acid, which begins with its oxidation by the enzyme 5-lipoxygenase

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1 Philippe Racine Fragrance Division Development Laboratory, Robertet SA, 37 Avenue Sidi Brahim, Grasse, France. Tel.: ; Fax: ; Inhibition of 5-lipoxygenase by essential oils and other natural fragrant extracts Sabine Baylac and Philippe Racine Aromatherapy utilises a number of essential oils in the treatment of certain inflammatory disorders. The mechanisms of inflammation involve complex cascades of events in which the metabolism of arachidonic acid, which begins by its oxidation by the enzyme 5-lipoxygenase, plays an important role. The prime objective of this study was to assess in vitro, the potential of essential oils, absolutes and natural or nature-identical chemicals to inhibit this enzyme. Thirty-two essential oils, 10 absolutes and 26 chemicals were screened. The results are reported and discussed. Ó 2003 Elsevier Ltd. All rights reserved. Introduction Inflammation is the normal body response when it is invaded by infectious agents, stimulated by antigens or submitted to physical, chemical or traumatic change. When this response is exaggerated it becomes the source of a variety of ailments which necessitate an appropriate treatment and a number of essential oils known in aromatherapy for their reported antiinflammatory activity are used for this purpose. The mechanisms of inflammation involve complex cascades of events where chemical messengers play an important role. An important class of messengers are the pro-inflammatory leukotrienes resulting from the metabolism of arachidonic acid, which begins with its oxidation by the enzyme 5-lipoxygenase (5-LOX). Essential oils are composed of lipid soluble, low molecular weight molecules which can diffuse through the skin barrier and reach the underlying tissues. These molecules belong to many chemical families and it is therefore not unreasonable to think that some may interfere with enzymatic reactions in the epidermis and beyond. The prime objective of this study was therefore to evaluate the ability of a number of essential oils and of some of their components to inhibit, in vitro, the enzyme 5-lipoxygenase. The comparison of these results with the reported anti-inflammatory (AI) action of certain essential oils may contribute to a better understanding of their mode of action. Another category of products extracted from aromatic plants are absolutes and resinoids. Virtually nothing is known on their activity and potential usefulness for aromatherapy. For this reason a number of these absolutes prepared in-house were included in this screening. Material and methods Reagents 5-Lipoxygenase from Solanum tuberosum (EC ) and Nordihydroguaiaretic acid (NDGA) from Cayman Chemical Company. Linoleic acid (P99%), dimethylsulphoxide (DMSO) and Tween â 20 from Fluka /$ - See Front Matter Ó 2003 Elsevier Ltd All rights reserved DOI: /S (03)

2 Potassium phosphate buffer 0.1 M, ph 6.3, was prepared with analytical grade reagents purchased from standard commercial sources. Essential oils or extracts were either from our own production or from producers with certificates of authenticity. All the essential oils studied were analysed by GC/MS and their profile validated before being tested for their activity. Instruments Spectrophotometer: UV/Vis Lambda 25 (Perkin Elmer Instruments) linked to a PC by the UV Winlab software. Thermostated bath at 25 C. Principle 5-Lipoxygenase is known to catalyse the oxidation of unsaturated fatty acids containing 1 4 pentadiene structures. Arachidonic acid is the biological substrate of 5-LOX, but the enzyme accepts linoleic acid too and the latter being easier to handle, was chosen in this study. The initial reaction rate was measured by spectrophotometry and the inhibitory activity of a substance measured by the decrease of this initial rate. Essential oils are not significantly water soluble and the assay was made in phosphate buffer. Therefore a small amount of a nonionic surfactant (Tween 20) was necessary to disperse the oil in this aqueous medium. Preliminary assays had determined the maximum concentration of surfactant that did not interfere with the enzyme kinetics. Spectrophotometry assay The assay mixture in 3 ml potassium phosphate buffer contained linoleic acid 0.1 mm, DMSO with Tween â 20 (control) or the compound under test dissolved in DMSO with Tween â 20, and sufficient enzyme to give an easily measurable initial reaction rate. The enzymatic reaction was started by adding the enzyme, and the increase in absorption at 234 nm arising from the modification of the unsaturation of linoleic acid (diene 1 4! diene 1 3) was measured for 10 min at 25 C. The initial reaction rate was determined from the slope of the straight line portion of the curve and the percentage inhibition of enzyme activity is calculated by comparing with the control. The concentration that gave 50% inhibition (IC 50 ) for the active compounds was then calculated. Each test compound and control was run in triplicate at each concentration and the results averaged. Results Previous studies (Safayhi et al., 1992; Abad et al., 1995; Hope et al., 1983) have shown that nordihydroguaiaretic acid (NDGA) could be used as reference compound for studies of 5- lipoxygenase inhibition, due to its widely reported strong inhibitory activity on this enzyme. Reported IC 50 values for this compound are highly variable, depending on the precise source of 5-lipoxygenase, and range from 0.26 lm (rat peritoneal cell system) to 44 lm (soybean lipoxygenase). In our study, its IC 50 value was found to be ppm ( lm), a value which falls within the range of figures already published. The in vitro activity of the E.O. and chemically defined products were compared to NDGA activity and an arbitrary scale of relative in vitro activity was defined as follows: ++++ IC 50 < 10 ppm IC ppm < IC ppm + 51< IC ppm Inactive above 100 ppm Table 1 summarises the results obtained with 32 essential oils by decreasing order of activity. With a few exceptions they are utilised both in aromatherapy and in fragrances. Essential oils rich in citral, like lemongrass and Litsea cubeba that are reported in aromatherapy to have anti-inflammatory activity could not be evaluated due to a strong absorption of citral (neral + geranial) at 234 nm, which rendered the spectrophotometric measurement impossible. Ten absolutes and resinoids prepared in-house were also screened for inhibitory activity. It must be noted that the mode of preparation of these naturals leaves a small amount of ethanol (less than 3%) and a few ppm of extraction solvents. The results by decreasing activity are in shown in Table 2. Aromatherapy treatises (Franchome and Penoel, 1985; Hadji Minaglou and Pierron, 1998) emphasise structure activity relationships to define broad categories of activities for the different classes of molecules naturally found in essential oils. For instance, aliphatic aldehydes are said to be predominantly anti-inflammatory and aromatic aldehydes antimicrobial. For this reason we have evaluated the activity of a number of aromatic materials that are also found in essential oils and these results are shown in Table 3. Contrary to the two previous tables, the materials are not listed in decreasing order of activities but grouped by chemical families. Natural ambrettolide is an important component (ffi15%) of ambrette seed colourless. Discussion It is interesting to observe that some essential oils used for their anti- 139

3 Table 1 activity by essential oils as compared to their reported anti-inflammatory (AI) effects in aromatherapy Designation In vitro activity Reported AI in aromatherapy NDGA reference inhibitor ++++ Myrrh (Commiphora myrrha) +++ Yes Copaiba balsam (Copaifera officinalis) +++ Yes Opoponax (Commiphora erythraea) +++ Yes Himalayan cedar (Cedrus deodara) +++ No Sandalwood (Santalum spicatum) +++ Yes Amyris (Amyris balsamifera) +++ No Salvia stenophylla +++ No Lemon (Citrus limon) peel +++ No Curcuma (Curcuma longa) +++ No Sweet Orange (Citrus sinensis) +++ No Mandarin (Citrus reticulata) peel oil +++ No Cedar Atlas (Cedrus atlantica) ++ No Blue Cypress (Callitropsis intratropica) ++ Not known Schinus (Schinus molle) ++ No Sandalwood (Santalum album) ++ No Patchouly (Pogostemon cablin) ++ Yes Helichrysum italicum ++ Yes Vetiver Java (Vetiveria zizanioides) ++ No Juniper berry (Juniperus communis) ++ Yes German chamomile (Matricaria recutita) inflammatory activity in aromatherapy have low IC 50 against 5-LOX in this in vitro test. This suggests that ++ Yes Tansy (Tanacetum annuum) ++ Yes Virginian cedar (Juniperus virginiana) Lemon Balm (Melissa officinalis) (France) ++ No ++ Yes Neroli (Citrus aurantium ssp. amara) + No Parsley leaves (Petroselinum sativum) + No Rose (Rosa damascena) (Turkey) Rosemary (Rosmarinus officinalis ct) I,8-cineole + No + No Roman chamomile (Anthemis nobilis) + Yes Clove leaf (Sygyzium aromaticum) + No Mint (Mentha arvensis) (China) No Peppermint (Mentha piperita) (Italy) No Monarda (Monarda fistulosa ct geraniol) No their good inhibitory action on 5- LOX contributes to their anti-inflammatory activity. This is the case for myrrh, opoponax, copaiba balsam, Helichrysum, German chamomile and juniper berry. Surprisingly other essential oils like sweet orange, lemon and mandarin peel oils appear to have a good activity in vitro but are not reportedly used in aromatherapy for their anti-inflammatory virtues. On the other hand Roman chamomile essential oil at best inhibits very weakly 5-LOX in vitro, which points toward other modes of action for its reported antiinflammatory activity. Absolutes and resinoids are not used in aromatherapy. There are at least two reasons for this: 1. They are prepared by solvent extraction which is anathema for aromatherapists because of residual solvents. This fear of residual solvents was justified when benzene and chlorinated solvents were in use. This is no longer true today. Furthermore the manufacturing process of most absolutes requires the dissolution of the concrete in ethanol to precipitate the waxes before distilling the ethanol out. An absolute contains less than 3% of residual ethanol and at most a few ppm of the primary volatile extraction solvent if it were not ethanol itself. 2. Contrarily to essential oils whose compositions are for the majority well described nowadays, those of absolutes and resinoids are generally poorly documented. This reflects the fact that their volatile constituents, which can be analysed by GC, are only a fraction of their total composition and that the remainder is mostly unknown. The only notable exceptions are incolores obtained by molecular 140

4 Table 2 Designation activity by absolutes and resinoids distillation of absolutes thus eliminating the nonvolatile constituents. In this preliminary screening it was found that four absolutes and resinoids had good to moderate activity. In particular, ambrette incolore appeared to be the best whereas the corresponding absolute did not inhibit 5-LOX. On the other hand, myrrh resinoid obtained by ethanol extraction of the gum had an in vitro activity comparable to the essential oil. The nonvolatile part of an absolute may very well possess components with activity. It is not the first time that absolutes have been shown to possess an activity potentially useful for aromatherapy and active cosmetics. Etienne et al. (2000) have shown that Rosa centifolia absolute had no action on human leucocyte elastase but an absolute obtained from a variety of rose called Sutter s Gold could inhibit in vitro this enzyme, thus indicating a potential usefulness in the treatment of disorders implicating an excessive activity of this enzyme. In an unpublished study, we found that Jasmine (Jasminum grandiflorum ) absolute had in vitro genoprotective potential against UV-B In vitro activity Ambrette seed (Hibiscus abelmoschus) colourless +++ Myrrh (Commiphora myrrha) resinoid +++ Immortelle Pays (Helichrysum stoechas) absolute +++ Orris root (Iris pallida) absolute ++ Jasmin (Jasminum grandiflorum) absolute + Heather (Erica multiflora) absolute + Osmanthus (Osmanthus fragrans) absolute Ambrette seed (Hibiscus abelmoschus) absolute Tuberose (Polyanthes tuberosa) absolute Carnation (Dianthus caryophyllus) absolute induced damage on human keratinocytes, due to its ability to act as a free radical (OH ) scavenger. This could find use in topical applications to protect the skin against sun damage. It seems regrettable therefore that aromatherapy has so far ignored the potential usefulness of absolutes and it is hoped that further studies will fill this gap. Some of the natural and natureidentical molecules tested demonstrated a very good activity against 5-LOX. The terpenes and sesquiterpenes evaluated appeared to be good inhibitors of 5-LOX in vitro. Aromatherapy reports the anti-inflammatory activity of terpenes and our data suggests that an ability to inhibit 5-LOX contributes to this property. There is a good correlation here between the activity of d-limonene with those of the citrus oils evaluated. This should not be surprising since d-limonene is the main component of sweet orange, mandarin and lemon oils. Although they have not been tested yet, it can be expected that essential oils rich in d-limonene like grapefruit, lime and celery will be good inhibitors of 5-LOX too. The two sesquiterpenes studied here strongly inhibited 5-LOX and like d-limonene with the citrus oils, we found a good correlation between the activity of copaiba balsam oil and that of b-caryophyllene, which is its main constituent (ffi70%). Alpha-bisabolol, which is widely reported to have a skin soothing action, strongly inhibited 5-LOX in vitro and essential oils containing important proportions of this sesquiterpenic alcohol like Salvia stenophylla can be expected to inhibit it too. The other sesquiterpenic alcohols tested also showed a good activity whereas the alcohols appeared to be inactive. Similar to a-bisabolol, it is expected that essential oils rich in trans-nerolidol and trans trans farnesol will demonstrate a good inhibiting capacity of 5-LOX. Aromatherapy reports variable anti-inflammatory activity for esters. Here we found that some, like benzyl salicylate, are quite good inhibitors and others are not. Ambrette seed incolore has been found to have the same strong activity against 5-LOX as the macrocyclic lactone ambrettolide which is found in this extract but represents only 11% of its composition. It seems likely, therefore, that the sesquiterpenic ester trans trans farnesyl acetate, which is the major component of this extract, will also be a good inhibitor of this enzyme. Although this is not directly related to aromatherapy, this data suggests an intriguing possibility for the skilled perfumer; the harmonious combination of aromatic materials (essential oils, absolute, natural and synthetic molecules) to create fragrances with a strong capacity to inhibit 5-LOX and therefore have the dual function of perfuming and to contribute to skin soothing 141

5 Table 3 Designation activity by aromatic materials In vitro activity aromatherapy and to active cosmetic preparations. Terpenic hydrocarbons d-limonene* +++ c-terpinene* +++ a-pinene* ++ Sesquiterpenic hydrocarbons b-caryophyllene* +++ Germacrene D* +++ Alcohols Geraniol Linalool Phenylethyl alcohol Sesquiterpenic alcohols a-bisabolol* +++ trans-nerolidol* +++ Farnesol (synthetic mixed isomers) +++ Aliphatic aldehydes trans-2-decenal +++ Dodecanal +++ Decanal ++ Aromatic aldehydes Vanillin Anise aldehyde Ketones a-ionone + b-ionone + Esters Benzyl salicylate +++ Citronellyl acetate +++ Methyl benzoate ++ Geranyl acetate ++ Linalyl acetate + l-bornyl acetate Benzyl acetate Macrocyclic lactone Ambrettolide (mixed isomers) +++ Those of natural origin are indicated with an *. activity in cosmetic or galenic lute and molecules used in products. fragrances possess activities (free Other studies in progress indicate that certain essential oils, abso- radical scavenging, anti-elastase and lipolytic) that are interesting to both Acknowledgements The authors are grateful to Robertet s management to allow the publication of this work. Thanks are due to Mr. J.D. Dor, D. Joulain and Ms C. Gadras for helpful discussions and criticisms. References Abad, M. J. et al. (1995). The activity of flavonoids extracted from Tanacetum microphyllum (Compositae) on soybean lipoxygenase and prostaglandin synthetase. General Pharmacology, 26(4): Etienne, J. J. et al. (2000). New and unexpected properties of perfumes. Effect upon free radicals and enzymes induced by essential oils, absolutes and fragrant compounds. International Journal of Cosmetic Science, 22(5): Franchome, P. and Penoel, D. (1985). L Aromatherapie exactement. Roger Jollois Editeur. Hadji Minaglou, F. and Pierron (1998). Index therapeutique, V1.0 CD ROM format. Hope, W. C. et al. (1983). In vitro inhibition of the biosynthesis of slow reacting substance of anaphylaxis (SRS-A) and lipoxygenase activity by quercetin. Biochemical Pharmacology, 32(2): Safayhi, H. et al. (1992). Boswellic acids. Novel, specific, nonredox inhibitors of 5-lipoxygenase. Journal of Pharmacology and Experimental Therapeutics, 261(3):