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First isolation of a flavonoid from Juniperus procera using ethyl acetate extract
*Corresponding author amujwah@ksu.edu.sa (Adil A. Mujwah)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Available online 10 Februray 2010
Abstract
Phytochemical investigation of the ethyl acetate extract of the leaves of Juniperus procera growing in south Saudi Arabia Enemas region led to the isolation of a new flavonoid using different chromatographic methods (i.e. paper, thin layer and column chromatography). The isolated flavonoid was identified and established by m.p., 1H-NMR, 13C-NMR, UV and MS spectral analysis. The isolated compound was identified as 3′,4′,3,7-tetrahydroxyflavone.
Keywords
Juniperus procera
Isolation
Extraction
Phenolic compound
Flavonoids
1 Introduction
Flavonoids are phenolic compounds widely spread in plants and foods of plant origin (Markham, 1982; Harborne, 1973; Nuutila et al., 2002; Argaez et al., 2007). They contain fifteen carbon atoms in their basic nucleus–flavan, arranged in a C6–C3–C6 configuration consisting of two aromatic rings (A and B) linked by a three carbon unit which may or may not form a third ring (C) Markham, 1982. Rings (A and C) are chromane ring bearing a second aromatic ring B in position 2, 3 or 4. Flavonoids encompass a large group of polyphenolic substances that has antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic and vasodilator effects (Zheng et al., 2007; Mediavilla et al., 2007; Coelho et al., 2006).
Juniperus procera called Arar commonly known in English as African Juniperus is a coniferous tree native to the mountain of eastern Africa from east Sudan to Zimbabwe, and southwest of the Arabian Peninsula (widely distributed throughout the southern part of Saudi Arabia) (Gaber et al., 1992). The Arar tree has two kinds of leaves, spreading needle-like and imprecated scale-like (Migahid et al., 1978). It is medium-sized tree reaching 20–25 m (rarely 40 m) (Migahid et al., 1978). It is used locally for the traditional remedy of tuberculoses and Jaundice (Gaber et al., 1992).
It was found in the literature, that a lot of work dealing with J. procera as general studies. While in this study we are focusing only on the isolation of flavonoids from leaves of J. procera. Only one flavonoid was isolated and could be identified as; 3′,4′,3,7-tetrahydroxyflavone 1. This flavones has been isolated from this plant for the first time.
2 Materials and methods
The leaves part of this plant was collected from Enemas region south of Saudi Arabia during November 2006 and identified by Dr. Jacob Thomas, Department of Botany, King Saudi University, Riyadh. The plant sample was collected, air dried in shade, separated, grinded to fine powder.
3 Phytochemical study
3.1 Extraction
Dry leaves of J. procera (3.75 kg) were percolated with 85% methanol (15 L) at ambient temperature for 5 days. The extract was filtered and the solvent was removed under vacuum. Then, the residue was collected by filtration. The solvent was evaporated in the vacuum to yield 150 g of crude extract (CE) dissolved in 500 ml aqueous mehanol. The aqueous suspension of this extract was partitioned successively with petroleum ether (40–60), ethyl acetate and n-butanol. TLC and PC investigation showed that ethyl acetate fraction contained mainly many flavonoids but some of them were traces and others present in butanol fraction.
3.2 Isolation
A sample of 8 g of ethyl acetate extract was chromatographed on silica gel (400 g, 60 mesh 70–230, Astem) for column (100 × 5 cm) using MeOH/CHCl3 in a ratio of 3:7 as eluent. The column fractions were combined on the bases of their PC and TLC patterns, fractions (F4–F12), 400 ml were collected and evaporated under reduced pressure to give 13 mg of solid compound 1, m.p. 250 °C.
3.3 Experimental
Analytical grade solvents were used. The UV spectra were recorded on a Perkin Elmer – Lambda 2 spectrophotometer and UV lamp used for localization of fluorescent spots on TLC and PC. The IR spectra were measured on a Shimadzu IR-8400 spectrophotometer. Nuclear magnetic resonance spectra were recorded on a JEOL DELTA ESP400 MHz NMR spectrophotometer. Melting points (Mps) were determined on a Kofler hot-stage apparatus and uncorrected Mass spectra were recordeded on a SHIMADZU GC/MS-GP5050 spectrophotometer.
3.3.1 3′,4′,3,7-Tetrahydroxyflavone
This was obtained as yellow crystals, 13 mg, Rf = 0.76, mp. 250 °C. UV–Vis λmax in MeOH: (nm) 248, 281 sh, 307 sh 318 sh 362; (AlCl3) 269 sh, 299, 319 sh, 423; (AlCl3/HCl) 264, 296 sh, 320, 458; (NaOAc) 252 sh, 292, 331, 379; (NaOAc/H3BO3) 263 sh, 315, 381; (NaOMe) 252, 299, 331, 361, 410. IR (KBr), ν = 605, 700 (C–H, Ar), 1097 (C–O, ethe), 1512, 1570 (C⚌C, Ar), 1604 (C⚌O) and 3354 cm−1 (OH). 1H-NMR (DMSO-d6), Fig. 1, δ = 6.89 (d, J = 8.8 Hz, 2H, H-6, H-8), 7.54 (dd, J = 2.2 Hz, J = 8.8 Hz, H5′, H-6′), 7.69 (d, J = 2.20 Hz, H-2′), 7.92 (d, J = 9.5 Hz, 1H, H-5), 10–14 ppm (broad singlet, 4-OH), Table 1.
The 1H-NMR chemical shift of flavonoid compound 1 in DMSO-d6.
Carbon no.
1H-NMR
13C-NMR
2
–
145.56
3
–
116.11
4
–
172.49
5
7.92 d, J = 9.5 Hz
156.82
6
6.87 d, J = 8.8 Hz
114.75
7
–
162.78
8
6.89 d, J = 8.8 Hz
102.36
9
–
147.77
10
–
115.20
1′
–
127.00
2′
7.68 d, J = 2.20 Hz
145.56
3′
–
115.49
4′
–
123.04
5′
7.55 dd, 2.20, 8.8 Hz
137.71
6′
7.96 dd, 2.20, 8.8 Hz
120.18
OH
9.03
–
The 1H–1H Cosy correlation between δH 6.89 and 7.69 and δH 7.19 and 7.52. 13C-NMR (DMSO-d6), Fig. 2: δ 172.49 (C-4); 162.78 (C-7); 156 (C-5); 147.77 (C-9); 145.56 (C-2 and C-2′); 137.71 (C-5′); 127.00 (C-1′); 123.04 (C-4′); 120.00 (C-6′); 116.11 (C-3); 115.49 (C-3′); 115.20 (C-10); 114.75 (C-6); 102.36 (C-8), Table 1.
The 13C-NMR chemical shift of flavonoid compound 1 in DMSO-d6.
4 Results and discussion
4.1 Phytochemical study
The flavonoid compound 1 shown in Fig. 3, which isolated from ethyl acetate extracts of J. procera, was identified by comparing its 1H-NMR, 13C-NMR, 1H–1H Cosy NMR and UV spectrum in methanol with different diagnostic shift reagents (NaOMe, AlCl3, AlCl3/HCl, NaOAc, NaOAc/H3BO3) with the published data in literature (Markham, 1982; Agrwal et al., 1989; Markham et al., 1989).
Isolated flavonoid compound 1.
The isolated compound 1 was identified as a flavonoid this was clear from its UV–Vis absorption spectra in methanol with shift reagents. It was a flavonol possess hydroxyl group at position 3; this was clear from the absorption pattern of bands I and II in the UV–Vis spectra. The absence of free carbon at passion 3 was confirmed by the absence of one singlet at δ 6.79 ppm in 1H-NMR spectrum.
The presence of 3′-OH, 4′-OH was observed from bathochromic shift (+48 nm) as appeared in band I with the decrease of intensity upon addition of sodium methoxide (NaOMe). From the 1H-NMR spectrum the presence of the hydroxyl substitution at 3′,4′ was provided by the presence of one signal doublet–doublet at δ 7.54 ppm J = 2.2 Hz, J = 8.8 Hz representing the proton at carbon 5′,6′, respectively and the presence of the other signal as doublet at δ 7.69 ppm J = 2.20 Hz representing carbon at 2′.
The presence of the hydroxyl substitute at 7-position this was provided by the presence of signal doublet at δ 6.89 ppm J = 8.8 Hz representing the proton at carbon 6 and 8, respectively. The presence of signal doublet at δ 7.92 ppm J = 9.5 Hz representing the proton at C-5, which appear at low field due to the deshielding effect of the 4-keto function.
The bathochrom shift (58+) nm at band I upon addition of aluminium chloride(AlCl3) which was indicating the presence (i) a catechol system, and when we added hydrochloric acid (HCl) we observed that no increasing of intensity of band I and II, this confirming the catechol system in B-ring.
Compound 1 substituted in position −7 as indicated by its UV–Vis spectra upon addition of diagnostic shift reagents (NaOAc). When we added boric acid (H3BO3) to methanolic sodium acetate bands 1 shifted (+19) nm indicating B-ring catechol system. However, flavonoids with 3′,4′ – hydroxylation pattern give two peaks in the UV–Vis spectrum band II has two peaks, the B-ring catechol moiety is probably located at C-3′ and C-4′. Further evidence in favour of the above structure accumulated from the mass spectrum (Fig. 4) where the molecular ion M+ 286.
Mass spectrum of flavonoid compound 1.
Compound 1 was identified as 3′,4′,3,7 tetrahydroxyflavone.
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