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Furanocoumarins their Properties & Distribution in Natural Aromatic Ingredients: an A-Z Listing.

Copyright Cropwatch April 2008 v 1.02

[N.B. Some references may be duplicated under different subject headings].

Ammi majus extract Ammi majus L. CAS n: 90320-46-0. Cropwatch summary: Described in older Arabic literature as local medicinal treatment for leukodermia fruits consumed orally and leukodermic patches exposed to sunshine (Dawood El-Antaki 1923). First used as source of xanthotoxin for vitiligo treatment (Mofty 1948). Ingredient hardly used in Western corporate cosmetics; Ammi majus & galenical preparations anyway banned under Annex II/35 of the Cosmetics Directive 76/768/EEC.

Dawood El-Antaki (1923) Tazkaret Oli El-Albab 3rd edition, Vol 1, 32.

Hehmann M., Lukacin R., Ekiert H. & Matern U. (2004) "Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-methyltransferase." Eur J Biochem. 271(5), 932-40. Abstract. Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the COMT activity remained stable. Furthermore, the recombinant AmBMT, which was most active in potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates. Dark-grown A. majus cells expressed significant BMT activity which nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation. Complementary Northern blotting revealed that the BMT transcript abundance increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT

transcript. The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens.

Ivie G.W. (1978) Linear furocoumarins (psoralens) from the seed of Texas Ammi majus L. (bishops weed). J Agric Food Chem 26, 1394-1403.

Kaminski M., Kartanowicz R., Kaminski., M., Krolicka A..; Sidwa-Gorycka M.; Lojkoska E. & Gorzen W. (2003) HPLC-DAD in identification and quantification of selected coumarins in crude extracts from plant cultures of Ammi majus and Ruta graveolens. J. Sep. Sci. 26, 1287-1291.

Ammi visnaga oil Ammi visnaga (L.) Lam. Cropwatch summary: [This entry is added for completeness: The coumarins in Ammi visnaga essential oil are mainly photochromones]. Ammi visnaga essential oil is produced in commercially, & has been used in aromatherapy. The essential oil may contain the spasmolytic furanochromone khellin & the pyranochromone visnagin, used to pigment skin via UV xposure in vitiligo treatment. Khellin said to form DNA- monoadducts and limited cross-links; photochemical cross-linkage to DNA disputed by photo-sensitised DNA cleavage study (Chen & Kagan 1993).

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khellin

Chen X. & Kagan J. Photo-sensitised cleavage & cross-linking of pBR322 DNA with khellin & visnagin J. Photochem. & Photobiol. B: Biology 20(2-3), 183-189.

Analysis for furanocoumarins. Cropwatch summary: Although rapid & qualitative analysis of citrus oils for furanocoumarins may be possible by GC techniques, without specific precautions, it is inevitable that thermal degradation of these somewhat fragile compounds will lead to quantitative inaccuracies. HPLC with spectrofluorometric detection is the most generally used method to determine coumarin derivatives after their separation from volatiles (Couchi & Barth 1994). In the past, isolation artifacts have confused the picture with regard to reported naturally occuring coumarins, as re-arrangements of side chain components can easily occur under acidic conditions i.e. from juice contact, or on silicic acid columns (Dugo & McHale 2002).

Frrot & Decorzant (2004) describe a combination of HPLC, and various detection methods (diode array, fluorescence & atmospheric pressure chemical ionization-mass spectrometry) to detect 15 major furanocoumarins in citrus oils. The authors conclude the method would be suitable for quality control laboratories, but the authors maybe forget that whereas capital for sophisticated analytical equipment may not be a problem for the larger corporate aroma companies, in the real world this type of equipment is not an option for SMEs. Any regulatory obligation to determine furanocoumarins levels in commercially

offered essential oils by this sort of methodology would therefore impose a level economic discrimination against SMEs.

An enzyme-linked immunoabsorbent assay technique for furanocoumarins was described by Saita et al. (2004), produced by immunizing rabbits with 6,7-dihydroxybergamottin conjugated with serum album. This latter technique appears sensitive for furocoumarins containing the geranyloxy side chain (e.g. bergamottin & 6,7-dihydroxybergamottin, the inhibitors of P450 3A4 enzymes in citrus fruits. But since the testing method employs the use of animals, presumably the method has no future in Europe.

Couchi D. & Barth D. (1994). "Rapid identification of some coumarin derivatives in deterpenated citrus peel oil by gas chromatography" J Chromatogr A. 672(1-2), 177-83. Abstract. Generally on the gas chromatogram of a volatile essential oil, terpenes, oxygenated compounds and sesquiterpenes appear. With temperature programming, it was shown that some non-volatiles are present with the volatiles. They are simple coumarin (2H-1-benzopyran-2-one) derivatives such as citropten (5,7-dimethoxycoumarin) and furocoumarins (psoralen, 7H-furo[3,2-g][1]benzopyran-7-one) such as bergapten (5-methoxypsoralen), some of which are phototoxic. Terpeneless oils are used in perfumes and cosmetics, so it is important to be able to establish rapidly if they contain phototoxic compounds.

Dugo P. & McHale D. (2002) Ch 15: The oxygen heterocyclic compounds of citrus oils eds G. Dugo & D. Giacomo D. (2002) The Genus Citrus CRC Press 2002.

Frrot E. & Decorzant E. (2004) "Quantification of total furocoumarins in citrus oils by HPLC coupled with UV, fluorescence, and mass detection." J Agric Food Chem 52, 6879-6886. Abstract. Furocoumarins or psoralens represent a class of photosensitizers whose use level is likely to be restricted to 1 ppm in cosmetic products by the EU. A reversed-phase HPLC method was developed to separate the 15 main furocoumarins present in citrus oils. Quantification by UV, fluorescence, or mass detectors was compared in terms of linearity and limit of detection. Cold-pressed oils of different citrus species were analyzed using this method. This method could be implemented in quality control laboratories equipped with an HPLC system and a UV diode array detector. Because of possible coelutions, the UV-spectral data should be carefully examined to avoid misleading interpretations of peaks.

Genkina G.L. (1981) "Methods of analysis of furocoumarins." Pharmaceutical Chemistry Journal 15(11), 819-824.

Mizuishi K., Kazama M., Nakamura Y., & Harada H. Hygenic chemical studies on irritants. III. High performance liquid chromatographic determination of bergapten. Tokyo-toritsu Eisei Kenkyuso Nempo 29-1, 118-120.

Peroutka R., Schulzov, V., Botek, P. & Hajlov J. (2007) "Analysis of furanocoumarins in vegetables (Apiaceae) and citrus fruits (Rutaceae)." Journal of the Science of Food and Agriculture 87(11), 2152-2163. Abstract. Several alternative approaches applicable for the analysis of furanocoumarins, toxic components occurring in some fruits and vegetables representing both Apiaceae and Rutaceae families, were tested in our study. Limits of detection (LODs) for angelicin, psoralen, bergapten, xanthotoxin, trioxsalen, isopimpinellin, sphondin, pimpinellin and isobergapten obtained by GC/MS (SIM) were in the range 0.01-0.08 g g1. Slightly higher LODs (0.02-0.20 g g1) were achieved by LC/MS-MS. The latter is the only alternative for analysis of bergamottin (LOD = 0.01 g g1) in citrus fruits because this furanocoumarin is unstable under GC conditions. Regardless of the determination step used, the repeatability of the measurements (expressed as RSD) did not exceed 10%. As shown in our study the levels of furanocoumarins in celery, celeriac, parsnip, carrot, lemon and other foods

obtained at a retail market varied over a wide range; the highest contents were determined in parsnip, while the levels of these toxins in carrots and citrus pulps were relatively low.

Saita T., Fujito H. & Mori M. (2004) "Screening of furanocoumarin derivatives in citrus fruits by enzyme-linked immunosorbent assay" Biol. Pharm. Bull. 27(7), 974-977 Abstract. This paper reports a sensitive and specific enzyme-linked immunosorbent assay for screening of furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in citrus fruits. Anti-6,7-dihydroxybergamottin antibody was obtained by immunizing rabbits with 6,7-dihydroxybergamottin conjugated with bovine serum albumin using the N-succinimidyl ester method. An enzyme marker was similarly prepared by coupling 6,7-dihydroxybergamottin with b -D-galactosidase. The enzyme-linked immunosorbent assay is capable of detecting as little as 800 pg/ml of 6,7-dihydroxybergamottin and 4 ng/ml of bergamottin. Cross-reactivity data showed that the antibody well recognizes both the furanocoumarin and 6,7-dihydroxy-3,7-dimethyloct-2-enyloxy moieties of the 6,7-dihydroxybergamottin, and is thus specific to the structure of furanocoumarin derivatives containing geranyloxy side chain as the cytochrome P450 3A4 inhibitors in grapefruit juice. The antibody was, therefore, used for screening a large number of citrus fruits for furanocoumarin derivatives such as 6,7-dihydroxybergamottin. Fifteen citrus fruits were examined and significant reactivity was observed in 8 of these: red pummelo, sweetie, melogold, banpeiyu pummelo, hassaku, sour orange, lime and natsudaidai. This enzyme-linked immunosorbent assay may be a powerful tool for screening for furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in grapefruit juice.

Xie Y, Chen Y, Lin M, Wen J, Fan G, Wu Y. (2007) "High-performance liquid chromatographic method for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma." J Pharm Biomed Anal. 44(1), 166-72. Abstract. A high-performance liquid chromatographic method was developed and validated for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma. The coumarin components and the internal standard isopsoralen were extracted from plasma samples with the mixture of tert-butyl methyl ether and n-hexane (4:1, v/v). Chromatographic separation was performed on a C(18) column (200 mm x 4.6mm, 5 microm) with the mobile phase acetonitrile-methanol-water-acetic acid (20:15:65:2, v/v/v/v) at a flow-rate of 1.0 ml/min. Only the peak of oxypeucedanin hydrate and byak-angelicin could be detected in dog plasma after oral administration of ethanol extracts of A. dahurica mainly containing xanthotoxol, osthenol, imperatorin, oxypeucedanin hydrate and byakangelicin. The calibration curves of oxypeucedanin hydrate and byak-angelicin were linear over a range of 22.08-8830.00 and 6.08-2430.00 ng/ml in dog plasma, respectively. The quantification limit of oxypeucedanin hydrate and byak-angelicin in dog plasma was 22.08 and 6.08 ng/ml, respectively. The intra- and inter-day precision was less than 7.6% and 8.5% and the accuracy was from 91.9% to 106.1%. The lowest absolute recoveries of oxypeucedanin hydrate and byak-angelicin were 85.7% and 87.0%, respectively. The method was successfully applied to the pharmacokinetic studies of oxypeucedanin hydrate and byak-angelicin in dog plasma after oral administration of ethanol extracts from A. dahurica.

Angelica oils. (A. archangelica spp. & subspp. only). CAS n: 8015-64-3; EINECS-CAS n: 84775-71-7 Cropwatch summary: Phototoxicity: Reportedly RIFM originally found angelica seed oil non-photo-toxic (?): Opdyke (1974) through Tisserand & Balacs (1995) p. 116. However it appears that in 1975, Opdyke (for RIFM) did find Angelica root oil phototoxic: (Opdyke 1975). The essential oil obtained by steam distillation of roots of A. archangelica was previously claimed free of furanocoumarins by EMEA (1996); root & seed essential oils were also claimed to be free of FCs, & therefore not phototoxic by Leung (1996). Against this confusion backdrop, IFRA (2001)

recommends 0.8% max.of Angelica root oil in leave-on products applied to skin then exposed to sunshine.

Composition: Harmala et al. (1992) identified the coumarins 2-angeloyl-3-isovaleryl vaginate, archangelicin, oxypeucedanin hydrate, bergapten, byakangelicin angelate, imperatorin, isoimperatorin, isopimpinellin, 8-[2-(3-methylbutroxy)-3-hydroxy-3-methylbutoxy]psoralen, osthol, ostruthol, oxypeucedanin, phellopterin, psoralen & xanthotoxin from a chloroform extract of the roots of Angelica archangelica L. ssp. archangelica.

Academic studies of lab. prepared root oils of Angelica archangelica L. ssp. archangelica var. sativa (Miller) Rikli reveal the presence of the angular furanocoumarins angelicin & archangelicin, as well as lesser amounts of linear furanocoumarins. Solvent extracts of Angelica spp. (often passed off as essential oils) contain angelicin, bergaptene, imperatorin, oxypeucedanin hydrate, xanthotoxin and xanthotoxol.

Little reliable information on the FC contents of aromatic commodities from the seeds & roots of the various species & subspecies and geographic origins is available. Angelica species used to produce aromatic commodities include:

Angelica archangelica L. ssp. archangelica var. sativa (Miller): European oil. A. sinensis (Oliv.) Diels. Chinese oil / extract. A. koreana Maxim. Contains imperatorin, isoimperatorin, oxypeucedanin, isooxypeucedanin, & oxypeucedanin methanolate (Woo et al. 1983; Seo et al. 2002). A. sylvestris L. Herb used in food flavourings.

Zobel & Brown (1991) show psoralen, xanthotoxin and bergapten in fruits & seeds of A. archangelica.

Exposure to Angelica furanocoumarins can occur via herbal medicine, & the EMEA considers that an exposure of 15g/day would not result in increased risk of photomutagenicity in consumers (EMEA 2007).

RIFM give the following data measured by the fragrance industry (??) for Angelica root oil (no botanical or geographical details given): psoralen up to 112ppm, bergapten up to 78ppm, angelicin 230 ppm; xanthotoxin, isopimpernellin & bergamottin were not detected (RIFM Fact Sheet No 3.).

Cropwatchs Perfumery Notes: (from Cropwatch Newsletter April 2007). Angelica qualities (root or seed oils of various geographic origins & species, solvent or CO2 extracts, absolutes etc.) are not widely used in perfumery, but their power is such, that when they are employed, they are generally present at relatively low levels e.g. 0.1%, rarely more. However, angelica oils have been used to good effect in chypres to enhance spicy peppery accords; they also find some uses in masculine fougeres. Angelica oils blend well with many aromatic raw materials, especially woody materials including patchouli, vetivert and clary sage, and many

workers consider that this property is, in part, due to synergistic effects imparted by the naturally occurring macrocyclic lactones in Angelica qualities

References: Carbonnier J. & Molho D. (1982). Contribution l'tude des furanocoumarines du genre Angelica. Distribution du beta-cyclolavanduliloxy-5-psoralne. Planta Medica 44, 162-165.

EMEA Medicines Agency, Committee on Herbal medicine Products (2007) Reflection paper on the risks of furocoumarins contained in preparations of Angelica archangelica L. EMEA/HMPC/317913/2006.

Harkar, S.; Razdan, T. K.; Waight, E. S. (1984) Steroids, chromones, and coumarins from Angelica officinalis. Phytochemistry 23, 419-426.

Harmala, P. (1991): Study on the isolation and chromatographic behaviour of coumarins from Angelica (Angelica archangelica) roots. Thesis, 58 p., University of Helsinki, J-Paino Ky, Helsinki.

Harmala P., Kaltia S., Vuorela H. & Hiltunen R. (1991) "A furanocoumarin from Angelica archangelica." Planta Med. Planta Med. 58(3), 287-9.

Harmala P., Vuorela H., Nyiredy S.Z., Tornquist K., Kaltia S., Sticher O. & Hiltunen R. (1992) "Strategy for the isolation and identification of coumarins with calcium antagonistic properties from the roots of Angelica archangelica." Phytochem. Anal. 3(1), 42-48.

Leung A.Y. & Foster S. Encyclopaedia of Common Natural Ingredients used in Food, Drugs & Cosmetics 2nd edn. Wiley-Interscience pub. 1996.

Piao X.L., Baek S.H., Park M.K. & Park J.H.(2004) "Tyrosinase-inhibitory furanocoumarin from Angelica dahurica." Biol Pharm Bull. 27(7), 1144-6. Abstract. An extract of the roots of Angelica dahurica Benth. et Hook. (Umbelliferae) showed potential tyrosinase inhibition against mushroom tyrosinase. Employing a bioassay-linked HPLC method, followed by semipreparative HPLC, the active principle was isolated and characterized as 9-hydroxy-4-methoxypsoralen.

Seo E.K., Kim K.H., Kim M.K., Cho M.H., Choi E., Kim K. & Mar W. (2002).. Planta Med. 68, 162163.

Steck W. & Bailey B.K. (1969). Leaf coumarins of Angelica archangelica. Canadian J. Chemistry 47, 2425-2430.

Tisserand R.B. & Balacs T. (1995) Essential oil safety; a guide for healthcare professionals. Churchill-Livingstone 1995. .

Woo W.S., Shin, K.H. & Lee C.K. (1983) Effect of naturally occurring coumarins on the activity of drug metabolizing enzymes. Biochem. Pharmacol. 32(11), 1800-1803.

Zobell A.M. & Brown S.A. (1991) Furanocoumarin concentrations in fruits and seeds of Angelica archangelica. Environmental and Experimental Botany 31(4), 447-452.

Angelicin (isopsoralen). CAS n: 523-50-2. Cropwatch Summary: Angelicin is an angular furanocoumarin. Angular furanocoumarins normally only produce DNA-adducts, whereas linear

furanocoumarins produce DNA-adducts and the potentially more toxic DNA-DNA interstrand cross links.

Occurrence: Angelicin occurs to 230ppm in angelica root oil & 430ppm in rue oil (IFRA 2007). Angelicin is absent in citrus oils (IFRA 2007).

Phototoxicity: There is limited evidence of carcinogenicity of angelicin to experimental animals in combination with UV radiation; inadequate evidence of carcinogenicity to experimental animals in absence of UV radiation (IARC 1986). No evaluation of carcinogenicity of angelicin could be made to humans (IARC 1986).

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IARC (1986) Angelicin & some synthetic derivatives. Vol 40 (1986), 291.

Aniseed oil. Pimpinella anisum L. CAS n: 8007-70-3; EINECS CAS No: 84775-45-1 Cropwatch summary: According to Leung (1996), plant tissues contain bergapten; however the steam distilled seed oil is not traditionally considered phototoxic.

Leung A.Y. & Foster S. Encyclopaedia of Common Natural Ingredients used in Food, Drugs & Cosmetics 2nd edn. Wiley-Interscience pub. 1996.

Anti-carcinogenic / anti-genotoxic effects of furanocoumarins. Imperatorin shows anti-carcinogenic effect against chemically induced cancer in a murine model (Prince et al. 2006), and, as does isopimpinellin, when administered in the diet (Kleiner et al. 2004). Kleiner et al. (2003) showed furanocoumarins are capable of inhibiting carcinogen-metabolising enzymes, & more specifically that bergamottin selectively inhibits tumor formation by benzo[]pyrene, and that imperatorin & isopimpinellin block tumor initiation by both benzo[]pyrene & 7,12-dimethylbenz[]anthracene.

Bakkali F, Averbeck S, Averbeck D, Idaomar M. (2008) "Biological effects of essential oils - a review." Food Chem Toxicol. 46(2),:446-75. Abstract Since the middle ages, essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries. Because of the mode of extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components. In vitro physicochemical assays characterise most of them as antioxidants. However, recent work shows that in eukaryotic cells, essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria. Depending on type and concentration, they exhibit cytotoxic effects on living cells but are usually non-genotoxic. In some cases, changes in intracellular redox potential and

mitochondrial dysfunction induced by essential oils can be associated with their capacity to exert antigenotoxic effects. These findings suggest that, at least in part, the encountered beneficial effects of essential oils are due to prooxidant effects on the cellular level.

Bakkali F., Averbeck S., Averbeck D., Zhiri A. & Idaomar M. (2005) "Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae." Mutat Res. 585(1-2):1-13. Abstract. In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum >Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.

Bode C.W., Zager A. & Hansel W. (2005) "Photodynamic and photo-cross-linking potential of bergamottin." Pharmazie. 60(1), 78-9. Abstract. Bergamottin (5-geranoxypsoralen) is a main component of bergamot and grapefruit oil. In order to investigate the photophysical and photochemical behaviour of bergamottin, absorption and fluorescence properties, production of singlet oxygen and superoxide radical anions and further cross-linking of DNA were studied. Strong photochemical reactions were not observed.

Cai Y., Baer-Dubowska W., Ashwood-Smith M. & DiGiovanni J. (1997a) Inhibitory effects of naturally occurring coumarins on the metabolic activation of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in cultured mouse keratinocytes. Carcinogen 18, 215-222. Abstract. Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and

coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons.

Cai Y., Kleiner H., Johnston D, Dubowski A., Bostic S., Ivie W. & DiGiovanni J. (1997b) Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo(a)pyrene and 7,12-dimethylbenz(a)anthrecene in SENCAR mice. Carcinogen 18, 1521-1527, 1997. Abstract. Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.

Kim Y-K., Kim Y.S & Ryu S.Y. (2006) "Anti-proliferative effect of furanocoumarins from the root of Angelica dahurica on cultured human tumor cell lines." Phytotherapy Research 21(3), 288-290 Abstract. A bioassay-guided fractionation of the root extract of Angelica dahurica (Umbelliferae) led to the isolation of six furanocoumarins as active ingredients responsible for the antitumoral property. The hexane soluble part of the extract demonstrated a signicant inhibition on the proliferation of cultured human tumor cells such as A549 (non small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nervous system) and HCT-15 (colon) in vitro, whereas the remaining water soluble part exhibited poor inhibition. Intensive investigation of the hexane soluble part of the extract yielded six furanocoumarins, i.e. isoimperatorin, cnidicin, imperatorin, oxypeucedanin, byakangelicol, oxypeucedanin hydrate, all of which exhibited a signicant inhibition on cell proliferation in a dose-dependent manner.

Kleiner H.E., Vuimiri S.V., Miller L., Johnson Jr. W.H., Whitman C.P. & DiGivanni J. (2001) Oral administration of naturally occurring coumarins leads to altered phase I & phase II enzyme activities & reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues

of SENCAR mice. Carcinogenesis 22(1), 73-82. Abstract. Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.

Kleiner H.E., Reed M.J. & DiGiovanni J. (2003). Naturally occurring coumarins inhibit human cytochromes P450 and block benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene DNA adduct formation in MCF-7 cells. Chem Res Toxicol. 16(3), 415-22. Abstract. Naturally occurring coumarins (NOCs) inhibit polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by blocking cytochrome P450 (P450)-mediated bioactivation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA). Bergamottin selectively inhibits tumor initiation by B[a]P, whereas imperatorin and isopimpinellin inhibit tumor initiation with both carcinogens. The goals of the current study were to examine the ability of NOCs to inhibit human P450s in vitro and to establish whether NOCs, which are anticarcinogenic in mice, can block carcinogen bioactivation in cultured human cells. For the initial experiments, incubations containing 5 microM P450, P450 substrate, an NADPH generating system, and NOCs were used to determine the concentrations of each inhibitor that blocked 50% of P450 activity (IC(50)). These results confirmed that NOCs are capable of inhibiting multiple human P450s and that they exhibit selectivity for certain isoforms of human P450s. In subsequent experiments, we examined the effects of bergamottin, imperatorin, and isopimpinellin on DMBA and B[a]P DNA adduct formation in the human breast MCF-7 adenocarcinoma cell line. Coincubation of cells with the three different NOCs significantly inhibited DMBA DNA adduct formation by 29-82% at doses ranging from 2 to 10 microM and significantly inhibited B[a]P DNA adduct formation by 37-80% at doses ranging from 20 to 80 microM. HPLC analysis of the DNA hydrolysates demonstrated that inhibition of DNA adducts corresponded to inhibition of the major B[a]P and DMBA diol-epoxide-derived adducts. Although bergamottin was not effective at blocking DMBA bioactivation in the mouse skin model, it was similar in effectiveness to imperatorin and isopimpinellin in MCF-7 cells. These results demonstrate that NOCs, which are present in citrus fruits and other components of the human diet, are capable of inhibiting carcinogen metabolizing enzymes and blocking bioactivation of both B[a]P and DMBA in MCF-7 cells.

Prince M., Campbell C.T., Robertson T.A., Wells A.J. & Kleiner H.E. (2006) "Naturally occurring coumarins inhibit 7,12-dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland." Carcinogenesis. 27(6), 1204-13. Abstract. Naturally occurring coumarins (NOCs) are anti-carcinogenic in the mouse skin model. To characterize the chemopreventive potential of NOCs

against breast cancer, we first examined their effects on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation in mouse mammary gland. We hypothesized that those NOCs that both inhibited cytochrome P450 1A1/1B1 and induced hepatic glutathione S-transferases (GSTs) would be the most effective in blocking DMBA-DNA adduct formation in mouse mammary gland. To address this hypothesis, simple coumarins (e.g. coumarin and limettin, which induced mouse hepatic GSTs but had little effect on P4501A1/1B1) and linear furanocoumarins (e.g. imperatorin and isopimpinellin, which induced hepatic GSTs and were potent inhibitors of P4501A1/1B1) were compared. Mice were pretreated with NOCs (150 mg/kg body wt, by gavage) prior to either a single dose of DMBA (50 microg) or multiple doses of DMBA (20 microg daily for 3 and 6 weeks). Mammary DMBA-DNA adduct formation was quantitated by the nuclease P1-enhanced 32P-postlabeling assay. With the single dose of DMBA, coumarin, limettin, imperatorin and isopimpinellin inhibited DMBA-DNA adduct formation by 50, 41, 79 and 88%, respectively. Coumarin, limettin and imperatorin blocked DMBA-DNA adduct formation by 36, 60, and 66% at 3 weeks, and by 0, 49 and 55% at 6 weeks of DMBA dosing, respectively. In a 6 week dose-response study of select NOCs and 7,8-benzoflavone (a potent P4501 inhibitor that had little effect on GSTs), DMBA-DNA adduct formation was inhibited by 0, 43 and 24% in the limettin groups; by 26, 26 and 69% in the isopimpinellin groups; and by 80, 96 and 97% in the 7,8- benzoflavone groups at 35, 70 and 150 mg/kg, respectively. Taken together, these results suggest that linear furanocoumarins had a greater inhibitory effect on DMBA-DNA adduct formation in mouse mammary glands compared with simple coumarins, and that the predominant effect may be P4501 inhibition.

Wang, X. (2006) "Cancer Preventative Agents. Part 6: Chemopreventative potential of furanocoumarins and related compounds Pharmaceutical Biology 44(2) 116-120.

Xuejun Y., Rende X., Dexiang L., Xinyong Z., Yonghua X., Ying H., Rui W. & Hechuan W. (1991) "A study on the antimutagenicity of chemical compounds extracted from Cnidium monniery (L.) Cuss. used in Chinese Traditional Medicine. Carcinogenisis, Teratogenesis & Mutagenesis 11(2), 55-71. Abstract. The antimutagenicity of 9 kinds of chemical compounds (osthol , bergaten , isopimpinellin , imperatorin , xanthotoxol , xanthotoxin , thymine , uracil and unknown one that need authenticating) was studied with the Ames test and the micronucleus and chromosomal aberretion assays in mice in vivo. These compounds were isolated from the water extract of Cnidium monnieri (L.) Cuss in Chinese traditional medicine. The results showed that osthol. , bergapten , isopimpinellin , imperatorin , xanthotoxol and xanthotoxin had inhibited activity of mutagenicity of aflatoxin B1 . And osthol , bergapten , isopimpinellin , imperatorin also had high activity in inhibiting the chromosomal aberrations and micronuclei that induced by cyclophosphamid in vivo. The other three compounds (thymine , uracil and unknown one that need authenticating) showed no antimutagenicity.

Bergamot oil - cold pressed (Sicily, Ivory Coast, S. America) Citrus aurantium L. subsp. bergamia (Risso et Poit.) Engl. CAS n : 8007-75-8 (or 900807-75-8); EINECS-CAS n 89957-91-5. Cropwatch summary: Phototoxicity. A string of older studies (see BoDD website) show that bergamot oil-containing perfumes are phototoxic. It must be remembered however that bergamot oil concentrations in fragrances were likely to be higher during that former period; there have been restrictions on bergamot oil concentrations deployed since then, & the use of so-called furanocoumarin-free types with lower total furanocumarin concentrations, may have subsequently reduced any adverse reaction frequency. Photoxicity of bergamot oil was demonstrated on mouse skin when irradiated with UV light (Opdyke 1973, part-based on a report from Forbes & Urbach 1972) Opdyke also reported that soaps, creams & lotions & perfumes normally contain 0.05% to 0.12%, 0.05% to 0.25%, and 0.20

to 3.00% of bergamot oil respectively in the finished products. Girard et al. (1979) compared the relative photosensitivity of guinea-pig & human skins to simulated solar radiation, finding human skin slightly more sensitive. The authors concluded that guinea-pig skin would serve as a good model for estimating the the phototoxicity of psoralen-containing oils. Girard et al. (1981) concluded that bergamot oils containing less than 5ppm bergapten did not cause adverse phototoxic effects to either guinea-pigs or humans.

IFRA restricts bergamot oil expressed to 0.4% concentration in fragrances not washed off skin, & exposed to sunshine.

On the other hand, there is a body of opinion which has long disputed the phototoxicity of bergamot oil. Campanella (1973) reported on a study of the bergamot-treated skin of human volunteers, the affected area then being subjected to UV radiation, and concluded there was little danger of phototoxicity from bergamot oil. Capua (1974) argued against many phototoxic findings for bergaptene & bergamot oil, but the scientific community founds these arguments inconvenient and ignored them. Bergamot oil samples can vary in phototoxic capacity Zaynoun et al. (1974) - through BoDD. Others have argued that bergamot oil is not cytotoxic or mutagenic in the absence of light (Dijoux et al. 2006) or have reported that acute phototoxic side effects from perfumes are rare, and that repeated exposure to sunlight after perfume application is uncommon. The fact that no reports of increases in epithelial tumour formation on areas of skin habitually exposed to perfume has also been reported (Dubertret et al. 1990 & Chouroulinkov et al. through Lawrence 1991).

Further despite its increasing application, there are only a few recent reports of phototoxic reactions to bergamot aromatherapy oil. - Kaddu et al. (2001). Bergamot oil was stated to be only known as a mildly phototoxic ingredient (Nathalie et al. 2006).

Composition. Porcaro & Shubiak (1974) determined begapten at between 0.162% to 0.357% in six commercial bergamot oils by HPLC; bergapten levels in two debergaptenised oils were determined at 0.017% to 0.018%. Shu et al. (1974) determined bergapten concentrations in bergamot oil at 0.28% to 0.33% by HPLC. Calabro & Di Giacomo & Calvarano (1978) analysed 125 samples of bergamot oil by HPLC where the bergapten content was found to be 0.211% to 0.456%; whereas after treatment to remove bergapten the bergapten content came in at 0.042% to less than 0.0004%. Curro (1979) determined the following FC concentrations in the non-volatle portion of bergamot oil by spectrofluorimetery: citropen 0.14% to 0.24%, 7-methoxy-5-geranoxycoumarin 0.12% to 0.15%; 5-MOP 0.15 to 0.33% & bergamottin 1.4% to 2.2%. The `same year Calvarano et al (1979) determined the FCs of 100 bergmat oils finding citropen 0.233% 0.03%, bergamottin 2.03% 0.38% & bergapten 0.247 0.048%. Again in 1979 Suzuki determined the bergapten contents of seven bergamot oils, finding values between zero and 1130ppm by a modified GC method. McHale & Sheridan (1989) found 1830

mg/100 g of bergamottin and 270 mg/100 g. of bergapten in expressed bergamot oil. Poiana et al. (1993) had determined the bergapten content of bergamot oil at 0.3-0.4%. Vernin et al. (1979) determined the citropen & bergapten content of various citrus oils, finding a citropen content of 0.50% to 0.75%, and a bergapten content of 0.60% to 0.87%. Di Giacomo (1981), reviewing the Italian bergamot industry, found the bergapten content of bergamot oils determined by HPLC in the range 0.204% to 0.475%.

Mondello et al. (1992) analysed 128 genuine bergamot oils from Calabria (1992 production) & found bergamottin (1.02% to 2.75%) 5-geranyloxy-7-methoxycoumarin (0.08% to 0.22%), citropten (0.14% to 0.35%) & bergapten (0.11 to 0.32%). Citropen & bergapten content was found to decrease seasonally from Jan. to Mar.

Duclos (2000) stated that the bergaptene content in bergamot oil (Italian) was 0.279% & that of bergamot oil Ivory Coast was 0.203-0.366%.

According to the SCC(NF)P 0740/03, bergamot oil - type, processing details, & origin not specified - contains 2.2% bergamottin,

RIFM give the following data measured by the fragrance industry (??) for bergamot oil (but no process or geographical source details given): psoralen 0-20ppm, bergapten up to 2300ppm, bergamottin up to 22,000ppm; xanthotoxin, isopimpinellin, oxypeucedanin & angelicin not detected (RIFM Fact Sheet No 3.).

Cropwatchs Perfumery Notes: (from Cropwatch Newsletter April 2007). Bergamot oil has both a citrus & herbaceous character, and mixes seamlessly with the palette of citrus oils and many herbaceous notes (especially lavender & basil) making it particularly useful in masculine fragrances. It is also is useful in fresh top note accords in floral fragrances. It also mixes well with mossy and ambery notes, and because it has this herbal dimension to its character, it is the cornerstone of the eau de cologne & chypre perfumes. Its place in perfumery is unique. Its employment in male fragrances is virtually ubiquitous and represents a lot of the fresh fragrance character - examples CK One (Calvin Klein 1994), Cool Water (Davidoff 1988), Eau Savage (Dior 1966), and in female fragrances it is also virtually ubiquitous as part of top note accords e.g. Chanel 19 (Chanel 1970), Anais-anais (Cacharel 1979), Rive-Gauche (Y. Saint-Laurent 1971), Obsession (Calvin Klein 1985). Bergamot oil is also of course used to flavour Earl Gray tea but no doubt suitable reasons will appear to prevent this flavoured beverage from being banned. However, in perfumery, bergamot oil is not capable of being replaced.

Calabro G. & Curro P. (1975) Spectrofluorometric determination of coumarins in bergamot essential oil. Essenz. Deriv. Agrum. 45, 246-262.

Calvarano I, Ferlazzo A. & Di Giacomo (1979) Coumarin & furocoumarin content of bergamot oil. Part I Bergamottin, citropen & bergapten. Rivista Ital. 61,.258-261.

Campanella P. (1973) Photosensitivity & bergamot. Essenze. Deriv. Agrum. 43, 32-38.

Capua M. (1974) Considerations on the phototoxicity of bergamot oil. Paper No !00, VIth Congress of Essential Oils, San Francisco 1974.

Chouroulinkov I., Lasne C. & Nguyen-Ba (1989) Study with 5-MOP, bergamot & Bergasol in mouse skin carcinogenicity tests. In Psoralens: Past, Present & Future of Photochemoprotection & other biological activities. eds: T.B. Fitzpatrick, F. Forlot, M.A. Pathak & F. Urbach pp345-355. John Libby Eurotext. Paris.

Clark S.M. & Wilkinson, S.M. Phototoxic contact dermatitis from 5-methoxypsoralen (bergapten) in aromatherapy oil. Contact Dermatitis (1998) 38 (5), 289-90

Corr S. (2005) 1,1,1,2-Tetrafluoroethane (R-134a): A selective solvent for generation of flavour & fragrance ingredients. In Natural Flavours & Fragrances, Chemistry, Analysis & Production eds Carl Frey & Russell L. Rouseff ACS Symposium Series No 908 ACS, Washington DC.

Di Giacomo A. & Calvarano I. (1974) Thin-layer chromatography & spectrofluorimetry of bergamot oil. Essenz. Deriv. Agrum. 44, 329-344.

Di Giacomo A. (1979) "Situation actuelle de l'industrie de la bergamot en Calabre" Rivista Ital 63, 300-305.

Dijoux N., Guingand Y., Bourgeois C., Durand S., Fromageot C., Combe,C. & Ferret P.J., (2006) "Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay." Toxicol. in Vitro 20, 480489. Abstract. When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.

Dubertret L., Morlire P., Averbeck D. & Young A.R. (1990) The photochemistry and photobiology of bergamot oil as a perfume ingredient: an overview. J Photochem Photobiol B. 7(2-4), 362-5.

Dubertret L., Serraf-Tircazes D., Jeanmougin M., Morliere P., Averbeck D. & Young A.R. (1990) Phototoxic properties of perfumes containing bergamot oil on human skin. Photoprotective effect of UVA and UVA substances. J. Photochem. Photobiol. B: Biology. 7, 251-259. Abstract. As part of an international cooperative study of the photophysical, photomutagenic and photocarcinogenic properties of bergamot oil and the effect of UVA and UVB sunscreens, the phototoxic properties of model perfumes containing 5, 15 and 50 ppm 5-methoxypsoralen (5-MOP) in bergamot oil with and without a sunscreen have been investigated on human skin. It has been confirmed that the photosensitivity of human skin is maximal 2 h after perfume application. Interestingly the addition of a UVA sunscreen is more efficient for decreasing the phototoxic properties of bergamot oil than is a UVB sunscreen. The addition of sunscreens in a model perfume containing 50 ppm 5-MOP in bergamot oil can reduce the phototoxic properties of this perfume to a toxicity equivalent to that produced by the application of a model perfume containing 15 ppm 5-MOP without sunscreens. However, despite their promising protective effect in vitro,

UVB and UVA sunscreens at low concentration (0.5%-1%) in perfumes cannot suppress the phototoxicity of bergamot oil on human skin.

Duclos T. (2000) Bergamot & other citrus from Ivory Coast International Citrus Symposium Orlando, February 15 - 18, 2000. [Quote:from Duclos: Bergaptene content Bergamot oil Italy 0.279%; Bergaptene content Bergamot oil Ivory Coast 0.203-0.366%]

Girard J., Unkovic J., Delahayes J. & Lafille C. (1979) Experimental study on bergamot oil phototoxicity correlations between man & guinea pig. Dermatologica 158, 229-243. Abstract. Phototoxicity of bergamot oil in solar simulating radiation (SSR greater than or equal to 290 nm) and in long ultraviolet radiation (LUV greater than or equal to 320 nm) has been compared by studying photoaugmentation of erythema in the guinea pig after 24 h and pigmentary photoaugmentation in man on the 8th day. The results show that a close relationship exists between guinea pig and human responses, with both radiations used, and that man seems to be slightly more sensitive to phototoxic effects of bergamot oil than the guinea pig. This difference of sensitivity necessarily implies the participation of UVA (320--400 nm) in the phototoxic reaction of bergamot oil with solar radiation. This UVA participation is particularly obvious in the guinea pig; in man, the results are less clear and a certain synergy of UVB rays (290--320 nm) may be involved in the phototoxic UVA-induced reaction of bergamot oil. Despite these slight differences, the erythematous reaction in the guinea pig appears to be a remarkable experimental model to show out potential phototoxic reactions of products containing psoralens in man.

Girard J., Unkovic J., Guimbard J.P. & Boccio E. (1981) Interest in the cosmetics of a non-phototoxic bergamot oil. Parfum, Cosm. Armes 38, 39-44.

Gattuso G, Barreca D, Caristi C, Gargiulli C. & Leuzzi U.(2007) "Distribution of flavonoids and furocoumarins in juices from cultivars of Citrus bergamia Risso." J Agric Food Chem. 55(24), 9921-7. Abstract. HPLC separation of flavonoids and furocoumarins in the crude juices of three cultivars of Citrus bergamia Risso ("Castagnaro", "Fantastico", and "Femminello") was carried out on a C18 reversed phase column. The analysis was performed in a single run using a DAD detector coupled with an ESI-MS-MS source. Two furocoumarins (bergapten and bergamottin) were detected and quantified simultaneously with the sixteen flavonoid components previously found in industrial bergamot juice. Full characterization of the furocoumarins was performed by (1)H NMR analysis on samples separated by means of preparative HPLC. The free-radical scavenging ability of cultivar juices was assessed by using DPPH radical. The data presented show that the "Femminello" cultivar, even though it is the least common of the three, is by far the richest in health-promoting bioactive compounds (both flavonoids and furocoumarins). Given the range of applications of furocoumarins, the preparative separation described herein is proposed as a simple and rapid method to obtain this class of compounds in good yield from crude juice.

Kaddu S., Kerl H., & Wolf P. (2001) Accidental bullous phototoxic reactions to bergamot aromatherapy oil. J. Am. Acad Dermatol 45(3), 458-461. Abstract. Oil of bergamot is an extract from the rind of bergamot orange (Citrus aurantium ssp bergamia) that has a pleasant, refreshing scent; until a few years ago it had been widely used as an ingredient in cosmetics but was restricted or banned in most countries because of certain adverse effects. More recently, oil of bergamot preparations have been gaining renewed popularity in aromatherapy. Oil of bergamot possesses photosensitive and melanogenic properties because of the presence of furocoumarins, primarily bergapten (5-methoxypsoralen [5-MOP]). However, 5-MOP is also potentially phototoxic and photomutagenic. Despite its increasing application, there are only a few recent reports of phototoxic reactions to bergamot aromatherapy oil. We describe two patients with localized and disseminated bullous phototoxic skin reactions developing within 48 to 72 hours after exposure to bergamot aromatherapy oil and subsequent ultraviolet exposure. One patient (case 2) had no history of direct contact with aromatherapy oil but developed bullous skin lesions after exposure to aerosolized (evaporated) aromatherapy oil in a sauna and subsequent UVA radiation in a tanning salon. This report highlights the potential health hazard related to the increasing use of psoralen-containing aromatherapy oils.

McHale, D. & Sheridan, J. B. (1989) "The Oxygen Heterocyclic Compounds of Citrus Peel Oils." In: Proceedings of 11th International Congress of Essential Oils, Fragrances, and Flavors eds Bhattacharyya S. C., Sen N., Sethi K. L. Oxford & IBH, New Delhi, India, 1989; Vol. 5, pp 63-71.

Mondello L., d'Alcontres I.S., Del Duce R., & Francesco Crispo (1993) "On the genuineness of citrus essential oils. Part XL. The composition of the coumarins and psoralens of Calabrian bergamot essential oil (Citrus bergamia Risso)." Flav & Frag J. 8(1), 17-24. Abstract. The content of bergamottin, 5-geranyloxy-7-methoxycoumarin, citropten and bergapten in Calabrian bergamot essential oil was determined by HPLC. 128 genuine bergamot essential oils, cold-pressed during the year 1992, were analysed; for each sample the date and area of production of the fruits is known. The percentage of bergamottin ranges between 1.02% and 2.75%. 5-geranyloxy-7-methoxycoumarin between 0.08 and 0.22%, citropten between 0.14% and 0.35%, bergapten between 0.11 and 0.32%. The mean content of bergamottin and 5-geranyloxy-7-methoxycoumarin did not vary during the production period, while that of citropten and bergapten decreased considerably from January to March. There does not seem to be any evident correlation between the production areas of the fruits and the coumarin composition. The presence of some peaks absent in genuine bergamot essential oils, has been detected in three bergamot oils, available on the market, probably because of lime oil addition.

Moysan A, Morlire P, Averbeck D. & Dubertret L. (1993) "Evaluation of phototoxic and photogenotoxic risk associated with the use of photosensitizers in suntan preparations: application to tanning preparations containing bergamot oil." Skin Pharmacol. 6(4), 282-91. Abstract. Bases for the elaboration of a standardized protocol are proposed for studying phototoxic effects of skin tanning preparations containing photosensitizing agents. The experimental procedure includes in vivo phototoxicity tests, evaluation of the photogenotoxic risk and determination of the photosensitizer concentration in plasma after topical application. This procedure was carried out with tanning preparations containing a well-known photosensitizer, 5-methoxypsoralen, as a component of bergamot oil. The whole study has been performed using topical application of the commercial suntan product, i.e. containing the sunscreens and all other components. Whereas the exposure to solar simulated radiation never triggered any phototoxic response, a photosensitizing effect was observed for skin type I volunteers exposed to high doses of ultraviolet A. The transepidermal penetration resulted in a 5-methoxypsoralen concentration of 1-4 ng/ml in the suction blister fluid. The photogenotoxicity of this suction blister fluid containing 5-methoxypsoralen and also other ingredients of the tanning preparation was assayed on yeast cells and was found to be rather low. 5-Methoxypsoralen was also detected in plasma after repeated applications but at low concentrations (about 1 ng/ml) which do not present a potential risk for systemic ocular effects.

Nathalie D., Yannik G., Caroline B., Sandrine D., Claude F., Corrine C., Pierre-Jacques F. Assessment of the Phototoxic Hazard of Some Essential Oils using Modified 3T3 Neutral Red Uptake Assay. Toxicol in Vitro 20(4), 480-489.

Nesci F.S. (1994). Il bergamotto: dall'azienda agraria al mercato problemi attuali e prospettive future.stazione sperimentale per l'industria delle essenze e dei derivati agrumari (Reggio Calabria). Collana di monogragie sugli oli essenziali e sui derivati agrumari, 10, 8-51.

Opdyke D.L.J. (1973) Bergamot oil expressed Food Cosm. Toxicol. 11, 1031.

Poiana M., Crispo F., Manziu E., Sicari V. & Mincione B. (1993) , in E. Reverchon & A. Schiraldi (eds) Atti de1 II Congresso i Fluidi Supercritici e le Loro Applicazioni, Ravello, 20-22 June 1993, p. 113.

Porcaro P.J. & Shubiak P. (1974) Liquid chromatographic determination of bergapten content in treated or natural bergamot oils. J.A.O.A.C. 57, 145-147.

Rouzet M.. Glandian R., Pouchus F. & Corneteau H. (1980): Paper presented at Symposium, Recherches et Techniques en Cosmetologie; Le Substances Naturelles. Orleans. 21-23 April, 1980.

Suzuki H (1979) Selective extraction & determination of bergapten, a photosensitive substance from bergamot oil as well as cosmetics containing bergamot oil. J.Soc. Cosm. Chem (Japan) 13, 57-61.

Verzera A., Trozzi A., Gazea F. Cicciarello G. & Cotroneo A. (2003) "Effects of rootstock on the composition of bergamot (Citrus bergamia Risso et Poiteau) essential Oil." J. Agric. Food Chem. 51 (1), 206 -210. Abstract. This paper reports the composition of bergamot oils obtained from plants grafted on the following rootstocks: sour orange, Carrizo citrange, trifoliate orange, Alemow, Volkamerian lemon, and Troyer citrange. The aim of this study is to evaluate the possibility of using rootstocks other than sour orange, checking their effect on the composition of the essential oil. Results are reported for analysis of 203 bergamot oils during the years 1997-1998, 1998-1999, and 1999-2000. The oils were analyzed by HRGC and HRGC/MS; 78 components were identified, and the results were in agreement with those reported in the literature for the Calabrian bergamot oils obtained from industry. Because of the quality of their essential oils, Alemow and Volkamerian lemon can be considered as substitutes for sour orange rootstocks.

Vernin G., Bianchini J.P. & Siouffi A. (1979) Methodes du dosage du bergaptene et du citropene dans les essences de bergamote tude comparative. Parfum, Cosm, Arm 30, 49-55.

Young A.R, Walker S.L., Kinley J.S. et al. (1990) "Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil. Evaluation and modiacation of risk of human use in an albino mouse skin model." J Photochem Photobiol. 7(2-4), 231-50. Abstract. The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle (i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations. Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations. Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.

Zaynoun S., Hall I., Johnson B.E. & Frain-Bell W. (1974) A study of bergamot sensitivity Brit J. Derm. 91(Suppl 10),14. Abstract. Using a modified open photopatch test technique, the phototoxic reactions produced by bergamot oil bergapten (5-methoxypsoralen) the active component of the oil and xanthotoxin (8-methoxypsaralen) was studied. The` reaction was affected by a range of factors such as the vehicle (PMF or ethanol), the concentration of ethanol in the vehicle, the skin site, the interval between application of the psoralen & irradiation, the hydration of the skin, and the degree of natural or sun-induced pigmentation. Repeated photopatch testing at the same skin site produced an increase in sensitivity. Eye colour, natural susceptibility to suntanning, age & sex had no effect on the phototoxic response to psoralens.

Zaynoun S.T., Johnson B.E. & Frain-Bell W. (1977) "A study of oil of bergamot and its importance as a phototoxic agent. I. Characterization and quantification of the photoactive component." Br J Dermatol. 96(5), 475-82. Abstract. It is important in the technique of photopatch testing, using oil of bergamot, to have determined the concentration of the active psoralen, bergapten, so as to avoid false negative responses in the assessment of phototoxic reactions. Techniques for assessing the phototoxic components of oil of bergamot are described and quantitative analyses of bergapten, the only significant photoactive compound in the samples examined, are reported. The phototoxicity of bergapten was found to be nearly the same as that of xanthotoxin (8-MOP) in tests on human skin.

Bergamot oil distilled. Cropwatch summary: Rectified oils may contain approx. 300ppm bergaptene according to one Italian manufacturer; however up to now distilled oils have generally been assumed to be non-phototoxic e.g. by S. Arctander (through Opdyke 1973). Distilled bergamot oil was determined to have: bergapten 0 to 41ppm; bergamottin 0 to 3017ppm; 5-geranyloxy-7-methylcoumarin 0 to 349ppm: see section below under Dugo (1999).

Bergamot oil FCF (FuranoCoumarin-Free). Cropwatch summary Bergamot FCF oils contain from zero to 9-10 ppm bergaptene when pure (Cropwatch consensus of several citrus manufacturers, 2007). However much of the commercially available bergamot oil FCF is adulterated, lowering the FC content further. Higher boiling fractions of distilled bergamot oil (which contain valuable odour compounds) may be treated by column chromatography, chemo-absorbtion techniques or by solvent extraction techniques to reduce the overall FC content. These FC-stripped higher boiling fractions may subsequently be added back to the bulk (thanks to Daniel Joulain & others for this information). Dugo et al. (1999) gave the following information for FuranoCoumarin Free bergamot oil:

(a) sodium hydroxide treated bergamot oil: bergapten 0 to 91ppm; bergamottin 11,726 to 16,250ppm, 5-geranyloxy-7-methylcoumarin 1539 to 1975 ppm. (b) distilled bergamot oil: bergapten 0-41ppm; bergamottin 0 to 3017ppm; 5-geranyloxy-7-methylcoumarin 0 to 349ppm.

Porcaro & Shubiack (1974) had previously determined the begapten content of FCF bergamot oil at 170 to 180ppm; Di Giacomo & Calvarano (1978) had determined the bergapten content at tr. to 420ppm; J. Anal. Stds Committee (1987) had determined the bergapten content at 1.7 to 5.7ppm.

Removal of photoxic furanocoumarins such as bergapten (and citropen) from natural oils such as bergamot said to have little impact on the compostion or fragrance: Corr S. (2005) Cropwatch comments: we doubt that this is true to the more discerning nose!

Corr S. (2005) 1,1,1,2-Tetrafluoroethane (R-134a): A selective solvent for generation of flavour & fragrance ingredients. In Natural Flavours & Fragrances, Chemistry, Analysis & Production eds Carl Frey & Russell L. Rouseff ACS Symposium Series No 908 ACS, Washington DC.

Di Giacomo & Calvarano (1978) The bergapten content in cold-extracted bergamot essential oil. Essez. Deriv. Agrum. 48, 51-83.

Dugo P., Mondello L. Proteggente A.R. & Dugp G. (1999) "Oxygen heterocyclic compounds of bergamot essential oils." EPPOS 27, 31-41.

Porcaro P.J. & Shubiack P. (1974) Liquid chromatic determination of bergapten content in treated or natural bergamot. J. Assocn Off Anal Chem 57, 145-147.

Bergamottin (5-geranoxypsoralen). CAS n: 7380-40-7; EC n: 200-782-5 Stability. Bergamottin is not particularly stable & tends to decompose during GC run conditions, making GC estimation unreliable. Morliere et al. (1990) found that not only was bergamottin responcible for about two-thirds of the absorption of UVA and UVB light by bergamot oil, but that bergamottin in bergamot oil was unstable to UV light in 80% ethanol. Under mildly acidic conditions bergamottin can be converted to bergaptol (Stanley & Vannier 1957).

Occurrence. The SCC(NF)P Opinion SCC(NF)P/0740/03 (adopted Oct 2003) quoted the bergamottin concentration at 2.2% in bergamot oil & 2.5% in cold-pressed lime oil (but with no clear references to the processing, geographical or botanical identity of the citrus species, or the source for this data provided). Bergamottin was found at 1253.37ppm by UV-DAD in cold-expressed Californian lemon oil Citrus limonum (L.) (N. L. Burman), at 428.6ppm in cold-pressed Israeli grapefruit oil Citrus paradisis Macf syn. Citrus decumana L., and at 111.6ppm in cold-pressed bitter orange oil Citrus aurantium L. subsp. amara. (Frrot & Decorzant 2004). Alkaline treatment of expressed bergamot oil to remove bergapten still leaves bergamottin as major furanocumarin (Frrot & Decorzant 2004).

Phototoxicity. The lack of interaction of bergamottin with DNA has been noted (Aubin et. al. 1994; Bode et al. 2005; Morliere et al. 1991). The SCC(NF)P Opinion 0740/03 notes the lack of phototoxicity when tested on rabbit & guinea pig skin (Naganuma et al. 1985), & that bergamottin inhibits cytochrome P450 CYP3A and CYP1A1/2 enzymatic activities and induction of the corresponding proteins and mRNAs (see Cai et al 1997a; Cai et al 1997b). The SCC(NF)P concludes from this: this finding may explain why bergamottin inhibited metabolic activation of benzo(a)pyrene in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from benzo(a)pyrene. The SCC(NF)P Opinion SCCPNFP/0740/03 concluded overall that there was insufficient information (as submitted by EFFA) on bertgamottins photo-toxic potential to come to an opinion regarding its safety in cosmetic products. RIFM subsequently commissioned David Kirkland of Covance Labs. to provide further studies where we are told (RIFM 2007, 2008) that that bergamottin induced structural chromosome aberrations in the presence of UV light, indicating a photoclastogenic response - however full experimental details are not in the public domain.

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Aubin F., Humbert P. & Agache P. (1994) "Effects of a new psoralen, 5-geranoxypsoralen, plus UVA radiation on murine ATPase positive Langerhans cells." J Dermatol Sci. 7(3), 176-84. Abstract. 5-geranoxypsoralen (5-GOP), commonly called bergamottin, is a highly photoreactive psoralen, which in contrast to most furocoumarins, does not strongly interact with DNA. 5-GOP gives the opportunity to study, in a more selective way, the mechanisms of phototoxic and immunological activities induced by psoralen and UVA radiation. We investigated the effects of repetitive treatments with 5-GOP plus UVA radiation (320-400 nm) on the number of ATPase+ epidermal Langerhans cells and on the induction of photoreactivity. These effects were compared with those of 8-methoxypsoralen (8-MOP) or 5-methoxypsoralen (5-MOP) plus UVA radiation and UVA radiation alone. C3H/HeN mice were treated topically with the psoralen three times/week for 4 consecutive weeks followed each time by 1 J/cm2 of UVA radiation. At the end of the treatment, mice treated with 8-MOP or 5-MOP plus UVA radiation exhibited severe gross phototoxicity and nearly total depletion of ATPase-stained Langerhans cells. Both treatments produced severe morphological alterations of Langerhans cells. No gross but a microscopic phototoxic effect was observed after 5-GOP plus UVA radiation treatment, while the number of ATPase+ Langerhans cells was also greatly reduced. Interestingly the latter treatment induced no morphological alterations of the remaining Langerhans cells in contrast to treatment with 8-MOP or 5-MOP plus UVA radiation. We conclude that phototoxicity and decrease in the number of ATPase-stained epidermal immune cells observed after treatment with 5-GOP plus UVA radiation are not related to the DNA binding activity of the psoralen.

Ballantyne M. (2002) Bergamotin: Reverse mutation in five histidine-requiring strains of Salmonella typhimurium, in the presence of ultra violet light. Final Report. Part of Submission I from EFFA, 2002. [listed as evidence in SCC(NF)P 0740/03 & in SCC(NF)P 0761/03. Cropwatch cannot find this document in the public domain].

Bode C.W., Zager A. &Hansel W. (2005) "Photodynamic and photo-cross-linking potential of bergamottin." Pharmazie. 60(1), 78-9. Abstract. Bergamottin (5-geranoxypsoralen) is a main component of bergamot and grapefruit oil. In order to investigate the photophysical and photochemical behaviour of bergamottin, absorption and fluorescence properties, production of singlet oxygen and superoxide radical anions and further cross-linking of DNA were studied. Strong photochemical reactions were not observed.

Cai Y., Baer-Dubowska W., Ashwood-Smith M., DiGiovanni J. (1997a) Inhibitory effects of naturally occurring coumarins on the metabolic activation of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in cultured mouse keratinocytes. Carcinogen 18, 215-222. Abstract. Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of

organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons.

Cai Y., Kleiner H., Johnston D., Dubowski A., Bostic S., Ivie W. & DiGiovanni J. (1997b) Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo(a)pyrene and 7,12-dimethylbenz(a)anthrecene in SENCAR mice. Carcinogen 18, 1521-1527, 1997. Abstract. Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.

Frrot E. & Decorzant E. (2004) "Quantification of total furocoumarins in citrus oils by HPLC coupled with UV, fluorescence, and mass detection." J. Agric Food Chem 52, 6879-6886.

Morlire P., Hppe G., Averbeck D., Young A.R., Santus R. & Dubertret L. (1990) J. Photochem Photobiol B. 7(2-4),199-208. Abstract. Bergamottin, which accounts for about two-thirds of the absorption of UVA and UVB light by bergamot oil, is shown to be fairly unstable on UV irradiation of solutions of bergamot oil (in ethanol-water, 80:20 (w/w)). Bergamottin photodegradation is

partly inhibited by molecular oxygen and also by a cinnamate sunscreen acting as a triplet excited state quencher. On UV irradiation of bergamot oil, type II photodynamic properties, i.e. singlet oxygen production, are observed, which can be mainly attributed to the excitation of bergamottin by light. Therefore bergamottin can be considered as a potential photosensitizer in the photobiological activity of bergamot oil.

Morliere P., Bazin M., Dubertret L., Santus R., Sa E Melo T, Huppe G., Haigle J, Forlot P & Bernard A. (1991) "Photoreactivity of 5-geranoxypsoralen and lack of photoreaction with DNA". Photochem Photobiol. 53(1),13-9. Abstract. 5-Geranoxypsoralen, commonly called bergamottin, a major furocoumarin contained in bergamot oil, is reported in vitro as a highly photoreactive psoralen. In ethanol, it exhibits quite a high triplet state quantum yield (approximately 0.37). The triplet state is involved in subsequent photochemistry which depends on the initial concentration and on the presence of oxygen. In contrast to most psoralens, absorption and fluorescence data suggest that 5-geranoxypsoralen does not interact with DNA in the dark. No UVA-induced interstrand cross-links in DNA were shown.

Naganuma M, Hirose S, Nakayama Y, Nakajima K, Someya T. (1985) A study of the phototoxicity of lemon oil.. Arch Dermatol Res 278: 31-36. Abstract. Lemon oil contains furocoumarin derivatives and is known to cause phototoxicity. In this study, lemon oil was fractionated, and its phototoxic activity was measured by means of a biological assay. The substances producing phototoxicity were identified by high-performance liquid chromatography as being oxypeucedanin and bergapten. The phototoxic potency of oxypeucedanin was only one-quarter of that of bergapten. However, the amounts of these two phototoxic compounds present in lemon oils produced in different regions of the world varied by a factor of more than 20 (bergapten, 4-87 ppm; oxypeucedanin, 26-728 ppm), and their ratio was not constant. The two compounds accounted for essentially all of the phototoxic activity of all lemon-oil samples. Among various other citrus-essential oils investigated, lime oil and bitter-orange oil also contained large amounts of oxypeucedanin. Oxypeucedanin was found to elicit photopigmentation on colored-guinea-pig skin without preceding visible erythema.

Occhiuto F. & Circosta C. (1996) "Antianginal & antiarrhythmic effects of bergamottine, a furnaocoumarin isolated from bergamot oil." Phytotherapy Research 10(6), 491-496. Abstract. The antianginal and antiarrhythmic activities of bergamottine, a furocoumarin, were investigated in various in vivo and in vitro test models and have been compared with those of verapamil, a well-known Ca-antagonistic drug. Bergamottine significantly decreased the typical electrocardiographic signs of coronary arterial spasm and the incidence of cardiac arrhythmias induced by pitressin in anaesthetized guinea-pigs. Bergamottine also increased the dose of ouabain required to cause ventricular premature beats, ventricular tachyarrhythmias and lethality. Bergamottine further reversed ouabain-induced persistent ventricular tachycardia and restored sinus rhythm in the guinea-pig. On isolated rat heart, bergamottine exerted a coronary dilator action and was able to reduce the hyperkinetic ventricular arrhythmias caused by post-ischaemic reperfusion. These results demonstrate that bergamottine possesses significant antianginal and antiarrhythmic properties and suggest that this furocoumarin is one of the active components responsible for the cardiovascular activity previously exhibited by non-volatile bergamot oil.

Occhiuto F. & Cicosta. C (1997) "Investigations to characterize the antiarrhythmic action of bergamottine, a furocoumarin isolated from bergamot oil." Phytotherapy Research 11(6), 450-453. Abstract. Bergamottine is a furocoumarin isolated from the non-volatile essential oil of Citrus bergamia Risso (Bergamot, Rutaceae) with antiarrhythmic properties. We investigated and classified its mechanism of action according to Vaughan Williams. We have found that bergamottine at a concentration range of 5-20 g/mL (14.7-59 M) decreased the frequency and the force of the contraction in spontaneously beating guinea-pig right atria and electrically paced left atria in a concentration-dependent manner. Furthermore this furocoumarin caused a parallel shift of the dose-response curve to CaCl2 similar to that of verapamil in the electrically driven left atrium, indicating a possible Ca-antagonistic property. Bergamottine (1 mg/kg i.v.) significantly prolonged the atrioventricular conduction time, the corrected sinus-node recovery time and the

corrected sinus-atrial conduction time in anaesthetized rabbits. Bergamottine did not inhibit isoprenaline-induced positive chronotropic responses in atrial muscle, indicating that it has no -blocking activity, and did not show significant local-anaesthetic property in the guinea-pig intradermal wheal test. These results demonstrate that bergamottine seems to be an antiarrhythmic drug having class IV (inhibitor of calcium transport) type activity..

Stanley W.L. & Vannier S.H. (1957) "Chemical composition of lemon oil. Isolation of a series of substituted coumarins." J. Amer Chem Soc. 79, 3488-3491.

Wen Y.H., Sahi J., Urda E., Kulkarni S., Rose K., Zheng X., Sinclair J.F., Cai H., Strom S.C., Kostrubsky V.E. (2002) Effects of bergamottin on human and monkey drug-metabolizing enzymes in primary cultured hepatocytes. Drug Metabolism Disposition 30, 977-984. Abstract. We investigated the effect of bergamottin, a major furanocoumarin in grapefruit juice, on phase I and phase II drug-metabolizing enzymes using cultured human and monkey hepatocytes. Both cultured systems were compared and evaluated for the direct effects of bergamottin as well as control treatments on liver enzymes. Treatment of hepatocytes with 0.1, 1, 5, and 10 microM bergamottin resulted in a concentration-dependent reduction in CYP3A4 activity (40-100%) in both human and monkey cells, as measured by testosterone 6 beta-hydroxylase activity. Bergamottin was potent at eliciting these inhibitory effects at both basal and induced states of CYP3A. Bergamottin (5 microM) completely inhibited alpha-naphthoflavone-induced ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in human hepatocytes and caused a 100% decrease in EROD activity in monkey hepatocytes. A 48-h exposure of cultured human hepatocytes to bergamottin resulted in increased levels of immunoreactive CYP3A4, CYP1A1, and CYP1A2 proteins, and CYP3A4, CYP1A1, CYP1A2, CYP2B6, and UDP-glucuronosyl transferase mRNAs. There was only a 20 to 30% reduction in glucuronidation and sulfation of 4-methylumbelliferone in human hepatocytes by 10 microM bergamottin and no effect on conjugation in the monkey hepatocytes. These results suggest that bergamottin causes both inhibition of CYP3A and CYP1A1/2 enzymatic activities and induction of correspondent proteins and mRNAs.

Bergapten (5-methoxypsoralen or 5-MOP). CAS n: 484-20-8 Phototoxicity: Marzulli & Maibach (1970) determined that the minimum amout of 5-MOP that would cause an adverse skin reaction in a perfume was 0.001% to 0.002%. Papadopoulo & Averbeck (1985) showed 5-MOP & 8-MOP produce genotoxic effects & DNA adducts in Chinese hamster V79 cells. About this time the IARC classified bergapten as probably carcinogenic to humans (IARC 1987).

Young et al. (1988) found that a 5-MOP induced tan is protective against the DNA-damaging effects of solar radiation. Following the finding that the use of 5-MOP applied in sunscreen enhanced the bodys natural protection for several weeks, even when the sunscreen + 5-MOP use was discontinued, funds were provided by the Cancer Research Campaign & Laboratoires Bergaderm to develop a lotion to improve the bodies natural defences (Anon 1992). This product, believed to contain some 30ppm 5-MOP, was eventually trialled as reported in the media (Hunt 1992). However worries about the furanocoumarin photocarcinogenicity led the EC to order Laboratoires Bergaderm not to release their bergapten-containing Bergasol product onto the market past July 1996 (Goldemberg 1996), eventually forcing Laboratoires Bergaderm into liquidation. Thanks to Martin Watt for supplying articles substantiating this information.

Young et al. (1990) found 5-MOP photocarcinogenic in humans, as did Dubertret et al. who decided it was photo-toxic to humans at 5 ppm (Dubertret et al. 1990a) & IFRA bases its Standards/restrictions for bergapten-containing essential oils on the published phototoxic effects of bergapten and the established dose-effect relationships - according to Young et al. (1990) & Dubertret et al. (1990a, 1990b).

Ocuurence: Bergapten was found at 1671.1 ppm in cold-pressed bitter orange oil Citrus aurantium L. subsp. amara by UV-DAD; at 531.2 ppm by UV-DAD in cold-pressed Israeli grapefruit oil Citrus paradisi Macf., and at 15.69 ppm by UV-DAD in cold-pressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004).

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bergapten aka 5-methoxypsoralen