Phenylpropanoid Glycosides from Orobanche caerulescens

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• LDL oxidation and screening for antioxidants
• Characterization of compounds 1, 1a, 2 and 2a
• Results and Discussion
• References

Abstract

Two new phenylpropanoid glycosides, caerulescenoside (1), and 3′-methyl crenatoside (2), as well as five known phenylpropanoid glycosides [acteoside (3), isoacteoside (4), campneoside II (5), crenatoside (6), and desrhamnosyl acteoside (7)] were isolated from the whole plant of Orobanche caerulescens. The antioxidative effects of compounds 1 - 7 on human low-density lipoprotein were evaluated. All these compounds suppress concentration-dependently conjugated diene formation with IC₅₀ values of 1.25 ± 0.06, 2.97 ± 0.31, 0.31 ± 0.01, 1.01 ± 0.05, 1.15 ± 0.04, 1.69 ± 0.15, and 0.64 ± 0.03 μM, respectively. Comparison of their antioxidative activities with that of resveratrol (IC₅₀ : 6.75 ± 1.05 μM), a natural phenolic antioxidant isolated from grape, demonstrated that the prolonged effect on lag-time and the damping effect on oxidative rate by compounds 1 - 7 were all more potent.

Introduction

Orobanche caerulescens Stephan (Orobanchaceae) is a parasitic herb growing on the roots of Artemisia spp. (Compositae) [1]. It is used as a tonic agent to treat impotence, an anti-inflammatory agent to treat cystitis, and a feces softener [2]. Previous chemical studies [3], [4], [5] of the genus Orobanche have shown the presence of a variety of phenylpropanoid glycosides, carotenoids, phytosterols and sugars.

The oxidative modification of low-density lipoproteins (LDL) may provide an important link between plasma LDL and the development of atherosclerosis [6]. If oxidized LDL contributes to atherosclerosis, the role of antioxidants in the prevention of the oxidative modification of LDL assumes great importance. We have recently found that a crude ethanolic extract of O. caerulescens possessed antioxidative activities on human low-density lipoprotein. From the ethanolic extract of O. caerulescens, seven phenylpropanoid glycosides have been isolated. This paper deals with the structure elucidation of two new phenylpropanoid glycosides by spectroscopic and chemical means. Furthermore, the results of the antioxidative activities of individual components are also presented.

Materials and Methods

General experimental procedures

M.p.s were determined with a Yanagimoto micro-melting point apparatus and are uncorrected. IR spectra were obtained as KBr pellets on a Nicolet Avatar 320 IR spectrometer. UV spectra were obtained on a Hitachi U-3200 spectrophotometer in MeOH. ¹H-, ¹³C- and 2D-NMR spectra were measured with a Varian Inova-500 spectrometer with deuterated solvent as internal standard. ESI-MS, FAB-MS and HR-FAB-MS were recorded on Finnigan LCQ and Finnigan/Thermo Quest MAT spectrometers, respectively.

Plant material

Orobanche caerulescens Stephan (Orobanchacea) was collected at Pali, Taipei, Taiwan, in September 2001, and identified by comparison with the voucher specimens (No. 175 047) deposited at the Herbarium of the Department of Botany of the National Taiwan University.

Extraction and isolation

The whole herbs of O. caerulescens (1.9 kg) were extracted with 95 % EtOH (20 L × 3). The combined ethanolic extracts were evaporated to dryness under vacuum to yield 470 g. The partial ethanolic extract (450 g) was subjected to Diaion HP-20 (2.5 kg) column chromatography using H₂O, 20 % MeOH/H₂O, 50 % MeOH/H₂O and MeOH as eluents (each 5 L) to yield I - IV fractions. Fr. II (36 g) was separated repeatedly by Sephadex LH-20 (10 - 50 % MeOH in H₂O) and CHP-20 column (10 - 50 % MeOH in H₂O) to give 1 (302 mg), 3 (3.92 g) and 5 (306 mg). Fr. IV (85 g) was rechromatographed repeatedly on a Sephadex LH-20 column eluting with 50 - 90 % MeOH in H₂O to yield compounds 2 (14 mg), 4 (1.20 g), 6 (5.71 g), and 7 (110 mg).

LDL oxidation and screening for antioxidants

Blood samples were collected from healthy male adults after 12-hour overnight fasting. Sera were fractionated by ultracentrifugation (Beckman L8 - 80M; R50 rotor) with the density adjusted by NaBr. LDL was obtained from the fractions corresponding to ρ = 1.019 - 1.063 [7]. To remove water-soluble antioxidants and NaBr, LDL containing fractions (3 - 5 mL) was dialyzed extensively at 4 °C/N₂ against phosphate buffer saline (PBS, 50 mM; pH 7.4) in the dark. Dialyzed LDL was used for assay as soon as possible. After dialysis, LDL was diluted with PBS to 100 μg/mL. Ninety μL aliquots of LDL in each well of a 96-well micro-titer plate were incubated with CuSO₄ (final concentration 10 μM) at 37 °C to induce lipid peroxidation [8]. In a routine assay, incubation was carried out in the atmosphere at 37 °C for 6 hours. For screening, LDL was pre-incubated with the test compounds at 37 °C for 30 minutes, and oxidation was then started by adding CuSO₄. Resveratrol was used as a positive control. Routinely, the time course of conjugated diene formation was also determined by following the increase in UV absorption at 234 nm [9]. The prolonged lag phase (min) was used as an index of antioxidant activity when an antioxidant was present in LDL oxidation with Cu²⁺.

Characterization of compounds 1, 1a, 2 and 2a

Caerulescenoside (1): Amorphous powder; [α]_D ²⁶: -98° (MeOH, c 0.49); UV (MeOH): λ _max (log ε ) = 333 (4.30), 292 (4.12), 246 (4.00), 232 (4.06), 219 (4.26) nm; IR (KBr): ν_max = 3400 (OH), 1703 (C = O), 1635, 1598, 1525 (C = C), 1451, 1283, 1062, 1031 cm^-1; ¹H-, ¹³C-NMR (Table [1]); FAB-MS: m/z = 785 [M - H]^-; HR-FAB-MS: m/z = 787.2657 [M + H]⁺ (calcd. for C₃₅H₄₇O₂₀ : 787.2661).

Dodecaacetate 1a: Treatment of 1 (20 mg) with Ac₂O (1 mL) and pyridine (1 mL) at room temperature overnight followed by the usual work-up afforded a crude acetate, which was purified by preparative TLC to give dodecaacetate 1a: ¹H-NMR (Table [1]); [α]_D ²⁶: -57° (MeOH, c 0.37); FAB-MS: m/z = 1313 [M + Na]⁺.

3′-Methyl crenatoside (2): Amorphous powder; [α]_D ²⁶: -23° (MeOH, c 0.44); UV (MeOH): λ _max (log ε ) = 329 (4.29), 290 (4.07), 247 (3.97), 233 (4.12), 219 (4.18) nm; IR (KBr): ν_max = 3400 (OH), 1715 (C = O), 1640, 1603, 1520 (C = C), 1445, 1278, 1120 cm^-1; ¹H-, ¹³C-NMR (Table [1]); ESI-MS: m/z = 635 [M - H]^-; HR-FAB-MS: m/z = 637.2134 [M + H]⁺ (calcd. for C₃₀H₃₇O₁₅ : 637.2132).

Heptaacetate 2a: Acetylation of compound 2 (Ac₂O/pyridine) gave 2a: ¹H-NMR (CDCl₃): δ = 1.11 (3H, d, J = 6.0 Hz, CH₃), 1.70, 1.90, 2.01, 2.12 (each 3H, OAc × 4), 2.29 - 2.33 (9H, aromatic OAc × 3), 3.87 (3H, s, OCH₃), 4.48 (1H, d, J = 8.5 Hz, H-1′′), 5.16 (1H, br s, H-1′′′), 6.39 (1H, d, J = 16.0 Hz, H-8′), 7.06 - 7.19 (6H, aromatic H), 7.74 (1H, d, J = 16.0 Hz, H-7′); FABMS: m/z = 953 [M + Na]⁺.

Results and Discussion

The ethanolic extract of the herbs of O. caerulescens was subjected to sequential column chromatography on Diaion HP-20 and Sephadex LH-20 to yield seven phenylpropanoid glycosides. The structures of 3 - 7 have been established as acteoside (3, verbascoside) [10], isoacteoside (4) [10], campneoside II (5) [11], crenatoside (6) [4], and desrhamnosyl acteoside (7) [12], on the basis of spectral analyses and by comparison with reported data.

Compound 1, for which we propose the name caerulescenoside, is a phenylpropanoid glycoside. It was obtained as a white amorphous powder, with a molecular weight of 786, consistent with the molecular formula C₃₅H₄₆O₂₀, which was confirmed by HR-FAB-MS. The ¹H-NMR spectrum of 1 revealed three anomeric protons at δ = 4.39 (1H, d, J = 8.0 Hz, H-1′′, 4.51 (1H, d, J = 7.5 Hz, H-1′′′′), and 5.23 (1H, br s, H-1′′′), consistent with the presence of two β-glucose and an α-rhamnose units. A set of signals of an aromatic ABX system and two trans olefinic protons in the spectrum suggested that caffeic acid was present. Other ABX aromatic protons, methylene protons, and oxygenated methylene protons were assigned to the 3,4-dihydroxyphenylethanol moieties. These observations indicated that the structure of 1 is closely related to that of rossicaside A [13], which contains the same molecular subunits. All proton and carbon resonances were assigned according to the results of ¹H-¹H COSY, TOCSY, HMQC and HMBC experiments. The caffeoyl group was positioned at C-4′′ of the glucose on the basis of the strong deshielding of the H-4′′ signal of the glucose (glucose-I) unit (δ = 4.95, t, J = 10.0 Hz). The carbon resonances assigned to the second glucose (glucose-II) unit showed no unusual chemical shifts, suggesting its terminal position. Acetylation of 1 with acetic anhydride/pyridine afforded a dodecaacetate (1a). A comparison of the ¹H-HMR spectra of 1 and 1a indicated that all the hydroxy groups of glucose-I, with the exception of that at C-2′′ and C-6′′, are not free because no significant shift had occurred upon acetylation. On the other hand, all the hydroxy groups of rhamnose are free, except C-1′′′ and C-3′′′, as deduced from the downfield shift of their protons upon acetylation. Finally, all connectivities within 1 were proven by an HMBC experiment, where correlations between C-9′ (δ = 168.3) and H-4′′ (δ = 4.95), C-1′′ (δ = 104.1) and H_{2 -} 8 signal (δ = 4.05, 3.73), C-1′′′ (δ = 102.6) and H-3′′ (δ = 3.82), and C-1′′′′ (δ = 105.5) and H-3′′′ (δ = 3.69) were observed. Therefore, the structure of 1 was established as 3,4-dihydroxy-β-phenylethoxy-O-β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→3)-4-O-caffeoyl-β-glucopyranoside, for which the name caerulescenoside is proposed.

Compound 2 has the molecular formula C₃₀H₃₆O₁₅, which was confirmed by HR-FAB-MS. The ¹H- and ¹³C-NMR data of 2 showed similarities to those of crenatoside (6) [4], except for the presence of a methoxy group. On acetylation with acetic anhydride/pyridine, 2 gave a heptaacetate derivative (2a). In the HMBC spectrum of 2, the C-3′ signal at δ = 149.4 was correlated with resonances at δ = 3.90 (OCH₃ - 3′), and 6.83 (H-5′), indicating that C-3′ was methoxylated. This assumption was also supported by the NOESY experiment, revealing a cross peak between signals δ = 3.90 and δ = 7.22 (H-2′). Therefore, compound 2 was characterized as 3′-methyl crenatoside.

The antioxidant effects of compounds 1 - 7 on human low-density lipoprotein were evaluated. Antioxidant activity was evidenced by the inhibition of conjugated diene formation. It has been documented that Cu²⁺-induced oxLDL exhibits biological and immunological properties similar to those in vivo. Cu²⁺-induced oxLDL is recognizable by scavenger receptors and causes cholesterol ester accumulation in macrophages. In screening for antioxidants to inhibit LDL oxidation, this method is commonly used. Resveratrol was used as a positive control in this study, which is a phenolic antioxidant isolated from red wine [14]. All these compounds suppress concentration-dependently conjugated diene formation with IC₅₀ values of 1.25 ± 0.06, 2.97 ± 0.31, 0.31 ± 0.01, 1.01 ± 0.05, 1.15 ± 0.04, 1.69 ± 0.15, and 0.64 ± 0.03 μM, respectively. The time courses of conjugated diene formation in the presence of compounds 1 - 7 (2.5 μM) and resveratrol (2.5 μM) are shown in Fig. [1]. Comparison of their antioxidant activities with that of resveratrol (IC₅₀ : 6.75 ± 1.05 μM) demonstrated that the prolonged effect on lag-time and the damping effect on oxidative rate by compounds 1 - 7 were all more potent. In addition, a prolonged lag phase with compounds 3 and 7 of six hours still maintained an inhibitory activity of 100 %. The relative antioxidative activities of compounds 1 - 7 are summarized in Table [2].

References

• 1 Yang Y P, Lu S Y. In Flora of Taiwan. 2nd Edition, Vol. IV Edited & Published by Editorial Committee of the Flora of Taiwan Taipei; 1998: pp 713-7
  Search in Google Scholar
• 2 Chiu N Y, Change K H. The Illustrated Medicinal Plants of Taiwan. Vol. 1 Southern Materials Center Inc Taipei; 1983: p 198
  Search in Google Scholar
• 3 Dini I, Iodice C, Ramundo E. Phenolic metabolites from Orobanche speciosa .  Planta Med. 1995;  61 389-90
  PubMedSearch in Google Scholar
• 4 Afifi M S, Lahloub M F, El-Khayaat S A, Anklin C G, Rueegger H, Sticher O. Crenatoside: A novel phenylpropanoid glycoside from Orobanche crenata .  Planta Med. 1993;  59 359-62
  PubMedSearch in Google Scholar
• 5 Dzhumyrko S F, Sergeeva N V. Carotenoid pigments from Orobanche owerinii .  Chem Nat Compd. 1985;  21 672-3
  PubMedSearch in Google Scholar
• 6 Steinberg D S. Oxidative modification of LDL and atherogenesis.  Circulation. 1997;  95 1062-71
  PubMedSearch in Google Scholar
• 7 Mackness M I, Durringtom P N. Lipoprotein separation and analysis for clinical studies. In: Converse CA, Skinner ER, eds Lipoprotein Analysis: A Practical Approach. Oxford; IRL Press 1992: pp 1-42
  Search in Google Scholar
• 8 Corongiu F P, Milla A. An improved and simple method for determining diene conjugation in autoxidized polyunsaturated fatty acids.  Chem Biol Interactions. 1983;  44 289-97
  CrossrefPubMedSearch in Google Scholar
• 9 Tsai J Y, Chou C J, Chen C F, Chiou W F. Antioxidant activity of piperlactam S: prevents copper-induced LDL peroxidation and ameliorates free radical-mediated oxidative stress of endothelial cells.  Planta Med. 2003;  69 3-8
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Kobayashi H, Oguchi H, Takizawa N, Miyase T, Ueno A, Usmanghani K, Ahmad M. New phenylethanoid glycosides from Cistanche tubulosa (Schrenk) Hook. f. I.  Chem Pharm Bull. 1987;  35 3309-14
  PubMedSearch in Google Scholar
• 11 Imakura Y, Kobayashi S, Mima A. Bitter phenylpropanoid glycosides from Campsis chinensis .  Phytochemistry. 1985;  24 139-46
  CrossrefPubMedSearch in Google Scholar
• 12 Shoyama Y, Matsumoto M, Nishioka I. Four caffeoyl glycosides from callus tissue of Rehmannia glutinosa .  Phytochemistry. 1986;  25 1633-6
  CrossrefPubMedSearch in Google Scholar
• 13 Konishi T, Narumi Y, Watanabe K, Kiyosawa S, Shoji J. Comparative studies on the constituents of a parasitic plant and its host. III. On the constituents of Boschniakia rossica Fedtsch. et Flerov.  Chem Pharm Bull. 1987;  35 4155-61
  PubMedSearch in Google Scholar
• 14 Frankel E N, Kanner J, German J B, Parks E, Kinsella J E. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine.  Lancet. 1993;  341 454-7
  CrossrefPubMedSearch in Google Scholar

Dr. Lie-Chwen Lin

National Research Institute of Chinese Medicine

155-1 Li-Nong Street, Section 2

Shih-Pai

Taipei 112

Taiwan

Republic of China

Phone: +886-2-28201999 ext. 8341

Fax: +886-2-28264276

Email: lclin@cma23.nricm.edu.tw

References

• 1 Yang Y P, Lu S Y. In Flora of Taiwan. 2nd Edition, Vol. IV Edited & Published by Editorial Committee of the Flora of Taiwan Taipei; 1998: pp 713-7
  Search in Google Scholar
• 2 Chiu N Y, Change K H. The Illustrated Medicinal Plants of Taiwan. Vol. 1 Southern Materials Center Inc Taipei; 1983: p 198
  Search in Google Scholar
• 3 Dini I, Iodice C, Ramundo E. Phenolic metabolites from Orobanche speciosa .  Planta Med. 1995;  61 389-90
  PubMedSearch in Google Scholar
• 4 Afifi M S, Lahloub M F, El-Khayaat S A, Anklin C G, Rueegger H, Sticher O. Crenatoside: A novel phenylpropanoid glycoside from Orobanche crenata .  Planta Med. 1993;  59 359-62
  PubMedSearch in Google Scholar
• 5 Dzhumyrko S F, Sergeeva N V. Carotenoid pigments from Orobanche owerinii .  Chem Nat Compd. 1985;  21 672-3
  PubMedSearch in Google Scholar
• 6 Steinberg D S. Oxidative modification of LDL and atherogenesis.  Circulation. 1997;  95 1062-71
  PubMedSearch in Google Scholar
• 7 Mackness M I, Durringtom P N. Lipoprotein separation and analysis for clinical studies. In: Converse CA, Skinner ER, eds Lipoprotein Analysis: A Practical Approach. Oxford; IRL Press 1992: pp 1-42
  Search in Google Scholar
• 8 Corongiu F P, Milla A. An improved and simple method for determining diene conjugation in autoxidized polyunsaturated fatty acids.  Chem Biol Interactions. 1983;  44 289-97
  CrossrefPubMedSearch in Google Scholar
• 9 Tsai J Y, Chou C J, Chen C F, Chiou W F. Antioxidant activity of piperlactam S: prevents copper-induced LDL peroxidation and ameliorates free radical-mediated oxidative stress of endothelial cells.  Planta Med. 2003;  69 3-8
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Kobayashi H, Oguchi H, Takizawa N, Miyase T, Ueno A, Usmanghani K, Ahmad M. New phenylethanoid glycosides from Cistanche tubulosa (Schrenk) Hook. f. I.  Chem Pharm Bull. 1987;  35 3309-14
  PubMedSearch in Google Scholar
• 11 Imakura Y, Kobayashi S, Mima A. Bitter phenylpropanoid glycosides from Campsis chinensis .  Phytochemistry. 1985;  24 139-46
  CrossrefPubMedSearch in Google Scholar
• 12 Shoyama Y, Matsumoto M, Nishioka I. Four caffeoyl glycosides from callus tissue of Rehmannia glutinosa .  Phytochemistry. 1986;  25 1633-6
  CrossrefPubMedSearch in Google Scholar
• 13 Konishi T, Narumi Y, Watanabe K, Kiyosawa S, Shoji J. Comparative studies on the constituents of a parasitic plant and its host. III. On the constituents of Boschniakia rossica Fedtsch. et Flerov.  Chem Pharm Bull. 1987;  35 4155-61
  PubMedSearch in Google Scholar
• 14 Frankel E N, Kanner J, German J B, Parks E, Kinsella J E. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine.  Lancet. 1993;  341 454-7
  CrossrefPubMedSearch in Google Scholar

Dr. Lie-Chwen Lin

National Research Institute of Chinese Medicine

155-1 Li-Nong Street, Section 2

Shih-Pai

Taipei 112

Taiwan

Republic of China

Phone: +886-2-28201999 ext. 8341

Fax: +886-2-28264276

Email: lclin@cma23.nricm.edu.tw