metabolic fate of depsides and alkaloid constituents in aqueous extracts from ...

Metabolic Fate of Depsides and Alkaloid Constituents in Aqueous Extractsfrom Mercurialis perennis L. during Fermentation

by Peter Lorenz*a), J�rgen Conradb), and Florian C. Stintzinga)

a) WALA Heilmittel GmbH, Section Phytochemical Research, Dorfstrasse 1, DE-73087 Bad Boll/Eckw�lden (phone: þ49-(0)7164930-6661; fax: þ49(0)7164930-228; e-mail: [email protected])

b) Institute of Chemistry, Hohenheim University, Garbenstrasse 30, DE-70599 Stuttgart

Dog�s mercury (Mercurialis perennis L.) is an old medicinal plant, nowadays used in complementarymedicine. Aqueous fermented extracts of the plant are being mainly applied in remedies to treat externalinflammations, but a thorough phytochemical characterization is still lacking. Therefore, the conversionof characteristic compound classes from M. perennis extracts during fermentation and storage wasinvestigated. The microbial transformation of the two main depsides phaselic acid (¼ (2R)-O-[(E)-caffeoyl]malic acid; 1) and mercurialis acid (¼ (2R)-[(E)-caffeoyloxy]glutaric acid; 2) was monitored byHPLC-DAD. The degradation followed a second-order kinetic, and the calculated half-life periods ofboth constituents were 67 and 30 months, respectively. Several depside metabolites were detected by GC/MS in AcOEt extracts as tBuMe2Si (TBDMS) derivatives after derivatization, mainly dihydrocinnamicacids. Moreover, numerous a-hydroxy acids were found, allegedly as degradation products from aminoacids or peptides. The microbial alteration of the main alkaloid hermidin was also examined. After threedays of fermentation, three novel N-metabolites were formed and thoroughly assigned in CH2Cl2 extractsas a mixture of 3-ethylhermidin, 3-ethylhermidin quinone, and (E/Z)-3-ethylidenehermidin by GC/MSand NMR methods, as well as by means of total synthesis. A mechanism for the formation of these N-metabolites starting from dimeric hermidin oxidation products is proposed. The obtained results revealthe complex pathways plant constituents may undergo during the fermentation of the extracts.

Introduction. – Lactic acid fermentation plays a pivotal role for preservation andmanufacturing of foods such as sourdough [1] [2] yogurt, kefir [3] [4], sauerkraut [5], orkimchi [6]. The most important genus of lactic acid-forming microorganisms (LAFO)for such fermentation processes is Lactobacillus, though other bacteria and yeasts mayalso be present [4]. The ability of the LAFO to generate lactic acid from glucose in amultistep pathway with a concomitant pH reduction is the key metabolic conversion.For aqueous extraction of medicinal plants, lactic acid fermentation is part of theproduction process according to an official procedure [7]. The outcome is amicrobiologically stable extract without using organic solvents. Therefore, thistechnology appears viable to be studied, as it may open interesting ramifications fora low-cost and environmentally friendly processing of plants both for food andpharmaceutical extracts. During the plant fermentation, primary constituents such aspolysaccharides, sugars [8] [9], amino acids [2] [10] [11], as well as polyphenolics[12] [13] are metabolized to yield compounds of lower molecular weight. Studying thedegradation pathways of phenolics by LAFO and investigating the biological effects ofthe resulting products on humans is a recent focus point for contemporary research[14] [15]. However, reports on the impact of fermentation upon secondary metabolites

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)1706

� 2013 Verlag Helvetica Chimica Acta AG, Z�rich

from medicinal plants are still rare [16 – 20]. Data on the bioactivity and pharmaco-logical potency of such fermented extracts are even more scarce [18] [21].

Mercurialis perennis L., belonging to the Euphorbiaceae family, has been used intraditional European medicine for a long time. In earlier times, the herbal parts of theplant were applied as a strong laxative, against anorexia, dropsy, bronchial catarrh,rheumatism, gout, and menstrual complaints [21] [22]. Because of putative toxic effects[23], an oral administration of Mercurialis became obsolete nowadays. Nevertheless,hydroalcoholic and fermented extracts from M. perennis are used in homeopathy andanthroposophic medicine, mainly for topical treatment of inflammations, hardlyhealing wounds, sores, suppurations, onychia, burns, and haemorrhoids. Also for thetherapy of conjunctivitis, eye drops containing Mercurialis preparations may beindicated [21]. According to an official procedure of the German HomeopathicPharmacopoeia [7] to 10 parts of fresh plant material (M. perennis), 7.5 parts purifiedwater, and 5 parts of whey are added at the beginning of the manufacturing process.After 3 d of incubation, the plant material is separated from the fermentation liquid byfiltration, and the resulting extract is further stored at 158 in the dark for at least sixmonths [7]. According to current conceptions, the natural microbial flora of the plant isgradually shifted towards a predominance of LAFO during fermentation with aconcomitant pH decrease [17] [20]. In parallel, leaching and biotransformation ofhydrophilic plant constituents proceed [7] [19] [20]. While a broad phytochemicalscreening of M. perennis has shown a wide spectrum of lipophilic and hydrophilicconstituents, such as alkaloids, n-alkyl resorcinols, hydroxycinnamic acid depsides, andflavonol glycosides [24 – 27], the objective of the current investigation was to monitorcompound profile alterations during the extraction, fermentation, and storage period of6 months. A special focus was given to the characteristic depside and alkaloidstructures. To assign the full spectrum of both lipophilic and hydrophilic metabolites ofthe fermented extract, two different extraction procedures were chosen.

Results and Discussion. – Monitoring of Depside Concentration during Fermenta-tion and Storage. Recent investigations on aqueous extracts from M. perennis haverevealed several cinnamic acid depsides (caffeic, p-coumaric, and ferulic acidsesterified with a-hydroxy acids, such as 2-hydroxyglutaric, malic, or glucaric acids)[26] [27], all of them being assigned by LC/MS/MS. In the present study, two maindepsides phaselic acid (¼ (2R)-2-O-[(E)-caffeoyl]malic acid; 1) and mercurialis acid(¼ (2R)-2-[(E)-caffeoyloxy]glutaric acid; 2) were analyzed as distinct marker com-pounds of aqueous fermented extracts from M. perennis (for a typical HPLC chromato-gram, see Fig. 1,a). Also several peaks of the previously analyzed glucaric acid depsides[26] were detected at tR 7 – 20 min (Fig. 1,a), but not further considered in this study.

For quality and authenticity control of the fermented extracts, it is relevant toinvestigate the amount and stability of the characteristic substances. To obtain purereference standards for HPLC quantification of 1 and 2, a facile total synthesis of 2 wasaccomplished using an established protection-group concept (Scheme 1), while 1 wassynthesized as decribed in [27]. Consequently, dimethyl (2RS)-2-hydroxyglutarate (4)obtained by esterification and reduction of 2-oxoglutaric acid (3) was treated with (E)-3,4-bis(acetyloxy)cinnamoyl chloride (5) to obtain (2RS)-6 as an intermediatecondensation product. Subsequently, the protection groups were removed from (2R)-

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 1707


Fig. 1. a) HPLC (328 nm) of an aqueous extract obtained from M. perennis. The asterisk (*) marks theregion of glucaric acid depsides with caffeic, p-coumaric, and ferulic acids, previously characterized [26]but not assigned in this study. b) Degradation kinetics for the main depsides phaselic and mercurialis acids(1 and 2, resp.) in two independent batches of aqueous extracts from M. perennis (n¼2, measured in

duplicate; �SD).

6 by treatment with 1n HCl/THF to yield the racemic mercurialis acid ((2RS)-2). Thechromatographic and NMR spectroscopic data of the synthetic (2RS)-2 were identicalto those of the natural (2R)-2, previously isolated from M. perennis [26]. For HPLCmonitoring, two separate batches of fermented extracts were prepared according to anestablished procedure (34 c) of the German Homeopathic Pharmacopoeia [7]. Thesamples were taken after 3 and 7 d, and 1, 3, 6, 9, 12, and 21 months (stored at �808)and investigated by HPLC-DAD, using external calibration derived from standarddilutions of 1 and 2. By plotting the concentration levels against time (Fig. 1, b),degradation graphs were obtained for 1 and 2. Curve fitting (regression analysis) of thedata points yielded the specific mathematical functions for the decay of 1 and 2. Alogarithmic slope of the curves indicated a second-order degradation kinetic, indicatingthat degradation of 1 and 2 accompanied the gradual decline of the microflora [28]. Thehalf-lives (t1/2) calculated were 67 and 30 months for 1 and 2, respectively. However, thelow-molecular-weight degradation products of the depsides were barely detectablewith the applied HPLC method.

Bioconversion of Depsides to Carboxylic Acids, Detection of Hydrophilic Biode-gradation Products. Since low-molecular-weight products from depside degradationescaped HPLC detection, AcOEt extracts of the fermented extracts were derivatizedwith N-[(tert-butyl)dimethylsilyl]-2,2,2-trifluoro-N-methylacetamide (MTBSTFA).The tBuMe2Si (TBDMS) derivatives thus obtained were analyzed by GC/MS. Bycomparison of the mass spectra (see Table 1) with the NIST database [29] and referencestandards, it was possible to assign a large number of aliphatic and phenolic carboxylicacids. Fig. 2 depicts a representative overview of the changes in the GC/MS profileduring fermentation. In the fresh unfermented extract, five major compounds were


Scheme 1. Synthesis of (2RS)-Mercurialis Acid (2)

a) 1. MeOH, H2SO4, reflux; 2. NaBH4/MeOH. b) Pyridine, CH2Cl2, 4-(dimethylamino)pyridine(DMAP). The arbitrary atom numbering is shown for the intermediate 6. c) THF/1n HCl 3 : 1 (v/v),

reflux.

detected (Fig. 2, a): 4-hydroxybutyric, succinic, fumaric, malic, and caffeic acids (13–15, 22, and 33, resp.). After 3 d of fermentation, numerous new peaks appeared, whichmarkedly increased after 6 months of storage (Fig. 2,b and c). In the chromatograms attR 18– 32 min, several small a-hydroxylated carboxylic acids were assigned such aslactic, 2-hydroxybutanoic, 3-hydroxypropanoic, 2-hydroxy-3-methylbutanoic, 2-hy-droxy-4-methylpentanoic, and (erythro/threo)-2-hydroxy-3-methylpentanoic acids (7 –11 and 12a/12b, resp.), as well as 2-hydroxyglutaric acid (24) at tR 37.2 min (Fig. 2 andTable 1). Furthermore, a number of newly formed aromatic phenolic acids weredetectable between tR 32 and 50 min: 3-phenyllactic, 4-hydroxybenzoic, dihydro-p-coumaric, dihydroferulic, (E)-p-coumaric, 3,4-dihydroxybenzoic, 4-hydroxyphenyllac-tic, and dihydrocaffeic acids (20, 23, 25, and 27– 31, resp.). The metabolites found cast alight on possible alteration pathways of the depsides and other constituents of theaqueous extract. Scheme 2 illustrates a presumable degradation pattern of 2 : in a firststep, the ester (depside) bond is cleaved by unspecific esterases. In the literature,several examples have been reported where depsides like rosmarinic, cichoric, orcaftaric acids are metabolized by Lactobacteria to 33 and the corresponding a-hydroxyacids [30] [31]. In the present study, the depside 2 was cleaved by hydrolases (esterases)to 33 and 24 (Scheme 2). Then, 33 was converted to 31 by hydrogenating enzymes. Thesame reaction afforded dihydro-p-coumaric and dihydroferulic acids (25 and 27, resp.)


Fig. 2. GC/MS Profile of AcOEt fractions from aqueous extracts of M. perennis during fermentation andstorage. a) Fresh extract. b) After 3 d of fermentation. c) After 6 months of storage. For constituent

identification (TBDMSi derivatives) see Table 1; *: silylating artifact (TBDMS)2NH.

from the corresponding cinnamic acids (Fig. 2 and Table 1). Incidentally, hydro-genation of natural cinnamic acids has been formerly reported from fermentations withLactobacillus fermentum and L. plantarum [32]. Besides dihydrocinnamic acids, whichrepresent the major conversion products of cinnamic acids, a parallel process catalyzedby Lactobacteria strains leads to 4-vinylphenols and 4-ethylphenols, via decarboxyla-tion and hydrogenation [33– 35]. In the same line, Rodriguez et al. have found 4-ethyl-and 4-vinylcatechol (18 and 34, resp.) as metabolic degradation products of 33 with astrain of L. plantarum [12]. In the fermented batches of M. perennis investigated, 18was detected only in low amounts (Fig. 2), but screening other production batchesrevealed that higher levels of 18 may be present, presumably depending on thedecarboxylase/hydrogenase activity of the LAFO flora. However, simultaneously tothe formation of 18, C�C-lyases are able to cleave and oxidize the Et group to yield 3,4-dihydroxybenzoic acid (29 ; Fig. 2 and Table 1), corroborating earlier findings by vanBeer and Priest [33]. Along the depletion of 33, the concurrently formed 24 can belikewise transformed by decarboxylation to yield 8 or converted to 19 by amidation and


Scheme 2. Putative Degradation Pathways of Mercurialis Acid (2) Effected by Enzymes

a) Esterase (hydrolase). b) Phenolic acid reductase. c) Phenolic acid decarboxylase. d) Decarboxylase.e) Vinylphenol reductase. f) C�C-Lyase. g) Decarboxylase. h) Amidase and dehydratase. For compound

assignments, see Table 1.


Tabl

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GC

/MS

Dat

aof

Car

boxy

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and

Alc

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TB

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No.

Con

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[min

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[M�

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[M�

TB

DM

Si]þ

Oth

erin

trin

sic

ions

7L

acti

cac

id18

.731

8.60

303

(2)

261

(81)

233

(58)

,189

(63)

,147

(100

),13

3(2

4),7

3(6

9)8

2-H

ydro

xybu

tyri

cac

id20

.633

2.63

317

(1)

275

(54)

247

(34)

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(35)

,147

(100

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3(1

3),7

3(4

8)9

3-H

ydro

xypr

opan

oic

acid

21.3

318.

6030

3(4

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1(1

00)

219

(20)

,189

(56)

,147

(51)

,133

(22)

,73

(70)

102-

Hyd

roxy

-3-m

ethy

lbut

anoi

cac

id21

.934

5.67

331

(1)

289

(57)

261

(38)

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(29)

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(100

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3(1

3),7

3(5

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2-H

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360.

6834

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6834

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303

(70)

275

(45)

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(23)

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24.0

332.

6331

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143

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Succ

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26.3

346.

6133

1(4

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189

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116

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(79)

15F

umar

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4.59

329

(3)

287

(100

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7(5

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(19)

16(Z

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enta

dec-

10-e

noic

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c )28

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4.64

339

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297

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3(1

1)17

Gly

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l29

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4.88

419

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thyl

cate

chol

30.6

366.

6935

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309

(22)

193

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179

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115

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carb

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7.64

342

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300

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98(2

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(82)

203-

Phe

nylla

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acid

33.3

394.

7037

9(0

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337

(68)

309

(53)

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(100

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3(1

3),7

3(5

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1-(4

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roxy

phen

yl)e

than

ol33

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6.69

351

(1)

309

(100

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5(3

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19(6

6),1

93(1

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26(1

2),7

3(3

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6.87

461

(2)

419

(68)

287

(36)

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(16)

,147

(41)

,133

(14)

,115

(32)

,73

(100

)23

4-H

ydro

xybe

nzoi

cac

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6.64

351

(0.4

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9(1

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265

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(11)

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126

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9)24

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ydro

xygl

utar

icac

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.249

0.90

475

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433

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221

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2.68

377

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335

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61(2

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27D

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40.9

424.

7240

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7(1

00)

295

(35)

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(26)

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(51)

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(16)

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2.68

377

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335

(100

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9(8

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43.8

496.

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439

(100

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5(5

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4.96

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133

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4.96

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467

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9450

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391

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dehydration (Scheme 2). It is also conceiveable that both 8 and 19 may stem frompeptide or amino acid degradation, like various other a-hydroxy acids beingsubstantially found in fermented M. perennis extracts (see Table 1). Similarily, a-hydroxy acids have been shown to be formed by L. plantarum fermentation of animalprotein or sourdough [2] [10].

Detection and Structure Elucidation of 3-Ethyl-4-methoxy-N-methylpiperidindiones(EMMPDs) as Microbial Metabolites from Hermidin. Hermidin (35), the mainalkaloid of M. perennis, is an air-sensitive molecule rapidly oxidizing in aqueoussolutions [36] [37]. Primarily, cyanohermidin 36, a transient-stable semiquinone anionradical is formed via a single-electron-transfer mechanism and has been formerlyconfirmed by ESR studies (see Scheme 3) [38]. Although 36 is presumably stabilized bya hydrate shell and mesomeric effects [24], the molecule is easily oxidized to hermidinquinone (37) or forms the dimer chrysohermidin (38) via free-radical dimerization(Scheme 3, step c). To investigate the metabolic conversion of 35, fresh and fermentedaqueous extracts from herbal parts of M. perennis were extracted with CH2Cl2 andanalyzed by GC/MS. A chromatogram of fresh extracts (exemplarily shown in Fig. 3, a)exhibited several peaks of N-containing constituents, perceptible by the odd-numberedmolecular-ion peaks in their mass spectra. By comparing the mass spectra withpreviously published data [24], signals of four known N-constituents (alkaloids) wereassigned, i.e., 35, 4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (42), 37, and 38. After3 d of fermentation, four new signals could be readily detected, whereas the dimer 38was completely degraded (Fig. 3,b). The mass spectra of the newly detected N-metabolites at tR 18.5, 20.2, 21.8, and 22.8 min (Fig. 3,b) exhibited molecular-ion peaksat m/z 199, and three times 197, respectively. The MS fragmentation patterns of these N-metabolites displayed [M�MeN]þ and [M�MeN�CO]þ fragments, indicating thatmost likely they are methoxy-N-methylpiperidindione derivatives, amplified byanother C2-unit. Since structural assignment by MS was not possible, an attempt wasmade to isolate these compounds. For this, a fermentation liquid (830 ml) from M.perennis was extracted with CH2Cl2 to yield a reddish-brown residue (0.6 g). Anextensive chromatographic purification of the residue (data not shown) afforded asemipure yellow gum-like compound (5 mg). The mass spectrum (from GC/MS) of theisolate showed a molecular-ion peak at m/z 197, and peaks resulting from subsequentfragmentations of [CH3]þ , twice [CO]þ , and [MeN]þ moieties, at m/z 182, 154, 126,and 97, respectively. The 1H-NMR spectrum of the compound revealed four different1H spin systems: a MeO group (d(H) 4.08 (s)), a MeN group (d(H) 3.30 (s)), and a CH2

unit (d(H) 2.60 (q, J¼7.5)) coupled with a Me group (d(H) 1.10 (t, J¼7.4)). Based onMS and NMR data, the unknown compound was tentatively assigned as 3-ethyl-hermidin quinone (39). A strong UV absorption at 271 nm (p!p*) and a weak one at338 (n!p*) substantiated its quinone structure. In virtue of the proposed structure, atotal synthesis of 39 was accomplished (see Scheme 4) starting from 3-oxoglutaric acid(43). After permethylation of 43 to obtain intermediate 44, an Et group was introducedat C(4) to yield the ester 45. Then, a cyclization was achieved by reaction with MeNH2,followed by MeONa treatment to give the piperidinedione 46a/46b as an isomermixture. Further reaction steps, i.e., oximation of 46a at C(5) and hydrolysis of theobtained oxime 47 with HCl/SnCl2 (Scheme 4, steps d and e), yielded the target product39, the structure of which was assigned by comprehensive NMR investigations



Scheme 3. Oxidation of Hermidin (35) and Conversion to the 3-Ethyl-4-methoxy-1-methylpiperidine-2,6-diones (EMMPDs)

a) Deprotonation and oxidation (�2 Hþ ; �e� ). b) Oxidation (�e� ). c) Dimerization and protonation(þ2 Hþ ). d) Cleavage. The waved lines in structure 38 mark the positions for possible microbial

cleavages.

Fig. 3. Sections of a GC/MS profile showing N-metabolites of CH2Cl2 fractions from aqueous M. perennisextracts. a) Fresh extract. b) After 3 d of fermentation. c) After 6 months of storage.

(Table 2). The chromatographic and spectroscopic data of the synthetic 39 were highlyconsistent with the N-metabolite isolated from the M. perennis fermentation liquid.Subsequently, an ethyl derivative of hermidin (35), compound 40, was readily obtainedby simple reduction of 39 with aqueous Na2S2O4 and extraction of the target 40 withCHCl3 under N2 (for NMR data, see Table 2 and Fig. 4). Compound 40 is much morestable in solution (CHCl3) than its analog 35 which undergoes rapid oxidation.Interestingly, like 35, the 3-ethyl derivative 40 easily formed a deep blue color when aCHCl3 solution was loaded on a SiO2 column, indicating the formation of a semistablesemiquinone anion radical. Nevertheless, further investigations by ESR spectroscopyare required to establish the formation of a radical. However, in the presence of SiO2 arapid oxidation of 40 was observed to yield again the quinone 39.

Finally, 3-ethylidenehermidin ((E/Z)-41) was synthesized via a Knoevenagel-typecondensation of 35 with MeCHO under reducing conditions (Scheme 5). Like 39, both40 and (E/Z)-41 could be unequivocally verified in the GC/MS of the fermented M.perennis extract by comparison with the synthesized reference compounds. To the bestof our knowledge, the 3-ethyl-4-methoxy-N-methylpiperidindiones (EMMPDs) havenot been previously described in the literature. Hence, the newly assigned N-metabolites 39, 40, and (E/Z)-41 beg the question for their biosynthesis. In theliterature, only a few examples have been reported, when microbial degradation ofalkaloids were studied during fermentation of plant material [16] [39] [40]. Asmentioned before (see above), the fermentation of the M. perennis depsides have


Scheme 4. Synthesis of 3-Ethylhermidin quinone (39) and 3-Ethylhermidin (40)

a) Trimethyl orthoformate ((MeO)3CH), MeOH, H2SO4, reflux. b) EtI, MeONa/MeOH, reflux. c) 1.MeNH2/H2O; 2. MeONa/MeOH. d) HNO2. e) HCl/SnCl2. f) Na2S2O4/H2O. The arbitrary atom

numberings are shown for 45 and 39.

yielded degradation products, indicating the activity of decarboxylating enzymes in thefermentation liquid. In case of alkaloid bioconversion, the originally detected dimer 38(Fig. 3,a) was completely depleted after 3 d (Fig. 3,b). For that reason, it is expectedthat 38 is the precursor molecule for the EMMPDs 39– 41, which are formed followingcleavage of the molecular framework of 38 by action of decarboxylating anddeoxygenating enzymes (Scheme 3, step d). Also a gradual depletion of 39– 41 duringstorage of the fermentated extract (see Fig. 3,c) reinforces the proposed degradationmechanism. However, further investigations are warranted to study this process indetail.

Conclusions. – The analysis of biotransformed products from different types ofplant constituents provide insights into the dynamic and complex of fermented extracts.Slow microbial degradation of the hydroxycinnamic depsides 1 and 2 during lactic acidfermentation of an aqueous extract from M. perennis and the subsequent storage


Table 2. 1H- and 13C-NMR Data of the Novel N-metabolites 39 and 40. In CDCl3, d in ppm.

Position 39 40

d(H) d(C) HMBC d(H) d(C) HMBC

2 – 163.56 (C¼O) – – 171.69 (C¼O) –3 – 135.26 (C) – 3.40 (dd, J¼

4.5, 4.9)47.28 (CH) C(2), C(4),

C(5), C(9),C(10)

4 – 155.14 (C) – – 143.21 (C) –5 – 172.07 (C¼O) – 5.67 (s) 123.62 (C) –6 – 155.96 (C¼O) – – 165.99 (C¼O) –7 3.30 (s) 27.31 (MeN) C(2), C(6) 3.22 (s) 26.81 (MeN) C(2), C(6)8 4.08 (s) 60.63 (MeO) C(4) 4.11 (s) 59.79 (MeO) C(4)9 2.60 (q, J ¼ 7.5) 18.89 (CH2) C(2), C(3),

C(4), C(10)2.17–2.09(ddq, J¼4.5,7.4, 13.7, Ha),2.06–1.98(ddq, J¼4.9,7.4, 13.8, Hb)

25.21 (CH2) C(2), C(3),C(4), C(10)

10 1.10 (t, J¼7.4) 13.12 (Me) C(3), C(9) 0.81 (t, J ¼ 7.5) 8.98 (Me) C(3), C(9)5.67 (OH) C(4), C(5),

C(6)

Scheme 5. Synthesis of (E/Z)-3-Ethylidenehermidin ((E/Z)-41) by Knoevenagel Condensation

a) Acetaldehyde/Na2S2O4/H2O.

primarily yielded dihydro caffeic acid (31) which was degraded further into 4-ethylcatechol (18) and 3,4-dihydroxybenzoic acid (29). Besides depside degradationproducts, several a-hydroxy acids derived from amino acid metabolism were analyzedin the fermentation batches. The high variability of the assigned metabolite spectrumled to the assumption of a �multi-enzyme� complex consisting of esterases, dihydro-genases, decarboxylases, and de-aminating and C�C-cleaving enzymes of the LAFOflora. Similar degradation products and bioconversion pathways have recently beenreported from the human gut [15] [41] or silage production [42]. In an analogous mannerto the microbial conversion of 33, the molecular framework of the alkaloid-type dimer38, an oxidation product of hermidin (35), is degraded by the same �multi-enzyme�complex to form the 3-ethyl-4-methoxy-N-methyl-piperidindiones (EMMPDs) 39, 40,and (E/Z)-41. The latter reaction is a novel example for plant alkaloid bioconversion.Herein, the side-chain Et group occurring both in the EMMPDs and 18 is presumably astable structural element in microbial degradation pathways. The results shown maybroaden our knowledge on the bioconversion of plant constituents during theproduction of fermented food or medicinal plant extracts, and constitute a basis forfuture bioactivity studies.


Fig. 4. gHMBC of 3-Ethyl-5-hydroxy-4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (40) recorded inCDCl3. The signal groups for Ha�C(9) and Hb�C(9) are expanded.

Experimental Part

General. Caffeic acid, p-coumaric acid, dimethyl 2-oxo-glutarate, 3,4-dihydroxyhydrocinnamic acid(dihydrocaffeic acid), 2-hydroxybutanoic acid sodium salt, d-a-hydroxyglutaric acid disodium salt, (þ)-(S)-2-hydroxy-3-methylbutanoic acid, (R)-2-hydroxy-4-methylpentanoic acid, 3-(4-hydroxyphenyl)pro-panoic acid (dihydro-p-coumaric acid), dl-(4-hydroxyphenyl)lactic acid, 3-hydroxypropanoic acid, dl-malic acid, 2-oxopyrrolidine-5-carboxylic acid, and N-[(tert-butyl)dimethylsilyl]-2,2,2-trifluoro-N-meth-ylacetamide (with 1% (tert-butyl)dimethylsilyl chloride i.e., MTBSTFA þ1% TBDMSCl) werepurchased from Sigma�Aldrich (D-Steinheim). 2-(4-Hydroxyphenyl)ethanol and (� )-3-phenyllacticacid were obtained from ABCR (D-Karlsruhe). 3-(4-Hydroxy-3-methoxyphenyl)propanoic acid(dihydroferulic acid) was from Alfa Aesar (D-Karlsruhe). 4-Hydroxybenzoic acid was purchased fromCarl Roth (D-Karlsruhe) and 3,4-dihydroxybenzoic acid from Fluka (CH-Buchs). 4-[(1E)-3-Chloro-3-oxoprop-1-en-1-yl]benzene-1,2-diyl diacetate, methyl 3-methoxypent-2-enoate, hermidin, 4-methoxy-1-methylpyridine-2,6(1H,3H)-dione, and phaselic acid (HPLC purity; >98.0%) were prepared accordingto published procedures [24] [27]. 2-Hydroxy-3-methylpentanoic acid (erythro/threo mixture) wassynthesized from d,l-isoleucine via reaction with HCl/NaNO2 (data not shown). All other chemicals ofanal. or synthetic grade were purchased from VWR (Merck Schuchardt OHG, D-Hohenbrunn).

Plant Material, Preparation of the Fermented Extracts and Sampling. Aerial parts of M. perennis werecollected in April 2010 in the beech forest above Bad Boll/Eckw�lden (Germany). Voucher specimens ofM. perennis were deposited with the Herbarium of the Department of Botany, Hohenheim University(Germany), and the plant material was identified by Prof. O. Spring (voucher Nos. HOH-011281, HOH-011282, and HOH-011283). Prior to extraction, the plant material (1000 g) was mechanically cleaned,rinsed with H2O, and processed according to an official procedure (GHP (¼German HomeopathicPharmacopoeia [7])), comprising comminution in H2O (750 g), addition of whey (500 g), fermentation,filtration, and storage. Samples were taken from the fresh extract, after 3 and 7d, and 1, 3, 6, 9, 12, and 21months, and immediately frozen at �808 until analyses.

Preparation of Standard Solns. from Phaselic and Mercurialis Acids (1 and 2, resp.) for HPLCQuantification. For standard calibration, a stock soln. was prepared. Ten mg of 1 and 20 mg of 2 wereexactly weighed, dissolved in H2O, and filled to a final volume of 50 ml in a volumetric flask (200 mg/ml of1 and 400 mg/ml of 2, resp.). Dilutions of 3 : 1, 1.66 : 1, 1 : 1, 1 : 1.66, 1 : 3, and 1 : 10 (v/v) with H2O wereprepared to obtain further working concentrations, resulting in the calibration range of 20–200 mg/ml for1 (R2¼1.0000) and 40–400 mg/ml for 2 (R2¼0.9999). The calibration curves were calculated from sampledata of seven reference standard dilutions (each measured in duplicate). For reasons of accuracy testing,one fermentation sample (900 ml) was spiked with the stock standard soln. (100 ml) and analyzed induplicate, resulting in a recovery of 100–101% for 1 and 2, resp.

HPLC Analysis and Quantification of the Depsides in the Fermented Extracts. For quantification of 1and 2, a Dionex Ultimate 3000 HPLC system (Dionex, D-Idstein) was used. A Reprosil-Pur C18-AQ� RPanal. column (250�4.0 mm i.d., 5 mm particle size; Dr. Maisch GmbH, D-Ammerbuch-Entringen) wasselected for chromatographic separation at 258 and a flow rate of 1.0 ml/min. The mobile phase consistedof HCOOH/H2O 0.2:99.8 (v/v ; mobile phase A) and MeCN (100%; mobile phase B). Starting with 10%B for 25 min, a linear gradient was followed to 23% B at 60 min, remaining isocratically for 2 min, thenincreasing to 100% B at 65 min, keeping for 5 min, before re-equilibration to starting conditions.Fermentation samples of M. perennis and reference standard dilutions were centrifuged (19064�g,5 min) before HPLC investigation. The injection volume was 20 ml, and monitoring of the depsides wasperformed at 328 nm (see Fig. 1, a). Curve fitting of the plotted measured concentration C [mg/ml] vs.fermentation time t [month] (Fig. 1, b), performed by Excel-regression analysis, resulted in the functions:C¼ �23.32 ln(T)þ238.98 and C¼ �9.456 ln(T)þ82.878 for 1 and 2, resp. By mathematicaltransformation, the half-lives (t1/2) values for 1 and 2 were calculated.

CH2Cl2 Extraction. GC/MS Analysis of Alkaloid Metabolites. For alkaloid analysis, aq. extracts fromM. perennis (5 ml each) were extracted with CH2Cl2 (2�5 ml), the extracts were dried (Na2SO4), thedesiccant was filtered off, washed with CH2Cl2 (5 ml), and the solvent was removed in vacuo by rotaryevaporation. Then, the residues were dissolved in AcOEt (0.2 ml) and injected into the GC/MS (1.0 ml).GC/MS was performed with a Perkin�Elmer Clarus 500 gas chromatograph with split injection (split


ratio 30 : 1) coupled to a mass detector. The column used was a Zebron ZB-5ms cap. column (60 m�0.25 mm i.d.�0.25 mm film thickness, 5% phenylpolysiloxane and 95% dimethylpolysiloxane coating;Phenomenex, Torrance, USA). He was the carrier gas at a flow rate of 1 ml/min. The injector used was aPSS (programmed-temp. split/splitless injector; temp.: 2508). The temp. program for the column ovenwas from 100 to 3208 at 48/min with a final hold time of 30 min. The mass spectrometer was run in theelectron ionization (EI) mode (70 eV).

AcOEt Extraction. Derivatization and GC/MS Profile of Fermentation Products (TBDMSderivatives). The fermentation samples (5 ml each, filtered over a 0.2 mm Nylon filter disk) wereextracted with AcOEt (2�10 ml), dried (Na2SO4), and the solvent was removed by rotary evaporationin vacuo. To azeotrope H2O and AcOH residues, toluene was added (2�10 ml) and distilled off again.The residues thus obtained (between 2.5 and 15 mg) were treated in capped headspace vials with amixture of MTBSTFA þ1% TBDMSCl (60 mg) and dry DMF (1.0 ml) each. After incubation at 1058(45 min), the solns. were injected into the GC/MS (1.0 ml).

NMR Spectroscopy. The NMR spectra were recorded in CDCl3 at 500 (1H) and 125 MHz (13C) with aVarian Unity Inova NMR spectrometer (D-Darmstadt), chemical shifts are reported in d [ppm]referenced to the residual solvent signal of CHCl3 (1H: d 7.27; 13C: d 77.00 ppm). 13C-NMR signalassignment of the novel compounds 6, 39, 40, and 47 were based on 2D-heteronuclear NMR experiments(gHMBC and gHSQC). For evaluation of the NMR spectra, the program SpinWorks 3.1.7. (Copyright�

2010, K. Marat, University of Manitoba, USA) was used.Synthesis of the Reference Compound (2RS)-[(E)-caffeoyloxy]glutaric Acid (¼ (2RS)-Mercurialis

Acid ; 2) . Dimethyl (2RS)-Hydroxyglutarate (4). Compound was 4 synthesized in 84% yield fromdimethyl 2-oxoglutarate (¼dimethyl 2-oxopentanedioate) according to a literature procedure [43]. GC/MS Purity: 91%; GC/MS* tR 12.0 min; GC/MS (70 eV): 145 (8, [M�MeO]þ ), 144 (6), 117 (51, [M�MeO�CO]þ ), 88 (13, [M�MeO�CO�OCH]þ ), 85 (100, [CCH2COOMe]þ ), 59 (16, [COOMe]þ ),57 (37, [CH2�CH2�CHO]þ ). Additionally, the compound was characterized via the TMDMS derivative,4-TBDMS: GC/MS* tR 21.2 min; GC/MS (70 eV): 275 (0.2, [M�Me]þ ), 259 (6, [M�MeO]þ ), 233 (22,[M�TBDMSi]þ ), 231 (13, [M�COOMe]þ ), 205 (9), 173 (100), 145 (19), 131 (8), 89 (84), 85 (30), 73(50). (*molecular ions were not observed).

Dimethyl (2RS)-2-({(2E)-3-[3,4-Bis(acetyloxy)phenyl]prop-2-enoyl}oxy)pentanedioate (6). A mix-ture of 4 (2.50 g, purity: 91%, 12.91 mmol), 4-[(1E)-3-chloro-3-oxoprop-1-en-1-yl]benzene-1,2-diyldiacetate (5 ; 3.65 g, 12.91 mmol), 4-(dimethylamino)pyridine (DMAP; 0.108 g, 0.88 mmol), pyridine(41 ml), and CH2Cl2 (82 ml) was stirred and heated under reflux (N2, 7 h). Subsequently, the solvent wasdistilled off in vacuo by rotoevaporation, and the residue was washed with hexane (3�50 ml, sonication3�1 min) to remove pyridine and starting material. The residue (7.97 g) was purified by vacuum LC(VLC; SiO2 (115 g); petroleum ether (PE)/AcOEt from 18 : 2–1 :1 (v/v) to yield 6 (3.67 g, purity: 86%;yield: 57%). The semipure 6 was used for the next reaction step. Further purification was achieved byanother VLC and washing 6 with PE. Rf (100% AcOEt) 0.62. UV/VIS (MeOH): 219 (4.13), 281 (4.29).GC/MS Purity: GC/MS (70 eV): 95% at tR 51.9 min. GC/MS (70 eV): 422 (3, Mþ ), 380 (11, [M�Ac]þ ),338 (55, [M�2 Ac]þ ), 247 (13), 205 (16), 163 (33), 162 (100), 159 (9), 134 (43). 1H-NMR (CDCl3,500 MHz): 7.67 (d, J¼16.0, H�C(7’)); 7.41 (dd, J¼1.7, 8.4, H�C(6’)); 7.37 (d, J¼1.7, H�C(2’)); 7.23 (d, J¼8.4, H�C(5’)); 6.42 (d, J¼16.0, H�C(8’)); 5.20 (dd, J¼4.7, 7.8, H�C(2)); 3.77 (s, H�C(7), MeO), 3.69 (s,H�C(6), MeO); 2.56–2.42 (m, H�C(4)); 2.31, 2.30 (2s, 2 Me of Ac); 2.28–2.17 (m, H�C(3)). 13C-NMR(CDCl3, 125 MHz): 172.7 (C(5)); 170.0 (C(1)); 168.0, 167.9 (C(10’), C(12’)); 165.6 (C(9’); 144.2 (C(7’);143.7 (C(4’)); 142.4 (C(3’)); 132.9 (C(1’)); 126.5 (C(6’)); 124.0 (C(5’)); 122.9 (C(2’)); 118.0 (C(8’)); 71.3(C(2)); 52.4 (C(6)); 51.8 (C(7)); 29.5 (C(4)); 26.4 (C(3)); 20.6, 20.6 (C(11’), C(13’)). C-Atomassignments by gHSQC and gHMBC (Fig. 5).

(2RS)-2-[(E)-Caffeoyloxy]glutaric Acid (¼ (2RS)-Mercurialis Acid ; (2RS)-2). Compound 6 (2.60 g,purity: 86%, 5.29 mmol) was dissolved under N2 in THF/1n HCl 1 : 3 (v/v, 150 ml). Then, the mixture wasstirred and heated under reflux for 6 h. Afterwards, the pH was adjusted to 3.0 by dropwise addition of 1nNaOH, and the soln. was saturated with NaCl and extracted with AcOEt (3�100 ml). Then, thecombined AcOEt extracts were dried (Na2SO4), and the solvent was removed in vacuo by rotaryevaporation to yield 2.78 g of crude material. By VLC (100 g TLC-grade C18-RP SiO2, preconditionedsubsequently with MeOH and MeOH/H2O 10/90 (v/v), the product was purified by elution with a linear


MeOH/H2O gradient from 10 : 90 to 20 : 70 (v/v). Fractions were analyzed by TLC showing that Frs. F7 –F12 (MeOH/H2O 20 : 70 (v/v)) contained the target product 2. These fractions were combined, and thesolvent was distilled off in vacuo: 0.975 g of (2RS)-2 ; cream colored foam; 59% of the theory. Rf (SiO2;CHCl3/AcOH/H2O 10 :8.4 :1.6 (v/v/v) 0.46. HPLC purity: >99.9%, tR 35.7 min. The NMR data of (2RS)-2 were consistent with those of the natural (2R)-2, previously isolated from M. perennis [26]. (2RS)-2 wasfurther characterized via the TBDMS derivative, 2-TBDMS*. GC/MS (70 eV): tR 74.9 min, m/z 767 (5,Mþ ), 752 (1, [M�Me]þ ), 709 (43, [M� tert-C4H9Si]þ ), 465 (23), 391 (64), 359 (81), 293 (11), 277 (7),261 (6), 219 (46), 191 (6), 129 (5), 115 (7), 73 (100). *Only semi-stable under GC/MS conditions, partialdecomposition into 33-TBDMS.

Synthesis of the Reference Compound 5-Ethyl-4-methoxy-1-methylpyridine-2,3,6(1H)-trione (39).Dimethyl (2Z)-4-Ethyl-3-methoxypent-2-enedioate (45). A soln. of MeONa, freshly prepared from Na(2.50 g, 108.74 mmol) and MeOH (53 ml), was treated with dimethyl (2Z)-3-methoxypent-2-enedioate(44 ; 19.60 g, 0.104 mmol) at reflux temp. Then, EtI (9.0 ml, 17.4 g, 111.56 mmol) was added dropwise, andthe mixture was heated under reflux for 12 h. Because of a flawed reaction turnover detected by TLC andGC, to the mixture were added again eight times fresh MeONa soln. and EtI (see above), and each soln.was refluxed for 6 h (58 h in total). After every second MeONa/EtI addition, the solvent was distilled offin vacuo, and the residual sirup was mixed with CHCl3 (250 ml). Then, precipitated KBr was filtered offby vacuum suction over a B�chner funnel and subsequently washed with CHCl3 (4�50 ml). From thecombined filtrates, the solvent was removed in vacuo by rotary evaporation. The crude material (21.6 g)thus obtained was subjected to VLC. In brief, TLC-grade SiO2 (100 g) was preconditioned with hexane(200 ml). Then, a soln. of half the crude material (10.8 g dissolved in 15 ml hexane) was loaded on thecolumn. By elution with CHCl3 (500 ml), several consecutive fractions were obtained, monitored by TLC(SiO2; CHCl3/AcOEt 19 : 1 (v/v)) and GC/MS. Fractions containing the target product 45 were combined,and the solvent was removed under reduced pressure. The second half of the crude was purified in thesame manner. In total, two fractions (1.98 and 11.22 g) were obtained (GC/MS purity: 93 and 82% 45,resp.; overall yield: 50%). For spectroscopic characterization, further purification of 45 was achieved byanother VLC. Rf (SiO2; CHCl3/AcOEt 19 : 1 (v/v)) 0.50. n20

D 1.4662. UV/VIS (MeCN): 234 (4.15). GC/MSPurity: GC/MS (70 eV): 97% at tR 16.9 min: 216 (8, Mþ ), 185 (46, [M�MeO]þ ), 184 (53, [M�MeO�H]þ ), 169 (37, [M�OOMe]þ ), 157 (34, [M�COOMe]þ ), 156 (100, [M�COOMe�H]þ ), 141 (83),128 (31), 125 (61, [M�CO�2 MeO�H]þ ), 115 (11), 111 (13), 97 (20). 1H-NMR (CDCl3, 300 MHz):5.18 (s, H�C(2)); 4.87 (dd, J ¼ 8.6, H�C(4)); 3.70, 3.69, 3.67 (3s, 3 MeO); 2.04–1.71 (m, CH2(5)); 0.92 (t,J ¼ 7.4, H�C(6)). 13C-NMR (CDCl3, 75 MHz): 171.9, 171.6 (C(1), C(7)); 167.7 (C(3)); 92.6 (C(2)); 55.9(C(9)); 52.0, 51.0 (C(8), C(10)); 47.5 (C(4)); 22.3 (C(5)); 11.6 (C(6)). For atom numbering, see Scheme 4.

5-Ethyl-4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (46a) and 3-Ethyl-4-methoxy-1-methylpyri-dine-2,6(1H,3H)-dione (46b ; ratio: 5 : 1 (w/w)). Compound 45 (11.22 g, purity: 82.7%; 42.55 mmol) wascooled (�108) and treated under N2 with a MeNH2/H2O soln. (40% (w/v); 30 ml). After stirring for10 min at �58 and 80 min at r.t., the mixture was stored overnight at þ48. Then, unreacted MeNH2 wasdistilled off by vacuum rotoevaporation, toluene (3�50 ml) was added and removed again underreduced pressure to azeotrope water. Afterwards, a soln. of MeONa, prepared from Na (2.0 g, 87.0 mmol)


Fig. 5. Key gHMBCs of 6

and MeOH (95 ml), was added, and the mixture was heated under reflux (90 min). Subsequently, thesolvent was distilled off, and the residual solid was dissolved in H2O (250 ml). Unreacted startingmaterial was recovered by extraction with Et2O (3�100 ml). Afterwards, the pH was adjusted to 5.3 bydropwise addition of AcOH (5.5 ml), and compound 46 was extracted with CHCl3 (3�100 ml). TheCHCl3 extract was dried (Na2SO4), and the solvent was removed in vacuo to yield an amber-colored sirup(8.3 g). Purification of the crude was achieved by VLC. Thus, TLC-grade SiO2 (100 g) was pre-conditioned with hexane (300 ml). Then, a soln. of the crude (dissolved in 30 ml CHCl3) was loaded onthe column, and elution was carried out with a linear gradient of hexane/CHCl3 (from 1 :1 to 100%CHCl3). Frs. 6–9 (100% CHCl3) containing 46 were combined, and the solvent was removed in vacuo byrotoevaporation to afford pure 46 as a bright-yellow sirup (2.66 g), which became crystalline at þ48(white crystals; 34% of the theory). M.p. 72–748. Rf (SiO2; CHCl3/AcOEt 19 : 1 (v/v)) 0.29. UV/VIS(MeCN): 212 (4.24), 254 (3.87). GC/MS Purity: GC/MS (70 eV): �99.9% at tR 20.0 min: 183 (4, Mþ ),168 (4, [M�Me]þ ), 155 (100, [M�CO]þ ), 140 (3, [M�Ac]þ ), 127 (16, [M�2 CO]þ ), 126 (15, [M�CO�MeN]þ ), 125 (11), 111 (58, [M�CO�N�2 Me]þ ), 98 (7). The isomers 46a and 46b were notseparated by GC but distinguished by 1H-NMR. Isomer 46a : 1H-NMR (CDCl3, 500 MHz): 3.78 (s,H�C(8)); 3.52 (s, H�C(5)); 3.17 (s, H�C(7)); 2.34 (q, J¼7.4, H�C(9)); 0.95 (t, J¼7.4, H�C(10)); isomer46b : 1H-NMR (CDCl3, 500 MHz): 5.43 (s, ¼CH, H�C(5)); 3.73 (s, H�C(8)); 3.31 (t, J¼4.8, H�C(3));3.16 (s, H�C(7)); 2.18–1.95 (m, H�C(9)) 0.76 (t, J¼7.4, H�C(10)).

(3Z)-5-Ethyl-3-(hydroxyimino)-4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (47). The mixture46a/46b (2.30 g, 12.55 mmol) was suspended under stirring in HCl (5.6%, (w/v); 180 ml). To the cooledmixture (� 58), a soln. of NaNO2 (1.12 g, 16.23 mmol dissolved in 40 ml of H2O) was added dropwiseunder cooling. After stirring below 08 (10 min), the mixture was extracted with CHCl3 (2�100 ml). Thecombined CHCl3 extracts were dried (Na2SO4), and the solvent was removed in vacuo to yield a brownsirup (3.17 g). The VLC purification on TLC-grade SiO2 (100 g; hexane/AcOEt gradient from 8 : 2 to 5 : 5(v/v)) yielded 47 as an orange gum (1.02 g; GC/MS purity: 91%) which became crystalline after a while.Recrystallization from AcOEt/hexane yielded pure 47 (0.17 g, purity: 99%) as orange-yellow crystals.From the mother liquor, further product was obtained (0.85 g, purity: 91%; total yield: 35%). M.p. 59 –618. Rf (SiO2; AcOEt/hexane 3 : 1 (v/v)) 0.60. UV/VIS (MeCN): 223 (3.76), 271 (4.04), 324 (3.62). GC/MS Purity: GC/MS (70 eV): 99% at tR 22.4 min: 212 (96, Mþ ), 195 (73, [M�OH]þ ), 180 (27, [M�NOH2]þ ), 169 (47, [M�CO�Me]þ ), 166 (35, [M�NOH�Me]þ ), 154 (35, [M�CO�2 Me]þ ), 139(46), 137 (51), 125 (40), 122 (35), 109 (42), 97 (73), 81 (100). 1H-NMR (CDCl3, 500 MHz): 15.98 (s,¼N�OH); 3.99 (s, H�C(8)); 3.30 (s, H�C(7)); 2.55 (q, J¼7.2, H�C(9)); 1.08 (t, J¼7.2, H�C(10)).13C-NMR (CDCl3, 125 MHz): 163.7 (C(2)); 162.1 (C(6)); 156.5 (C(4)); 137.3 (C(5)); 124.6 (C(3)); 62.3(C(8)); 26.1 (C(7)); 18.0 (C(9)); 13.5 (C(10)). C-Atom assignment by gHSQC and gHMBC (Fig. 6).

5-Ethyl-4-methoxy-1-methylpyridine-2,3,6(1H)-trione (39). A mixture of 47 (0.85 g; GC/MS purity:91%, 3.64 mmol), HCl (37% (w/w); 40 ml), and SnCl2 ·2 H2O (1.65 g, 7.31 mmol) was sonicated for 2 minand then stirred at r.t. (4 h). Afterwards, the mixture was extracted with CHCl3 (3�50 ml), thecombined extracts were dried (Na2SO4), and the solvent was removed in vacuo. The crude product(0.139 g) was finally purified by use of a Chromatotron� (2-mm layer, SiO2/gypsum 45 :18 (w/w);preconditioned with hexane). The elution of 39 was performed with a hexane/AcOEt linear gradient(80 : 20 to 50 : 50 (v/v)) and monitored by TLC. Yield: 0.103 g (13.5% of the theory). Yellow crystals. Rf

(SiO2; AcOEt/hexane 3 : 1 (v/v)) 0.61. UV/VIS (MeCN): 271 (3.92), 338 (3.04). GC/MS Purity: GC/MS(70 eV): >95% at tR 20.3 min: 197 (47, Mþ ), 182 (14, [M�Me]þ ), 154 (10, [M�Me�CO]þ ), 151 (5,


Fig. 6. Key gHMBCs of 47

[M�Me�MeO]þ ), 126 (12, [M�Me�2 CO]þ ), 112 (15, [M�MeN�2 CO]þ ), 97 (100), 84 (8), 69(32). For NMR data, see Table 2.

3-Ethyl-5-hydroxy-4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (¼ 3-Ethylhermidin ; 40). A soln.of 39 (0.029 g; 0.1486 mmol) in CHCl3 (10 ml) was treated with an aq. soln. of Na2S2O4 (9% (w/v); 10 ml)and bubbled with a vigorous stream of N2 (10 min). The GC/MS showed complete conversion of 39 to 40.After drying (Na2SO4), the solvent was removed in vacuo to yield 40 (0.026 g; 89% of the theory). GC/MS (70 eV): Purity: >99% at tR 18.5 min: 199 (34, Mþ ), 170 (100, [M�MeN]þ ), 142 (18, [M�MeN�CO]þ ), 127 (9, [M�MeN�CO�Me]þ ), 114 (10, [M�MeN�2 CO]þ ), 97 (10), 82 (15), 69 (19). ForNMR data, see Table 2 and Fig. 4.

Synthesis of the Reference Compound (3EZ)-3-Ethylidene-5-hydroxy-4-methoxy-1-methylpyridine-2,6(1H,3H)-dione ((E/Z)-41). A soln. of Na2S2O4 (0.059 g, 0.339 mmol) in H2O (12 ml) was degassedunder stirring with N2 (10 min). Then, 35 (0.05 g, 0.293 mmol) and MeCHO (4.8 ml, 3.78 g, 85.81 mmol)were added. After stirring overnight (N2 atmosphere), the mixture was extracted with CHCl3 (3�10 ml), and the combined extracts were dried (Na2SO4). A complete conversion of 35 to the (E)(Z)-41(E/Z ratio 1.6 : 1.0) was evidenced by GC/MS. Although (E/Z)-41 was stable in soln. at �808, an attemptto isolate the pure compound by VLC on SiO2 failed, because of stability reasons. Rf (SiO2; CHCl3/AcOEt 19 :1 (v/v)) 0.39. GC/MS purity: GC/MS (70 eV): >95% at tR 21.8 and 22.8 min: 197 (100, Mþ ),182 (61, [M�Me]þ ), 169 (6, [M�C2H4]þ ), 154 (18, [M�Me�CO]þ ), 151 (15, [M�Me�MeO]þ ),126 (67, [M�Me�2 CO]þ ), 113 (14), 108 (11), 95 (49, [M�Me�2 CO�MeO]þ ), 83 (18), 69 (59).Mass spectra of both isomers are very similar.

REFERENCES

[1] A. V. Moroni, F. Dal Bello, E. K. Arendt, Food Microbiol. 2009, 26, 676.[2] S. Weckx, R. Van der Meulen, D. Maes, I. Scheirlinck, G. Huys, P. Vandamme, L. De Vuyst, Food

Microbiol. 2010, 27, 1000.[3] K. H. Steinkraus, Antonie van Leeuwenhoek 1983, 49, 337.[4] K. H. Steinkraus, Compr. Rev. Food Sci. Food Safety 2002, 1, 23.[5] T. Xiong, Q. Guan, S. Song, M. Hao, M. Xie, Food Control 2012, 26, 178.[6] H. Lee, H. Yoon, Y. Ji, H. Kim, H. Park, J. Lee, H. Shin, W. Holzapfel, Int. J. Food Microbiol. 2011,

145, 155.[7] GHP, German Homoeopathic Pharmacopoeia, Homçopathisches Arzneibuch, Spezielle Herstel-

lungsvorschriften, Vorschrift 34c, Deutscher Apotheker-Verlag Stuttgart, Govi-PharamzeutischerVerlag Eschborn, 2008.

[8] O. Kandler, Antonie van Leeuwenhoek 1983, 49, 209.[9] M. G. G�nzle, C. Zhang, B.-S. Monang, V. Lee, C. Schwab, Food Microbiol. 2009, 26, 712.

[10] P. K. Hietala, H. W. Westermarck, M. Jaarma, Nutr. Metab. 1979, 23, 227.[11] M. G. G�nzle, N. Vermeulen, R. F. Vogel, Food Microbiol. 2007, 24, 128.[12] H. Rodr�guez, J. M. Landete, B. de las Rivas, R. Munoz, Food Chem. 2008, 107, 1393.[13] H. Rodr�guez, J. A. Curiel, J. M. Landete, B. de las Rievas, F. L. de Felipe, C. Gomez-Cordoves,

J. M. Mancheno, R. Munoz, Int. J. Food Microbiol. 2009, 132, 79.[14] A. M. Aura, Phytochem. Rev. 2008, 7, 407.[15] F. A. van Dorsten, S. Peters, G. Gross, V. Gomez-Roldan, M. Klingenberg, R. C. de Vos, E. E.

Vaughan, J. P. van Duynhofen, S. Possemiers, T. van de Wiele, D. M. Jacobs, Agric. Food Chem.2012, 60, 11331.

[16] A. Millet, F. Stintzing, I. Merfort, J. Pharm. Anal. 2009, 49, 1166.[17] A. Millet, F. Stintzing, I. Merfort, J. Pharm. Anal. 2010, 53, 137.[18] A. Millet, E. Lamy, D. Jonas, F. Stintzing, V. Mersch-Sundermann, I. Merfort, J. Agric. Food Chem.

2012, 60, 2148.[19] S. M. Duckstein, P. Lorenz, F. C. Stintzing, Phytochem. Anal. 2012, 23, 588.[20] M. Schwarzenberger, F. C. Stintzing, U. Meyer, U. Lindequist, Pharmazie 2012, 67, 331, 460.[21] P. Lorenz, C. Beckmann, J. Felenda, U. Meyer, F. C. Stintzing, Z. Phytother. 2013, 34, 40.


[22] G. Madaus, �Lehrbuch der biologischen Heilmittel 1979�, 2. Nachdruck der Ausgabe Leipzig 1939,Medimed Verlag, Hildesheim.

[23] F. Rugman, J. Meecham, J. Edmondson, Br. Med. J. 1983, 287, 1924.[24] P. Lorenz, M. Hradecky, M. Berger, J. Bertrams, U. Meyer, F. C. Stintzing, Phytochem. Anal. 2010,

21, 234.[25] P. Lorenz, M. Knoedler, J. Bertrams, M. Berger, U. Meyer, F. C. Stintzing, Z. Naturforsch., C 2010,

65, 174.[26] P. Lorenz, J. Conrad, J. Bertrams, M. Berger, S. Duckstein, U. Meyer, F. C. Stintzing, Phytochem.

Anal. 2012, 23, 60.[27] P. Lorenz, S. Duckstein, J. Conrad, M. Knçdler, U. Meyer, F. C. Stintzing, Chem. Biodiversity 2012,

9, 282.[28] S. K. Schmidt, S. Simkins, M. Alexander, Appl. Environ. Microbiol. 1985, 50, 323.[29] S. Stein, Y. Mirokhin, D. Tchekhovskoi, G. Mallard, �The NIST Mass Spectral Search Program�,

NIST/EPA/NIH Spectral Library, Vers. 2.0, distributed by National Institute of Standard andTechnology (USA), 2008.

[30] R. Bel-Rhlid, V. Crespy, N. Page-Zoerkler, K. Nagy, T. Raab, C. E. Hansen, J. Agric. Food Chem.2009, 57, 7700.

[31] R. Bel-Rhlid, N. Page-Zoerkler, R. Fumeaux, T. Ho-Dac, J. Y. Chuat, J. L. Sauvageat, T. Raab, J.Agric. Food Chem. 2012, 60, 9236.

[32] L. Svensson, B. Sekwati-Monang, D. Lopez Lutz, A. Schieber, M. G. G�nzle, J. Agric. Food Chem.2010, 58, 9214.

[33] S. van Beer, F. G. Priest, App. Environ. Microbiol. 2000, 66, 5322.[34] J. A. Curiel, H. Rodr�guez, J. M. Landete, B. de las Rivas, R. Munos, Food Chem. 2010, 120, 225.[35] N. Buron, M. Coton, C. Desmarais, J. Ledauphin, H. Guichard, D. Barillier, E. Coton, Food

Microbiol. 2011, 28, 1243.[36] G. A. Swan, Experientia 1984, 687.[37] G. A. Swan, J. Chem. Soc., Perkin Trans. 1 1985, 1757.[38] A. R. Forrester, Experientia 1984, 688.[39] D. A. Rathbone, N. C. Bruce, Curr. Opin. Microbiol. 2002, 5, 274.[40] W. H. Herath, D. Ferreira, I. A. Khan, Nat. Prod. Res. 2003, 17, 269.[41] H. Morishita, M. Ohnishi, Studies Nat. Prod. Chem. 2001, 25, 919.[42] S. M. Wittermer, M. Ploch, T. Windeck, S. C. M�ller, B. Drewelow, H. Derendorf, M. Veit,

Phytomedicine 2005, 12, 28.[43] A. R. Battersby, K. S. Cardwell, F. J. Leeper, J. Chem. Soc., Perkin Trans. 1 1986, 1565.

Received December 13, 2012


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