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A R O M A T I C E S T E R S F R O M 1SOTACH1S JAPON1CA 1023

Abb . 2 verdeutlicht, daß in den mittels der unter 5 . beschriebenen Methode gewonnenen Eluaten das 5., 6. und 10. eine biologische Aktivität am isolier-ten Rattenuterus zeigte. Außerdem erwies es sich, daß Bradykinin denselben RpWevt aufweist wie die hauptwirksame Fraktion (5 — 6) aus dem Haut-extrakt der Neunaugen.

Diskussion

Bereits der Zusatz des durch bloßes Abspülen der Tiere mit Methanol gewonnenen Schleimextraktes an den isolierten Rattenuterus und das Meerschwein-chenileum löste an diesen Organen Kontraktionen aus. Dieser stimulierende Effekt blieb auch nach Zugabe der Antagonisten gegenüber den üblichen in Organextrakten gelegentlich vorkommenden biolo-gisch aktiven Begleitsubstanzen voll erhalten. Da die Wirkung dieser aktiven Komponente durch Trypsin, Chymotrypsin und Carboxypeptidase B zer-stört wird, muß es sich um ein Peptid handeln. Die Wirkung der Carboxypeptidase B lieferte einen Hin-weis auf die Gruppenzugehörigkeit dieses Peptids, da dieses Enzym Bradykinin und Bradykinin-ähn-liche Peptide rasch, Tachykinine im allgemeinen je-doch nicht angreift.

Die Analyse des Wirkungsbildes des entfetteten Hautextraktes ähnelt dem von Bradykinin und Kal-lidin (Erschlaffung des Rattenduodenum). Eine blutdrucksenkende Wirkung an der Ratte war eben-falls vorhanden.

Dünnschichtchromatographisch ließen sich zwei aktive Komponenten nachweisen, wobei die Haupt-aktivität dem „Kin in " zukommt und der /?/-Wert von Bradykinin dem des Peptids entspricht. Die zweite, weniger wirksame Fraktion konnte einst-weilen nicht identifiziert werden. Da die Äquivalente aber an drei Präparaten nicht mit Bradykinin gleich waren, muß entweder eine Bradykinin-ähnliche Sub-stanz oder die Interferenz durch das zweite wirk-same Peptid neben „Bradykinin" angenommen werden.

Um auf eine Identität der beiden Substanzen schließen zu können, wären weitere chemische Unter-suchungen notwendig, welche aber wegen der un-genügenden Menge des zur Verfügung stehenden Materials aufgeschoben werden mußten.

Für wertvolle Hinweise sind die Verfasser Univ.-Prof. Dr. F. LEMBECK zu großem Dank verpflichtet.

Aromatic Esters from the Liverwort, Isotachis japonica1

AKIHIKO MATSUO, MITSURU NAKAYAMA , a n d SHÜICHI HAYASHI

Department of Chemistry, Faculty of Science, Hiroshima University, Hiroshima, Japan

(Z. Naturforsch. 26 b, 1023—1025 [1971] ; received July 24, 1971)

The oily portion of n-hexane and methanol extracts of the liverwort, Isotachis japonica Steph., was investigated, and three aromatic esters, benzyl benzoate, benzyl cinnamate and /?-phenylethyl cinnamate, were identified as major components. It was established that terpene compounds, which were main constituents in essential oils of other liverworts, were not contained in this liverwort in noticeable proportion.

The liverworts (Class Hepaticae), which are situated phylogenetically between the vascular plants and the algae, form an unique group of the plant kingdom together with mosses (Class M u s c i ) . Their haploid plant bodies (gametophytes) are grown f rom the spores as same as the pollen of higher

Reprints request to AKIHIKO MATSUO, Department of Che-mistry, Faculty of Science, Hiroshima University, Hiro-shima, Japan.

plants. Being the small size of the diploid plant bodies (sporophytes) , our chemical investigation performed mainly on the gametophytes. Owing to an interest in the chemical constituents of the game-tophytes, several phytochemical investigations have been systematically carried out in our laboratory.

1 Chemical constituents from Hepaticae, Part VII: Part VI, A . MATSUO, M . NAKAYAMA, Y . ISHIDA, and S . HAYASHI, Z . Naturforsch. 26 b, 32 [1971].

1024 A . M A T S U O , M . N A K A Y A M A , A N D S. H A Y A S H I

In connection with previous works on Bazzania pompeana2, Chiloscyphus polyanthus3 and Pellia fabbroniana 4 , the present work was carried out on chemical constituents of lsotachis japonica Steph. This leafy liverwort which is native in Yaku Island, Formosa and Southeast Asia, is placed in Herber-tineae and it has characteristic perfume; each cell of the leaf (gametophyte) contains ovoid oil bodies (8 — 1 0 - 5 — 6 jum in size) two by two.

Results and Discussions

The liverwort, after air-drying, was digested with 77-hexane and subsequently with methanol to obtain a semisolid and a dark green viscous substances, which were respectively subjected to filtration and/or elution chromatography to separate into oily and crystalline portions. The present paper deals with the identification of benzyl benzoate, benzyl cin-namate and /?-phenylethyl cinnamate from these oily portions.

Both oily portions f rom the n-hexane and metha-nol extracts showed resembling gas chromatographs consisting of six peaks as reproduced in Fig. 1.

Peak 1

Column: 10% DEGS on Diasolid L Column temperature: 225°

Peak 2

Retention time [min] >-

Fig. 1. Gas chromatogram of oily portion from methanol extract of lsotachis japonica.

Hereupon, the oily substances were tried to be separated into individual components by being sub-jected to elution chromatography and/or preparative gas chromatography; three components of peak 1, 5 and 6, were successfully isolated to be identified

2 S. HAYASHI, A . MATSUO, and T . MATSUURA, Experientia [Basel] 25, 1139 [1969] ; S. HAYASHI and A. MATSUO, ibid. 2 6 , 3 4 7 [1970].

3 S . HAYASHI, A . MATSUO, and T . MATSUURA, Tetrahedron

as benzyl benzoate, benzyl cinnamate and /?-phenvl-ethyl cinnamate.

Benzyl benzoate: The first component showing peak 1 had a molecular ion at m/e 212, and the IR spectrum contained several absorption bands at 3070 , 3035, 1605 , 1590, 1500, 715, 700, 1725, 1275 and 1115 c m - 1 . On the bases of this spectrum the component can be characterized as an aromatic ester, besides the N M R spectrum definitely indicated that the ester is benzyl benzoate (c) 5 .37 , 2H, singlet; 7 . 4 0 - 7 . 6 0 , 8H, complex ; 8 . 0 5 - 8 . 2 5 , 2H, com-plex) .

This assignment was also supported by the mass spectrum which had the characteristic fragment ions at m/e 77, 91 , 105 (base) , 133, 167, 184 and 212 ( M e ) .

Benzyl cinnamate: A component of peak 5 was isolated by preparative gas chromatography. In comparison with the above compound the IR and N M R spectra clearly exhibited that the component was also an benzyl ester of aromaitc acid (1590 , 1505, 700, 1730 , 1170 and 1015 c m " 1 ; <S 5.11, 2H, singlet; 7 . 1 5 - 7 . 4 0 , 10H, c o m p l e x ) ; the ap-pearance of an additional olefinic doublet at d 6.34 (1H, d, 7 = 1 6 . 5 Hz) and <S 7 .62 (1H, d, J = 16.5 H z ) , which can be ascribed to a trans-oriented double bond locating between carbonyl and phenyl groups, indicates the acid moiety to be cinnamic acid. Series of fragment ions appearing in the mass spectrum along with two characteristic ions at m/e 238 ( M @ ) and 192 ( [ M — H C O ^ H ] ®) are reason-ably explained by the structure of benzyl cinnamate {m/e 77, 91 (base ) , 103, 107, 131, 147 and 2 2 0 ) .

/?-Phenylethyl cinnamate: The third component showing peak 6 was isolated as a crystalline sub-stance (mp. 5 6 ° ) by preparative gas chromatogra-phy. The IR and N M R spectra exhibited the exi-stence of an ester linkage (1725 and 1170 c m - 1 ) , two monosubstituted benzene rings (1590 , 1500 and 700 c m " 1 ; <S 7 . 1 7 - 7 . 4 0 , 10H, complex) and trans-oriented double bond ( 1 6 5 0 and 8 7 0 c m - 1 ; (5 6 .32, 1H, doublet, 7 = 16.5 Hz ; 7 .60, 1H, doublet. 7 = 1 6 . 5 Hz ) .

By these evidence the component was charac-terized as an aromatic ester of cinnamic acid, and

Letters [London] 1 9 6 9 , 1 5 9 9 ; A . MATSUO and S. HAYASHI. ibid. 1970, 1289.

4 A . MATSUO, M . NAKAYAMA, and S. HAYASHI, Phytochemi-stry 10 ,430 [1971] .

A R O M A T I C E S T E R S F R O M 1SOTACHIS JAPONICA 1025

the alcohol moiety of this ester is estimated to be /5-phenylethyl alcohol on the bases of the remaining proton signals at <5 2 .94 (2H, triplet, J = 7.0 Hz) and d 4 .31 (2H, triplet, / = 7 .0 H z ) , which are reasonably assigned to two methylene groups locating between carbonyl and phenyl groups. A series of fragment ions at m/e 77, 91 , 104 (base) , 131, 147, 206 , 207 , 234 and 252 (M®) indicated the ester is /?-phenylethyl cinnamate.

The identity of the three esters was confirmed in comparison of the IR, N M R and mass spectra with those of the authentic samples.

Thus, it was firmly established that the major oily substances f rom Isotachis japonica are mainly constituted of benzyl benzoate ( 5 2 . 0 % ) , benzyl cin-namate (14 .6%) and /5-phenylethyl cinnamate ( 1 2 . 0 % ) , and that the characteristic aroma of the liverwort come f rom these aromatic esters. The per-centage of each ester in the total oily substances was calculated f rom the peak area of the gas chro-matogram. Although these aromatic esters have been isolated f rom many kinds of higher plants5 , this report is the first case in which aromatic esters were isolated f rom the liverworts.

The identification of the remaining minor com-ponents of peak 2, 3 and 4 was not achieved, be-cause the sufficient amount of each component could not be isolated. Each IR spectrum of the isolated minor components, although it was defect one, indi-cated the existence of ester bands.

Isotachis japonica contained no terpene com-pounds in noticeable proportion.

Experimental

The IR and NMR (60 MHz) spectra were obtained in CC14 solutions, and the mass spectra were taken at 80 eV ionization voltage, 1500 eV ion accelerating voltage and 200° ionization chamber temperature. The gas chromatograms were measured by using a TCD type apparatus in connecting with two kinds of separa-tion columns (each 3 mm x 3 m) , which were packed with Diasolid L (60 —80 mesh) impregnated by 10% DEGS or 10% SE-30, under 20 ml/min flow rate of helium.

Isotachis japonica was collected at Yaku Island in S.W. Japan. After the liverwort was dried in the shade for a several days, it (2800 g) was digested with n-hexane (20 I) and subsequently with methanol (17 / ) at room temperature. From the hexane solution a dark green semisolid substance (4.9 g) was obtained together with a crystalline substance (220 mg) which was pre-cipitated during the concentration, and the methanol solution affored viscous substance (4 g) only.

Each of both extracted substances was stepwise eluted over a silica gel column with petroleum ether and benzene to be separated into oily and crystalline substances. Oily substances thus obtained as main por-tions from both extracts were examined in the present investigation. As to the crystalline substances, paraf-fins, secondary alcohols and plant sterols, it will be reported elsewhere.

The authors are indebted to Dr. T A R O SEKI and Dr. KUNITO NEHIRA, D e p a r t m e n t of B o t a n y , H i r o s h i m a University, for identifying the liverwort.

5 Regarding with benzyl benzoate: E. DOMOLE and E. LEDE-RER, Bull. Chem. Soc. France 1958, 1128. Y.-R. NAVES, Helv. chim. Acta 42, 1692 [1959] ; 44, 1121 [1961]. Ben-zyl cinnamate: L. G. ENSMINGER, J. Assoc. Offic. Agr. Chem. 40, 423 [1957]. /?-Phenylethyl cinnamate: A. GORIS and H. CANAL, Bull. Chem. Soc. France 1936, 1982.