HIV gp41 Binding Phenolic Components from Fraxinus sieboldiana var. angustata

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

By means of HIV gp41 binding affinity directed chromatographic fractionation, three phenylethanoid glycosides; calceolarioside A (1), calceolarioside B (2) and acteoside (3), along with four hydroxycoumarins; esculin (4), fraxin (5), fraxetin (6) and esculetin (7), and one lignan, (-)-olivil (8) were isolated from the n-butyl alcohol fraction of Fraxinus sieboldiana var. angustata. Among the isolated compounds, calceolarioside B (2) and esculetin (7) showed moderate binding affinity on HIV gp41 with IC₅₀ values of 0.1 mg/ml and 0.5 mg/ml, respectively. Calceolarioside A (1), calceolarioside B (2), acteoside (3), and (-)-olivil (8) were isolated for the first time from this plant.

HIV (human immunodeficiency virus) is a retrovirus that causes acquired immune deficiency syndrome (AIDS). The HIV envelope protein consists of gp120 surface and gp41 transmembrane glycoproteins that play critical roles in virus-cell fusion and virus entry in the host cell. The interaction of gp120 with receptors on the target cell triggers a conformational change in the gp41 and allows a small part of gp41 to penetrate the cell membrane and cause fusion of HIV-cell [1]. During the conformational change the coiled-coil region of gp41 would be exposed, and molecules that bind potently to the coiled-coil region of gp41 can be potential anti-HIV agents [2]. Compounds that inhibit the conformational change of gp41 can be measured using the associating property of the coiled-coil and the membrane proximal helical domains of gp41 [3]. An assay system to detect gp41-binding molecule using the interaction between these two domains has been established [4], and applied to measure the inhibitory activity of peptide based gp41 inhibitors [5].

In our continued search for biologically active compounds from traditional medicine, we wished to identify potent gp41 inhibiting small molecules from natural products. This paper describes the isolation and characterization of phenolic components from Fraxinus sieboldiana var. angustata Nakai (Oleaceae). The bark of this plant has diverse pharmacological activities such as soothing, expectorant and anti-inflammatory activity [6]. The isolations of hydroxycoumarins (e. g., esculin, fraxin, fraxetin, fraxinol, isofraxetin and esculetin) from this plant have been reported from several research groups over the past two decades [7].

By means of HIV gp41 binding affinity directed chromatographic fractionation, three phenylethanoid glycosides; calceolarioside A (1) [8], calceolarioside B (2) [9] and acteoside (3) [10], along with four hydroxycoumarins; esculin (4) [11], fraxin (5) [7], fraxetin (6) and esculetin (7) [12], and one lignan; (-)-olivil (8) [13] were isolated from the n-butyl alcohol fraction of Fraxinus sieboldiana var. angustata. The structures of these compounds were fully elucidated on the basis of spectroscopic methods.

Table [1] summarizes the results of the inhibitory activity of the isolated compounds on the binding of the two helical regions of gp41. Since the non-peptide inhibitor of HIV gp41 as a reference compound is not available, the peptide inhibitor C51, a peptide from the CαH region of gp41, was used as a positive control [4]. Among the test compounds, calceolarioside B (2) and esculetin (7) showed moderate activity with IC₅₀ values of 0.1 mg/ml and 0.5 mg/ml, respectively. Calceolarioside A (1) and fraxetin (6) showed comparable activities to those of compounds 2 and 7, respectively. Although the IC₅₀ values of these compounds are higher than the peptide inhibitor C51 (IC₅₀ = 1.0 μg/ml, data not shown in Table), these small molecules still possess moderate binding affinity on the HIV gp41. On the other hand, two coumarins (4 and 5), acteoside (3) and (-)-olivil (8) exhibited no inhibitory activity when tested up to the concentration of 0.5 mg/ml. The poor binding affinities of these compounds (3-5) on the HIV gp41 are probably due to the presence of a sugar moiety such as a rhamnosyl group in the phenylethanoid (3) and glucosyl group in the coumarins (4 and 5), respectively. Based on the above results, two phenolic components (2 and 7) play the dominant role on the inhibitory activity of n-butyl alcohol fraction of Fraxinus sieboldiana var. angustata on the HIV gp41. Among the isolated compounds, calceolarioside A (1), calceolarioside B (2), acteoside (3), and (-)-olivil (8) have not been reported previously in this plant.

Materials and Methods

The stems of Fraxinus sieboldiana var. angustata were collected from Mt. Chiri, Korea, in August 1996 and the species was identified by Dr. Jong Hwan Kwak at Korea Institute of Science and Technology. Voucher specimens (676 - 25C) have been deposited in our laboratory.

The dried stems of Fraxinus sieboldiana var. angustata (246 g) were cut into small pieces and extracted three times with MeOH at room temperature to afford a dark-brown residue (12.6 g). The methanol extract was suspended in water and then partitioned in turn with CH₂Cl₂, EtOAc, n-BuOH. The active n-BuOH extract (4.00 g) was divided into seven fractions on Sephadex LH-20 (3.5 × 35 cm) eluting with MeOH (850 ml). Fraction 3 (667.5 mg) was recrystallized from MeOH to afford compound 5 (206.3 mg). Fraction 4 (1.38 g) was chromatographed on silica gel (3.5 × 40 cm) eluting with CH₂Cl₂/MeOH/H₂O (5 : 1:0.1 → 3 : 1:0.1) to give compound 3 (107.0 mg) and five fractions (Fr.4a ∼ Fr.4e). Fraction 4d (252.8 mg) was purified on LiChroprep^® RP-18 (2 × 23 cm) eluting with MeOH/H₂O (4 : 6 → 6 : 4) gradient system to give compound 4 (544.6 mg). Fraction 5 (297 mg) was chromatographed over LiChroprep^® RP-18 (2 × 25 cm) eluting with MeOH/H₂O (3 : 7 → 7 : 3) gradient system to give compound 1 (72.2 mg) and ten fractions (Fr. 5a ∼ Fr. 5j). Fraction 5c (32.3 mg) was recrystallized from MeOH to afford compound 7 (14.0 mg). Fraction 5d (24.6 mg) was purified by preparative TLC on RP-18 (0.25 mm, 20 × 20 cm) developed with MeOH/H₂O (45 : 55) to afford compound 6 (2.7 mg). Fraction 6 (17 mg) was purified by preparative TLC on RP-18 (0.25 mm, 5 × 10 cm) developed with MeOH/H₂O (50 : 50) to afford compound 2 (54.3 mg).

Gp41 binding assay: The binding affinity of the tested compound to the coiled-coil region of gp41 was measured using two chimeric proteins, Trx-N and GST-C. Trx-N has the coiled-coil region of gp41 at the C-terminus of thioredocin, and GST-C has the membrane proximal helical region of gp41 at the C-terminus of glutathione S transferase. These two proteins interact with each other, and binding of a compound to the coiled-coil region of gp41 was measured as previously described. Briefly, one hundred μl of Trx-N protein (2 μg/ml) in 10 μM Tris·HCl, pH 8.0 were placed overnight in a 96-well cell culture plate. The surface of the plate coated with Trx-N was treated with 5 % skim milk solution and washed 6 times with PBS solution containing 0.5 % Tween-20 (PBST). Then 100 μl of 1.0 μg/ml GST-C in PBS solution were incubated in the plate for 1 h at room temperature. After washing with PBST solution 6 times, the GST-C bound at the surface of the plate was detected by successive treatment with 2000-fold diluted anti-GST antibody and HRP-labeled anti-goat antibody in PBS solution. The peroxidase reaction was initiated by adding 100 μl of the substrate solution (0.1 % of OPD, 1 × Stable Peroxide Substrate Buffer). After 3 - 5 min, the reaction was terminated by adding 100 μl of 2.5 M sulfuric acid and the absorbance at 490 nm was measured using a Spectra Max 340 spectrophotometer (Molecular Device Corp., USA).

Acknowledgements

This work was supported by the Ministry of Science and Technology, Korea (2N23260). The authors thank Dr. J. H. Kwak for identification of the specimen and Dr. Y. J. Kim for measurements of ES/MS.

References

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  PubMedSearch in Google Scholar

Yong Sup Lee, Ph. D.

Korea Institute of Science and Technology

P.O.Box 131

Cheongryang

Seoul 130-650

Korea

Phone: +82-2-958-5167

Fax: +82-2-958-5189

Email: yslee@kist.re.kr

References

• 1 Chan D C, Kim P S. HIV entry and its inhibition.  Cell. 1998;  93 681-4
  CrossrefPubMedSearch in Google Scholar
• 2 Judice J K, Tom J YK, Huang W, Wrin T, Vennari J, Petropoulos C J, McDowell R S. Inhibition of HIV type 1 infectivity by constrained α-helical peptides: Implication for the viral fusion mechanism.  Proc Natl Acad USA. 1997;  94 13 426-30
  PubMedSearch in Google Scholar
• 3 Chan D C, Fass D, Berger J M, Kim P S. Core structure of gp41 from the HIV envelope glycoprotein.  Cell. 1997;  89 263-73
  CrossrefPubMedSearch in Google Scholar
• 4 Ryu J R, Lee J, Choo S, Yoon S H, Woo E R, Yu Y G. Development of an in vitro assay system for screening of gp41 inhibitory compounds.  Mol Cells. 1998;  8 717-23
  PubMedSearch in Google Scholar
• 5 Jin B -S, Ryu J -R, Ahn K, Yu Y G. Design of a peptide inhibitor that blocks the cell fusion mediated by glycoprotein 41 of human immunodeficiency virus type 1.  AIDS Research and Human Retroviruses. 2000;  16 1797-804
  CrossrefPubMedSearch in Google Scholar
• 6 Ahn D K. Illustrated book of Korean medicinal herbs. Kyohak Publishing Co 1998: 309
  Search in Google Scholar
• 7 Han D R, Lee M W. Coumarin glycoside from the stembark of Fraxinus sieboldiana var. angustata .  Kor J Pharmacogn. 1983;  14 1-3
  PubMedSearch in Google Scholar
• 8 Shimomura H, Sashida Y, Ogawa K. Iridoid glucosides and phenylpropanoid glycosides in Ajuga species of Japan.  Phytochemistry. 1987;  26 1981-3
  CrossrefPubMedSearch in Google Scholar
• 9 Shimomura H, Sashida Y, Adachi T. Phenolic glucosides from Prunus grayana .  Phytochemistry. 1987;  26 249-51
  PubMedSearch in Google Scholar
• 10 Calis I, Lahloub M F, Rogenmoser E, Sticher O. Isomartynoside, a phenylpropanoid glycoside from Galeopsis pubescens .  Phytochemistry. 1984;  23 2313-5
  CrossrefPubMedSearch in Google Scholar
• 11 Matsuda N, Kikuchi M. A coumarin glycoside from Lonicera gracilipes var. glandulosa .  Phytochemistry. 1995;  38 803-4
  CrossrefPubMedSearch in Google Scholar
• 12 Razdan T K, Qadri B, Harkar S, Waight E. Chromones and coumarins from Skimmia laureola .  Phytochemistry. 1987;  26 2063-9
  CrossrefPubMedSearch in Google Scholar
• 13 Tsukamoto H, Hisada S, Nishibe S. Lignans from bark Fraxinus mandshurica var. japonica and F. japonica .  Chem Pharm Bull. 1984;  32 4482-9
  PubMedSearch in Google Scholar

Yong Sup Lee, Ph. D.

Korea Institute of Science and Technology

P.O.Box 131

Cheongryang

Seoul 130-650

Korea

Phone: +82-2-958-5167

Fax: +82-2-958-5189

Email: yslee@kist.re.kr