SciELO - Scientific Electronic Library Online

 
vol.45 issue4CARACTERIZACION Y SEPARACION DE UNA ESTILBITA ORIGINARIA DE LA REGION DE ATACAMA, CHILENEUTRAL COMPLEXES OF SILVER HALIDES AND TETRAMETHYLTHIURAM MONOSULFIDE author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Boletín de la Sociedad Chilena de Química

Print version ISSN 0366-1644

Bol. Soc. Chil. Quím. vol.45 n.4 Concepción Dec. 2000

http://dx.doi.org/10.4067/S0366-16442000000400008 

BIOLOGICALLY ACTIVE COMPOUNDS FROM
CHILEAN PROPOLIS

LUIS ASTUDILLO S1., FERNANDO AVILA R.1, ROBERTO MORRISON A.1, MARGARITA GUTIERREZ C. 1, JAUME BASTIDA2, CARLOS CODINA2
AND GUILLERMO SCHMEDA-HIRSCHMANN1*

1Laboratorio de Química de Productos Naturales, Instituto de Química de Recursos
Naturales, Universidad de Talca, Casilla 747, Talca, Chile.
2Departamento de Productos Naturales, Facultad de Farmacia, Universidad de Barcelona,
Avda. Diagonal 643, 08028 Barcelona, Cataluña, España.
(Received: June 7, 2000 - Accepted: July 31, 2000)

ABSTRACT

A propolis sample from the western Andean slopes of the Maule Region, Chile, showed free radical scavenging activity and inhibited the enzyme xanthine oxidase "in vitro". Six flavonoids were isolated as the main active constituents of the sample. 5,7-dihydroxyflavone 4, 3,5,7-trihydroxyflavone 6 and 5,7-dihydroxyflavanone 1 displayed a strong free radical scavenging effect. At 50 mg/ml, compounds 6 and 1 inhibited the enzyme xanthine oxidase by 95 and 86%, respectively.

KEY WORDS: propolis, flavonoids, free radical scavenging activity, DPPH, xanthine oxidase.

RESUMEN

Una muestra de própolis producida en los Andes orientales de la Región del Maule, Chile, mostró actividad atrapadora de radicales libres e inhibió la enzima oxidasa de la xantina "in vitro". Se aislaron e identificaron seis flavonoides como los componentes activos mayoritarios de la muestra. La 5,7-dihidroxiflavona 4, 3,5,7-trihidroxiflavona 6 y 5,7-dihidroxiflavanona 1 exhibieron un fuerte efecto como atrapadores de radicales libres. A la concentración de 50 mg/ml, los compuestos 6 y 1 inhibieron la enzima oxidasa de la xantine en 95 y 86%, respectivamente.

PALABRAS CLAVES: propolis, flavonoides, actividad atrapadora de radicales libres, DPPH, xantina oxidasa.

INTRODUCTION

Propolis is a product collected by bees and formed by the resinous excretions of buds and bark of trees and shrubs1). Propolis is widely used in the northern hemisphere for its medicinal properties1) .

Several biological activities have been reported for propolis including antiinflammatory2), antibacterial3), antifungal and/or antiviral 4-6), immunomodulatory properties7), suppression of HIV-1 replication and immunoregulatory effect8), cytotoxicity9-10), hepatoprotection11) and free radical scavenging activity12).

In Chile, propolis is considered of limited interest by beekeepers and very little is known on its biological activities and chemical composition. Following our studies on bioactive products from Chilean biodiversity, a study on a propolis sample collected in Central Chile was conducted.

EXPERIMENTAL

The sample under study was collected at San Clemente, Región del Maule, Chile, late June 1997. The crude propolis (926 g) was sonicated four times with MeOH (4 x 1.5 L) at room temperature. The remaining unsolubles, including plant residues, insects and waxes were separated by filtration.

The MeOH-soluble part was concentrated under reduced pressure to a syrup, resuspended in MeOH:H2O 1:1 (1 L) and partitioned against petroleum ether (PE) (2 x 500 mL), dichloromethane (DCM) (4x 500 mL) and EtOAc (4 x 500 mL). The percentual weight/weight yield was 0.20. 32.25 and 0.27 % for the PE (1.86 g), DCM (298 g) and EtOAc-solubles (2.49 g), respectively.

Part of the DCM extract (8 g) was permeated in Sephadex LH-20 with MeOH. 65 fractions of 20 mL each were collected. After TLC comparison, fractions 1-13 contained mainly waxes whereas most of the UV-active compounds eluted in fractions 14-30.

Fractions 14/30 were chromatographed on silica gel with a PE - EtOAc; EtOAc; EtOAc:MeOH gradient. 19 fractions were thus obtained. Fractions with similar TLC patterns were combined. Further purification by medium pressure chromatography yielded: Fr. 2: 18 mg 5-hydroxy-7-methoxyflavanone 2; Fr. 3: 20 mg of a mixture of 2 and 5-hydroxy-7-methoxyflavone 5, Fr. 4: 10 mg 5; Fr. 7: 87 mg 5,7-dihydroxyflavone 4 with ca. 2 mg 5,7-dihydroxyflavanol 3. From Fr. 8, 2.58 g 5,7-dihydroxyflavanone 1 were obtained while Fr. 36/40 from the Sephadex column yielded 6 mg 3,5,7-trihydroxyflavone 6.

Isolated compounds were identified by comparison of their spectroscopic data, mainly 13C NMR with literature values 13-14). The 1H- and 13C-NMR data of the isolated compounds is presented in Tables I and II.



BIOASSAYS

Xanthine oxidase activity. Xanthine oxidase (XO) derived from cow’s milk, xanthine and the standard inhibitor allopurinol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The XO activities with xanthine as substrate were measured spectrophotometrically as previously reported using a Shimadzu UV-160A equipment15). Extracts and products were evaluated at 50 mg/ml. Results are presented in Table III.


The quenching of free radicals by extracts and compounds was evaluated against the diphenyl picryl hydrazyl radical (DPPH, Aldrich). The free radical scavenging effect of both crude extracts and pure compounds was assessed by the decoloration of a methanolic solution of DPPH16). The degree of decoloration indicates the free radical scavenging efficiency of the substances. A methanolic solution of DPPH served as a control.

RESULTS AND DISCUSSION

Six major flavonoids were isolated from the propolis sample under study.

The compounds showed an unfunctionalyzed B ring with hydroxy and/or methoxy functions in the A ring. A good correlation was observed with the position of the functional groups and the bioactivity. Compounds 4-6 which were active in the DPPH assay bears a 5,7-dihydroxy system. The structure-antioxidant activity relationship of flavonoids and phenolic acids has been revised17-18). For flavone and flavonols, OH groups at C-5 and C-7 and the double bond between C-2 and C-3 were essential for a high activity on XO17-18).

The chemical composition of propolis samples is highly variable depending on their origin and plant sources. Prenylated chromane derivatives were reported from the EtOAc-soluble fraction of the MeOH extract of Brazilian propolis10). From Cuban propolis, polyisoprenylated benzophenone derivatives were isolated5) and clerodane diterpenes reported from a Brazilian propolis sample9). Di-O-caffeoyl quinic acid derivatives were isolated from the water-soluble extract of propolis and displayed a strong hepatoprotective activity11). Propolis from the Canary Islands showed a different chemical profile with furofuran lignans as their main aromatic constituents19). The antibacterial activity of the sample and isolated compounds at 400 mg/spot, however, was similar to propolis from other geographic origin.

Samples of propolis from different geographic origins were all active against the bacteria Staphylococcus aureus and Escherichia coli and displayed antifungal effect on Candida albicans. Most of them also showed antiviral effect against Avian influenza virus4).

The antibacterial activity of essencial oils from Brazilian propolis has been reported20). The samples originate from native South American stingless bees contained low amounts of essential oils and showed a weak activity against Staphylococcus aureus 209 and were inactive towards Escherichia coli WF+ at 0.4 mg/spot.

Bioassay-guided isolation using the DPPH and XO assays allowed the isolation of the powerful antioxidant propol (3-[4-hydroxy-3-(3-oxo-but-1-enyl)-phenyl]-acrylic acid) from the water extract of Brazilian propolis. In both bioassays, propol was more active than common antioxidants such as vitamin C and vitamin E12).

A review on the biological properties and toxicity of propolis has been published21). The author reports that propolis is relatively non-toxic, with a no-effect level (NOEL) of 1400 mg/kg body weight/day in mice.

CONCLUSIONS

The sample of propolis under study was produced in the western Andean slopes. It consisted mainly in flavonoids with an underivatized B ring with substitution at C-5 and C-7. The flavonoids identified belongs to the flavanones, flavones and flavonols. The free radical scavenging effect as well as the antioxidant activity of the propolis under study and that of the isolated compounds was assessed using two different models. Both crude propolis and pure compounds showed a strong free radical scavenging effect, and some of the flavonoids were also active as XO inhibitors.

Lignans have been reported as components of Chilean propolis22) , but they have not been detected as main constituents in the present study. The botanical sources of our propolis sample remain to be established.

The biological activity found in the our sample of propolis and the establishment of its chemical composition opens the possibility to implement chemical and biological quality controls for its commercialization and human uses.

ACKNOWLEDGEMENTS

We acknowledge the finantial support from the Dirección de Investigación, Universidad de Talca, Programa "Desarrollo de Productos Bioactivos".

*To whom correspondence should de addressed.

REFERENCES

1. B. König. Bee World 66, 136 (1985).        [ Links ]

2. O.K. Mirzoeva and P.C. Calder. Prostaglandins Leuko. Essent. Fatty Acids 55, 441(1996).        [ Links ]

3. Y.K. Park, M.H. Koo, J.A. Abreu, M. Ikegaki, J.A. Cury and P.L. Rosalen. Curr. Microbiol. 36, 24 (1998).        [ Links ]

4. A. Kujumgiev, I. Tsvetkova, Y. Serkedjieva, V. Bankova, R. Christov and S. Popov J. Ethnopharmacol. 64, 235 (1999).         [ Links ]

5. O.C. Rubio, A.C. Cuellar, N. Rojas, H.V. Castro, L. Rastrelli and R. Aquino. J. Nat.Prod. 62, 1013 1999).        [ Links ]

6. B. König and J. H. Dustmann. Naturwissenschaften 72, 659 (1985).         [ Links ]

7. R. Volpert and E. F. Elstner. Arzneimittelforschung 46, 47 (1996).        [ Links ]

8. Z. Harish, A. Rubinstein, M. Golodner, M. Elmaliah and Y. Mizrachi. Drugs Exp. Clin. Res. 23, 89 (1997).        [ Links ]

9. T. Matsuno, Y. Matsumoto, M. Saito and J. Morikawa. Z Naturforsch [C] 52, 702 (1997).        [ Links ]

10. A.H. Banskota, Y. Tezuka, J.K. Prasain, K. Matsushige, I. Saiki and S. Kadota. J. Nat. Prod. 61, 896 (1998).        [ Links ]

11. P. Basnet, K. Matsushige, K. Hase, S. Kadota and T. Namba. Biol. Pharm. Bull. 19, 1479 (1996).        [ Links ]

12. P. Basnet, T. Matsuno and R. Neidlein. Z. Naturforsch. [C] 52, 828 (1997).        [ Links ]

13. T.J. Mabry, K.R. Markham, M.B. Thomas. The Systematic Identification of Flavonoids. Springer Verlag, New York (1970).        [ Links ]

14. P. K. Agrawal. Carbon -13 NMR of Flavonoids. Studies in Organic Chemistry 39, Elsevier (1989).        [ Links ]

15. G. Schmeda-Hirschmann, J. Zuñiga, M. Dutra-Behrens and G. Habermehl. Phytother. Res. 10, 260 (1996).        [ Links ]

16. C. Viturro, A. Molina and G. Schmeda-Hirschmann. Phytother. Res. 13, 422 (1999).        [ Links ]

17. C.A. Rice-Evans, N.J. Miller and G. Paganga. Free Radical Biology and Medicine 20, 933 (1996).        [ Links ]

18. P. Cos, L. Ying, M. Calomme, J.P. Hu, K. Cimanga, B. Van Poel, L. Pieters, A. J. Vlietinck and D. Vanden Berghe. J. Nat. Prod. 61, 71 (1998).        [ Links ]

19. R. Christov, V. Bankova, I. Tsvetkova, A. Kujumgiev and A. Delgado Tejera.Fitoterapia 70, 89 (1999).        [ Links ]

20. V. Bankova, R. Christov, S. Popov, M.C. Marcucci, I. Tsvetkova and A. Kujumgiev. Fitoterapia 70, 190 (1999).        [ Links ]

21. G.A. Burdock. Food Chem. Toxicol. 36, 347 (1998).        [ Links ]

22. S. Valcic, G. Montenegro and B.N. Timmermann. J. Nat. Prod. 61, 771 (1998).        [ Links ]

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License