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-rhamnopyranosyl-catalpol 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-1-picrylhydrazyl (DPPH). The five compounds isolated were also evaluated as anti-inflammatory, where scropheanoside-III was the most active among all compounds tested.

Experimental

General Experimental Procedures

Optical rotation  were measured on a Zeiss polarimeter Model 53187 using a sodium lamp. UV spectra were obtained on a Varian CARY 2290 spectrophotometer in Me OH. IR spectra were recorded on a Perkin –Elmer Model 337 spectrometer in K Br discs. FABMS were recorded with an MS 2500 high-resolution spectrometer ( Kratos , Manchester, UK) with 70 eV, an ion source temperature of 180 ^o C and a direct inlet using meta nitrobenzyl alcohol   and/ or thioglycerin as matrix. NMR spectra were measured on CD₃OD and pyridine-d₅  and recorded at 300 MHz for ¹ H-NMR and 75 MHz for ¹³C-NMR using TMS as internal standard on a Varian XL300  (Darmstadt, Germany ). Chemical shifts were expressed in δ values (¹H) and ppm (¹³C-NMR). Precoated thin–layer chromatographic (TLC) plates coated with silica gel 60 F₂₅₄ ,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-H₂SO₄ solution followed by heating at 100 ^o C for 10 minutes. Solvent systems used for TLC were: I. CHCl₃-CH₃OH-H₂O (15:7.5:2.5, lower phase); II. Et OAc –  CH₃OH   – n-C₃H₇OH – H₂O (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 water-insoluble 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, 4.5×120 cm ) using CHCl₃ – MeOH –H₂O  gradient elution and repeated purification columns  to afford the following compounds:

Compound 1

Obtained as a white amorphous powder; [α]_D²⁵, -100^O (CH₃OH, c =1 ) with R _{f s} 0.51 (system I ) and 0.56 (system II). IR ( K Br ) : _{√ max}  cm^-1  3400, 2900, 1700, 1660, 1630, 1580, 1510, 1440, 1260, 1030. Positive FABMS: m/z  691  [M +Na ]⁺, 669 [ M +H ]⁺ , 323 [ C₁₆H₁₈O₇ ]⁺, 177 [C₁₀H₉O₃]⁺.¹H-NMR (300 MHz, CD₃OD,   δ  , ppm, J= Hz ): 4.9 (d, J=7 Hz, 1- H ), 6.3 (dd, J=6.5 & 2 Hz, 3-H ), 5.1 (dd, J=6.5 & 4 Hz,  4-H ), 2.8 (dddd, J =7.5, 5.5 , 4 & 2 Hz, 5- H ), 4.4 (m ,6- H ), 5.7  (dddd, J =2,2,2&2 Hz, 7-H ),  2.88 (dd, J=7.5 & 7 Hz, 9-H ), 4.23  (d, J=16 Hz , 10 _a -H), 4.34 ( d, J= 16 Hz, 10 _b -H), 4.7 (d, J=8 Hz, 1′-H), 3.2 ( dd, J=9 & 8 Hz, 2′-H ), 3.4 (dd, J= 9 & 9 Hz , 3′-H), 3.26 – 3. 34 (m, 4′-H ) ,  3.26-3.34  (m, 5′-H), 3.6 (dd, J= 12 & 6 Hz, 6′_a– H ), 3.8 (dd, J= 12 & 2 Hz, 6′_b-H ), 4.8 (d, J=2 Hz, 1″-H ), 3.83 (dd , J =2 & 3.5 Hz , 2″-H ), 3.86 (dd, J =9.5 & 3.5 Hz , 3″-H ), 5 (d, J=9.5 Hz, 4″-H), 3.9 (dd, J = 9. 5 & 6.5 Hz, 5″-H ), 1.2 ( d, J =6.5 Hz , 6″-H ), 7.09 (d, J =2 Hz, 2′”-H ), 6.9 (d, J= 8 Hz, 5″‘-H ), 7.06 (dd, J = 8 & 2 Hz, 6″‘-H ), 6.35  (d, J =16 Hz, α-H ), 7.6 (d, J= 16 Hz, β-H ), 3.8 (s, OCH₃). ¹³C-NMR (75 MHz, CD₃OD   , δ ,  ppm) see Table 1.

Compound 2

Obtained as white amorphous powder; [α]_D ²⁵ -180⁰ (CH₃OH, c=1) with R _{f s} 0.47 ( system I) and 0.51  (system II ). IR ( KBr ) : _{√ max} cm^-1  3400, 2900, 1630, 1610, 1600, 1580, 1500. Positive FABMS : m/z 685 [M+H]⁺ Calcd for the formula (C₃₁H₄₀O₁₇+H ), 707 [M+Na]⁺, 323 , 177. ¹H-NMR (300 MHz, CD₃OD,  δ  , ppm, J =Hz ): 5.1 (d, J=9.5 Hz,1-H), 6.37 (dd, J=6.5 &2 Hz, 3-H), 5 (dd, J=6.5 &4 Hz, 4-H), 2.4 (dddd, J=8,8,4&2 Hz, 5-H), 4 (dd, J=8 &2 Hz, 6-H) , 3.7 (d, J=2 Hz ,7-H), 2.7 (dd J=8 &9.5 Hz, 9-H), 3.8 (d, J=13 Hz, 10_a-H), 4.2 (d, J=13 Hz, 10_b-H), 4.8 (d, J=8 Hz, 1′-H), 3.3 (dd, J=9 &8 Hz, 2′-H), 3.4 (dd, J=9 &9 Hz,3′-H), 3.2 (dd, J=9 &8 Hz, 4′-H), 3.3 (m, 5′-H), 3.6 (dd, J= 6 &12 Hz, 6′_a-H), 3.9 (dd, J=12 &6 Hz, 6′_b-H), 5 (d, J= 2 Hz, 1″-H), 3.9 (dd, J= 4 & 2 Hz, 2″-H ), 3.8 (dd, J=9.5 &4 Hz, 3″H), 5 (dd, J=9.5 &9.5 Hz, 4″-H), 3.89 (dd, J= 6 &9.5 Hz,5″-H), 1.2 (d, J= 6 Hz, 6″-H ), 7 (d, J=2 Hz, 2″‘H), 6.9 (d, J= 8.5 Hz, 5″‘-H), 7 (dd, J=8.5 &2 Hz, 6″‘-H ), 6.4 (d, J=16 Hz, α- H) , 7.6 (d, J=16 Hz, β-H) ,3.9  ( s, OCH₃). ¹³C-NMR (75 MHz, CD₃OD , δ,   ppm ) see Table 1.

Compound 3

Obtained as white amorphous powder; [α]_D ²⁵ -89⁰  (CH₃OH ,c=1) with R _{f s}  0.43 (system I) and o.47 (system II ). IR ( KBr ) : _{√ max} cm^-1 3400 ,1745 , 1715 ,1630 1575  .Positive FABMS :m/z 740 [M +NH₄]⁺, 745 [M+Na]⁺ Calcd for the formula  (C₃₄H₄₂O₁₇), 361 (M- C₁₅H₁₂O₁₀) .¹H-NMR (300 MHz, CD₃OD , δ , ppm , J=Hz): of  the catalpol nucleus and the glucose moiety are in good agreement with those of compound 2 isolated from the same plant,  5.1 (d, J=2 Hz, 1″-H) ), 5.3 (dd, J= 4 & 2 Hz, 2″-H ), 5.4 (dd, J=9.5 &4 Hz, 3″-H), 5 (dd, J=9.5 &9.5 Hz, 4″-H), 4 (dd, J= 6 &9.5 Hz, 5″-H), 1.2 (d, J= 6 Hz, 6″-H ), 7.4 (m , 2″‘-H), 7.6 (m, 3″‘H), 7.4 (m,4″‘-H), 7.6 (m, 5″‘-H) 7.6 (m, 6″‘-H), 6.4 (d , J=16 Hz, α-H), 7.6 (d, J=16 Hz, β-H) , 2 ( s, COCH₃) , 2.1 (s, CO-CH₃) . ¹³C-NMR (75 MHz, CD₃OD , δ , ppm ) see Table 1.

Compound 4

Obtained as yellowish-white amorphous powder; [α]_D ²⁵ -31⁰ (CH₃OH , c=1) with R _{f s} 0.33 (system I) and 0.36 (system II ). IR ( KBr ) : _{√   max} cm^-1  3380 , 2920 , 1795 ,1630 ,1600,1510. .Positive FABMS : m/z 625 [M +H]⁺, 642 [M+NH₄]⁺, 647 [M+Na]⁺ Calcd for the formula  ( C₂₉H₃₆O₁₅).  The UV (MeOH,   λ_max  ,nm) 320, 280 and 230 nm.  ¹H-NMR (300 MHz, CD₃OD , δ ,  ppm , J=Hz): 6.6 (d, J=2 Hz, 2-H) , 6.7 (d, J=8 Hz, 5-H) , 6.5 (dd, J=2 & 8Hz, 6-H) ,3.7(dd, J=7 & 17 Hz, α-H_a), 4 (dd, J=7 &17 Hz, α-H_b) ,2.8 (dd, J=7 &7, β –H_{a, b} ), 4.4 (d, J= 8 Hz,1″-H), 3.4 (dd, J = 8 & 9 Hz, 2″ –H ), 3.8 (d, J = 8 Hz, 3″- H ), 4.8 (m, 4″-H ), 3.6 (m, 5″-H , 6″-H _{a, b} ), 6.3 (d, J=16 Hz, α’-H ), 7.6 (d, J= 16Hz, β’-H), 7 (d, J= 2 Hz, 2H), 6.8 (d, J=8 Hz, 5′-H ), 6.9 (dd, J= 2 &8 Hz,6′-H ), 5.2 (d, J=2 Hz, 1″‘-H ), 3.9 (dd, J= 2 &3.5 Hz, 2″‘-H ), 3.5 (dd, J= 9.5 &9.5 Hz, 3″‘-H ), 3.3 (dd, J= 9.5 &9.5 Hz, 4″‘-H ), 3.4 (m, 5″‘-H ), 1 (d, J= 6 Hz, 6″‘-H ) . ¹³C-NMR (75 MHz, CD₃OD ,  δ , ppm ): 131 (C-1 ), 117 ( C-2 ), 144 (C-3 ), 146 (C-4 ), 116 (C-5 ), 121 (C-6 ), 72 (C-α ), 36 (C-β ), 104 (C-1″), 76 (C-2″), 81 (C-3″), 70 (C-4″), 76 (C-5″), 62 (C-6″), 114 (C-α’), 147 (C-β’), 168 (C=O), 127 (C-1′),115 (C-2′), 149 (C-3′), 147 (C-4′), 116 (C-5′), 123 (C-6′), 102 (C-1″‘), 72 (C-2″‘), 71 (C-3″‘), 73 (C-4″‘), 70 (C-5″‘), 18 (C-6″‘).

Compound 5

Obtained as white amorphous powder; [α]_D²⁵ -71⁰ (CH₃OH , c=1) with R _{f s} 0.29 (system I) and o.31 (system II ). IR ( K Br ) : _{√ max} cm^-1 3400 ,2920 , 1695 ,1630 ,1600,1520. .Positive FAB MS :m/z 653 [M +H]⁺, 670 [M+NH₄]⁺, Calcd for the formula (C₃₁H ₄₀O₁₅). The UV (Me OH,  λ_max , nm) :328, 288. ¹H-NMR (300 MHz, CD₃OD , δ , ppm ,J=Hz) of the compound showed close similarity to the ¹HNMR of compound 4 isolated from the same plant, except for the presence of two methoxyl groups  [δ _H 3.8 (s, 4- OCH₃ )  and δ _H 3.9 (s, 3′- OCH₃ ) ]. ¹³C-NMR (75MHz, CD₃OD, δ, ppm): 132 (C-1), 116( C-2 ), 147 (C-3) , 147 (C-4 ), 116 (C-5 ), 121 (C-6), 72 (C-α ), 36 (C-β ),  56 (4- OCH₃ ), 104 (C-1″), 76 (C-2″), 81 (C-3″), 70 (C-4″), 76 (C-5″), 62 (C-6″), 115 (C-α’), 147 (C-β’), 168 (C=O), 127 (C-1′),111 (C-2′), 150 (C-3′), 149 (C-4′), 116 (C-5′), 124 (C-6′),  56 ( 3′- OCH₃ ), 102 (C-1″‘), 72 (C-2″‘), 72 (C-3″‘), 73 (C-4″‘), 70 (C-5″‘), 18 (C-6″‘)

 Acid Hydrolysis

Compounds isolated (15 mg each) were refluxed, separately, with 0.1 N HCl (30 ml) for 6 hr. Then H₂O was added and the mixtures were extracted with CHCl₃. The aqueous layer, in each case was neutralized with Ag₂CO₃ and filtered. The filtrate was evaporated in vacuo and the residue was examined by comparison with authentic sugar  samples  using silica gel F₂₅₄ plate, n- BuOH – PrOH – H₂O (10:5:4) as developer and anisaldehyde – H₂SO₄  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 1.5×10^-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±2⁰C with a 12-h 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.

Results and Discussion

Compound 1, Scropheanoside-I, with formula C₃₁H₄₀O₁₆ through the positive FABMS, with   ion beak at m/z 669 [M+H]⁺ , 691 [M +Na]⁺ , 323 [C₁₆H₁₈O₇] for isoferuloyl-rhamnose fragment and m/z 177 [C₁₀H₉O₃]⁺ for isoferuloyl fragment. The IR spectrum of the compound showed absorption bands at 3400 cm^-1 (hydroxyl groups), 1700 cm^-1 (α,β-unsaturated ester), and 1630 cm^-1( conjugated double bond ). ¹H-NMR data indicated an olefinic  proton at C-3 at, δ 6.3 (1 H, dd) with vicinal coupling J_3,4=6.5 with H-4 (δ 5.1 ppm ) and an allylic coupling J_3,5 =2 Hz with H-5 ( δ   2.8 ppm ). The presence of H-7 at δ 5.7 ppm and the three homoallylic coupling of H-6 with protons at C-9 and C-10   indicated the presence of carbon, carbon –double bond between C-7 and C-8 of the iridoid nucleus of the compound. The ¹H-NMR data showed full agreement with the reported data of aucubin [Abbas and Zayed, 2004, and Pachaly et al., 1994]. The ¹H-NMR spectrum exhibited signals for three aromatic protons (δ _H 7.09, d, J = 2 Hz, 6.9, d, J = 8 Hz and 7.06, dd , J = 8 &2 Hz) and two olefinic protons ( δ _H 6.35, d,  and 7.6, d, AB system, J_AB =16 Hz ) substantiating the presence of trans isoferuloyl moiety , one methoxy signal (δ _H 3.8, s) , one methyl signal (δ _H 1.2, d, J = 6.5 Hz ) and two anomeric protons ( δ _H 4. 7, d, J = 8 Hz, 4.8, d , J =2 Hz ) indicating the presence of two sugar moieties in the molecule.

Scheme 1   Click here to View Scheme

On acid hydrolysis, the compound afforded D-glucose and L-rhamnose as sugar moieties. The chemical shifts of protons and carbon atoms were compared with the reported data [Pachaly et al., 1994] which indicated the presence of β-D-glucose and α –L- rhamnose, respectively. The H-1 of the aglycone exhibited long range coupling in the heteronuclear multiple bond connectivity ( HMBC ) spectrum with C-1′ of the glucose unit, H-6 correlated with C-1″ of the rhamnose moiety which indicated that glucose is connected with ether linkage at position 1 and rhamnose with position 6.

The assignments of glucosyl and rhamnosyl protons were also made with the help of ¹H-¹H –COSY experiments starting with their anomeric protons ( δ _H 4.7, d, J = 8 Hz,  glucosyl  H -1,   δ _H 4.8, d,  J = 2 Hz, rhamnosyl H-1 )  and methylene protons (  δ _H  3.6 , dd, J = 12 & 6 Hz, 6′_a– H; 3.8  ,dd, J = 12 & 2 Hz,  6′_b-H ) in case of glucose and methyl proton (δ _H 1.2, d, J = 6.5 Hz, Me-6″ ) in case of rhamnose.  The   long –rang couplings in the HMBC spectrum also confirmed the above assignments. The ¹³C-NMR (75 MHz, CD₃OD , 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 C₃₁H₄₀ ). The sequential assignments of protons and carbon atoms were made with the help of ¹H-¹H-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-O-isoferuloyl) –rhamnopyranosyl aucubin.

Compound 2, Scropheanoside-II, showed positive FABMS at m/z 685 [M+H]⁺, 707 [M+Na]⁺, for molecular formula C₃₁H₄₀O₁₇. The fragment ion peaks at m/z 323 and 177 due to isoferuloyl- rhamnose residue similar to compound 1. Acid hydrolysis of compound 2 gave D-glucose and L-rhamnose. Interpretation of ¹H and ¹³C-NMR indicated an iridoid nucleus with C-9  aglycone  of catalpol  derivative [ Pachaly et al.,1994]. The ¹HNMR 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 epoxy- function between C-7 and C-8. The ¹H-NMR also showed signals for β –glucose, α- L-rhamnose and isoferuloyl residues similar to compound 1.  Analysis of the ¹³C-NMR spectrum, which showed 31 carbon signals, nine corresponding to aglycone, twelve for sugar moieties and ten for isoferuloyl moiety. Full assignments of the ¹H and ¹³C-NMR signals were accomplished using ¹H-¹H-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.

Table 1: ¹³C- NMR Spectral Data of  Scropheanoside I-III (75 MHz, CD₃OD).

The ¹³C-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 ¹³C-NMR spectrum of the compound, where two anomeric carbons at  δ c 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- L-rhamnose. The significant deshielding of C-4″ of rhamnose (δ c 75) confirmed the placement of isoferloyl residue to C-4″ of rhamnose. ¹³C-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 ¹H and ¹³C-NMR spectral data with those of previously reported data [ Pachaly et al., 1994].

Compound 3, Scropheanoside-III, with molecular formula C₃₄H₄₂O₁₇, positive FABMS at m/z 740 [M+NH₄]⁺, m/z 745 [M+Na ]⁺. The fragment ion peak at m/z 361 [M-C₁₅H₁₂O₁₀] ⁺ for acetylcinnamoyl rhamnosyl residue. The ¹³C-NMR spectrum showed 34 carbon atoms for the molecule containing three methyls, two methylenes,24 methines , two quaternary and three carbonyl carbon atoms (in total C₃₄H₄₂ ). The sequential assignments of protons and carbon atoms were made with the help of ¹H-¹H (COSY) and HETCOR experiments starting with the easily distinguishable acetal methin proton at δ 5.1 assigned to position 1 (δ c 95) , H-9 ( δ _H 2.7,  δ c 43) and  H-5 ( δ _H 2.4 , δ c  37 ).

The  ¹H- and ¹³C-NMR spectra exhibited signals for five aromatic protons substantiating the presence of a trans– cinnamoyl moiety, two acetoxy signals (δ _H  2.03, s ; 2.15, s ;  2 x Me; δ c  171.5, 171.8,  2 C=O ) and two anomeric protons  δ _H  4.8, d, J = 8 Hz, δ c 100  ;  δ _H 5.1, d, J = 2 Hz,  δ c 98 ), indicating the presence of two sugar  moieties in the molecule. The chemical shifts of proton and carbon atoms were compared with the values reported in the literature [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,and 1993] which showed a  close  resemblance.

Compound 4, Acetoside, showed UV at  λ_max 320, 280 and 230 nm. The molecular formula C₂₉H₃₆O₁₅ established by positive FABMS at m/z 625 [M+H]⁺, m/z 642 [M+NH₄]⁺ and m/z 647 [M+Na ]⁺. ¹³C-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 ¹H-NMR spectrum showed the presence of (E) –caffeic acid and 3,4- dihydroxyphenyl 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 ¹H and ¹³C –NMR data were in full agreement with those reported for acetoside  [Pachaly et al., 1994, Li et al., 2000,and Akdemir et al.,1991].

Compound 5,  Martynoside, with. molecular formula (C₃₁H₄₀H₁₅)   established  by  positive  FABMS  m/z 653 [ M+H]⁺ and m/z 670 [ M+NH₄ ]⁺; The ¹³C-NMR spectrum of the compound exhibited 31 carbon resonances. The ¹HNMR exhibited six aromatic  for one ferulic acid and phenylethyl aglycon part;. 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 β – glucose unit and  α- rhamnose unit, respectively. The ¹H-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 ¹H-¹H 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 anti-inflammatory activities

Anti-oxidant Activity [Molgard and Ravn, 1988, Jimenez  and  Riguera , 1994,  Xiang et al., 1996,and   Gao et al., 2000]

Martynoside and acetoside have potent radical  scavenging activity which is more than dl– α- -tocopherol as natural  antioxidant. Their IC₅₀ values are as follows: Martynoside 3.6×10 ^-4 M; acetoside 2.4×10 ^-4 M  and dl-α- tocopherol  4.8×10 ^-4 M .  Inhibitory activity of acetoside was higher than that of martynoside .

Table 2: Effect of Compounds 1-5 on Right Rat Paw Swelling Induced By Carrageenin.

Values are expressed as ± S.E.M.

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 anti-inflammatoryactivity. 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.

Acknowledgements

The author is deeply thankful to Dr. Marie- Luise Koch, Institute of Pharmaceutical Biology, Bonn University, Germany, for carrying the spectroscopic measurement.  The author would also like to thank   Dr. Kamal EL Tahir, Professor of Pharmacology, College of Pharmacy, KSU, Saudi Arabia for the pharmacological studies.

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