Translate this page into:
Flavonoids from Algerian propolis
⁎Corresponding author at: Laboratoire de phytopharmacologie, Département de biologie, Université de Jijel, 18000 Jijel, Algeria. Tel.: +213 772465125; fax: +213 213 81 88 62. zellaguia@yahoo.com (Amar Zellagui)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Highlights
• The chemical study of propolis collected in the northern-east part of Algeria has been carried out. • The study afforded five flavones: pectolinarigenin, pilosin, ladanein, Chrysin and Apigenin. • The structures of the flavones were elucidated by spectroscopic analysis, including mass spectrometry, 1D and 2D NMR.
Abstract
The investigation of propolis collected from Jijel, located in the northern-east part from Algeria afforded five flavones: pectolinarigenin (1), pilosin (2), ladanein (3), Chrysin (4) and apigenin (5). The structures were elucidated by spectroscopic analysis, including mass spectrometry, 1D and 2D NMR.
Keywords
Propolis
Algeria
Flavones
1 Introduction
Propolis is a natural substance collected by bees from buds and exudates of plants and trees. Bees use this product to protect their hives from enemies. Propolis is used in folk medicines in many regions of the world (Ghisalberti, 1979). It has been reported to have various biological activities such as antibacterial, antiviral, anti-inflammatory and anticancer (Ammaros et al., 1994; Almeida and Menezes, 2002; Kimoto et al., 1998; Kujumgiev et al., 1999). Recent research has highlighted that propolis prevents such illnesses such as heart disease, diabetes and cancer (Burdock, 2000).
The chemical composition of propolis depends upon the vegetation of the collection site (Bankova et al., 2001; Marcucci, 1995). For example, Propolis from Europe contains many kinds of flavonoids and phenolic esters. In contrast, the major components in Brazilian propolis were terpenoids and prenylated derivatives of p-coumaric acids (Marcucci and Bankova, 1999; Tazawa et al., 1999).
In previous studies we have demonstrated that Algerian propolis prevents hepatic toxicity of some cancer therapy (Lahouel et al., 2004). Hence, it is worthy of consideration to carry out a chemical study dealing with the chemical composition of Algerian propolis collected from Jijel.
2 Experimental
2.1 Material
Propolis was collected from the north-east of Algeria (Jijel) in 2006 by scraping the “bee glue” of walls, frames and entrance of the hive.
3 Extraction and isolation
Propolis (500 g) was extracted with CH2Cl2:MeOH (1:1). The extract was concentrated to dryness, the residue was then extracted with MeOH:H2O (70:30 v/v) and concentrated under reduced pressure. The extract (70:30 v/v) was dissolved in boiling water, stored in the cold and filtered after 24 h. The filtrate was extracted successively with EtOAc to yield (2.7 g) and n-BuOH to yield (8.3 g).
Two-dimensional paper chromatography using 15% AcOH and BAW (n-BuOH:AcOH:H2O 4:1:5 upper phase) as solvents shows that the MeOH–CH2Cl2 and EtOAc extracts contain different compounds representing flavonoids and phenolic acids.
The CH2Cl2:MeOH (1:1) extract (10 g) was fractionated by silica gel CC eluted with n-hexane, followed by a gradient of n-hexane–CH2Cl2 up to 100% CH2Cl2 and CH2Cl2–MeOH up to 15% MeOH, 28 fractions were collected and analyzed by TLC. Fractions 13, 14 and 15 were concentrated and yellow precipitates were obtained. Recrystallisation of fraction 13 in CHCl3 yielded a mixture of compounds 1 and 2 as yellow crystals (Fig. 1). Due to the small quantity of the mixture, we have not attempted to separate both compounds in order to avoid any loss of material. However, NMR data were discernable since they furnished different intensities of the signals for both compounds. Recrystallisation of fractions 14 and 15 in MeOH yielded compounds 3 and 4 (Fig. 1).
C–H long range correlation found in HMBC.
EtOAc extract was subjected to column chromatography on silica gel eluting with a gradient of CH2Cl2–MeOH with increasing polarity; 47 fractions were collected and analyzed by TLC. Fraction 5 was separated on thin layer chromatography using CH2Cl2:EtOAc (9:1) as an eluting system to offer compound 5 (Fig. 1). Purification was carried out using MeOH over Sephadex LH20. Compounds 1 and 2 were identified by spectroscopic techniques (UV–visible, 1H NMR, 13C NMR, Dept, COSY, HMQC and HMBC), while compound 3 and 4 were identified by UV–visible, 1H NMR and 13C NMR. Compound 5 was identified by UV–visible and 1H NMR and compared with the reported data (Livinenko et al., 1969; Maisashvili et al., 2009).
3.1 Compound 1, C17H14O6; mp 210–211 °C
UV (λmax in MeOH): gives bands at 321 and 267 nm for band I and II, addition of NaOH; 385, 329, 267 and AlCl3: 338, 299; and HCl: 340, 299; and NaOAc: 331, 272; while H3BO3: 331, 272. Mass spectrum EI/MS m/z (rel. int): 314 [M]+ (100), 299 [M-Me] (70), 271 [M-Me-CO] (76), 183 [A1+H]+ (10), 167 [A1−Me]+ (90), 133 [B1+H]+ (40).
1H NMR spectrum (300 MHz, DMSO-d6), δ (ppm): δ 13.01 (1H, s, 5-OH), 10.67 (1H, s, 7-OH), 8 (2H, d, J = 8.9 Hz, H-2′ and H-6′), 7.08 (2H, d, J = 8.9 Hz, H-3′ and H-5′), 6.83 (1H, s, H-3), 6.59 (1H, s, H-8), 3.84 (3H, s, OMe), 3.75 (3H, s, OMe).
13C NMR (75 MHz, DMSO-d6), δc (ppm): 182.60 (C-4), 163.30 (C-2), 160.93 (C-4′), 15.23 (C-7), 152.15 (C-5), 151.9 (C-9), 131.29 (C-6), 128.75 (C-2′ and C-6′), 123.70 (C-1′), 11.48 (C-3′ and C-5′), 104.10 (C-10), 103.9 (C-3), 94.25 (C-8), 60.40 (6-OMe), 55.80 (4-OMe).
Compound 1 appeared purple on TLC. Methanol spectra of 1 showed two major absorption peaks at 321 and 267 nm. The peaks are characteristics of flavones skeleton. The UV spectral behavior with diagnostic reagents indicated the presence of free 5 and 7 hydroxyl groups. 13C NMR spectrum showed 17 carbon signals including two methoxy carbons (55.80 and 60.40 ppm), 14 aromatic carbons and a carbonyl carbon (182.60 ppm), indicative of flavones structure of compound 1.
EI–MS spectrum showed m/z 314 as base ion and fragment ions of 167 and 133, which could be obtained by retro Diels–Alder reaction (rDA) Lee et al., 1994 of a flavone with one methoxyl group and two hydroxyl groups on A ring (m/z 167) and one methoxyl group on B ring (m/z 133).
The 1H NMR spectrum of 1 showed an A2X2 system at δ 7.08 and 8 ppm corresponding to 7.08 (2H, d, J = 8.9 Hz, H-3′/H-5′) and H-2′, H-6′ (δ = 8 ppm, J = 8.9 Hz), two singlets of aromatic protons (6.59 and 6.83 ppm) that could be assigned for the two protons of C-8 and C-3 of a flavone compound and two hydroxyl groups protons (10.67 and 13.01 ppm). The latter proton is assignable as hydroxyl group with a hydrogen bond and could be assigned as a hydroxyl group on C-5 position of the flavone compounds. In order to clarify the position of the methoxyl groups and to assign all the chemical shifts of the carbon, we carried out HSQC and HMBC experiments. The chemical shifts of all the protonated carbons were assigned firmly based on the cross peaks found in HSQC. Based on C–H long range correlation found in the HMBC experiment, all the carbons were assigned as shown in Fig. 2.
Chemical structures of compounds 1–5 from Algerian propolis.
The HSQC and HMBC correlation between C10-H8, C6-H8 and C6- the methoxyl protons and between C7-H8 and C9-H8 assigned the substitution on C-6 by the methoxyl group (δH = 3.84 ppm, δC = 60.4 ppm). The substitution on C-4′ by the second methoxy group (δH = 3.75 ppm, δC = 60.8 ppm) was deduced by the HMBC and HSQC spectrums, which showed the correlation between C4′-H3′ and C4′-methoxyl protons.
Compound 1 was identified as 5,7-dihydroxy-6,4′-dimethoxyflavone (pectolinarigenin) (Imre et al., 1977).
3.2 Compound 2
UV (λmax in MeOH): gives bands at 321 and 267 nm for band I and II, addition of NaOH: 385, 329, 267; and AlCl3: 338, 299; and HCl: 340, 299; and NaOAc: 331, 272; while H3BO3: 331, 272. Mass spectrum EI/MS m/z (rel. int): 330 [M]+ (100), 316 [M-Me+H]+ (46), 287 [M-Me−CO] (90), 199 [A1+H]+ (50), 155 [A1-CO-Me] (20), 133 [B1]+ (32).
1H NMR spectrum (300 MHz, DMSO-d6), δ (ppm): δ 12.8 (1H, s, 5-OH), 8.02 (2H, d, J = 8.9 Hz, H-2′ and H-6′), 7.08 (2H, d, J = 8.9 Hz, H-3′ and H-5’), 6.83 (1H, s, H-3), 3.84 (3H, s, OMe), 3.75 (3H, s, OMe).
13C NMR (75 MHz, DMSO-d6), δc (ppm): 182.60 (C-4), 162.74 (C-2), 160.93 (C-4′), 153.18 (C-7), 151.9 (C-9), 146.84 (C-5), 136.7 (C-6), 128.7 (C-2′ and C-6′), 123.70 (C-1′), 115.02 (C-3′ and C-5′), 103.9 (C-10), 102.2 (C-3), 129.7 (C-8), 60.45 (6-OMe), 55.99 (4′-OMe) (Bankova et al., 2001).
EI–MS spectrum of 2 showed m/z 330 as base ion and fragment ions of 199 and 133 which could be obtained by retro Diels–Alder reaction (rDA) (Lee et al., 1994) of a flavone with one methoxyl group and three hydroxyl groups on A ring (m/z 199) and one methoxyl group on B ring (m/z 133).
The 1H NMR and 13C NMR spectra of 2 showed similar signals of compound 1 except for the absence of the H-8 signal and the presence of the signal at δ = 129.7 ppm, that confirmed the substitution of hydroxyl at C-8. Compound 2 was identified as 5, 7, 8-trihydroxy-6,4′-dimethoxyflavone (pilosin) (Christine et al., 2004).
3.3 Compound 3, C17H14O6; mp 215–217 °C
UV (λmax in MeOH): gives bands at 331 and 277 nm for band I and II, addition of NaOH: 363, 275; and AlCl3: 351, 294, 261; and HCl: 352, 301, 260; and NaOAc: 365, 276; while H3BO3: 334, 278. Mass spectrum EI/MS m/z (rel. int): 314 [M]+ (100), 296 [M-H2O] (84), 268 [M-H2O-CO] (24), 182 [A1]+ (15), 152 [A1-OMe+H]+ (23), 139 [153-Me+H] (15), 133 [B1]+ (33).
1H NMR spectrum (250 MHz, CDCl3), δ (ppm): 12.88 (1H, s, 5-OH), 10.94 (1H, s, 6-OH), 8.03 (2H,d, J = 8.9 Hz, H-2′ and H-6′), 7.10 (2H, d, J = 9 Hz, H-3′ and H-5’), 6.91(1H, s, H-3), 6.8 (1H, s, H-8), 3.90 (3H, s, OMe), 3.84 (3H, s, OMe).
13C NMR (62.5 MHz, DMSO-d6), δc (ppm): 182.54 (C-4), 163.83 (C-2), 162.70 (C-4’), 157.73 (C-7), 152.84 (C-9), 151.88 (C-5), 131.78 (C-6), 128.69 (C-2′ and C-6′), 123.19 (C-1′), 114.98 (C-3′ and C-5′), 104.53 (C-10), 103.38 (C-3), 94.72 (C-8), 60.40 (7-OMe), 55.94 (4′-OMe).
The comparison of EI–MS spectrum of 3 with those of 1 showed the same spectrum. The UV absorption maxima of 3 in MeOH at 331 and 277 nm were typical of flavonoid derivatives. Addition of NaOAc caused a shift in the UV absorption maxima of 49 nm on band I with a decrease in intensity of absorption, confirming the substitution of C-4′ by the methoxyl group. The absence of bathochromic shift of band II in the NaOAc spectrum indicates the absence of free 7-hydroxyl group, this suggest the substitution of C-7 by the second methoxyl group. The addition of acid in AlCl3 containing flavonoid solution produced a bathochromic shift of 21 nm in band I indicating the presence of free hydroxyl group on C-5 and a substitution on C-6. The chemical shift of C-6 (δc = 131.78 ppm) confirmed the presence of hydroxyl group on C-6. Compound 3 was identified as 6,7-dihydroxy-7,4′-dimethoxyflavone (ladanein) (Toth et al., 2007).
3.4 Compound 4, C15H10O4; mp 258–268 °C
UV (λmax in MeOH): gives bands at 315 and 269 nm for band I and II; addition of NaOH: 368, 277 and AlCl3: 384, 330, 280, 252; and HCl: 390, 328, 280, 251; and NaOAc: 354, 273; wile H3BO3: 320, 269. Mass spectrum EI/MS m/z (rel. int): 254 [M]+ (100), 226 [M-CO] (21), 152 [A1]+ (15), 124 [A1-CO]+ (10), 102 [B1]+ (17).
1H NMR spectrum (250 MHz, DMSO-d6), δ (ppm): δ 12.81 (1H, s, 5-OH), 10.92 (1H, s, 7-OH), 8.05 (2H, dd, J = 7.6 and 1.6 Hz, H-2′ and H-6’), 7.57 (3H, m, H-3′, H-4′ and H-5′), 6.93 (1H, s, H-3), 6.50 (1H, d, J = 2.09 Hz, H-8), 6.20 (1H, d, J = 2.09 Hz, H-6) (Chen et al., 2003; Toth et al., 2007).
13C NMR (62,5 MHz, DMSO-d6), δ (ppm): 182.31 (C-4), 164.9 (C-7), 163.61 (C-2),161.88 (C-5), 157.9 (C-9), 132.47 (C-4′), 131.13 (C-1′), 129.58 (C-3′ and C-5′), 126.85 (C-2′ and C-6′), 105.10 (C-10), 104.38 (C-3), 99.57 (C-6), 94.57 (C-8).
The UV spectral data with diagnostic reagents indicated the presence of free 5 and 7 hydroxyl groups. The mass spectrum of 4 showed a base ion at m/z 254 suggesting the absence of methoxyl group which is confirmed by the 1H NMR and 13C NMR spectra. Compound 4 was identified as 5,7-dihydroxyflavone (chrysin) (Chen et al., 2003).
3.5 Compound 5, C15H10O5
UV (λmax in MeOH): gives bands at 333 and 268 nm for band I and II; addition of NaOH: 392, 326, 275; and AlCl3: 382, 347, 302, 275; and HCl: 381, 341, 301, 277; and NaOAc: 387, 308, 275; wile H3BO3: 339, 270. Mass spectrum EI/MS m/z (rel. int): 270 [M]+ (100), 242 [M-CO] (40), 152 [A1]+ (75), 124 [A1-CO]+ (50), 118 [B1]+ (50).
1H NMR spectrum (250 MHz, DMSO-d6), δ (ppm): δ 13.01 (1H, s, 5-OH), 7.08 (2H, d, J = 8.4 Hz, H-2′ and H-6’), 6.9 (2H, d, J = 9 Hz, H-3’and H-5′), 6.58 (1H, s, H-3), 6.4 (1H, d, J = 1.26 Hz, H-8), 6.20 (1H, d, J = 1.26HZ, H-6) (Maisashvili et al., 2009; Lee et al., 1994). Compound 5 was identified as 5,7,4′-trihydroxyflavone (apigenin) (Nagao et al., 2002).
All compounds are isolated from Algerian propolis for the first time. Moreover, compounds 1 and 3 were identified from propolis for the first time.
Acknowledgment
Partial financial support by ANDRS (Agence Nationale pour le Développement en Santé) and MESRES (Ministère de l’Enseignement Supérieur et de la Recherche Scientifique) are gratefully acknowledged.
References
- Propolis extract anti-inflammatory activity: review. J. Venem. Anim. Toxins. 2002;8:191-212.
- [Google Scholar]
- Comparison of anti-herpes simplex virus activities of propolis and 3-methylbut-2-eny-caffeate. J. Nat. Prod.. 1994;64:235-240.
- [Google Scholar]
- Review of the biological properties and toxicity of bee propolis (propolis) Food Chem. Toxicol.. 2000;36:347-363.
- [Google Scholar]
- J. Chromatogr.. 2003;988:95-105.
- Nat. Prod. Rep.. 2004;21:539-573.
- Phytochemistry. 1977;16:799.
- Apoptosis and suppression of tumor growth by artepillin C extracted from Brazilian propolis. Cancer Detect. Prev.. 1998;22:506-515.
- [Google Scholar]
- Antibacterial, antifungal and antiviral activities of propolis of different geographic origin. J. Ethnopharmacol.. 1999;64:235-240.
- [Google Scholar]
- The flavonoids effect against vinblastine, cyclophosphamide and paracetamol toxicity by inhibition of lipid-peroxidation and increasing liver glutathione concentration. Pathologie Biologie. 2004;52:314-322.
- [Google Scholar]
- J. Am. Chem. Soc.. 1994;116:2163.
- Apigenin and its glycosides from Gratiola officinalis. Chem. Nat Compd.. 1969;5(4):328-329.
- [Google Scholar]
- Flavonoids and coumarins from Allium rotundum. Chem. Nat. Compd.. 2009;45(1):87-88.
- [Google Scholar]
- Chemical composition, biological properties and therapeutic activity. Apidologie. 1995;26:83-88.
- [Google Scholar]
- Chemical composition, plant origin and biological activity of Brazilian propolis. Curr. Topics Phytochem.. 1999;2:115-123.
- [Google Scholar]
- Biol. Pharm. Bull.. 2002;25(7):875-879.
- Studies on the constituents of Brazilian propolis. Chem. Pharm. Bull.. 1999;47:1388-1392.
- [Google Scholar]
- Biochem. System Ecol.. 2007;35:894-897.
