Type Culture Collection (ATCC) (Manassas, VA, USA) were inoculated into a 96-well culture plate at 6 × 103 cells/well and incubated in 5% CO2/air at 37 °C for 24 h. As medium, Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 10 mL/L penicillin-streptomycin (5000 IU/mL and 5000 μg/mL, Gibco), and 37 mg/L ascorbic acid (Sigma-Aldrich) was used. Cells were washed with OPTIminimum essential medium (OPTI-MEM) and transfected with pM-hPPAR-γ and p4× UASg-tk-luc using LipofectAMINE PLUS (Gibco). In a mock control, pM and p4 × UASg-tk-luc were transfected into CV-1 cells. After 24 h of transfection, the medium was changed to DMEM containing 10% charcoaltreated FBS and each sample, and the cells were further cultured for 24 h. Then, the cells were washed with Ca 2 +and Mg 2 +-containing phosphate-buffered saline (PBS+), to which Luc-Lite (Perkin-Elmer, Wellesley, MA, USA) was added. The intensity of emitted luminescence was determined using a TopCount microplate scintillation/luminescence counter (Perkin-Elmer). The luminescence intensity ratio (test group/control group) was determined for each sample, and PPAR-γ ligand-binding activity was expressed as the relative luminescence intensity of the test sample to that of the control sample (Table 3). 2.6. Determination of sugar configuration Compounds 1 and 2 (0.5 mg) were hydrolyzed by heating in 0.5 M HCl (0.1 mL) and neutralized with 1.0 M NH4OH. After drying in vacuo, the residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. A 0.1 mL solution of

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Table 2 Antimicrobial activities of compounds 1–25 (each conc. 200 mg/L in DMSO). Compounds

Inhibition zone diameter (mm) S. aureus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

E. coli

S. typhi

Gentamicin (control) S. dysenteriae

K. pneumonia

P. aeruginosa 11

16

19

20

30 32

22

11

17

35

17

14 09

31

13

40 39 34

10 19 14

15

12

16

12

11

13

33

15

26 36 42

22

16

37 39

13 11

18

31 29 39

10 21

phenylisothiocyanate (0.5 mg) in pyridine was added to the mixture, which was heated at 60 °C for 1 h. The reaction mixture was directly analyzed by reversed-phase HPLC. The peaks at tR 10.59, 21.34 and 21.52 min were coincided with derivatives of D-glucose, L-rhamnose and D-xylose respectively. The configurations were determined by comparing their retention times (tR D-glucose 10.58 min, tR L-rhamnose 21.34 min and tR D-xylose 23.52 min) with acetylated thiazolidine derivatives prepared in a similar way from standard sugars. 3. Results and discussion The dried plant material (root and aerial parts) of E. elatum (1.00 kg) was extracted with 95% EtOH (4 L, 4×, 48 h) at 45 °C. Each extract was combined and concentrated under reduced

pressure to give a residue (62 g). The residue was chromatographed on a silica gel eluted with CHCl3–MeOH in a gradient way and subjected to further series of chromatographic separations to obtain 1 (4.5 mg), 2 (3.2 mg), 3 (18.0 mg), 4 (16.3 mg), 5 (12.4 mg), 6 (8.6 mg), 7 (18.5 mg), 8 (4.9 mg), 9 (7.3 mg), 10 (30.2 mg), 11 (5.4 mg), 12 (17.3 mg), 13 (14.1 mg), 14 (17.4 mg), 15 (5.8 mg), 16 (2.5 mg), 17 (42.5 mg), 18 (28.1 mg), 19 (37.1 mg), 20 (13.1 mg), 21 (11.4 mg), 22 (24.7 mg), 23 (13.7 mg), 24 (10.7 mg), and 25 (28.8 mg). Compound 1 was isolated as yellow amorphous solid. The molecular formula C41H52O23 was deduced from the HR-ESI with molecular ion peak at m/z 935.2789 [M+ Na] +. The UV absorption maxima (270, 314, 352 nm) indicated the flavonol skeleton. The IR spectra showed absorption bands for OH groups (3352 cm− 1), flavonol carbonyl group (1738 cm− 1) and ester groups (1650 and 1597 cm− 1). A comparison of

Table 3 PPAR-γ activities of compounds 1–25. IC50 (μM) Compound

PPAR-γ

Compound

PPAR-γ

Compound

PPAR-γ

Compound

PPAR-γ

1 2 3 4 5 6

na 16 na 21 18 17

7 8 9 10 11 12

na 13 na na na na

13 14 15 16 17 18

14 29 31 na 42 10

19 20 37 22 23 24 25

21 33 37 51 13 28 11

PPAR-γ ligand-binding activity was expressed as the relative luminescence intensity (IC50) of the test sample to that of the control sample. Troglitazone at 2.0 μM was used as a positive control. Dimethyl sulfoxide at 0.5 mL/L as a solvent control. Values are means ± SD, n = 2 experiments. Statistical significance is indicated as p b 0.05 as determined by Dunnett's multiple comparison test.

M.A. Tantry et al. / Fitoterapia 83 (2012) 665–670 1 H- and 13C NMR data of 1 with those of epimedin B [20] suggests that the structure of both compounds were similar but 1 showed an additional methoxyl signal, a substituted ring B and presence of an acetoxy group (Fig. 1, R=CH3). The 1H NMR spectrum confirmed the presence of a 3,3Dimethylallyl and one acetyl group at δH 1.98, two methoxyls (δH = 3.78, 3.82). The 13C NMR spectrum of 1 showed the characteristic signals of a one glucose, one rhamnose and acetylated xylose at C-5 as δC 170.2 (CO) and 21.0 (CH3). The position of the three sugar units, two methoxyls and the acetoxy group was confirmed by HMBC experiments. Thus, the correlation H-1″ (δH 5.00)/C-7 (δC 160.1), indicated that a glucose unit attached to C-7. The correlation of C-3 (δC 133.4)/ H-1‴ (δH 5.15) and the cross peak H-1⁗ (δH 5.03)/C-2‴ (δC 69.9) suggested the attachment of the rhamnose unit at C-3 and xylose unit at C-2‴ of the rhamnose, supporting skeleton to be the same as that of epimedin B [16]. The COSY also revealed that the sugar proton H-4‴ (δH 3.03) correlated with δC 17.2 (C-6‴) confirming rhamnose. The attachment of acetate at C-5 of xylose was confirmed by HMBC correlation of H-5⁗ (δH 5.45) with δC 170.2 (C-6⁗) indicating the linkage of acetyl group with CH2 of xylose. ESI-MS n supported the structure which gave molecular ion peaks at m/z 877.2739 [MOCOCH3 + Na] +, m/z 723.2499 [M-OCOCH3-xylose + H] +, m/z 599.1734 [M-OCOCH3-xylose-rhamnose + Na] + and m/z 415.1389 [M-OCOCH3-xylose-rhamnose-glucose + H] +. Thus the structure of 1 was determined to be 8-(3,3Dimethylallyl)-3′,5,7-trihydroxy-4′,5′-dimethoxyflavonol 3-[O-5-O-Acetyl-β-D-xylopyranosyl-(1→2)-α-L-rhamnopyranoside] 7-(β-D-glucopyranoside) and was assigned the name Elatoside A (Fig. 1, R=CH3). The molecular formula of compound 2 was established as C40H50O23 by means of its HR-ESI [M+ H]+ peak at m/z 899.2819, which in association with its 13C NMR supports this molecular formula. The 13C NMR spectrum showed 40 resonances into five methyl, two methylene, 19 methines and 14 quaternary carbons same as that of 1 with one methyl short. The 1H NMR spectrum of 2 (Table 1) contained aromatic, glycosidic, methyl and acetyl protons. Two meta coupled and ortho

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substituted resonances at δH 6.99 and 6.96 were assigned to H-2′ and H-6′ respectively, of the flavonoid ring B. The singlet at δH 6.69 was assigned to H-6 of ring A. The 1H and 13C NMR resonances assignable to three aromatic moieties, β-D-glucopyranose [δH 4.99 (d, J=6.4 Hz, H-1″) and δC 100.5 (C-1″), 73.4 (C2″), 76.6 (C-3″), 70.2 (C-4″), 76.5 (C-5″) and 60.5 (C-6″)], α-Lrhamnopyranose [δH =5.17 (br, s) and δC 101.7 (C-1‴), 69.8 (C-2‴), 77.2 (C-3‴), 71.1 (C-4‴), 69.9 (C-5‴) and 17.2 (C-6‴)], β-D-xylopyranose [δH =5.02 (d, J =7.1 Hz, H-1⁗), δC =104.1 (C-1⁗), 73.5 (C-2⁗), 75.8 (C-3⁗), 71.5 (C-4⁗), 90.2 (C-5⁗)]. Analysis of correlation in the HQSC and HMBC spectra provided the full assignment for the aglycone part i.e. epimedin B except further substituted ring B as hydroxyls δC 152.4 (C-3′) and δC =153.2 (C-5'). The 1H and 13C NMR spectra do coincide with the glycone part of epimedin B as well, with α-L-rhamnose and β-D-xylopyranose attached at C-3 of the aglycone part and β-D-glucopyranose at C-7. The HMBC correlation H-5⁗ of xylose showed a correlation with δC =170.2 and its downfield chemical shift (δC = 90.2) is indicative of the fact that the lone acetoxy group is attached to C-5 of xylose. (Fig. 1, R=OH). ESI-MSn again supported the structure which gave molecular ion peaks at m/z 899.2819 [M+H]+, 863.2579 [M-OCOCH3 + Na]+, m/z 563.1760 [M-OCOCH3-xylose-rhamnose+H]+. Based on 1H and 13C NMR and conclusive evidence of fragment ions observed in positive ion mode compound 2 was found to be substituted with epimedin B, thus the structure of 1 was formulated as 8-(3,3-Dimethylallyl)-3′,5′,5,7-tetrahydroxy-4′methoxyflavonol 3-[O-5-O-Acetyl-β-D-xylopyranosyl-(1→2)α-L-rhamnopyranoside] 7-(β-D-glucopyranoside) and was assigned the name Elatoside B. The significant HMBC correlations are illustrated in Fig. 2. From the antimicrobial test results (Table 2), it appears that compounds 2, 4, 6, 14, 19 and 24 exhibit antimicrobial activity against S. aureus. Compounds 2, 4, 8, 12 and 15 exhibited antimicrobial activity against E. Coli and S. typhi, compounds 10, 12, 16 and 22 showed activity against S. dysenteriae while compounds 9, 12, 20 and 23 showed the same activity against K. pneumoniae and compounds 1, 7, 12 and 22 exhibited antimicrobial activity against P. aeruginosa. All compounds were analyzed for PPAR-γ ligand binding activity. Compounds anhydroicaritin (3), 8-isoprenylkaempferol (4), breviflavone B (5), epimedokoreanin B (8), desmethylanhydroicaritin (13), epimedoside A (14), epimedoside B (15), icariin (17), epimedin B (18), epimedin C (19), icariside II (20), icaritin (21), ikarisoside A (22), isoliquiritigenin (23), caohuoside F (24) and diphylloside A (25), showed the most potent ligand-binding activity. The activity of these compounds implies that the compounds having prenyl units are necessary for the appearance of the potent activity in these compounds. Considering all of the data together, PPAR-γ ligand-binding activity in the phenolic compounds are affected by differences of substitution groups on the aromatic ring. 4. Conclusion

Fig. 1. Structure of compounds 1 and 2.

The isolation of these compounds forms the first report of the chemical profile of E. elatum. The isolated compounds apart from prenylated flavonols for which the genus Epimedium is known for contain other classes of flavonoids, steroids etc. The existence of acetylated glycosides is in coincidence with the similar type of glycosides isolated from Epimedium

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OH

OH O

OH

O

OH O

O

O

O

O

HO HO

OH

O

O OH

HO HO

OH

O OH

O

O

OH

O HO HO O O HO HO

O O

O

HO HO O

O

O OH

HO HO

O

O OH

Fig. 2. Significant HMBC correlations of 1 and 2.

koreanum. But the isolation of two new compounds as Elatosides A and B which showed substitution at C-3′ and C-5′ of flavonol ring-B can serve as the chemotaxonomic markers of E. Elatum.

Acknowledgements MT would like to thank The University of Mississippi, United States, for providing fellowship as Postdoctoral Research Associateship and ASS thanks the Center for Scientific and Industrial Research (CSIR), India for Emeritus Scientistship.

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