The effect of propolis and its components on eicosanoid production during the inflammatory response

Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(6), 441-449 © Pearson Professional Ltd 1996

The effect of propolis and its components on eicosanoid production during the inf lammatory response

O. K. Mirzoeva, 1 P. C. Calder 2

1Department of Biochemistry, University of Oxford, UK 2School of Biological Sciences, University of Southampton, UK

Summary To investigate the possible mechanism of the therapeutic action of propolis, we studied: (a) the effect of propolis, its components, caffeic acid phenethyl ester (CAPE), caffeic acid (CA), quercetin and naringenin, as well as the synthetic compounds indomethacin (IM) and nordihydroguaiaretic acid (NDGA), and a novel lipoxygenase inhibitor N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)isourea (DCHCU) on eicosanoid production by mouse peritoneal macrophages in vitro; (b) the effect of IM, NDGA, CA, CAPE, DCHCU and propolis on eicosanoid production during acute inflammation in vivo; and (c) the ex vivo and in vivo effect of dietary propolis on arachidonic acid metabolism. The ethanol extract of propolis suppressed prostaglandin and leukotriene generation by murine peritoneal macrophages in vitro and during zymosan-induced acute peritoneal inflammation in vivo. Dietary propolis significantly suppressed the lipoxygenase pathway of arachidonic acid metabolism during inflammation in vivo. CAPE was the most potent modulator of the arachidonic acid cascade among the propolis components examined.

INTRODUCTION

The release and oxygenation of arachidonic acid is a critical event in regulating key processes in host defence, inflammation and hemodynamics. 1,2 Oxygenation of arachidonic acid catalysed by either cyclooxygenase (COX) or lipoxygenases (LO) initiates the biosynthesis of potent bioactive mediators: prostaglandins, thrombo- xanes, prostacyclins, mono- and dihydroxy acids, leukotrienes and lipoxins, collectively termed eicosanoids. 3 Eicosanoids can be synthesized by all cells of the immune system, especially by monocytes and macrophages which are the first line of defence against infection. 4 Macrophages contain both COX and LO activities and are capable of generating large amounts of prostanoids, leukotrienes and different hydroxy fatty acids. 4,5 The generation of eicosanoids by macrophages is relevant to their activity in, and regulation of, the immune and

Received 1 May 1996

Accepted 16 May 1996

Correspondence to: Dr P. C. Calder, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, Hants, SO16 7PX, UK; Tel. 01703- 594223; Fax. 01703- 594383.

inflammatory responses. The eicosanoids are important intercellular signalling agents which affect cell behaviour and cell-to-cell interactions and they are also postulated to be key mediators of inflammation. 6,7 Eicosanoids par- ticipate in a variety of normal physiological processes such as maintaining blood pressure and body temperature, and protecting organs from damage caused by disease, traumatic injury and stress, s A deficiency of these compounds results in progressive impairment of functions, while excessive or imbalanced production may result in inflammation, allergic reactions, carcinogenesis, asthma, psoriasis, immunosuppression and other patho- physiological states. 4,9 Since eicosanoids possess important biological activities, the control or inhibition of their biosynthesis has been an area of intense investigation and could open new approaches to the treatment of a variety of human diseases, l°,u

There are many natural and synthetic effective inhibitors of COX and LO which act both in vitro and in vivo. 12-1~ In recent years there has been considerable interest in the medical properties of natural products, in particular plant and bee products, such as honey and propolis. Propolis is well known for its medical effects,

441

442 Mirzoeva and Calder

including anti-inflammatory, antiviral, immunostimulato- ry and carcinostatic activities, lz-2° akhough as for any drug with strong activity, some problems related to local reactions involving individuals with a basic allergy have been reportedY ,22

Crude extracts of propolis have very complicated com- positions, resulting from variation in geographical and botanical origin. 23 For some time the wide spectrum of activity of propolis was attributed mostly to the large number of flavonoid compounds it contains. 2°, 24 These are reported to have antioxidant, anti-inflammatory, anti- allergic and antiviral activities, as well as an anticancer effectY -2z In addition to flavonoids, propolis contains cinnamic derivatives such as caffeic acid (3,4-dihydroxycin- namic acid) and its esters 2z,2s,29 and also sesquiterpene quinones and coumarins, z° In recent years a very active propolis ingredient has become available: caffeic acid phenethyl ester (CAPE). This is a biologically active compound with anti-inflammatory, antimutagenic, antioxidant, cytostatic and anticancer activities. 31-33

The present study was designed to investigate the effect of propolis and its components on the production of lipid- derived mediators (leukotrienes and prostaglandins) by macrophages and during the inflammatory response. To evaluate the action of the most potent propolis ingredi- ents, we used flavonoids (quercetin and naringenin) and cinnamic derivatives (caffeic acid (CA) and CAPE). These natural compounds were studied in comparison with the well-known COX inhibitor indomethacin (IM) and the LO inhibitor nordihydroguaiaretic acid (NDGA). Of particular interest is a new synthetic compound also used in this study: N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl) isourea (DCHCU). This is an intermediate in the chemical synthesis of CAPE and was shown previously to exhibit similar antioxidant properties and inhibitory action on plant lipoxygenases, a4 The structures of CAPE and DCHCU are depicted in Figure 1.

In this study we investigated (a) the effect of propolis and its components CAPE, CA, quercetin and naringenin, the synthetic compounds IM, NDGA and DCHCU on the secretion of LO and COX products by mouse peritoneal macrophages in vitro; (b) the effect of IM, NDGA, CA, CAPE, DCHCU and propolis on eicosanoid production during acute infammation in vivo; and (c) the ex vivo and in vivo effect of dietary propolis on arachidonic acid metabolism.

CAPE

0 NHC6Hll

DCHCU Fig. 1 The chemical structures of caffeic acid phenethyl ester (CAPE) and N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)isourea (DCHCU).

acid, quercetin and naringenin were obtained from Sigma Chemical Co. (Poole, UI 0. CAPE and DCHCU were synthesized in A. N. Belozersky Inst. of Phys.-Chem. Biology (Moscow State University, Moscow, Russia) by Drs G. A. Korshunova and N.V. Sumbatyan by the method described elsewhere) 4 ELISA kits for measuring eicosanoid concentration were purchased from Cayman Chemical (Ann Arbor, MI, USA). The ethanol extract of propolls was obtained from Bee Health Ltd (Scarborough, UK).

Animals and diets

Male C57BL6 mice (weighing between 20 and 25 g) were housed in the Department of Biochemistry. Apart from experiments involving feeding a propolls-enriched diet, mice were allowed access ad libitum to standard labora- tory chow (SDS No. 1; Special Diet Services, Witham, Ut 0. In experiments investigating the effect of dietary propolis, 7-week-old mice were fed for 3 weeks on a diet containing 0.2% by weight of propolis. This diet was prepared by mixing powdered diet base (250/0 high nitrogen casein, 35% sucrose, 25% corn starch, 6% non-nutritive bulk), sunflower oil (to give 50/0 by weight) and propolis ethanol extract (to give 0.2% by weight); control animals were fed on an identical diet with no added propolis. The diets were moistened with water, pelleted and dried at room temperature.

MATERIALS AND METHODS

Chemicals

Zymosan, RPMI, glutamine, fetal calf serum (FCS), antibiotics, bacterial lipopolysaccharide (LPS), calcium ionophore A23187, nordihydroguaiarefic acid, indomethacin, caffeic

In vitro experiments with mouse peritoneal macrophages

Thioglycollate-elicited mouse peritoneal macrophages were isolated as described elsewhere ss and were purified by adhesion to the surface of Petri dishes treated for tissue culture use. Macrophages (1 × 106 cells/ml) were


The effect of propolis and its components on eicosanoid production during the inflammatory response 443

incubated in I ml RPMI supplemented with 10% (v/v) FCS, 2 mlVI glutamine and antibiotics, in the presence of 20 gg/ml Ca 2+ ionophore for 2 h (for leukotriene synthesis) or 100 gg/ml LPS for 24 h (for prostaglandin synthesis) in a 19:1 air: CO2 atmosphere at 37°C. The ionophore and LPS were dissolved in sterile phosphate-buffered saline (PBS) and unstimulated cells were incubated with the equivalent volume of PBS. Inhibitors or propolis, in ethanolic solution, were added to the cells 10 rain before the stimuli and the equivalent volume of ethanol was added to the control cells (both unstimulated and stimulated). The final ethanol concentration was not more than 1%. The ethanol extract of propolis was evaporated, the solid resinous material was weighed and dissolved in ethanol up to 10% concentration. This stock solution was used for all experiments with propolis.

Induction of acute inflammation

The model of acute inflammation used was that described by LetkowithY Mice received an intraperitoneal (i.p.) injection of 1 mg of zymosan in 1 ml of sterile PBS and were sacrificed 30 min later. The peritoneal cavities were washed with exactly 2 ml of PBS. The lavaged fluid was collected and centrifuged at 800 x g at 4°C for 5 min to remove cells and then frozen at -70°C until analysis. To estimate the basal level of eicosanoids secreted in response to injection, a group of animals received i.p. the equivalent volume (1 ml) of PBS. In studies using inhibitors or propolis, mice were injected with 6 mg/kg of inhibitor or 80 mg/kg of propolis added to the zymosan administered intraperitoneally (i.p.). Stock solutions of the inhibitors or propolis were prepared in ethanol, an equivalent amount of ethanol was included in the zymosan and PBS injected into control animals. The final concentration of ethanol used was not more that 1%.

Analysis of eicosanoid production

Medium from cultured macrophages and peritoneal lavage fluids were analysed quantitatively for LTB4, LTC4 and PGE2 content using enzyme-linked immune specific assay (ELISA) with acetylcholinesterase coupled to different eicosanoid antisera as label. 3z The concentrations of eicosanoids were determined according to the instruc- tions given by the manufacturer of the ELISA kits.

Data analysis

Data are represented as the mean + standard error of the mean (SEM). Where multiple comparisons were made, a one-way analysis of variance (ANOVA) followed by an unpaired t-test was performed; P values of 0.05 or less were considered significant.

RESULTS

The in vitro effect of propolis extract and its components on eicosanoid production by murine peritoneal macrophages

When incubated with the calcium ionophore A23187 for 2 h, mouse peritoneal macrophages showed an enhanced release of LTB 4 ( a b o u t 100pg/106 cells) and LTC 4 (about 5 ng/106 cells). LPS stimulation for 24 h greatly enhanced the production of PGE 2 (about 40 ng/106 cells) and only slightly increased leukotriene synthesis (data not shown). This enhanced secretion of LTB4, LTC4 and PGE2 by isolated macrophages was completely prevented by the addition of propolis extract in concentrations as low as 0.002-0.02% (Fig. 2). Taking into consideration that propolis is a very complex and heterogeneous mixture of components which may have antagonistic actions, we carried out further in vitro experiments with separate components of propolis: CAPE, caffeic acid and flavonoids (quercetin and naringenin) and synthetic LO inhibitors (NDGA and DCHCU) and a COX inhibitor (IM). CAPE strongly suppressed LTB 4 and LTC 4 synthesis by A23187- stimulated macrophages in a dose-dependent manner with ICs0 about 0.5 gM (Fig. 3A,B). CAPE had similar inhibitory potency to NDGA and DCHCU; these compounds were more potent inhibitors of leukotriene production than CA. Quercetin was an effective LO inhibitor only at concentration > 2.5 glVl, whereas naringenin displayed its action only o n LTC 4 production and was the weakest 5-LO inhibitor among the examined compounds (Fig. 3A, B). The inhibitory action of the compounds on LTB 4 production in vitro decreased as follows: CAPE _> NDGA = DCHCU > CA > quercetin. A slightly different order of potency upon LTC 4 production in vitro was observed: NDGA = CAPE = DCHCU > CA = quercetin > naringenin (Fig. 3A, B). IM did not affect leukotriene synthesis, but it was the most potent inhibitor of PGE2 production (ICs0 less than 0.25 gM). CAPE also had dose- dependent inhibitory action on prostaglandin synthesis (Fig. 3C). DCHCU, NDGA and quercetin suppressed PGE2 generation by macrophages at concentrations greater than 2.5 gM (Fig. 3C). The action of the examined compounds on PGE 2 synthesis was as follows: IM >> CAPE > DCHCU > quercetin > NDGA. Thus, CAPE, DCHCU, quercetin and NDGA decreased both COX and LO activities, indi- cating their poor specificity. This may be because these compounds are antioxidants and act as free radical scavengers of peroxy systems relevant to arachidonate metabolism.

The effect of propolis and its components on in vivo synthesis of eicosanoids during zymosan-induced acute inflammation

To validate the efficacy of propolis and its components

© Pearson Professional Ltd 1996 Prostaglandins, L eukotrienes and Essential Fatty Acids (1996) 55(6), 441-449


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Fig. 2 The effect of propolis on (A) LTB4, (B) LTC4, and (C) PGE2 production by murine peritoneal macrophages. Leukotriene or PGE2 synthesis was stimulated by 20 ~g/ml of Ca 2÷ ionophore A23187 for 2 h or by 100 I~g/ml of LPS for 24 h, respectively. Ethanol propolis extract was added to the cells 10 rain before the stimuli. Results are expressed as a percentage of the maximal eicosanoid production and are the mean _+ SEM of 3-5 experimental values.

in vivo, the ability of the compounds to inhibit acute peritoneal inflammation in the mouse was examined. Eicosanoid production in the peritoneal cavity following an i.p. injection of an inflammatory challenge (1 mg zymosan) was markedly depressed if propolis at a concentration of 80 mg/kg was included with the zymosan injection (Fig. 4). Propolis administered i.p. decreased LTB4 production up to 950/0, LTC4 production up to 90% and PGEa production by up to 70% (Fig. 4A,B,C). The examined cinnamic components of propolis (CAPE and CA) as well as the synthetic inhibitors (DCHCU, NDGA and IM) were injected at a dose of 6 mg/kg together with zymosan. CAPE and DCHCU decreased LTB4 synthesis by up to 40-50% (Fig. 5A). DCHCU was a more potent inhibitor of LTC4 synthesis in vivo than CAPE and depressed LTC4 generation in the murine peritoneal cavity by up to 90% (Fig. 5B). Caffeic acid affected LTC4 pro-

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duction in vivo (up to 50% inhibition) and did not affect LTB4 generation (Fig. 5A, B). NDGA was the most potent inhibitor of leukotfiene production in vivo and suppressed LTB4 and LTC4 synthesis by up to 900/0. The inhibitory action of the compounds on leukotriene production in vivo was as follows: NDGA > DCHCU > CAPE > CA. The cyclooxygenase inhibitor IM caused decreased PGE2 production by up to 50%, while NDGA, CAPE,



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DCHCU, CA did not affect PGE2 production at the concentrations used (Fig. 5C).

Effect of dietary propolis treatment on in vivo generation of eicosanoids during acute peritoneal inflammation in the mouse

To investigate the effect of dietary propolis on inflammation in vivo we compared the generation of eicosanoids in routine peritoneal fluid in response to zymosan in groups of 10 animals fed for 3 weeks on a control diet or on a diet containing 0.2% propolis. The mice fed on the propolis- containing diet had decreased production of LTB 4 (by about 30%) and LTC4 (by about 50%) in comparison with the control animals (Fig. 6A, B). The amounts of PGE2 generated did not differ between mice fed on propolis or the control diet (Fig. 6C). Dietary propolis administration did not affect the basal level of eicosanoids present in the peritoneal fluid of mice injected with PBS instead of zymosan (Fig. 6A,B,C).

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Effect of dietary propolis treatment on ex vivo production of eicosanoids by isolated murine peritoneal macrophages

Peritoneal macrophages were affected by including propolis in the diet. The ex vivo production of eicosanoids was examined using macrophages obtained from five mice fed for 3 weeks either a control diet or a diet containing 0.2% propolis. In contrast to the in vivo observa- tions (Fig. 6), macrophages isolated from animals fed on the propolis diet had an increased response to the stimuli in comparison with control animals. Dietary propolis significantly increased the production of LTB4 and LTC4 (2- fold), as well as the synthesis of PGE2 (1.6-fold) by isolated

© Pearson Professional Ltd 1996 Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(6), 441-449


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Fig. 6 The effect of dietary propolis on (A) LTB4, (B) LTC4 and (C) PGE2 production during acute inflammation. Mice were fed on control or 0.2% propolis containing diet for 3 weeks and then were injected i.p. with either PBS (I I) or 1 mg/ml zymosan (TYC~) to induce acute inflammation. Results are expressed as the mean + SEM from 5 animals for each treatment (triplicate measurements were carried out). *P < 0.05 compared to zymosan-injected animals fed on control diet (control).

peritoneal macrophages stimulated with A23187 or LPS. The basal level of eicosanoid production was unaffected (Fig. 7A, B,C).

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Macrophages secrete a variety of biochemically different substances, including proteins, lipids, nucleotide metabolites and oxygen metabolites. Macrophage-derived products are important in the physiological and pathological functions of these cells in inflammation, tissue repair, lipoprotein metabolism, acute phase response, as well as in antimicrobial, cytostatic and immunoregulatory activitiesY Acute inflammatory responses are of impor-

tance during early host-microbial interactions, especially in non-immune hosts at a time when specific humoral or cellular immunity is not sufficiently protective. 39 Macrophage-secreted mediators markedly affect functions of other participants in acute inflammation, such as polymorphonuclear leukocytesP 8,4° While macrophages are highly integrated with other immune cells, obser- vations in selected pathological models allow insights into the role of one or more specific cell functions in processes associated with inflammation. We used mouse



peritoneal macrophages as a practical system for studying the effects of propolis on the secretion of lipid derivatives and for assessing the inflammatory response.

The results obtained in this study have shown that both propolis and its active component, CAPE, inhibit eicosanoid production by macrophages. The ethanol extract of propolis markedly suppressed the generation of both lipoxygenase and cyclooxygenase products by murine peritoneal macrophages in vitro and during acute inflammation in vivo. This indicates the presence in propolis of both lipoxygenase and cyclooxygenase inhibitors. The effect on COX by propolis could be due to a large number of flavonoids, which have been reported to inhibit prostaglandin endoperoxide synthase with various potency depending on their structure and hydrophilicity. 12 Quercetin (3,5,7,3',4'-pentahydroxyflavone) inhibits the LO pathway, although at high concentrations it also blocks the COX pathways. 41 Cinnamic components of propolis (caffeic acid and its ester) are predominantly LO inhibitorsY Therefore it is difficult to predict qualitative- ly the mode of propolis action on the arachidonic acid cascade in vitro and in vivo. In this study CAPE was a very potent inhibitor of leukotriene synthesis by peritoneal macrophages in vitro, while other propolis components (caffeic acid, naringenin and quercetin) were weaker inhibitors than CAPE and its synthetic analogue DCHCU. CAPE also had an inhibitory action on PGE2 generation by macrophages in vitro, probably due to its antioxidant properties, although it was much weaker than the known cyclooxygenase inhibitor IM. Among the other propolis components studied, quercetin at the high concentrations (above 2.5 ~tM) also decreased PGE2 production.

CAPE affected only leukotriene synthesis in vivo. CAPE was a less potent inhibitor of LTC4 synthesis in vivo than its synthetic analogue DCHCU, although their activities in vitro were about the same. This apparent difference in the inhibitory action on LTC4 and LTB4 production in vivo between the two analogues is an interesting finding. Since both CAPE and DCHCU are assumed to share a common mode of action (on the stage of arachidonic acid conversion to the LTA4), there is no simple explanation for this difference. Possibly their activity is influenced by their different hydrophobicity and so their ability to penetrate through the cell membrane.

Dietary propolis markedly affected the inflammatory response. Mice fed on a 0.2% propolis-containing diet produced decreased amounts of LTB4 and LTC4 in response to inflammatory challenge in vivo. Propolis sup- plementation did not affect the generation of PGE2 in zymosan-injected mice. However, peritoneal macrophages from propolis-fed mice showed increased production of both leukotrienes and prostaglandins in response to the stimuli used in vitro. To explain this paradoxical result we must assume that the response of macrophages can

adapt to the long-term presence of an inhibitor. Possibly, the inhibitory action of propolis was counterbalanced by an enhancement of some aspect of the functioning of macrophages. It is possible that ceils of the immune system can adapt to the presence of inhibitor in such way that the inhibitory effect can be diminished. We infer from our results that macrophages which developed in the presence of inhibitors acquired an ability to increase eicosanoid production to compensate for the inhibitory effect. This ability was displayed once the cells were cultured ex vivo in the inhibitor-free medium. Such adaptation, if it did occur, was only partial, since dietary propolis resuked in inhibition of leukotriene production in vivo. There was no action on PGE2 production in vivo, possibly because the adaptation at the level of prostaglandin synthesis was more effective. The other possible reason explaining the absence of the effect on prostaglandin production in vivo is that the concentration of the propolis in the diet was enough to cause lipoxygenase inhibition but was insufficient to suppress cyclooxygenase activity. This is in agreement with reports, that nonsteroidal anti-inflammatory agents, even at subtoxic doses, inhibit cyclooxygenase activity in vivo by 40-60% and that even in the face of long-term medication suffi- cient arachidonic acid metabolism remains to fulfil normal immunological roles. 42'43

The study of purified constituents allows more precise interpretation of data, but the additive or antagonistic effect of numerous contaminating compounds may be lost. The various components have different metabolic effects and their action may depend upon the route of applica- tion. An interesting finding was that propolis administrated i.p. suppressed production of all three eicosanoids studied, although dietary propolis decreased only the LO pathway during acute peritoneal inflammation in vivo. Evidently, the route of administration and the duration of treatment were critical for the final effect of propolis, which is in agreement with other work. 44 Most of the individual compounds of propolis are reported to posses various therapeutical properties but the complete insolu- bility of the mixture generally prevents the administration of propolis as a parenteral, and possibly a more effective, remedy. 3°

The commonly used non-steroid anti-inflammatory drugs (e.g. aspirin, indomethacin) selectively inhibit the COX pathway of arachidonic acid metabolism. Propolis causes inhibition of leukotriene production and possibly also of prostaglandin formation and so may have an important anti-inflammatory effect. Propolis or/and its purified components are worthy of further investigations of therapeutic efficacy. In addition, preparations for clini- cal use need to be investigated. It will also be important to study the effectiveness of interactions of propolis with other pro-inflammatory biochemical pathways so

© Pearson Professional Ltd 1996 Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(6), 441-449


t h a t t h e m o d e of ac t ion of this p r o d u c t can be m o r e clearly

clar/fied.

This i n v e s t i g a t i o n c lear ly d e m o n s t r a t e s t h a t p ropol i s

con t a in s v e r y p o t e n t i nh ib i to r s o f e i c o s a n o i d p r o d u c t i o n

w h i c h can s t rong ly affect t h e i m m u n e a n d i n f l a m m a t o r y

response . T h e e x a c t ef fec t o f p ropo l i s d e p e n d s o n t h e

r o u t e o f a d m i n i s t r a t i o n a n d t h e d u r a t i o n of t r e a t m e n t .

P ropol i s p r o v i d e s an ac t ive c o m p o n e n t , CAPE, w h i c h is

p o t e n t m o d u l a t o r o f t h e a r a c h i d o n i c ac id cascade . CAPE

m a y h a v e p o t e n t i a l as a n o v e l t h e r a p e u t i c a g e n t con-

t r i b u t i n g to an a n t i - i n f l a m m a t o r y a c t i o n o f propol is .

ACKNOWLEDGEMENTS

We are grateful to Drs G.A. Korshunova and N.V. Sumbatyan (A. N. Belozersky Inst. of Phys.-Chem. Biology, Moscow State University, Moscow, Russia) for the synthesis of compounds CAPE and DCHCU. We thank Dr G. F. Sud'ina (A. N. Belozersky Inst. of Phys.-Chem. Biology) for the helpful discussion of the manuscript.

REFERENCES

1. Serhan C. N. Lipoxin biosynthesis and its impact in inflammatory and vascular events. Biochim Biophys Acta 1994; 1212: 1-25.

2. Samuelsson B., Dahlen S. E., Lindgren J. A., Rouzer C. A., Serhan C. N. Leukotrienes and lipoxins: structures, biosynthesis and biological effects. Science 1987; 237:1171-1176.

3. Samuelsson B. An elucidation of the arachidonic acid cascade discovery of prostaglandins, thromboxane and leukotrienes. Drugs 1987; 33: 2-9.

4. Kinsella J. E., Lokesh B., Broughton S., Whelan J. Dietary polyunsaturated fatty acids and eicosanoids: potential effects on the modulation of inflammatory and immune cells: an overview. Nutrition 1990; 6: 24-44.

5. Hwang D. Essential fatty acids and immune response. FASEBJ 1989; 3: 2052-2061.

6. Samuelsson B. Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 1983; 220: 568-575.

7. Salmon J. A., Higgs G. A. Prostaglandins and leukotrienes as inflammatory mediators. Br IVied Bull 1987; 43: 285-296.

8. Ninnemann J. L Prostaglandins, leukotrienes and the immune response. Cambridge University Press, 1988: 3.

9. Marx J. L The leukotrienes in allergy and inflammation. Science 1982; 215: 1380-1383.

10. Vane J. The evolution of non-steroidal anti-inflammatory drugs and their mechanism of action. Drugs 1987; 33:18-27.

11. Ford-Hutchinson A. W. Leukotrienes: their formation and role as inflammatory mediators. Fed Proc 1985; 44: 25-29.

12. Kalkbrenner F., Wurm G., von Bruchhausen F. In vitro inhibition and stimulation of purified prostaglandin endoperoxide synthase by flavonoids: structure-activity relationship. Pharmacology 1992; 44: 1-12.

13. Cucurou C., Battioni J. P., Thang D. C., Nam N. H., Mansuy D. Mechanism of inactivation of lipoxygenases by phenidone and BW755C. Biochemistry 1991; 30: 8964-8970.

14. Cryer B., Feldman M. Effects of nonsteroidal anti-inflammatory drugs on endogenous gastrointestinal prostaglandins and therapeutic strategies for prevention and treatment of

nonsteroidal anti-inflammatory drug-induced damage. Arch Intern Med 1992; 152:1145-1155.

15. Higgs G. A., Hower R. J., Vane J. R. A new approach to anti- inflammatory drugs. Biochem Pharmaco11979; 28: 1959-1961.

16. Wheeler E. L., Berry D. L. In vitro inhibition of mouse epidermal cell lipoxygenase by flavonoids: structure-activity relationships. Carcinogenesis 1986; 7: 33-36.

17. Grunberger D., Banerjee R., Eisinger K. et al. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 1988; 44: 230-232.

18. Khayyal M. T., EIGhazaly M. A., E1Khatib A. S. Mechanisms involved in the anti-inflammatory effect of propolis extract. Drug Exp Clin Res 1993; 19: 197-203.

19. Dobrovolski J. W., Vohora S. B., Sharma K., Shah S. A., Nagvi S. A. H., Dandiya P. C. Antibacterial, antifungal, antiamoebic, anti-inflammatory and antipyretic studies on propolis bee products. J Ethnopharmaco11991; 35: 77-82.

20. Grange J. M., Davey R. W. Antibacterial properties of propolis (bee glue). J Roy Soc Med 1990; 83:159-160.

21. Hausen B. M., Wollenweber E., Senff H., Post B. Propolis allergy (I). Origin, properties, usage and literature review. Contact Dermatitis 1987; 17: 163-170.

22. Hausen B. M., Wollenweber E. Propolis allergy. (Ill). Sensitization studies with minor constituents. Contact Dermatitis 1988; 19: 296-303.

23. Ghisalberti E. L. Propolis: a review. Bee World 1979; 60: 59-84. 24. Vennat B., ArvouetGrand A., Gross D., Pourrat A. Qualitative

and quantitative analysis of flavonoids and identification of phenolic acids from a propolis extract. JPharm Belg 1995; 50: 438-444.

25. Middleton E., Kandswami C. Effects of flavonoids on immune and inflammatory cell functions. Biochem Pharmacol 1992; 43: 1167-1179.

26. Rapta P., Misik V., Stasko A., Vrabel I. Redox intermediates of flavonoids and caffeic acid esters from propolis: an EPR spectroscopy and cyclic voltammetry study. Free Rad Biol Med 1995; 18: 901-908.

27. Das A., Wang J. H., Lien E.J. Carcinogenicity, mntagenicity and cancer preventing activities of flavanoids: A structure-system-activity relationship (SSAR) analysis. Prog Drug Res 1994; 42: 133-166.

28. Ghisalberti E. L., Jeffeties P., Lanteri R. Potential drugs from propolis. In: Frigefio A., Ghisalberti E. L., eds Mass Spectrometry in Drug Metabolism. Plenum Press: New York, 1977:111.

29. Greenaway W., Scaysbrook T., Whatley F. R. The analysis of bud exudate of Populus x euramericana, and of propolis, by gas chromatography-mass spectrometry. Proc Roy Soc Lon 1987; B232: 249-272.

30. Ivanovska N. D., Dimov V. B., Pavlova S., Bankova V. S., Popov S. S. Immunomodulatory action of propolis. V. Anticomplementary activity of a water-soluble derivative. J Ethnopharmaco11995; 47: 135-143.

31. Ran C. V., Desai D., Simi B., Kulkarni N., Amin S., Reddy B. S. Inhibitory effect of caffeic acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci formation in rat colon. CancerRes 1993; 53: 4182-4188.

32. Chiao C., Carothers A. M., Grunberger D., Solomon G,, Preston G. A., Barter J. C. Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cancer Res 1995; 55: 3576-3583.

33. Sud'ina G. F., Mirzoeva O. K., Pushkareva M. A., Korshunova G. A., Sumbatyan N. V., Varfolomeev S. D. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lelt 1993; 329:21-24.

34. Mirzoeva O. K., Sud'ina G. F., Pushkareva M. A., Korshunova



G. A., Sumbatyan N. V., Varfolomeev S. D. Lipophilic derivatives of caffeic acid as lipoxygenase inhibitors with antioxidant properties. BiaargKhim 1995; 21: 143-151.

35. Yaqoob P., Calder P. Effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages. Ceil Immuna11995; 163: 120-128.

36. Lefkowith J. B. Essential fatty acid deficiency inhibits the in vivo generation of leukotriene B 4 and suppresses levels of resident and elicited leukocytes in acute inflammation. J Immuna11988; 140: 228-233.

37. Pradelles P., Grassi J., Maclouf J. Enzyme immunoassays of eicosanoids using acetylcholinesterase. Methad Enzyma11990; 187: 82-89.

38. Takemura R., Werb Z. Secretory products of macrophages and their physiological functions. Am J Physia11984; 246: C 1-C9.

39. Anderson D. C. The role of phagocytic cells in host defense and inflammatory disease. Nutrition 1990; 6: 5-15.

40. Martin T. R., Rangi G., Merritt T. L., Henderson W. R. Relative

contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage. J Clin Ivest 1987; 80:1114-1124.

41. Landolfi R., Mower R. L., Steiner M. Modification of platelet function and arachidonic acid metabolism by bioflavonoids. Structure-activity relations. Biochem Pharmacol 1984; 33: 1525-1530.

42. Fitzpatrick F. A., Wynalda M. A. In vivo suppression of prostaglandins biosynthesis by nonsteroidal antiinflammatory agents. Prost~glandins 1976; 12: 1037-1051.

43. Naden R. P., lliya C. A., Arant B. S., Gant N. F., Rosenfeld C. R. Hemodynamic effects of indomethacin in chronically instrumented pregnant sheep. Am J Obstet Gyneco11985; 151: 484-494.

44. Ivanovska N. D., Dimov V. B., Bankova V. S., Popov S. S. Immunomodulatory action of propolis. VI. Infuence of a water soluble derivative on complement activity in vivo. J Ethnopharmaco11995; 47: 145-147.

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The effect of propolis and its components on eicosanoid production during the inflammatory response

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