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Inhibition of human leukocyte elastase by natural fragrant extracts of aromatic plants
The International Journal of Aromatherapy (2004) 14, 179–182
The International Journal of
Aromatherapy intl.elsevierhealth.com/journals/ijar
Inhibition of human leukocyte elastase by natural fragrant extracts of aromatic plants S. Baylac*, P. Racine Fragrance Division Development Laboratory, ROBERTET SA, 37 avenue Sidi-Brahim, 06130 Grasse, France
KEYWORDS
Summary The influence of 37 essential oils, absolutes, resinoids, oleoresins and natural plant extracts were tested on the enzymatic activity of human leukocyte elastase HLE (EC 3.4.21.37). Among them, poplar bud absolute, rosemary extract, benzoin resinoid and turmeric oleoresin had an inhibitory activity significantly higher than the reference, ursolic acid. Specifically, turmeric oleoresin was the most potent inhibitor of HLE. Essential oils tested were in their majority totally inactive. Because of the specific role of human leukocyte elastase in the inflammatory process, its inhibition by such absolutes, resinoids, oleoresins or plant extracts should encourage their use in Aromatherapy and in cosmetic products for irritated, reactive and/or senescent epidermis. c 2004 Elsevier Ltd. All rights reserved.
Human leukocyte elastase; 5-Lipoxygenase; Essential oils; Resinoids; Absolutes; Oleoresins
Introduction In a previous study (Baylac and Racine, 2003), it was shown that natural fragrant extracts of aromatic plants (essential oils, absolutes, resinoids) could inhibit in vitro an important enzyme of the inflammatory cascade, 5-lipoxygenase (5-LOX). Pursuing our investigation on the properties of extracts of aromatic plants on the actors of inflammation, we have studied whether natural extracts presently utilised for their olfactive properties may also influence in-vitro the activity of human leukocyte elastase.
*
Corresponding author. Fax: +33 4 93 70 68 09. E-mail address: [email protected] (S. Baylac).
Human leukocyte elastase (HLE) and cathepsin G are the two main proteases in neutrophils and play an important role in the pathogenesis of many inflammatory disorders (Lo ¨ser et al., 2000; Melzig et al., 2001; Mitaine-Offer et al., 2002; Safayhi et al., 1997; Ying et al., 1991). They are stored in high quantities in the azurophilic granules of leukocytes and are responsible for the neutrophil-mediated degradation of matrix proteins such as elastin and different types of collagen. A transient increase in elastase activity is part of the normal body response when it is aggressed; however a typical characteristic of active and chronic inflammation is an elevated plasma level of neutrophil elastase (Lo ¨ser et al., 2000; Melzig et al., 2001). It has also been established that elastase activity in the epidermis and the dermis is increased after UV irradiation (activity peaks 24 h after exposure).
0962-4562/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ijat.2004.09.008
180 This helps understand why chronic exposure to UV leads to increased wrinkling and sagging of the skin. The purpose of this study was therefore to screen a number of essential oils, absolutes and extracts to evaluate their capacity to inhibit in vitro the enzyme HLE. Ursolic acid is a known inhibitor of HLE (Ying et al., 1991) and was chosen as a benchmark in this study.
Experimental Reagents HLE (EC 3.4.21.37) was supplied by ICN Biomedicals (USA); N-(Methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide and ursolic acid were purchased from Sigma (France). Extracts from aromatic plants 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/Visible Lambda 25 (Perkin– Elmer Instruments). Data processing by UV Winlab software.
Enzyme assay The determination of leukocyte elastase activity was performed according to Mitaine-Offer (2002). Briefly, HLE activity was measured using N-(Methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide as substrate in Tris buffer 50 mM, pH 8, containing 150 mM NaCl at 25 C. Enzyme (7 nM) was preincubated for 5 min in buffer, in the presence of test compound or vehicle (DMSO) for controls. The hydrolysis was started by the addition of substrate (500 lM) in DMSO and release of p-nitroanilide was monitored by change in absorbance at 405 nm. The initial reaction rate was determined from the slope of the straight line portion of the curve and the percentage inhibition of enzyme activity calculated by comparing with controls. The concentration of the test substance that gave 50% inhibition (IC50) was then calculated. Each test compound and the control were run in triplicate at each concentration and the results averaged.
S. Baylac, P. Racine
Results With the above described protocol, the IC50 of ursolic acid was found to be 2 ± 0.5 ppm at 25 C. A scale of relative in vitro activity was defined as follows: ++++++ +++++ ++++ +++ ++ + 0
IC50 < 0.1 ppm 0.1 6 IC50 6 1 ppm 1.1 6 IC50 6 5 ppm 5.1 6 IC50 6 10 ppm 10.1 6 IC50 6 15 ppm 15.1 6 IC50 6 20 ppm Inactive above 20 ppm
We considered that an IC50 greater than 10 times that of the reference inhibitor was not significant. Thirty six essential oils, absolutes, resinoids, oleoresins and a non odorous rosemary extract were evaluated and the results are shown in Table 1.
Discussion Of the 37 products evaluated, 11 were essential oils, 25 were fragrant extracts (absolutes, resinoids and oleoresins) and one, a non odorous extract of rosemary obtained from the aerial part of the plant after elimination of the essential oil by steam distillation. Only two essential oils (myrrh and ylang ylang) showed a weak activity but the others were totally inactive whereas all these essential oils had been found to be good to moderate inhibitors in vitro of 5-LOX. On the other hand, 12 of the 25 extracts were as or more active than ursolic acid itself. More precisely, it is interesting to note that poplar bud absolute, rosemary extract, benzoin resinoid and turmeric oleoresin had an inhibitory activity significantly higher than the reference compound, ursolic acid. Among these extracts, turmeric oleoresin was the most potent inhibitor of HLE. Indeed we have found its IC50 to lie between 40 and 50 ppb (0.04–0.05 ppm), which is between 40 and 50 times lower than ursolic acid. These four extracts have also demonstrated excellent free radical scavenging capacity. The volatile fragrant components of absolutes and resinoids can be identified by GC/MS but they represent only a fraction of the total extract. Unfortunately the other components of higher molecular weights have been very seldom determined quantitatively and even qualitatively. For these reasons, it is difficult to explain the
Inhibition of human leukocyte elastase by natural fragrant extracts of aromatic plants Table 1
181
In vitro inhibition of human leukocyte elastase by aromatic extracts (by decreasing order of activity)
Extracts/compound
In vitro activity
Ursolic acid (reference) Turmeric (Curcuma longa) oleoresin Poplar (Populus balsamifera) buds absolute Rosemary (Rosmarinus officinalis) extract Benzoin (Styrax tonkinense) resinoid Gentian (Gentiana lutea) absolute Linden (Tillia spp.) absolute Violet (Viola odorata) leaf absolute Wheat (Triticum aestivum) bran absolute Myrh (Commiphora myrrha) resinoid Cocoa (Theobroma cacao) absolute Artichoke (Cynara scolymus) absolute Fucus (Fucus vesiculosus) resinoid Jasmine (Jasminum officinalis) absolute Blackcurrant (Ribes nigrum) buds absolute Rice (Oryza sativa) absolute Tea (Thea sinensis) resinoid Rose (Rosa damascena) absolute Myrrh (Commiphora myrrha) essential oil Ylang ylang (Cananga odorata) essential oil Mate (Ilex paraguarensis) absolute Champaca (Michelia champaca) absolute Spanish broom (Spartium junceum) absolute Fig (Ficus carica) absolute Honey absolute Frangipani (Plumeria acutifolia) absolute Osmanthus (Osmanthus fragrans) absolute Jasmine (Jasminus Sambac) Sambac absolute Spilanthes (Spilanthes acmella) oleoresin Turmeric (Curcuma longa) essential oil Marjoram (Origanum marjorana) essential oil Patchouly (Pogostemon cablin) essential oil Cabreuva (Myrocarpus frondosus) essential oil Copaiba balsam (Copaifera officinalis) essential oil Atlas cedar (Cedrus atlantica) essential oil Cade wood (Juniperus oxycedrus) essential oil Sweet orange (Citrus sinensis) essential oil Rosemary (Rosmarinus officinalis) essential oil
++++ ++++++ +++++ +++++ +++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ ++ + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
difference in inhibitory activity of the various absolutes, resinoids and oleoresins that we have tested. Some general comments can still be made in view of the fact that the essential oils tested were in their majority totally inactive. Essential oils contain only low molecular weight volatile components (terpenes, sesquiterpenes, alcohols, aldehydes, esters, ketones, etc.) whereas plant extracts like absolutes, resinoids and oleoresins in addition to these volatiles contain higher molecular weight components of C20 and higher. From these experimental results, it seems more likely that good inhibitors of HLE are to be found in molecules of C20 and higher rather than in the ‘lighter’ molecules found in essential oils. In the rosemary extract that was found to be about 10 times more potent than ursolic acid, we have iden-
tified besides ursolic acid itself (10–15%), oleanolic acid (@5%), carnosic acid and carnosol (@20%) and betulinic acid (@5%). It probably also contains flavonoids and other phenolic compounds. It is not known if the stronger anti-elastase activity of this extract is due to one of its components or as a result of synergies between them. On the other hand, rosemary essential oil (a- and b-pinene, camphene, 1,8-cineole, limonene, camphor, l-borneol and a-terpineol) has absolutely no inhibitory activity against HLE. The striking difference between the exceptionally strong inhibitory activity of the oleoresin of Curcuma longa and the complete lack of activity of the essential oil on HLE does not contradict this hypothesis. Turmeric oleoresin is known to contain curcuminoids, a blend of three complex diones
182 (Verghese, 1993) with reported antioxidant and anti-inflammatory activity (Majeed et al., 1995), whereas they are absent in the essential oil. Turmeric extracts are already known for their anti-inflammatory activity and free radical scavenger activity. We have also found that the oleoresin is a strong free radical scavenger in the DPPH test. In conclusion, this study points to the fact that extracts of aromatic plants primarily used for perfumery purpose (absolutes, resinoids, oleoresins) can also be considered in Aromatherapy. These results should encourage practitioners to investigate their usefulness. One can also surmise that topical applications of these extracts in conjunction with natural actives with additional activities like inhibitors of 5-LOX and of reactive oxygen species could be beneficial in cosmetics for irritated, reactive and/or senescent epidermis.
Acknowledgement We thank the management of Robertet for allowing the publication of this work.
References Baylac S, Racine P. Inhibition of 5-lipoxygenase by essential oils and other natural fragrant extracts. Int J Aromatherapy 2003;13(2/3).
S. Baylac, P. Racine Lo ¨ser B, Kruse S, Melzig M, Nahrstedt A. Inhibition of neutrophil elastase activity by cinnamic acid derivatives from Cimicifuga racemosa. Planta Med 2000;66:751. Majeed M, Badmaev V, Shivakumar U, Rajendran R. Curcuminoids antioxidant phytonutrients. New Jersey: NutriScience Publishers; 1995. Melzig MF, Lo ¨ser B, Ciesielski S. Inhibition of neutrophil elastase activity by phenolic compounds from plants. Pharmazie 2001;56:12. Mitaine-Offer A, Hornebeck W, Sauvain M, Ze `ches-Hanrot M. Triterpenes and phytosterols as human leucocyte elastase inhibitors. Planta Med 2002;68:930. Safayhi H, Rall B, Sailer ER, Ammon H. Inhibition by boswellic acids of human leukocyte elastase. J Pharmacol Exp Therapeutics 1997;281:460. Verghese J. Isolation of curcumin from Curcuma longa L. rhizome. Flavour Fragr J 1993;8:315. Ying QL, Rinehart A, Simon S, Cheronis J. Inhibition of human leukocyte elastase by ursolic acid. Biochemistry 1991;277: 521.
Glossary of ISO norms Concrete: extract with a characteristic odour from a fresh vegetable raw material by a non aqueous solvent. Resinoid: extract with a characteristic odour from a dried vegetable raw material by a non aqueous solvent. Absolute: product with odour obtained from a concrete or a resinoid by extraction with ethanol at room temperature, cooled and filtered to remove the waxes, the ethanol is then removed by distillation. Oleoresin: extract of spice or aromatic herbs with a characteristic odour and/or flavour. The residual solvent content should be limited to food use.
The International Journal of
Aromatherapy intl.elsevierhealth.com/journals/ijar
Inhibition of human leukocyte elastase by natural fragrant extracts of aromatic plants S. Baylac*, P. Racine Fragrance Division Development Laboratory, ROBERTET SA, 37 avenue Sidi-Brahim, 06130 Grasse, France
KEYWORDS
Summary The influence of 37 essential oils, absolutes, resinoids, oleoresins and natural plant extracts were tested on the enzymatic activity of human leukocyte elastase HLE (EC 3.4.21.37). Among them, poplar bud absolute, rosemary extract, benzoin resinoid and turmeric oleoresin had an inhibitory activity significantly higher than the reference, ursolic acid. Specifically, turmeric oleoresin was the most potent inhibitor of HLE. Essential oils tested were in their majority totally inactive. Because of the specific role of human leukocyte elastase in the inflammatory process, its inhibition by such absolutes, resinoids, oleoresins or plant extracts should encourage their use in Aromatherapy and in cosmetic products for irritated, reactive and/or senescent epidermis. c 2004 Elsevier Ltd. All rights reserved.
Human leukocyte elastase; 5-Lipoxygenase; Essential oils; Resinoids; Absolutes; Oleoresins
Introduction In a previous study (Baylac and Racine, 2003), it was shown that natural fragrant extracts of aromatic plants (essential oils, absolutes, resinoids) could inhibit in vitro an important enzyme of the inflammatory cascade, 5-lipoxygenase (5-LOX). Pursuing our investigation on the properties of extracts of aromatic plants on the actors of inflammation, we have studied whether natural extracts presently utilised for their olfactive properties may also influence in-vitro the activity of human leukocyte elastase.
*
Corresponding author. Fax: +33 4 93 70 68 09. E-mail address: [email protected] (S. Baylac).
Human leukocyte elastase (HLE) and cathepsin G are the two main proteases in neutrophils and play an important role in the pathogenesis of many inflammatory disorders (Lo ¨ser et al., 2000; Melzig et al., 2001; Mitaine-Offer et al., 2002; Safayhi et al., 1997; Ying et al., 1991). They are stored in high quantities in the azurophilic granules of leukocytes and are responsible for the neutrophil-mediated degradation of matrix proteins such as elastin and different types of collagen. A transient increase in elastase activity is part of the normal body response when it is aggressed; however a typical characteristic of active and chronic inflammation is an elevated plasma level of neutrophil elastase (Lo ¨ser et al., 2000; Melzig et al., 2001). It has also been established that elastase activity in the epidermis and the dermis is increased after UV irradiation (activity peaks 24 h after exposure).
0962-4562/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ijat.2004.09.008
180 This helps understand why chronic exposure to UV leads to increased wrinkling and sagging of the skin. The purpose of this study was therefore to screen a number of essential oils, absolutes and extracts to evaluate their capacity to inhibit in vitro the enzyme HLE. Ursolic acid is a known inhibitor of HLE (Ying et al., 1991) and was chosen as a benchmark in this study.
Experimental Reagents HLE (EC 3.4.21.37) was supplied by ICN Biomedicals (USA); N-(Methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide and ursolic acid were purchased from Sigma (France). Extracts from aromatic plants 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/Visible Lambda 25 (Perkin– Elmer Instruments). Data processing by UV Winlab software.
Enzyme assay The determination of leukocyte elastase activity was performed according to Mitaine-Offer (2002). Briefly, HLE activity was measured using N-(Methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide as substrate in Tris buffer 50 mM, pH 8, containing 150 mM NaCl at 25 C. Enzyme (7 nM) was preincubated for 5 min in buffer, in the presence of test compound or vehicle (DMSO) for controls. The hydrolysis was started by the addition of substrate (500 lM) in DMSO and release of p-nitroanilide was monitored by change in absorbance at 405 nm. The initial reaction rate was determined from the slope of the straight line portion of the curve and the percentage inhibition of enzyme activity calculated by comparing with controls. The concentration of the test substance that gave 50% inhibition (IC50) was then calculated. Each test compound and the control were run in triplicate at each concentration and the results averaged.
S. Baylac, P. Racine
Results With the above described protocol, the IC50 of ursolic acid was found to be 2 ± 0.5 ppm at 25 C. A scale of relative in vitro activity was defined as follows: ++++++ +++++ ++++ +++ ++ + 0
IC50 < 0.1 ppm 0.1 6 IC50 6 1 ppm 1.1 6 IC50 6 5 ppm 5.1 6 IC50 6 10 ppm 10.1 6 IC50 6 15 ppm 15.1 6 IC50 6 20 ppm Inactive above 20 ppm
We considered that an IC50 greater than 10 times that of the reference inhibitor was not significant. Thirty six essential oils, absolutes, resinoids, oleoresins and a non odorous rosemary extract were evaluated and the results are shown in Table 1.
Discussion Of the 37 products evaluated, 11 were essential oils, 25 were fragrant extracts (absolutes, resinoids and oleoresins) and one, a non odorous extract of rosemary obtained from the aerial part of the plant after elimination of the essential oil by steam distillation. Only two essential oils (myrrh and ylang ylang) showed a weak activity but the others were totally inactive whereas all these essential oils had been found to be good to moderate inhibitors in vitro of 5-LOX. On the other hand, 12 of the 25 extracts were as or more active than ursolic acid itself. More precisely, it is interesting to note that poplar bud absolute, rosemary extract, benzoin resinoid and turmeric oleoresin had an inhibitory activity significantly higher than the reference compound, ursolic acid. Among these extracts, turmeric oleoresin was the most potent inhibitor of HLE. Indeed we have found its IC50 to lie between 40 and 50 ppb (0.04–0.05 ppm), which is between 40 and 50 times lower than ursolic acid. These four extracts have also demonstrated excellent free radical scavenging capacity. The volatile fragrant components of absolutes and resinoids can be identified by GC/MS but they represent only a fraction of the total extract. Unfortunately the other components of higher molecular weights have been very seldom determined quantitatively and even qualitatively. For these reasons, it is difficult to explain the
Inhibition of human leukocyte elastase by natural fragrant extracts of aromatic plants Table 1
181
In vitro inhibition of human leukocyte elastase by aromatic extracts (by decreasing order of activity)
Extracts/compound
In vitro activity
Ursolic acid (reference) Turmeric (Curcuma longa) oleoresin Poplar (Populus balsamifera) buds absolute Rosemary (Rosmarinus officinalis) extract Benzoin (Styrax tonkinense) resinoid Gentian (Gentiana lutea) absolute Linden (Tillia spp.) absolute Violet (Viola odorata) leaf absolute Wheat (Triticum aestivum) bran absolute Myrh (Commiphora myrrha) resinoid Cocoa (Theobroma cacao) absolute Artichoke (Cynara scolymus) absolute Fucus (Fucus vesiculosus) resinoid Jasmine (Jasminum officinalis) absolute Blackcurrant (Ribes nigrum) buds absolute Rice (Oryza sativa) absolute Tea (Thea sinensis) resinoid Rose (Rosa damascena) absolute Myrrh (Commiphora myrrha) essential oil Ylang ylang (Cananga odorata) essential oil Mate (Ilex paraguarensis) absolute Champaca (Michelia champaca) absolute Spanish broom (Spartium junceum) absolute Fig (Ficus carica) absolute Honey absolute Frangipani (Plumeria acutifolia) absolute Osmanthus (Osmanthus fragrans) absolute Jasmine (Jasminus Sambac) Sambac absolute Spilanthes (Spilanthes acmella) oleoresin Turmeric (Curcuma longa) essential oil Marjoram (Origanum marjorana) essential oil Patchouly (Pogostemon cablin) essential oil Cabreuva (Myrocarpus frondosus) essential oil Copaiba balsam (Copaifera officinalis) essential oil Atlas cedar (Cedrus atlantica) essential oil Cade wood (Juniperus oxycedrus) essential oil Sweet orange (Citrus sinensis) essential oil Rosemary (Rosmarinus officinalis) essential oil
++++ ++++++ +++++ +++++ +++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ ++ + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
difference in inhibitory activity of the various absolutes, resinoids and oleoresins that we have tested. Some general comments can still be made in view of the fact that the essential oils tested were in their majority totally inactive. Essential oils contain only low molecular weight volatile components (terpenes, sesquiterpenes, alcohols, aldehydes, esters, ketones, etc.) whereas plant extracts like absolutes, resinoids and oleoresins in addition to these volatiles contain higher molecular weight components of C20 and higher. From these experimental results, it seems more likely that good inhibitors of HLE are to be found in molecules of C20 and higher rather than in the ‘lighter’ molecules found in essential oils. In the rosemary extract that was found to be about 10 times more potent than ursolic acid, we have iden-
tified besides ursolic acid itself (10–15%), oleanolic acid (@5%), carnosic acid and carnosol (@20%) and betulinic acid (@5%). It probably also contains flavonoids and other phenolic compounds. It is not known if the stronger anti-elastase activity of this extract is due to one of its components or as a result of synergies between them. On the other hand, rosemary essential oil (a- and b-pinene, camphene, 1,8-cineole, limonene, camphor, l-borneol and a-terpineol) has absolutely no inhibitory activity against HLE. The striking difference between the exceptionally strong inhibitory activity of the oleoresin of Curcuma longa and the complete lack of activity of the essential oil on HLE does not contradict this hypothesis. Turmeric oleoresin is known to contain curcuminoids, a blend of three complex diones
182 (Verghese, 1993) with reported antioxidant and anti-inflammatory activity (Majeed et al., 1995), whereas they are absent in the essential oil. Turmeric extracts are already known for their anti-inflammatory activity and free radical scavenger activity. We have also found that the oleoresin is a strong free radical scavenger in the DPPH test. In conclusion, this study points to the fact that extracts of aromatic plants primarily used for perfumery purpose (absolutes, resinoids, oleoresins) can also be considered in Aromatherapy. These results should encourage practitioners to investigate their usefulness. One can also surmise that topical applications of these extracts in conjunction with natural actives with additional activities like inhibitors of 5-LOX and of reactive oxygen species could be beneficial in cosmetics for irritated, reactive and/or senescent epidermis.
Acknowledgement We thank the management of Robertet for allowing the publication of this work.
References Baylac S, Racine P. Inhibition of 5-lipoxygenase by essential oils and other natural fragrant extracts. Int J Aromatherapy 2003;13(2/3).
S. Baylac, P. Racine Lo ¨ser B, Kruse S, Melzig M, Nahrstedt A. Inhibition of neutrophil elastase activity by cinnamic acid derivatives from Cimicifuga racemosa. Planta Med 2000;66:751. Majeed M, Badmaev V, Shivakumar U, Rajendran R. Curcuminoids antioxidant phytonutrients. New Jersey: NutriScience Publishers; 1995. Melzig MF, Lo ¨ser B, Ciesielski S. Inhibition of neutrophil elastase activity by phenolic compounds from plants. Pharmazie 2001;56:12. Mitaine-Offer A, Hornebeck W, Sauvain M, Ze `ches-Hanrot M. Triterpenes and phytosterols as human leucocyte elastase inhibitors. Planta Med 2002;68:930. Safayhi H, Rall B, Sailer ER, Ammon H. Inhibition by boswellic acids of human leukocyte elastase. J Pharmacol Exp Therapeutics 1997;281:460. Verghese J. Isolation of curcumin from Curcuma longa L. rhizome. Flavour Fragr J 1993;8:315. Ying QL, Rinehart A, Simon S, Cheronis J. Inhibition of human leukocyte elastase by ursolic acid. Biochemistry 1991;277: 521.
Glossary of ISO norms Concrete: extract with a characteristic odour from a fresh vegetable raw material by a non aqueous solvent. Resinoid: extract with a characteristic odour from a dried vegetable raw material by a non aqueous solvent. Absolute: product with odour obtained from a concrete or a resinoid by extraction with ethanol at room temperature, cooled and filtered to remove the waxes, the ethanol is then removed by distillation. Oleoresin: extract of spice or aromatic herbs with a characteristic odour and/or flavour. The residual solvent content should be limited to food use.