Acyclic diterpenoid from the red alga Gracilaria foliifera

 

Walied, M. Alarif ^a*, Seif–Eldin,. N. Ayyad^b, Sultan, S. Al–lihaibi^b

 

^a Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, PO. Box 80207, Jeddah 21589, Saudi Arabia. Tel (work): +966–2–6952383, (mob.): +966–56–0352034, Fax: +966–2–6401747. E–mail address: walied1737@yahoo.com

^b Department of Chemistry, Faculty of Science, King Abdulaziz University, PO. Box 80203, Jeddah 21589, Saudi Arabia.

 

Received January 2010.
Accepted April 2010.

 

ABSTRACT

The dichloromethane/methanol extract from the red alga Gracilaria foliifera was subjected to a column chromatography which afforded a new (naturally) acyclic diterpenoid 3,7,11,15 tetramethyl–3–hexadec–en–1–ol (1) in addition to two C₂₉ ethylidene steroids fucosterol, (24E)–Stigmasta–5,24(28)–diene–3β–ol (2) and isofu–costerol, (24Z)–Stigmasta–5,24(28)–diene–3β–ol (3) which are reported for the first time from the genus Gracilaria. The structures were assigned mainly on the basis of ¹H and ¹³C NMR experiments.

Keywords: Red Sea, Gracilaria foliifera, Acyclic diterpenoid, Fucosterol, Isofucosterol.

 

RESUMEN

Del extracto de diclorometano/methanol de la alga roja Gracilaria foliifera se obtuvo por cromatografía en columna un nuevo diterpenoide aciclico (natural) el 3,7,11,15 tetrametiyl–3–hexadec–en–1–ol (1) y también dos esteroides C₂₉ fucosterol etilideno: (24E)–estigmasta–5,24(28)–diene–3β–ol (2) y el isofucosterol, (24Z)–estigmasta–5,24(28)–diene–3β–ol (3). Es el primer reporte de estos compuestos en el género Gracilaria. Las estructuras de los compuestos fueron asignadas a través de las técnicas ¹H y ¹³C RMN.

Keywords: Gracilaria foliifera, diterpenoide aciclico, Fucosterol, Isofucosterol.

 

INTRODUCTION

Gracilaria foliifera (Gracilariales, Rhodophyta) red alga is probably confined to the Red, Arabian and Indian Seas (Guiry & Freamhainn, 2009). Red algae of the genus Gracilaria are known for the production of agar (Rodriguez et al., 2009), polysaccharides (Chattopadhyay et al., 2008) toxins (Yotsu–Yamashita et al., 2007), Lipids (Hwang et al., 2007) and steroids (Govindan et al., 1993; Das, Srinivas, 1992a; Das, Srinivas, 1992b). Investigation of the dichloromethane/methanol extract of the red alga Gracilaria foliifera has resulted in the isolation of a new acyclic diterpenoid 3,7,11,15 tetramethyl–3–hexadec–en–1–ol (1), (compound 1 was synthesized by Kulkarni et al., 1988) in addition to two C₂₉ ethylidene steroids fucosterol, (24E)–Stigmasta–5,24(28)–diene–3β–ol (2) and isofucosterol, (24Z)–Stigmasta–5,24(28)–diene–3β–ol (3) which are reported for the first time from the genus Gracilaria.

 

EXPERIMENTAL

Apparatus and materials

Column chromatography was carried out using silica gel (60G Merck). Thin layer chromatography was carried out on aluminum– backed silica gel (F254 Fluka). EI/MS analyses were carried out on a Shimadzu–QP 2010, NMR measurements were performed for solutions in CDCl₃, the ¹³C and ¹H NMR data were assigned on the basis of ¹H–¹H COSY (correlated spec–troscopy), ¹³C–¹H HMQC (heteronuclear multiple quantum coherence spectroscopy) and ¹³C–¹H HMBC (heteronuclear multiple bond correlation spectroscopy) measured on BRUKER 600MHz NMR spectrometer.

Chemical shifts were recorded as δ values in part per million (ppm) relative to tetra–methylsilane. All J values are given in Hz.

Extraction and isolation

Gracilaria foliifera was collected in Nov. 2007, at the southern coast of the Red Sea of Saudi Arabia near Yemen. After air–drying, samples (300g) were ground and extracted with Dichlromethane/methanol (v/v). The extract was concentrated under reduced pressure to yield 7. 6 g of crude material, which was then subjected to chro–matography over a silica gel column using a gradient of n–hexane–ethyl acetate as gradient solvent. Purification of compounds was monitored by sulfuric acid/ethanol as a spray reagent.

Compound 1

The fraction eluted with n–hexane/EtOAc ,9:1, was further purified by preparative TLC on silica gel using n–hexane/EtOAc (7:3) to give compound 1 (60mg, 0.02%) as a colorless oil R_f 0.71 (n–hexane/EtOAc ,32:6); [α]_D= +0.18° (c= 1.2, CHCl₃). IR (CHCl₃) cm^–1:3450, 2955, 2920 and 1660. EI–MS^m/z: 296 [m, C₂₀H₄₀O]+, 278 [M– H₂O]⁺,263 [M–CH₃–H₂O]⁺,71 [C₅H₁₁]⁺, 57 [C₄H₉]⁺. ¹H NMR and ¹³CNMR (CDCl₃), δ (ppm): (table 1).

Compounds 2 and 3

The fraction eluted with a mixture of n–hex–ane/EtOAc (8:2) is further purified by repeated column chromatography and PTLC on silica gel using n–hexane/EtOAc (7:1) led to the isolation of compounds 2 and 3 as a mixture (80mg) as a white solid of nearly equal R_f = 0. 45 (n–hexane/EtOAc , 32:6) but with slightly different color response up on spraying with sulfuric acid/ethanol and heating at 110°C for few minutes. Running out 2D TLC several times we could separate them into pure forms.

Compound 2 (major): white solid m.p. 121–123°C.EI–MS^m/z: 412 [M, C₂₉H₄₈O]⁺, 397 [M– CH₃]⁺, 379 [M–CH₃–H₂O]⁺, 314 (100) [M–C₇H₁₄]⁺, 299 [M–C₇H₁₄–CH₃]⁺, 296 [M–C₇H₁₄–H₂O]⁺¹, 281 [M–C₇H₁₄–H₂O–CH₃]⁺¹, 281 [M–side chain + 2H]⁺¹. H NMR (CDCl₃),δ (ppm): 0.86–0.88 (d, J=6.6 Hz, 6H, 26–CH₃ and 27–CH₃ ), 0.91 (d, J=6.6 Hz, 3H,21–CH₃), 0.68 (s, 3H,18–CH₃), 1.00 (s, 3H,19–CH₃), 1.58 (d, J=6.6 Hz 3H,29–CH₃), 3.53 (m,H,H–3), 5.35 (m,H,H–6), 5.18 (q,H,H–28), 2.32 (sep, J= 6.6 Hz, H–25).¹³C NMR (CDCl₃), δ (ppm): (39.75, C–1), (35.20, C–2), (71.80, C–3), (42.33, C–4), (140.74, C–5), (121.70, C–6), (36.14, C–7), (35.78, C–8), (50.10, C–9) , (39.72, C–10), (28.23, C–11), (42.28, C–12), (42.3, C–13), (56.74, C–14), (31.64, C–15), (34.78, C–16), (56.72, C–17), (11.85, C–18), (19.40, c–19), (39.50, c–20), (24.32, c–21), (37.23, C–22), (31.90, C–23), (146.60, C–24), (36.50, C–25), (22.23, C–26), (22.50, C–27), (115.54, C–28), (18.70, C–29).

Compound 3 (minor): white solid m.p. 133–135°C.EI–MS^m/z: 412 [M, C₂₉H₄₈O]⁺, 397 [M– CH₃]⁺, 379 [M–CH₃–H₂O]⁺, 314 (100) [M–C₆H₁₀O]⁺.¹H NMR (CDCl₃),δ (ppm): 0.860.88 (d, J=6.6 Hz, 6H, 26–CH₃ and 27–CH₃ ), 0.91 (d, J=6.6 Hz, 3H,21–CH₃), 0.68 (s, 3H,18–CH₃), 1.00 (s, 3H,19–CH₃), 1.59 (d, J=6.6 Hz 3H,29–CH₃), 3.53 (m,H,H–3), 5.35 (m,H,H–6), 5.11 (q,H,H–28), 2.22 (sep, J= 6.6 Hz, H,H–25).¹³C NMR (CDCl₃), δ (ppm): ( 39.75, C–1), (35.20, C–2), (71.80, C–3), (42.33, C–4), (140.74, C–5), (121.70, C–6), (36.14, C–7), (35.78, C–8), (50.10, C–9), (39.72, C–10), (28.23, C–11), (42.28, C–12), (42.3, C–13), (56.74, C–14), (31.64, C–15), (34.78, C–16), (56.72, C–17), (11.85, C–18), (19.40, c–19), (39.50, c–20), (24.32, c–21), (37.23, C–22), (31.90, C–23), (146.00, C–24), (33.90, C–25), (22.50, C–26), (22.23, C–27), (115.94, C–28), (18.90, C–29).

 

RESULTS AND DISCUSSION

Compound 1

Compound 1 was obtained as colorless oil and showed a molecular ion peak in EI–MS at m/z 296 which together with ¹³C NMR data, suggested a molecular formula of C₂₀H₄₀O which indicates 1 degree of unsaturation that can be deduced to be a double bond by examining IR (v 1660 cm^–1) absorption spectrum, ¹H NMR one olefinic proton at δ 5.41 ppm and ¹³CNMR two signals at δ 123.02 and 140.38 ppm. The DEPT spectra showed that compound 1 contains five methyl, ten methylene, four methine and one quaternary carbons. The nature of oxygen atom was found to be primary alcoholic from both ¹H NMR δ 3.53 (t, J= 6.5) ppm and ¹³CNMR δ 59.44 ppm. So compound 1 should be acyclic diterpene alcohol which can be deduced to be phytol (4) but comparing our present spectral data with that of phytol (Bang et al., 2002) (table 1) we can observe a difference in the position of the double bond. Localization of the hydroxyl group at C–1, methyl groups and the double bond at C₃–C₄ were deduced from HMQC, HMBC correlations and COSY experiment. Positions of the methyl groups also can be explained by the obtained spectral data (table 1) which is in agreement with the biogenetic rule of terpenoids. These data of compound 1 allowed to be assigned the following structure, 3,7,11,15 tetramethyl–3–hexadec–en–1–ol.

Compounds 2 and 3

GC/MS of the purified steroidal fraction (Liebermann–burchard's reaction gave the typical slow reacting green color of a Δ⁵ sterol) (Gibbons et al., 1968) gave two closely peaks of nearly equal retention times but with identical MS fragmentation pattern of a parent ion peak at m/z 412 which together with ¹³C NMR data, suggested a molecular formula of C₂₉H₄₈O. The loss of part of the side chain (C₇H₁₄) is characteristic of sterols with a Δ²⁴⁽²⁸⁾ or Δ^24(24') (Gibbons et al., 1968). ¹H NMR data were typical of sterols, compound 2 gave a doublet at δ 1.59 ppm (J= 6.6 Hz) for the proton at C–29 (δ_C 18.70), a quartet δ 5.18 ppm (J= 6.6 Hz) at C–28 proton (δ_C 115.54), a multiplet at 5.35 for C–6 proton (δ_C 121.70) and a multiplet at 3.53 for C–3 proton (δ_C 71.80), while compound 3 gave a doublet at δ 1.58 ppm (J= 6.6 Hz) for the proton at C–29 (δ_C 18.90), a quartet δ 5.11 ppm (J= 6.6 Hz) at C–28 proton (δ_C 115.94), a multiplet at 5.35 for C–6 proton (δ_C 121.70) and a multiplet at 3.53 for C–3 proton (δ_C 71.80). ¹³C NMR for both compounds indicated 29 carbons, of which two double bonds and a secondary alcohol (cf exp.) and the DEPT spectra showed that compounds 2 and 3 both contain six methyl, ten methylene, nine methine and four quaternary carbons. The stereochemistry at the side chain double bond can be explained by observing the ¹HNMR spectra at the C–25 proton which resonates at δ 2.32 ppm (δ_C 36.50) in case of (E) isomer and at δ 2.22 ppm (δ_C 33.90) in case of (Z) isomer (Frost, ward, 1968). The position of the ethylidene group was proved from HMQC, HMBC correlations and COSY experiment. These data are in agreement with literature (John Goad, Akihisa, 1997) which can allow us to assign compound 2 as fucosterol and compound 3 as isofucosterol.

 

CONCLUSIONS

Phytol (3,7,11,15 tetramethyl–2–hexadec–en–1–ol) is a common diterpenoid in plant kingdom. This report of its isomer 3,7,11,15 tetramethyl–3–hexadec–en–1–ol adds to that on the capability of such red alga of isomerization . In addition, we reported two C₂₉ ethylidene steroids fucosterol and isofucos–terol, which are reported for the first time from the genus Gracilaria.

 

ACKNOWLEDGMENTS

The authors are thankful to the researcher's assistants for their help in the laboratories work. Deanship of Scientific Research (DSR) is also acknowledged for the financial support (Grant No. 8–003/429).

 

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