New Phenylethanoid Glycosides From Cistanche Tubulosa

In our series of investigations on the chemical constituents of Cistanche spp. (Orobanchaceae), the phenylethanoid glycosides1-3) and iridoide4) from Cistanche salsa (C. A. MEY.) G. BECK has been reported. The present paper deals with the phenylethanoid glycosides from Cistanche tubulosa (SCHRENK) HOOK. f. collected in Pakistan. Cistanche tubulosa (SCHRENK) HOOK. f.5) is a parasitic plant growing on the roots of Salvadora and Calotropis spp., and occurs widely in North Africa, Arabia, West Asia Pakistan, and India. The whole plant is used medicinally in Pakistan as a remedy for diarrhea and sores.6) We now wish to report the isolation of four new phenylethanoid glycosides, named tubulosides A (II), B (VI), C (VII) and D (VIII), as well as four known phenylethanoid glycosides, echinacoside (I), acteoside (III), acteoside isomer (IV) and 2'-acetylacteoside (V). The structures of these compounds were determined based on chemical evidence and spectroscopic studies. 

The ethanolic extract of the whole plant was suspended in water. This suspension was extracted with ethyl acetate and then with n-butanol saturated with water. The n-butanol-soluble fraction was chromatographed on polyamide and silica gel columns and subjected to high-performance liquid chromatography (HPLC) successively, to give eight phenylethanoid glycosides (I-VIII). 

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Compounds I, III, IV, and V were isolated as amorphous powders, showing similar spectra to those of echinacoside,1) acteoside,1) acteoside isomer7) and 2'-acetylacteoside,2) respectively, and were identified by direct comparison with authentic samples [thin layer chromatography (TLC), infrared (IR), proton nuclear magnetic resonance (1H-NMR), and carbon-13 nuclear magnetic resonance (13C-NMR) spectra]. 

Tubuloside A (II) was isolated as an amorphous powder, [ƒ¿]D-103.7•‹ (MeOH), C37H48O21.3/2 H2O. The IR spectrum suggested the presence of hydroxyl groups (3440 cm-1), a conjugated ester (1705 cm-1), a double bond (1634 cm-1), and aromatic rings (1608, 1522 cm-1), and the ultraviolet (UV) spectrum showed absorption maxima at 220, 250 sh, 292 sh and 334 nm. The 1H-NMR spectrum of II showed signals of a methyl group of rhamnose [6 1.07 (3H, d, J= 6 Hz)], a methyl signal of an acetoxy group [6 1.98 (3H, s)], benzylic methylene protons [6 2.70 (2H, t, J= 7 Hz)], two glucose-anomeric protons [6 4.32, 4.54 (1H each, d, J= 8 Hz)], a rhamnose-anomeric proton [6 5.11 (1H, br s)], two trans olefinic protons [6 6.25, 7.64 (1H each, d, J= 16 Hz)] and aromatic protons [6 6.5 7 .2 (6H)]. On acetylation, II afforded the undecaacetate (IIa), which was identical to the dode-capacitate of echinacoside (I). The 13C-NMR spectrum of II was almost identical to that of I, except for the signals due to the glucose bonded directly to the aglycone and the acetoxyl group [6 20.9 (CH3), 171.5 (C =0)], suggesting that the acetoxyl group is attached to the glucose moiety. In the 13C-NMR spectrum of II, the acylation shifts81 [-2.3 (C-1),-0.9 (C-2) and-1.0 (C-3)] were observed at C-1, C-2, and C-3 of the inner glucose by detailed comparison with the spectrum of I, indicating that the acetoxy group is linked to the C-2 hydroxyl group of the glucose moiety in II. On methanolysis of II with acetyl chloride in methanol, methyl caffeate, and 3,4-dihydroxyphenethyl alcohol were detected by TLC and HPLC. Acid hydrolysis of II with 10% sulfuric acid afforded glucose and rhamnose in a ratio of 2 to 1. 

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Based on the above-mentioned evidence, the structure of tableside A was determined to be 2-(3,4-dihydroxy phenyl)ethyl 0-a-L-rhamnopyranosyl-(1•¨3)-[ƒÀ-D-glu-copyranosyl-(1-6)]-(4-O-caffeoy1)-2-O-acetyl-fl-D-glucopyranoside (II). 

Tubuloside B (VI) was isolated as an amorphous powder, [a]D,- 39.0•‹ (MeOH), C31 H38016, whose 1H-NMR spectrum showed the presence of an aliphatic acetoxyl group [6 1.98 (3H, s)]. The 13C-NMR spectrum of VI was very similar to that of acteoside isomer (IV) but differed slightly in the signals due to the glucose moiety and the presence of the acetoxy group [6 20.9 (CH3), 171.6 (C =0)]. The location of the acetoxy group in the glucose moiety of VI was determined from its 13C-NMR spectrum by detailed comparison with that of IV.

The signals of C-1, C-2, and C-3 of the glucose moiety showed acylation shifts (-2.5 (C-1), －0 .6 (C-2) and-1.4 (C-3)], as in the case of II, indicating that the acetoxy group is linked to the C-2 hydroxyl group of the glucose moiety in VI. On acetylation, VI afforded the octaacetate (VIa) which was identical with the nonaacetate of IV. On methanolysis of VI with acetyl chloride in methanol, methyl caffeate, and 3,4-dihydroxyphenethyl alcohol were detected by TLC and HPLC. Acid hydrolysis of VI with 10% sulfuric acid afforded glucose and rhamnose in a ratio of 1 to 1. These results led us to conclude that the structure of tubuloside B is 2-(3,4-dihydroxyphenyl)ethyl 0-a-L-rhamnopyranosyl-(1-3)-(6-0-caffeoy1)- 2-0-acety143-D-glucopyranoside (VI). 

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Tubuloside C (VII) was isolated as an amorphous powder, [a],-104.8•‹ (MeOH) , C43H54024 H20, whose 1H-NMR spectrum showed the presence of four aliphatic acetoxyl groups [6 1.80, 1.92, 1.95 and 2.08 (3H each, s)]. The "C-NMR spectrum of VII was almost identical with that of tubuloside A (II), which possesses an aliphatic acetoxyl group in the inner glucose, except for the signals due to the rhamnose moiety. Furthermore, in the 13C-NMR spectrum of VII, acylation shifts') were observed in the signals due to C-2, C-3, and C-4 of the rhamnose moiety by detailed comparison with the data for II. Consequently, the locations of the four acetoxy groups were determined to be C-2 of the inner glucose and C-2, C-3, and C-4 of the rhamnose moiety in VII. On acetylation, VII afforded the octaacetate which was identical with tubuloside A undecaacetate (IIa). On methanolysis of VII with acetyl chloride in methanol, methyl caffeate, and 3,4-dihydroxyphenethyl alcohol were detected by TLC and HPLC. Acid hydrolysis of VII with 10% sulfuric acid afforded glucose and rhamnose in a ratio of 2 to 1. 

From the above results, the structure of tableside C was determined to be 2-(3,4- hydroxyphenyl)ethyl 2,3,4-tri-O-acetyl-a-L-thamnopyranosyl-(1 -+ 3)-[/9-D-glucopyranosyl- (1 -+ 6)]-(4-0-caffeoy1)-2-0-acetyl-fl-D-glucopyranoside (VII). 

Tubuloside D (VIII) was isolated as an amorphous powder, [oc],-91.4° (MeOH), C43H54023. H2O, whose 1H-NMR spectrum showed the presence of four aliphatic acetoxy groups [6 1.81, 1.93, 1.96, and 2.09 (3H each, s)]. On acetylation, VIII afforded the heptaacetate (VIIIa), whose 1H-NMR spectrum showed eight aliphatic [6 1.87, 1.94, 1.96, 1.99, 2.10 (3H each, s) and 2.02 (9H, s)] and three aromatic [6 2.27, 2.30 and 2.32 (3H each, s)] acetoxyl methyl signals. The 13C-NMR spectrum of VIII was almost identical to that of tubuloside C (VII), except for the signals due to the p-coumaric acid moiety. On methanolysis of VIII with acetyl chloride in methanol, methyl p-coumarate, and 3,4-dihydroxyphenethyl alcohol were detected by TLC and HPLC. Acid hydrolysis of VIII with 10% sulfuric acid afforded glucose and rhamnose in a ratio of 2 to 1. 

Based on these results, the structure of tableside D was determined to be 2-(3,4- dihydroxy phenyl)ethyl 2,3,4-tri-O-acetyl-a-Lrhamnopyranosyl-(1•¨3)-[ƒÀ-D-glucopyranosy-] (1→6)} (4-0-p-coumary1)-2-0-acetyl-fl-D-glucopyranoside (VIII).

Many phenylethanoid glycosides such as forsythoside A,'°) leucosceptoside A,7 isomar-tynosidell) and so on, having a rhamnose moiety as the terminal sugar has been reported. In these cases, the rhamnose moiety is not acetylated. Tubulosides C (VII) and D (VIII) contain an acetylated rhamnose moiety and are the first naturally occurring compounds having a triacetylrhamnose moiety to be reported.

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