Chemical Analysis and Antimicrobial Activity of Greek Propolis

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Material
• Extraction and Isolation of compounds
• Antimicrobial bioassay
• Results and Discussion
• Acknowledgements
• References

Abstract

One new 2,3-dihydroflavone derivative, 7-O-prenylstrobopinin, and 25 known diterpenes and phenolic compounds were identified from the n-butanol extract of Greek propolis. This is the first time that diterpenes have been isolated from propolis of European origin, while six of the known compounds are reported as propolis constituents for the first time. The structures of the isolated compounds were determined by spectroscopic methods, mainly by the concerted application of 1D, 2D NMR techniques (HMQC, HMBC, NOESY) and mass spectrometry. The studied sample and the isolated compounds were tested for their antimicrobial activity against Gram (±) bacteria and fungi and five of them exhibited strong activities.

Introduction

Propolis is a resinous material produced by bees. It contains sticky compounds coming from several plants mixed with waxes and other bee excretions. The use of propolis in traditional medicine has been known since 3000 BC in Egypt. The word propolis is derived from the Greek ”pro”, for, in front of, at the entrance to, and ”polis” the city or community; a substance that is for or in defence of the city or hive. Propolis is widely used in folk medicine, in cosmetology and in the food industry for health foods, beverages and nutrition supplements. It is claimed to improve human health and prevent diseases such as inflammation, heart disease, diabetes and cancer. Among the several biological activities that have been reported for propolis and its constituents, the most important are antimicrobial, anti-inflammatory, antioxidant and antiproliferative [1], [2]. In contrast to the beneficial uses of propolis, it also possesses some disadvantages, exerting allergenic and, in some cases, toxic effects [2].

The chemical consistency of propolis is highly dependent on the flora of the region from which it is collected. The Greek flora presents a generally known biodiversity with a high percentage of endemic plants and consequently the chemical investigation of Greek propolis appeared to be very interesting. Although propolis is a natural product that is produced and used in Greece for thousands of years its chemical consistency and its pharmacological activities have never been studied. The aim of this work is the isolation, the structure elucidation and the biological testing of the constituents of Greek propolis.

Materials and Methods

General experimental procedures

Optical rotations were measured with a Perkin-Elmer 341 polarimeter. NMR spectra were recorded on Bruker DRX 400 and Bruker AC 200 spectrometers [¹H (400 and 200 MHz) and ¹³C (50 MHz)]; chemical shifts are expressed in ppm downfield from TMS. The ¹H-¹H- and the ¹H-¹³C-NMR experiments were performed using standard Bruker microprograms. GC-MS analysis was carried out on a Hewlett-Packard 6890 - 5973 system operating in the EI mode, equipped with a 30 m × 0.25 mm i. d.; 0.25 μm HP-5 MS capillary column; temperature program: 60 °C (5 min) to 280 °C at a rate of 3 °C/min; injection temperature 200 °C. CI-mass spectra were determined on a Finnigan GCQ Plus ion-trap mass spectrometer using CH₄ as the CI ionization reagent and HR-MS on an AEI MS-90 spectrometer. Medium pressure liquid chromatography (MPLC) was performed with a Büchi model 688 apparatus on columns containing silica gel 60 Merck (20 - 40 μm). Thin layer chromatography (TLC) was performed on plates coated with silica gel 60 F₂₅₄ Merck, 0.25 mm.

Material

The sample of propolis was collected in North-West Greece (Preveza region) in May 2001. The sample has been subjected to pollen analysis, resulting in a 90 % concentration of pollen from Pinaceae, 2.5 % from Asteraceae, and 2 % each from Ericaceae, Cistaceae and Oleaceae, respectively. A voucher sample (MEL03) has been deposited in the Herbarium of the Laboratory of Pharmacognosy-Chemistry of Natural Products, School of Pharmacy, University of Athens, Greece.

Extraction and Isolation of compounds

Propolis (400 g) was cut in small pieces, and extracted for 48 h with CH₂Cl₂ (3 × 2 L) and then with n-butanol (3 × 2 L). After evaporation of the solvent from the n-butanol, the residue (4.2 g) was submitted to column chromatography (4.0 cm) on silica gel 60 Merck (130 g, 40 - 63 μm) with CH₂Cl₂:MeOH (from 100 : 0 to 95 : 5 gradient) as eluent to afford 35 fractions of 100 mL each: frs. A1 - A20, CH₂Cl₂ eluate; frs A21 - A26, CH₂Cl₂:MeOH (99 : 1) eluate; frs A27 - A30, CH₂Cl₂:MeOH (97.5 : 2.5) eluate; and frs A31 - A35 CH₂Cl₂:MeOH (95 : 5) eluate.

Fractions A1 - A4 (246 mg) were re-chromatographed on MPLC (1.8 × 23 cm) with cyclohexane:EtOAc (99 : 1 to 93 : 7) to afford 100 fractions of 10 mL each: frs. B1 - B15, cyclohexane:EtOAc (99 : 1), frs. B16 - B35, cyclohexane:EtOAc (97 : 3), frs. B36 - B55, cyclohexane:EtOAc (95 : 5), frs. B56 - B100, cyclohexane:EtOAc (93 : 7). Frs. B8 - B14 afforded totarol (1) (25 mg, [α]_D ²⁵: + 52°) [3],[4], frs B17 - B29 afforded 7-O-prenylstrobopinin (2) (20 mg) [5], frs B42 - B54 afforded 7-O-prenylpinocembrin (3) (21 mg, [α]_D ²⁵: -18.7°) [6], frs B60 - B81 afforded pinostrobin (4) (44 mg, [α]_D ²⁵: -48.0°) [6].

GC-MS analysis of the frs A1 - A4 also revealed the presence of ferruginol, cinamyl cinnamate, benzyl cinnamate, butyl cinnamate, benzyl benzoate and butyl vanillate. Fractions A5 - A15 (144 mg) were rechromatographed on MPLC (1.8 × 23 cm) with cyclohexane: EtOAc (99 : 1 to 93 : 7) to afford 100 fractions of 10 mL each: frs. C1 - C35, cyclohexane:EtOAc (99 : 1), frs. C36 - C60, cyclohexane:EtOAc (95 : 5), frs. C61 - C100, cyclohexane:EtOAc (93 : 7). Frs. C80 - C94 afforded copalol (5) (10 mg, [α]_D ²⁵: + 15°) [7].

Fractions A16 - A20 (65 mg) were rechromatographed on MPLC (1.8 × 23 cm) with cyclohexane: EtOAc (97 : 3 to 85 : 15) to afford 100 fractions of 10 mL each: frs. D1 - D10, cyclohexane:EtOAc (97 : 3), frs. D11 - D40, cyclohexane:EtOAc (90 : 10), frs. D41 - D100, cyclohexane:EtOAc (85 : 15). Frs. D6 - D10 afforded 13-epi-torulosal (6) (7 mg, [α]_D ²⁵: + 29°) [8], frs. D21 - D30 afforded pinocembrin (7) (6 mg, [α]_D ²⁵: -50°) [9], frs D33 - D39 afforded isoagatholal (8) (10 mg, [α]_D ²⁵: + 19°) [10], frs D42 - D55 afforded sakuranetin (9) (3 mg, [α]_D ²⁵: -21°) [11], frs D60 - D74 afforded pinobanksin (10) (7 mg, [α]_D ²⁵: + 10°) [12] and frs D81 - D90 afforded pinobanksin 5-methyl ether (11) (6 mg, [α]_D ²⁵: + 6°) [13].

Fractions A32 - A35 (735 mg) were re-chromatographed on MPLC (3 × 23 cm) with cyclohexane:EtOAc (97 : 3 to 85 : 15) to afford 150 fractions of 10 mL each: frs. E1 - E10, cyclohexane:EtOAc (97 : 3), frs. E11 - E25, cyclohexane:EtOAc (90 : 10), frs. E26 - E150, cyclohexane:EtOAc (85 : 15). Frs. E18 - E25 afforded: benzoic acid (17) (90 mg), frs. E28 - E37 afforded caffeic acid (18) (80 mg), frs E42 - E48 afforded chrysin (12) (10 mg) [14], frs. E55 - E71 afforded 13-epi-cupressic acid (13) (64 mg, [α]_D ²⁵: + 55°) [9], frs. E73 - E85 afforded 13-epi-torulosol (14) (12 mg, [α]_D ²⁵: + 41°) [9], frs. E90 - E120 afforded isocupressic acid (15) (100 mg, [α]_D ²⁵: + 40°) [9], [15], frs. E122 - E134 afforded agathadiol (16) (23 mg, [α]_D ²⁵: + 29°) [16], and frs E135 - E150 afforded p-coumaric acid (19) (215 mg). All the known compounds were identified by comparison of their NMR and MS data with the literature values. All collected fractions, were estimated by TLC using eluent systems similar to those used in the relative column chromatographies.

7-O-Prenyl-strobopinin (2): colourless solid; m. p. 97 - 100 °C; [α]_D ²⁵: -35.4° (c 0.2, CDCl₃); ¹H-NMR (CDCl₃/TMS, 400 MHz): δ = 12.10 (1H, s, OH-5), 7.48 (5H, m, ArH), 6.10 (1H, s, H-8), 5.45 (1H, t, J = 6.5, Hz, H-2′′), 5.41 (1H, dd, J = 12.5, 3 Hz, H-2), 4.55 (2H, d, J = 6.5, H-1′′), 3.11 (1H, dd, J = 17, 12.5 Hz, H-3a), 2.82 (1H, dd, J = 17, 3 Hz, H-3b), 2.03(3H, s, CH₃ - 6), 1.80 (3H, s, CH₃ - 3′′), 1.73 (3H, s, CH₃ - 3′′); ¹³C-NMR (CD₃OD, 50 MHz): δ = 195.73 (C-4), 165.14 (C-7), 160.99 (C-9), 160.44 (C-5), 138.57 (C-3′′, C-1′), 128.86 (C-3′′, C-4′, C-5′), 126.14 (C-2′, C-6′), 118.98 (C-2′′), 106.29 (C-6), 102.69 (C-10), 91.67 (C-8) 79.35 (C-2), 65.46 (C-1′′), 43.51 (C-3), 25.76 (CH₃ - 3′′), 18.26 (CH₃ - 3′′), 6.98 (CH₃ - 6); EI-MS (70 eV): m/z = 338 (M⁺), 193, 270 (100); HR-EI-MS: m/z = 338.1518 [M]⁺(Δ 0.6 mmu).

Although totarol (1) is a well-known compound, its detailed ¹H-NMR in CDCl₃ has been described only once [3]. The obtained ¹H-NMR spectrum was completely different from that described by Yank et al. but the ¹³C-NMR and all the other data were identical. The ¹H-NMR data given below are in good agreement with those reported incompletely by Kuo et al [4].

Totarol (1): ¹H-NMR (CDCl₃/TMS, 400 MHz) δ = 0.89 (3H, s, CH₃ - 19), 0.92 (3H, s, CH₃ - 18), 1.15 (3H, s, CH₃ - 20), 1.16 (1H, overlapped, H-5), 1.19 (1H, overlapped, H-3ax), 1.31 (3H, d, J = 7.0 Hz, CH₃ - 16), 1.32 (1H, overlapped, H-1ax), 1.33 (3H, d, J = 7.0 Hz, CH₃ - 17), 1.44 (1H, br d, J = 13.3 Hz, H-3eq), 1.52 (1H, overlapped, H-2ax), 1.65 (1H, overlapped, H-2eq), 1.73 (1H, overlapped, H-6ax), 1.89 (1H, dd, J = 13.3, 7.9 Hz, H-6eq), 2.20 (1H, br d, J = 12.4 Hz, H-1eq), 2.73 (1H, ddd, J = 17.0, 11.6, 7.9 Hz, H-7ax), 2.92 (1H, dd, J = 17.0, 6.2 Hz, H-7eq), 3.27 (1H, hept, J = 7.0 Hz, H-15), 4.42 (1H, s, -OH), 6.50 (1H, d, J = 8.5 Hz, H-12), 6.98 (1H, d, J = 8.5 Hz, H-11).

Antimicrobial bioassay

In vitro antibacterial studies were first carried out by the disc diffusion method [17] by measuring the zone of inhibitions against the two Gram-positive bacteria: Staphylococcus aureus (ATCC 25 923), Staphylococcus epidermidis (ATCC 12 228), the four Gram-negative bacteria: Escherichia coli (ATCC 25 922), Enterobacter cloacae (ATCC 13 047), Klebsiella pneumoniae (ATCC 13 883) and Pseudomonas aeruginosa (ATCC 227 853) as well as the pathogenic fungi Candida albicans (ATCC 10 231), C. tropicalis (ATCC 13 801) and C. glabrata (ATCC 28 838). In addition, the tests were performed against the oral pathogenic Gram-positive bacteria Streptococcus mutans and S. viridans, obtained from the culture collection of the Laboratory of Microbiology of the Anticancer Hospital of Athens ”St. Savvas”. Standard antibiotics netilmicin and 5-flucytocine (Sanofi, Diagnostics Pasteur) were used in order to control the sensitivity of the tested bacteria and fungi, respectively, while the standard sanguinarine (Sigma Chemical Co.) was used especially for the oral pathogens. The test compounds were dissolved in MeOH. For each experiment a control disc with pure solvent was used as blind control. All the paper discs had a diameter of 6 mm and were deposited on the surface of the seeded trypticase soy agar Petri dishes. The plates were inoculated with the tested organisms to give a final cell concentration of 10⁷ cell/mL and were incubated for 48 h at 37 °C. The fungi were grown on Sabouraud’s agar at 25 °C for 48 h. The experiments were repeated three times and the results (diameters in mm) are expressed as average values.

The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method [17] in 96-well plates. The results of these tests (Table [1]) showed interesting and promising antimicrobial activity.

Results and Discussion

A sample of propolis, collected from the Preveza region, was extracted with dichloromethane and then with n-butanol. The n-butanol extract after several column chromatographic separations afforded diterpenic and phenolic compounds like: totarol (1), 7-O-prenylstrobopinin (2), 7-O-prenylpinocembrin (3), pinostrobin (4), copalol (5), 13-epi-torulosal (6), pinocembrin (7), isoagatholal (8), sakuranetin (9), pinobanksin (10), pinobanksin 5-methyl ether (11), chrysin (12), 13-epi-cupressic acid (13), 13-epi-torulosol (14), isocupressic acid (15), agathodiol (16), benzoic acid (17), caffeic acid (18), p-coumaric acid (19). Additionally ferruginol, cinamyl cinnamate, benzyl cinnamate, butyl cinnamate, benzyl benzoate, and butyl vanillate were identified by GC-MS. From those constituents, 2 is a new natural product, while 3, 5, 6, 8, 14, and 16 have never been reported as constituents of propolis.[]

The molecular formula of 2 was determined by HRMS as C₂₁H₂₂O₄. The EI-MS showed a quasi-molecular ion at m/z = 338. Its ¹H-NMR spectrum showed a multiplet at 7.48 ppm integrating for five protons and corresponding to a monosubstituted benzene ring and a singlet at 6.10 ppm which integrated for one proton (H-8). Based on the HMQC and DEPT spectrum it was found to be an aromatic methine. This singlet in the HMBC spectrum was correlated with two oxygenated carbons (C-7, C-9). Additionally, the ¹H-NMR spectrum of 2 showed a hydrogen-bonded phenolic proton at 12.10 ppm (corresponding to 5-OH) as well as one double doublet at 3.11 ppm (J = 17, 12.5 Hz), one double doublet at 2.82 ppm (J = 17, 3 Hz) and one double doublet at 5.41 ppm (J = 12.5, 3 Hz), each integrating for one proton. The COSY spectrum showed that the last three protons were cross-coupled, confirming the presence of a flavanone structure. The ¹H-NMR spectrum also showed three three-proton singlets at 2.03 ppm, 1.80 ppm and at 1.73 ppm corresponding to one aromatic methyl group and two other methyl groups, placed on a double bond. In the HMBC spectrum the protons of the methyl groups at 1.80 ppm and at 1.73 ppm had the same (strong ² J,³ J) correlation with two olefinic carbons at 118.98 ppm and 138.57 ppm. Furthermore, the ¹H-NMR spectrum showed a two-proton doublet at 4.55 ppm and a one-proton triplet at 5.45 ppm. From the HMQC, DEPT spectra it was observed that these two protons corresponded to an oxygenated methylene (65.46 ppm, C-1′′) and to an olefinic methine (118.98 ppm C-2′′). The COSY spectrum showed that all three protons were cross-coupled, revealing the presence of a prenyl group. From all these data it is suggested that 2 is a flavanone having an unsubstituted ring B and a ring A bearing an aromatic methyl group, a prenyloxy and a hydrogen bonded hydroxy group. The arrangement of these groups on the A ring was established by the HMBC spectrum. In the HMBC spectrum, the protons of the aromatic methyl group had a strong ³ J correlation with the aromatic oxygenated carbon bearing the prenyl group and a strong ³ J correlation with the aromatic oxygenated carbon bearing the hydroxy group. Consequently, the methyl group was placed in an ortho position between the prenyl and hydroxy groups as depicted in Fig. [1]. Interestingly, a compound with identical spectroscopic data had been isolated from Platanus acerifiolia buds and its structure had been reported as 8-C-methyl-7-O-prenylpinocembrin [4]. This structure had been assigned without 2D NMR and the placement of the aromatic methyl group was uncertain. Besides, it is noteworthy that 7-prenylpinocembrin (3) a rare natural product [4], [18], has also been isolated from P. acerifolia and is now reported for the first time as a propolis constituent.

With regard to the isolation of diterpenes, it should be noted that it is generally considered as a characteristic of tropical propolis. Although there is an indication for diterpenic acids in a sample of propolis from Sicily (Italy) [19], this is the first time that diterpenes are isolated from propolis of European origin. Diterpenes have also been occasionally identified (only through GC, GC/MS) from propolis of Mediterranean origin (Turkey, Algeria). In all these cases, the plant origins of the studied propolis samples, have not been determined and the presence of diterpenes has just been considered as unexpected [19]. In our case, pollen analysis of propolis showed that 90 % was coming from Coniferae trees and especially from Pinus sp. Populus sp. which has been considered as the major source of European propolis was not identified, not even in traces, revealing the special character of the Greek propolis sample.

The isolated compounds were studied for their antimicrobial activity against six Gram-negative and Gram-positive bacterial strains, two oral pathogens (S. mutans, S. viridans) and three human-pathogen fungi (Candida albicans, C. tropicalis, C. glabrata). The results of these tests (Table [1]) showed interesting and promising antimicrobial activity. Some of the isolated compounds such as 1, 2, 3, 7 and 12 exhibited very strong antimicrobial activity. Especially totarol (1), previously known as an antimicrobial agent, showed a specific activity against S. aureus and S. epidermidis comparable to standard antibiotics. Chrysin (12) previously known mostly for its anti-inflammatory activity [20], showed a very strong activity against the tested pathogenic fungi, while pinocembrin (7), appeared as the most active compound against the oral pathogens S mutans and S. viridans. These results could explain the activity, which has been published from the ethanolic extracts of Brazilian propolis [21], rich in galangin and pinocembrin, which have displayed strong activities against both glucosyltransferase activity and growth of tested S. mutans.

In conclusion, our studied sample of propolis as well as most of the isolated chemical compounds, have shown antimicrobial activity, confirming its traditional reputation as an antimicrobial agent.

Acknowledgements

The authors wish to thank Mrs S. Karabournioti (Director of the Chemical and Analytical Laboratory of ”Attiki” Bee-Culturing Company) for the pollen analysis of our propolis samples, and Dr. E. Chinou from the Laboratory of Microbiology of the Anticancer Hospital of Athens ”St. Savvas”, for her help concerning the oral pathogenic bacteria. This study was supported, by the General Secretariat of Research and Technology of Greece (PENED project), as well as by ”Korres” Natural Products, s. a.

References

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Prof. Asssoc. Dr. Ioanna B. Chinou

Department of Pharmacognosy

Chemistry of Natural Products

University Campus of Zografou

School of Pharmacy

University of Athens

157 71 Athens

Greece

Phone: +30-210-7274-595

Fax: +30-210-7274-115

Email: chinou@pharm.uoa.gr

References

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  PubMedSearch in Google Scholar
• 2 Castaldo S, Capasso F. Propolis, an old remedy used in modern medicine.  Fitoterapia. 2002;  73 (Suppl. 1) 1-6
  CrossrefPubMedSearch in Google Scholar
• 3 Yang O H, Suh D Y, Han B H. Isolation and characterization of platelet-activating factor receptor binding antagonists from Biota orientalis .  Planta Med. 1995;  61 37-40
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Kuo Y -H, Chen W-C. Three new diterpenes, 1,3-dioxototarol, isototarolenone, and 1-oxo-3β-hydroxytotarol, from the roots of Juniperus chinensis Linn.  Chem Pharm Bull. 1994;  42 1774-6
  PubMedSearch in Google Scholar
• 5 Kaouadji M, Ravanel P, Mariote A -M. New prenylated flavanones from Platanus acerifolia buds .  J Nat Prod. 1986;  49 153-5
  CrossrefPubMedSearch in Google Scholar
• 6 Haberlein H, Tschiersch K P. Triterpenoids and flavonoids from Leptospermum scoparium .  Phytochemistry. 1994;  35 765-8
  CrossrefPubMedSearch in Google Scholar
• 7 Toshima H, Oikawa H, Toyomasu T, Sassa T. Total synthesis of both enantiomers of copalol via optical resolution of a general synthetic intermediate for drimane sesquiterpenes and labdane diterpenes.  Tetrahedron. 2000;  56 8443-50
  CrossrefPubMedSearch in Google Scholar
• 8 Su W, Fang J, Cheng Y. Labdanes from Cryptomeria japonica .  Phytochemistry. 1994;  37 1109-14
  CrossrefPubMedSearch in Google Scholar
• 9 Sirat H M, Rahman A A, Itokawa H, Morita H. Constituents of the rhizomes of two Alpinia species of Malaysia.  Planta Med. 1996;  62 188-9
  PubMedSearch in Google Scholar
• 10 Hasegawa S, Hirose Y. A Diterpene glycoside and lignans from seed of Thujopsis dolabrata .  Phytochemistry. 1980;  19 2479-81
  CrossrefPubMedSearch in Google Scholar
• 11 Vasconcelos J M, Silva A MS, Cavaleiro J AS. Chromones and flavanones from Artemisia campestris subsp. maritima .  Phytochemistry. 1998;  49 1421-4
  CrossrefPubMedSearch in Google Scholar
• 12 Kuroyanagi M, Yamamoto Y, Fukushima S, Ueno A, Noro T, Miyase T. Chemical studies on the constituents of Polygonum nodosum .  Chem Pharm Bull. 1982;  30 1602-8
  CrossrefPubMedSearch in Google Scholar
• 13 Bankova V S, Popov S S, Marekov N L. A study on flavonoids of propolis.  J Nat Prod. 1983;  46 471-4
  CrossrefPubMedSearch in Google Scholar
• 14 Usia T, Banskota A, Tezuka Y, Midorikawa K, Matsushige K, Kadota S. Constituents of Chinese propolis and their antiproliferative activities.  J Nat Prod. 2002;  65 673-6
  CrossrefPubMedSearch in Google Scholar
• 15 Gardner D, Molyneux R, James L, Stegelmeier B. Ponderosa pine needle-induced abortion in beef cattle: Identification of isocupressic acid as the principal active compound.  J Agric Food Chem. 1994;  42 756-61
  CrossrefPubMedSearch in Google Scholar
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Prof. Asssoc. Dr. Ioanna B. Chinou

Department of Pharmacognosy

Chemistry of Natural Products

University Campus of Zografou

School of Pharmacy

University of Athens

157 71 Athens

Greece

Phone: +30-210-7274-595

Fax: +30-210-7274-115

Email: chinou@pharm.uoa.gr