Phenylpropanoid and phenylethanoid glycosides from Scrophularia xanthoglossa and their antioxidative and antiinflammatory activities

Repeated column chromatography of the butanol extract of Scrophularia xanthoglossa yielded three phenylpropanoid glycosides identified as scropheanoside-I, scropheanoside-II, scropheanosideIII together with two phenylethanoid glycosides identified as, acetoside, and martynoside. Their structures were determined by spectroscopic analysis, chemical evidence ®as well as comparison with literature values. The biological activity was also studied where acetoside and martynoside showed significant antioxidative activity, while scropheanoside-III showed significant antiinflammatory activity.


INTRODUCTION
In our previous study [Abbas and Zayed, 2004], we reported the isolation and structure determination of five iridoid glycosides; aucubin, harpagid, 6-O-α-L-rhamnopyranosyl-aucubin, harpagoside and 6-O-α-L-rhamnopyranosylcatalpol from Scrophularia xanthoglossa Bioss. A survey of literature showed that no phytochemical and pharmacological works have been reported on phenylpropanoid and phenylethanoid glycosides from Scrophularia xanthoglossa Bioss., a perennial plant growing in Yemen and southwestern Arabia [Miagahid,1989]. In continuation to our studies on the constituents of Scrophularia xanthoglossa, we investigated the butanol extract of the plant to furnish the isolation and identification of three phenylpropanoid glycosides scropheanosides I-III, and two phenylethanoid glycosides; acetoside and martynoside. The antioxidative activity of the five glycosides was evaluated against 2, 2-diphenyl-1picrylhydrazyl (DPPH). The five compounds isolated were also evaluated as anti-inflammatory, where 60 F 254 ,0.25 mm layer thickness and silica gel ( 60-120 mesh ) for column chromatography were obtained from Merck ( Darmstadt, Germany ). The spots were visualized by spraying with 1% vanillin-H 2 SO 4 solution followed by heating at 100 o C for 10 minutes. Solvent systems used for TLC were: I. CHCl 3 -CH 3 OH-H 2 O (15:7.5:2.5, lower phase); II. Et OAc -CH 3 OH -n-C 3 H 7 OH -H 2 O (15:10:2:7, lower phase).

Plant Material
Scrophularia xanthoglossa Bioss. is a glabrous perennial plant ( up to one meter high ) that grows in Yemen and Southwestern Arabia [Miagahid,1989]. The sample of aerial parts for the present study was collected from road and hill sides in Sanaa region (Wadi Zahr) in Yemen in March 1997. Identity of the plant was confirmed by Professor Sultan Abdeen, Faculty of Pharmacy, King Saud University. A certified specimen has been deposited at the Pharmacognosy Department, Faculty of Pharmacy, King Saud University, Saudi Arabia. The aerial parts of the plant were air-dried and powdered before extraction.

Extraction and Isolation
The air-dried powdered plant material of Scrophularia xanthoglossa Bioss. (500g) was exhaustively extracted with ethanol (95%, 5 x 2 L) by cold maceration. The solvent was distilled off under reduced pressure, then the residue (60 g) was suspended in distilled water (500ml), the waterinsoluble material was removed by filtration. The aqueous solution was successively extracted with petroleum ether, ether, chloroform, ethyl acetate, and finally with butanol saturated with water (3 L each ) .The solvent in each case was distilled off under reduced pressure to afford ( 13 , 7, 2 , 11, 17 g , respectively ) . TLC examination of the butanol extract using solvent system II revealed the presence of five spots having R f s 0.56, 0.51, 0.47, 0.36.and 0.31. About 10 g of the dried butanol extract were subjected to flash silica gel column (Merck, 500 g, 4.5x120 cm ) using CHCl 3 -MeOH -H 2 O gradient elution and repeated purification columns to afford the following compounds:

Compound 1
Obtained as a white amorphous powder;   Table 1.

Acid Hydrolysis
Compounds isolated (15 mg each) were refluxed, separately, with 0.1 N HCl (30 ml) for 6 hr. Then H 2 O was added and the mixtures were extracted with CHCl 3 . The aqueous layer, in each case was neutralized with Ag 2 CO 3 and filtered. The filtrate was evaporated in vacuo and the residue was examined by comparison with authentic sugar samples using silica gel F 254 plate, n-BuOH -PrOH -H 2 O (10:5:4) as developer and anisaldehyde -H 2 SO 4 for detection. All compounds gave glucose ( R f 0.3 ) and rhamnose ( R f 0.55 ).

Measurement of DPPH radical scavenging activity
Each EtOH solution (100µl) of compounds 1-5 at various concentrations was added to 1.5x10 -5 M DPPH/EtOH solution. The reaction mixture was shaken vigorously and the absorbance of remaining DPPH was measured at 530 nm after 30 min. The radical scavenging activity was determined by subtracting the absorbance with that of blank (100%) containing only DPPH and solvent. The samples were prepared using the same dilution procedures and dl -α-tocopherol was used as standard [Abourashed, 2005].

Experimental animals
Wister rats of either sex weighing 200-250 g were used to determine anti-inflammatory activity. The animals were maintained at 23±2°C with a 12h light and dark cycle, fed a Purina rat chow diet supplied by Grain Silos and Flour Mills Organization, Riyadh, Saudi Arabia, and had free access to food and water.

Determination of Anti-inflammatory Activity
Six rats each were allotted to different treatment groups. Edema was induced in the rats by injecting carrageenin (0.05ml, 1% w/v in normal saline) into the subplantar tissue of the right hind paw [Winter et al.,1962]. Paw volume (mm) was measured with a plethysmometer (7140, Ugo Basile) before carrageenin injection and 0, 1, 2 and 3h thereafter. The edema was reported as the difference between the initial and the final volume. The anti-inflammatory effect was expressed as the percentage inhibition compared with vehicle-treated animals with respect to a reference group treated with phenylbutazone (100 mg/kg). The test compounds (10mg/kg) with distilled water (0.1ml/ 100g rat) were administered orally 1h before injection of the phlogistic agent. The long -rang couplings in the HMBC spectrum also confirmed the above assignments. The 13 C-NMR (75 MHz, CD 3 OD , Table 1) and DEPT spectra of the compound showed 31 carbon atoms for the molecule consisting of two methyls, two methylenes, twenty two methines, four quaternary and one carbonyl carbon atom (in total C 31 H 40 ). The sequential assignments of protons and carbon atoms were made with the help of 1 H-1 H-COSY and HETCOR spectra. The spectral data of the compound were also compared with those of scropheanoside-I, isolated from Scrophularia koraiensis [Pachaly et al., 1994] which showed a close resemblance. Unequivacally, the structure of the compound was elucidated to be 6-O-α-L-(4-Oisoferuloyl) -rhamnopyranosyl aucubin. HNMR showed absorption at δ 6.37 (1 H, dd, J = 6.5 & 2 Hz ) of olefinic H-3 for iridoid nucleus. The resonance of H-7 at δ 3.7 ppm indicated the epoxyfunction between C-7 and C-8. The 1 H-NMR also showed signals for β -glucose, α-L-rhamnose and isoferuloyl residues similar to compound 1. Analysis of the 13 C-NMR spectrum, which showed 31 carbon signals, nine corresponding to aglycone, twelve for sugar moieties and ten for isoferuloyl moiety. Full assignments of the 1 H and 13 C-NMR signals were accomplished using 1 H-1 H-COSY and H-C correlation. The absorption at δ c 95 and 84 ppm were assigned to C-1 and C-6, respectively, indicating glycosidation at positions 1 and 6 of the iridoid skeleton.

RESULTS AND DISCUSSION
The 13 C-NMR data also showed two olefinic carbons of the iridoid skeleton at C-3 (δ c 142) and C-4 (δ c 103 ). The diglycosidic structure was confirmed by the 13 C-NMR spectrum of the compound, where two anomeric carbons at δc 100 and 101 were observed. All protons of the two sugar units were assigned unambiguously from the shift correlation spectroscopy (COSY) spectrum, and the two sugars were found to be β -glucose and α-Lrhamnose. The significant deshielding of C-4" of rhamnose (δ c 75) confirmed the placement of isoferloyl residue to C-4" of rhamnose. 13 C-NMR also showed signals corresponding to trans isoferuloyl at δ c 116 (C-α ), 146 (C-β ), 168 (C=O), signals for six aromatic carbons ( δ c 128., 114, 148, 151, 112 & 122) and signals for methoxy function at 56. Finally the compound was proved to be scropheanoside-II by comparing 1 H and 13 C-NMR spectral data with those of previously reported data [ Pachaly et al., 1994].   [Pachaly et al., 1994, Agarwal, 1992, and Miyase and Mimatsu,1999. The spectral data of the compound were also compared with those of scropolioside -A and scropolioside-D, previously isolated from Scrophularia scopolii and, Scrophularia ilwensis, respectively [Calis et al.,1988[Calis et al., ,and 1993 13 C-NMR experiments showed three methylene, eighteen methane, one methyl and seven quaternary carbons with two characteristic anomeric carbons at δ c 104 and 102. The 1 H-NMR spectrum showed the presence of (E) -caffeic acid and 3,4dihydroxyphenyl ethanol moieties confirmed by the six aromatic proton signals between δ 6.6-7.0 for 2ABX system, two olefinic protons (AB system, d, J AB = 16 Hz ) at δ 6.3 and 7.6, a benzylic methylene at δ 2.8 (2H, dd, J = 7 &7 Hz) and two non-equivalent protons at δ 3.7 and 4.0 (each 1H, m). Additionally, two doublets of anomeric protons were observed at δ 4.4 (d, J = 8 Hz) and at δ 5.2 (d, J = 2 Hz ) indicating diglycosidic structure. The significant deshielding of C-4" of the glucose at δ c (70.) confirmed that the caffeoyl residue was attached to C-4" of glucose . A downfield shift of C-3" of glucose δ c (81) indicated that rhamnose unite was terminal and attached to C-3" of glucose. The rest of 1 H and 13 C -NMR data were in full agreement with those reported for acetoside [Pachaly et al., 1994, Li et  . two olefinic protons (d, each, H-α' and β') with J A,B =16 Hz for a trans ferulic acid unit; two methoxy groups; ethylene protons and one secondary methyl group as typically found for rhamnose were assigned. Additionally, two anomeric protons were observed at δ 4.4 (d, J = 8 Hz ) and δ 5.2 (d, J = 2 Hz ) which was consistent with βglucose unit and αrhamnose unit, respectively. The 1 H-NMR spectrum also confirmed placement of the feruloyl moiety at C-4" of the glucose moiety (deshielding of H-4" glucose resonances at δ 4.8, t, J = 9.6 Hz). The signals attributed to the aglycone and an acyl moiety was consistent with the presence of 3-hydroxyl -4 methoxy phenylethanol and ferulic acid, respectively. Because no substituent chemical shift was observed for the rhamnose unit, the deoxy sugar was proven to be terminally linked to the glucose moiety. The complete assignment of most protons and carbons were based on the 1 H-1 H COSY, HMQC and HMBC experiments. The structure of the compound was confirmed to be martynoside by comparison with published data [Pachaly et al., 1994].

Biological activity
The isolated glycosides were also screened for their anti-oxidative and antiinflammatory activities Anti-oxidant activity [Molgard and Ravn, 1988, Jimenez and Riguera , 1994, Xiang et al., 1996.

Anti-inflammatory activity
The results are presented in Table 2.The phenylpropanoid glycosides decreased edema in the range of 22-31 % at a dose of 10 mg/kg after 3 h with respect to the control group treated only with carrageenin, against phenylbutazone (60 % decrease) at a dose of 100 mg/kg, which indicated that the test compounds had moderate antiinflammatoryactivity. Scropheanoside-III (31% decrease) and Scropheanoside-I (26 % decrease). It may be concluded that compound containing a cinnamoyl moiety as found in Scropheanoside-III has significant activity.