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Isolation and functional characterization of CYP71AJ4 encoding for the

first P450 monooxygenase of angular furanocoumarin biosynthesis.

Larbat R, Hehn A, Hans J, Schneider S, Jugdé H, Schneider B, Matern U, Bourgaud F.

UMR 1121 Nancy Université-Institut National de la Recherche Agronomique Agronomie Environnement

Nancy-Colmar, 2 Avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy, France.

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with

only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from

CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa

(CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen

synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and

negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully

characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin

synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14

min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete

"metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms

that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence

comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70%

identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition

sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown

functionality by gene duplication and subsequent molecular evolution.

Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-


methyltransferase.

Hehmann M, Lukacin R, Ekiert H, Matern U.

Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Germany.

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.

Cations modulate the substrate specificity of bifunctional class I O-


methyltransferase from Ammi majus.

Lukacin R, Matern U, Specker S, Vogt T.

Institut für Pharmazeutische Biologie der Philipps-Universität Marburg, Deutschhausstr. 17A, D- 35037

Marburg/Lahn, Germany.

Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae)

cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The

translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved

class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For
biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-

affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on

sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-

methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of

Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced

by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities

in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-


methyltransferase.

Hehmann M, Lukacin R, Ekiert H, Matern U.

Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Germany.

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.

Coumarin compounds in Ammi majus L. callus cultures.

Ekiert H, Gomółka E.

Department of Pharmaceutical Botany, Collegium Medicum, Jagiellonian University, Kraków, Poland.

mfekiert@cyf-kr.edu.pl

Callus cultures of Ammi majus L. (bishop's weed), Apiaceae, were maintained on variants of Linsmaier-

Skoog's (L-S) medium differing in the content of the phytohormones, alpha-naphthaleneacetic acid (NAA)

and 6-benzylaminopurine (BAP) (0.1-10.0 mg/l). The increments of callus tissue fresh weight showed

considerable differences (1.4 bis 4.4-fold) during 4-week subcultures on the media tested. HPLC analysis

revealed the presence of six compounds in the extracts of callus tissues, which are known metabolites in

plants growing under natural conditions: the linear furanocoumarins psoralen, bergapten, xanthotoxin,

isopimpinellin, imperatorin and their precursor umbelliferone. The total contents of coumarin compounds

under examination showed marked differences dependent on the phytohormone concentrations in the

medium. They ranged from 40.95 to 871.05 mg/100 g of dry weight. Imperatorin was the metabolite

dominating among the furanocoumarins in calli from almost all tested variants of L-S medium (maximum

content of 169.27 mg/100 g). Callus tissue cultured on one of the variants (0.1 mg/l NAA, 0.1 mg/l BAP)

yielded in high content of xanthotoxin (145.33 mg/100 g). On the majority of media, umbelliferone was also

accumulated at high quantities (maximum content of 536.29 mg/100 g). The medium containing 0.1 mg/l

NAA and 0.1 mg/l BAP was the best for the accumulation of analysed coumarins. This medium favoured the

formation of embryogenic callus. Xanthotoxin also dominated quantitatively among the furanocoumarins in

the tested vegetative organs and fruits of the plant grown under natural conditions (leaves: 26.10 mg, roots:

5.55 mg, fruits: 3010.41 mg/100 g). Maximum contents of this metabolite in in vitro culture were many times

higher than those found in vegetative plant organs but manifold lower than in fruits. On the other hand,

maximum contents of imperatorin obtained in in vitro culture were many times higher in comparison with
those detected in vegetative organs and fruits (leaves: 14.10 mg, roots: 3.30 mg, fruits: 94.70 mg/100 g of

dry weight.). The A. majus L. callus culture, established in the course of the present experiments, can be

considered a valuable model for studies of the biosynthesis of coumarin compounds, and a potential source

of the psoralen 8-alkoxy derivatives imperatorin and xanthotoxin and their precursor umbelliferone.

Occupational allergic rhinitis and contact urticaria caused by bishop's


weed (Ammi majus).

Kiistala R, Mäkinen-Kiljunen S, Heikkinen K, Rinne J, Haahtela T.

Department of Allergic Diseases, Helsinki University Central Hospital, Finland.

Bishop's weed (Ammi majus) has been known to induce toxic phytophotodermatitis. We now describe IgE-

mediated rhinitis and contact urticaria caused by exposure to bishop's weed in a 31-year-old atopic female

florist. A skin prick-prick test with bishop's weed flowers gave an 8-mm wheal, and the bishop's weed-

specific IgE level in the patient's serum was 9.7 PRU/ml (RAST class 3). In an immunoblotting experiment

with the patient's serum, nine IgE-binding protein bands with the molecular weights 19, 34, 39-41 (doublet),

52-61 (doublet), and >67 (triplet) kDa were detected in bishop's weed extract. The patient became

symptomless after she had ceased to work as a florist.

Psoralen photobiology and photochemotherapy: 50 years of science and


medicine.

Bethea D, Fullmer B, Syed S, Seltzer G, Tiano J, Rischko C, Gillespie L, Brown D, Gasparro FP.

Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107,

USA. francis.gasparro@mail.tju.edu

In 1998 it is appropriate to commemorate the 50th anniversary of el Mofty's use of purified 8-

methoxypsoralen (8-MOP) in the treatment of vitiligo (el Mofty AM. A preliminary clinical report on the

treatment of leukoderma with Ammi majus linn. J R Egypt Med Assn 1948,31:651 65. el Mofty AM, el

Sawalhy H, el Mofty M. Clinical study of a new preparation of 8-methoxypsoralen in photochemotherapy. Int

J Dermatol 1994;8:588 92). Two young American dermatologists (Aaron Lerner and Thomas Fitzpatrick)
were intrigued by the potency of this material. After Lerner determined that artificial long wavelength

ultraviolet (320-400 nm, UVA) radiation was the most efficient for activating 8-MOP. the development of

artificial sources enabled the efficient delivery of these photons to skin containing 8-MOP. Their initial

studies for vitiligo led to further development of this therapy for the treatment of psoriasis (Parrish JA,

Fitzpatrick TB, Tannenbaum L, et al. Photochemotherapy of psoriasis with oral methoxsalen and long-wave

ultraviolet light. New Engl J Med 1974;291:1207-11. Honigsmann H, Fitzpatrick TB, Pathak MA, et al. Oral

photochemotherapy with psoralen and UVA (PUVA): principles and practice. In: Fitzpatrick TB, Eisen AZ,

Wolf K, editors. Dermatology in General Medicine. New York: McGraw-Hill, 1987:1728-54). This

photochemotherapy came to be called 'PUVA' (psoralen + UVA). The position PUVA holds today as one of

the most common procedures performed in dermatology can be traced to their initial curiosity and their

subsequent ingenuity. Further developments in more recent years capitalized on their seminal work. The

therapy met with unprecedented success from the outset, leaving little perceived need to understand

underlying science. However, in recent years there has been a new found interest in the basic aspects of

psoralen photobiology and molecular mechanistic events contributing to therapeutic responses as well as to

the development of skin cancers in PUVA patients. These will be surveyed in this review commemorating

the 50 years of modern psoralen photobiology and photomedicine.

Topical Calendula officinalis L. successfully treated exfoliative cheilitis: a


case report.

Roveroni-Favaretto LH, Lodi KB, Almeida JD.

Department of Biosciences and Oral Diagnosis, São Paulo State University - UNESP, São José dos

Campos, São Paulo, Brazil.

Authors describe a case of recurrent exfoliative cheilitis that responded to treatment with a standardized

topical preparation of Calendula officinalis L. An eighteen-year-old man was referred to UNESP - São Paulo

State University, Department of Biosciences and Oral Diagnosis, São José dos Campos Dental School to

investigate a chronic dry scaling lesion on his lips. The patient's main chief was aesthetic compromising.

Corticoid therapy was suspended and Calendula officinalis ointment 10% for ad libitum use has been
prescribed. The results presented allow the authors to consider Calendula officinalis L. as a potential

therapy in cases of cheilitis exfoliative.

Protective effect of Calendula officinalis extract against UVB-induced


oxidative stress in skin: Evaluation of reduced glutathione levels and
matrix metalloproteinase secretion.

Fonseca YM, Catini CD, Vicentini FT, Nomizo A, Gerlach RF, Fonseca MJ.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n,

14040-903 Ribeirão Preto, São Paulo, Brazil.

BACKGROUND AND PURPOSE: Calendula officinalis flowers have long been employed time in folk

therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The

main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory

diseases of several origins. The recommended doses are a function both of the type and severity of the

condition to be treated and the individual condition of each patient. Therefore, the present study investigated

the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin.

METHODS: Firstly, the physico-chemical composition of marigold extract (ME) (hydroalcoholic extract) was

assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the

cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect

of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining

reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. RESULTS

AND CONCLUSIONS: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6mg/g,

18.8mg/g, 1.6mg/g and 12.2mg/g, respectively and evaluation of the in vitro antioxidant activity

demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments

demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of

15mg/mL. However, concentrations greater than or equal to 30mg/mL, toxic effects were observed. Finally,

oral treatment of hairless mice with 150 and 300mg/kg of ME maintained GSH levels close to non-irradiated

control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9

(MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have
a complete understanding of the protective effects of ME for skin. Copyright © 2009 Elsevier Ireland Ltd. All

rights reserved.

New promising natural immunocorrective agents]

[Article in Russian]

[No authors listed]

The influence of Sorbus sibirica, Calendula officinalis and Althaea officinalis extracts on the humoral immune

response and nonspecific resistance of mice to immunosuppression by cyclophosphan was studied. It was

shown that these extracts are not inferior to Echinacea purpurea tincture in terms of stimulation of humoral

immune response, phagocytic and bactericidal activity of peritoneal macrophages but exceed effect of E.

purpurea on phagocytic activity of peripheral blood neutrophils.

Determination of the wound healing effect of Calendula extracts using


the scratch assay with 3T3 fibroblasts.

Fronza M, Heinzmann B, Hamburger M, Laufer S, Merfort I.

Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany.

PHARMACOLOGICAL RELEVANCE: Presentation of the scratch assay as a convenient and inexpensive in

vitro tool to gain first insights in the wound healing potential of plant extracts and natural compounds. AIM

OF THE STUDY: The present study deals with the optimization of the scratch assay which can be used as

an in vitro model for quantification of fibroblast migration to and proliferation into the wounded area. It is

suitable for the first evaluation of the wound re-epithelialization potential of crude herbal extracts, isolated

compounds and pharmaceutical preparations. As a proof of concept three preparations from traditional

medicinal plants were investigated. MATERIALS AND METHODS: Swiss 3T3 albino mouse fibroblasts were

used in monolayers and platelet derived growth factor as positive control. Hexane and ethanolic extracts

from Calendula officinalis and Matricaria recutita, Hypericum oil as well as the triterpenoids faradiol myristate

and palmitate were studied. To differentiate between proliferation and migration antimitotic mitomycin C was

added. RESULTS: Both extracts of Calendula officinalis stimulated proliferation and migration of fibroblasts
at low concentrations, e.g. 10 microg/ml enhanced cell numbers by 64.35% and 70.53%, respectively.

Inhibition of proliferation showed that this effect is mainly due to stimulation of migration. Faradiol myristate

and palmitate gave comparable stimulation rates at an almost 50 microg/ml concentration, indicating that

they contribute partially, but not most significantly to the wound healing effects of Calendula preparations.

Extracts from Matricaria recutita were only moderately active. Hypericum oil was cytotoxic at concentrations

higher than 0.5 microg/ml. CONCLUSIONS: The scratch assay in the present form can be used as a

promising scientific approach and platform to differentiate between plant extracts known for their wound

healing and their anti-inflammatory properties.

Investigations into the antibacterial activities of phytotherapeutics


against Helicobacter pylori and Campylobacter jejuni.

Cwikla C, Schmidt K, Matthias A, Bone KM, Lehmann R, Tiralongo E.

School of Pharmacy, Griffith University, Gold Coast Campus, Queensland 4222, Australia.

The prevalence of gastric diseases is increasing with H. pylori, the causative agent of acute and chronic

gastritis, being a major predisposing factor for peptic ulcer disease and gastric carcinoma. C. jejuni is the

most common cause of enteric infections, particularly among children, resulting in severe diarrhoea.

Increasing drug resistance of these bacteria against standard antibiotics, and the more widespread use of

herbal medicines, favours investigations into additional anti-Helicobacter and anti-Campylobacter effects of

phytotherapeutics that are already used for their beneficial effects on bowel and digestive functions.Twenty-

one hydroethanol herbal extracts and four essential oils were screened for antibacterial activity using a

modification of a previously described micro-dilution assay and compared with the inhibitory effects of

antibiotics. The herbal extracts showing the highest growth inhibition of C. jejuni were Calendula officinalis,

Matricaria recutita, Zingiber officinale, Salvia officinalis, Foeniculum vulgare and Silybum marianum.

Agrimonia eupatoria, Hydrastis canadensis, Filipendula ulmaria and Salvia officinalis were the most active

herbal extracts in inhibiting the growth of H. pylori. This study provides evidence for additional beneficial

effects of phytotherapeutics marketed for their gastrointestinal effects and identifies new beneficial

antibacterial effects for some herbal medicines not currently recommended for gastrointestinal problems.

Copyright (c) 2009 John Wiley & Sons, Ltd.


Wound healing activity of flower extract of Calendula officinalis.

Preethi KC, Kuttan R.

Department of Biochemistry, Amala Cancer Research Centre Amala Nagar, Thrissur-680555, Kerala, India.

The effects of oral and topical application of Calendula officinalis flower extract on excision wounds made in

rats were checked. The parameters assessed were the days needed for re-epithelization and percentage of

wound closure. The hydroxy proline and hexosamine content in the granuloma tissue of the wound was also

measured. The percentage of wound closure was 90.0% in the extract-treated group, whereas the control

group showed only 51.1% on the eighth day of wounding (p < .01). The days needed for re-epithelization

were 17.7 for the control animals; extract treatment at a dose of 20 or 100 mg/kg b.wt reduced the period to

14 and 13 days, respectively. A significant increase was observed in the hydroxy proline and hexosamine

content in the extract-treated group compared with the untreated animals. The data indicate potent wound

healing activity ofC. officinalis extract.

PMID: 19601397 [PubMed - indexed for MEDLINE]

Antioxidant activity of Calendula officinalis extract: inhibitory effects on


chemiluminescence of human neutrophil bursts and electron
paramagnetic resonance spectroscopy.

Braga PC, Dal Sasso M, Culici M, Spallino A, Falchi M, Bertelli A, Morelli R, Lo Scalzo R.

Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy. piercarlo.braga@unimi.it

There is growing interest in natural chemical compounds from aromatic, spicy, medicinal and other plants

with antioxidant properties in order to find new sources of compounds inactivating free radicals generated by

metabolic pathways within body tissue and cells, mainly polymorphonuclear leukocytes (PMNs) whose

overregulated recruitment and activation generate a large amount of reactive oxygen species (ROS) and

reactive nitrogen species (RNS), leading to an imbalance of redox homeostasis and oxidative stress. The

aim of this study was to examine whether a propylene glycol extract of Calendula officinalis interferes with

ROS and RNS during the PMN respiratory bursts, and to establish the lowest concentration at which it still
exerts antioxidant activity by means of luminol-amplified chemiluminescence. Electron paramagnetic

resonance (EPR) spectroscopy was also used in order to confirm the activity of the C. officinalis extract. The

C. officinalis extract exerted its anti-ROS and anti-RNS activity in a concentration-dependent manner, with

significant effects being observed at even very low concentrations: 0.20 microg/ml without L-arginine, 0.10

microg/ml when L-arginine was added to the test with phorbol 12-myristate 13-acetate and 0.05 microg/ml

when it was added to the test with N-formyl-methionyl-leucyl-phenylalanine. The EPR study confirmed these

findings, 0.20 microg/ml being the lowest concentration of C. officinalis extract that significantly reduced 2,2-

diphenyl-1-picrylhydrazyl. These findings are interesting for improving the antioxidant network and restoring

the redox balance in human cells with plant-derived molecules as well as extending the possibility of

antagonizing the oxidative stress generated in living organisms when the balance is in favor of free radicals

as a result of the depletion of cell antioxidants. Copyright 2009 S. Karger AG, Basel.