Bot. Meg. Tokyo 83: 263-269 (August 25, 1970)

Flavonoid Pattern in the Pterid4ceae, III. Constituents in the Fronds of Dennstaedtia quitf ordii

Yoko AKABORI* and Masao HASEGAWA*

Received April 11, 1970

Abstract

Flavonoid in the fronds of Dennstaedtia wilfordii were isolated to study the flavonoid pattern of the genus Dennstaedtia. The fronds contained pinocembrin 7-rhamnoglucoside (III), pinocembrin 7-glucoside (II), isoquercitrin (VII) and astragalin (V). Among them pinocembrin 7-rhamnoglucoside (III) was obtained as crystal and the sugar component was confirmed as neohesperidose (VIII) by NMR, enzymic hydrolysis and chromatographic behaviors. Other were identified by paper and thin layer chromatography. The flavonoid pattern of D. wilfordii was different from that of the four species belonging to the genus Adiantum, which had been already reported.

In the previous papers1'2' we reportedd the flavonoid patterns of four species of the genus Adiantum A. aethiopicum, A. monochlamys, A. capillus-veneris and A. cuneatum. They contained flavonol glycosides as the main flavonoids and their structural differences were observed in the glycosidec sugars. The genus Dennstaedtia is characterized3' by the presence of indisia, exteriorly opened indisia, undimidiated and uncuneif ormic ultimate pinnules and marginal son. While the genus Adiantum can be distinguished from Dennstaedtia by the presence of indisia consisting of the reflexed margin of the segment, scaly rhizomes and soni borne on the reflexed margin of the pinnules. The morphological differences are of great to taxonomy, so some taxonomists have elevated both genera to the rank of families4'5,s', i. e., Dennstaedtiaceae and Adiantaceae. So we deal with the flavonoid constituents of D. wilfordii concerning to taxo- nomical studies in this report.

Experiments and. Results

Isolation of flavonoids. Fronds of D. wilfordii were collected at Nippara on the west side of Tokyo in early November, and 290 g of fresh leaves were extracted as described in the previous papery. After addition of a small amount of ethanol to the ethyl acetate extract, it was allowed to stand for several days. White crystall mass appeared was gathered by filtration; yield, 0.5 g. This crude pinocembrin 7- rhamnoglucoside (III) was recrystallized from aqueous ethanol and from 99% ethanol to give colorless needles of m. p. 265-266°. * Department of Biology, Faculty of Science, Tokyo Metropolitan University, Setagaya Ku, Tokyo, Japan. 264 AKABORI,Y. and HASEGAWA,M. Vol. 83

Pinocembrin 7-glucoside (II) was isolated from the mother liquor of recrystalli- zation using thin layer and paper chromatography. Solvent systems were ECFW (TLC), BAW and 6% AA (abbreviations are in Table 1). After filtration of pinocembrin 7-rhamnoglucoside (III), the mother liquor was subjected to a large scale paper chromatography with the solvent systems of BAW and 6%AA. Two brown bands on the chromatogram under a UV lamp were ex- tracted with 50°o ethanol. Each extract was hydrolyzed with a mixture of 2 N HCl and ethanol (1:1)7'. On hydrolysis, the lower gave (VI) and glucose, while the aglycone of upper glycoside was not sufficiently determined be- cause of its minute amount. Rf values and absorption maxima of the two glycosides showed their identity with isoquercitrin (VII) and astragalin (V). Pinocembrin 7-rhamnoglucoside (III). An ethanolic solution gave a brown color- ation with ferric chloride and an aqueous solution gave a reddish brown coloration. In alcoholic solution it gave a yellow orange coloration with magnesium powder and conc. hydrochloric acid, but no coloration was given with zinc powder and conc. hydrochloric acid. Absorption : /ZM°H330 ma, 286 mp (log =4.10) Specific rotation : [a]D=-112.7° (2.9% in pyridine), [a]D=- 75.0° (0.4% in 80% aqueous acetone). Anal. Calc. for C27H32O132H2O:C, 56.53: H, 5.75 Found : C, 56.73: H, 5.43. Rf values and IR spectrum are shown in Table 1 and Fig. 2, respectively. The acetate was prepared as usual with pyridine and acetic anhydride, m. p. 121-122°. Hydrolysis of pinocembrin 7-rhamnoglucoside (III). The pinocembrin glycoside (69.8 mg) was hydrolyzed with 5% hydrochloric acid on a water bath, and 30.7 mg (43.9°0) of the aglycone was obtained. When it is posturated as pinocembrin 7- rhamnoglucoside (III) the theoretical yield of pinocembrin (I) will be 45.3%. The aglycone was recrystallized from aqueous ethanol to give colorless needles of m. p. 192-194°. After subliming at 165-175°/3 mm Hg, it gave a melting point of 195-196°. No depression was observed by admixture with authentic pinocembrin (I)$' obtained from Prunus verecunda.

Table 1. Rf values x 100 of pinocembrin 7-rhamnoglucoside and its hydrolyzed products. August, 1970 Flavonoid Pattern in the Pteridaceae III 265

Fig. 1. Structures of flavonoids isolated from D. wilfordii and neohesperidose.

The filtrate was treated with Amberlite IRA-410 and concentrated to a syrup. This was paperchromatographed, and glucose and rhamnose were identified by com- parison with authentic sugars. Quantitative analyses of sugars by wilson's method9j and of pinocembrin (I) by spectrophotometric method showed that the ratio of

Fig. 2. IR spectra of pinocembrin and pinocembrin 7-rhamnoglucoside. (KBr) A = pinocembrin 7-rhamnoglucoside, B = authentic pinocembrin, C = pinocembrin 266 AK.ABORI,Y. and HASEGAWA,M. Vol. 83 pinocembrin, glucose and rhamnose was to be 1 : 1 : 1. Rf values of these three components are listed in Table 1. IR spectra of the authentic pinocembrin and the= aglycone obtained by hydrolysis are shown in Fig. 2. Structure of glycosidic sugar of Pinocembrin 7-rhamnoglucoside (III). a. Enzymic hydrolysis. Pinocembrin 7-rhamnoglucoside (III) was treated in the' same procedure as Okada10' with a mixture of naringinase and hesperidinase which was obtained from Aspergillus niger. This enzyme mixture split rhamnose from the glycoside. In parallel, and were also tested for comparison. The glycoside gave rhamnose and pinocembrin 7-glucoside (II) by hydrolysis with hesperidinase, which was prepared by acid treatment'0' from the mixture of both enzymes. Chromatographic behaviors of the hydrolyzed products are shownn diagramatically in Fig. 3. b. NMR spectrum of the glycoside acetate. Rosier et a111' reported that the flavonoids, rutinoside and neohesperidoside, were distinguished by NMR spectra of their acetates in CDC13. They have described that the ratio of integrated proton numbers in the region 4.5-5.5 ppm (int. TMS) and 3.4-4.4 ppm was 7 : 5 in the case of neohesperidoside, and 8:4 in the case of rutinoside. From the chart (see Fig. 4) of pinocembrin 7-rhamnoglucoside acetate, we could read a proton ratio between the above regions. It showed a ratio of 7: 5. c. Hydrolysis with acetic acid. Pinocembrin 7-rhamnoglucoside (III), naringin and hesperidin were hydrolyzed with 10% acetic acid for 14 hours12', respectively. On cooling, a solidified mass in each flask was filtered, and the filtrate was evapo- rated using a rotary evaporator. Each of the residue was paperchromatographed and then subjected to paper electr- ophoresis13' in 0.2 M borate buffer, pH 10. From hesperidin ( 7-rutinoside) glucose, rhamnose and rutinose were detected, but in the case of naringin and pinocembrin 7-rhamnoglucoside (III) gave glucose and rhamnose. From the results obtained above, we could think of this glycoside as pinocem- brin 7-neohesperidoside. Pinocembrin 7-glucoside (II). Pinocembrin monoglycoside was compared on paper chromatogram with pinocembrin 7-glucoside (II) obtained by partial hydrolysis of

Fig. 3. Enzymic hydrolysis of pinocembrin 7-rhamnoglucoside and related compounds. Abbreviations : H = hesperidin, N = naringin, P = , S = pinocembrin 7-rhamno- glucoside. H', N' and S' inicate reaction products of H, N and S, respectively. August, 1970 Flavonoid Pattern in the Pteridaceae III 267

Fig. 4. NMR spectrum of acetylated pinocembrin 7-rhamnoglucoside.

-pinocembrin 7-rhamnoglucoside (III) with naringinase. Rf values of the aglycone, the glycoside and the sugar are summarized in Table 2. Astragalin (V) and isoquercitrin (VII). Rf values of astragalin (V) and iso- quercitrin (VII) and its aglycone and sugar were also shown in Table 2. Their absorption maxima were coincident with those of the authentic samples.

Table 2. Rf values x 1.00 of pinocembrin 7-glucoside, isoquercitrin, astragalin and their hydrolyzed products.

Discussion

As for pinocembrin 7-rhamnoglucoside (III), Ribas et a114' isolated it from Sarothamnus commutatus and gave the name sarotanoside (sarantoside, Chem. Abst. 54. 2502 (1960)). Matas15' also isolated a pinocembrin rhamnoglucoside (III) from Cytisus commutatus, but he thought it was an isomer of sarotanoside by comparison of their melting points of acetates, specific rotations and color reactions with zinc 268 AKABORI, Y. and HASEFAWA, M. Vol. 83 and hydrochloric acid. But unfortunately, dissaccharide components of both glyco- sides have not been studied in detail. The glycoside obtained by us resembles closely to sarotanoside in melting point, but it differs in melting point of the acetate and specific rotation as shown in Table 3. A slight difference was also observed in melting point of the acetate and specificc rotation of an isomer described by Matas. Chopin and Dellamonica16' have synthesized pinocembrin 7-rhamnoglucoside,, pinocembrin 7-neohesperidoside, which has the melting point of 237° and the specific. rotation of-112° in pyridine. However, this compound is different from our glycoside in its melting point (cf., Table 3). Disagreements as such will be studied later in_n detail. Concerning the relationship between taste and structure of the glycosides Horowitz17j describes that neohesperidosides of have a bitter taste in general. Our glycoside has slightly bitter taste, so this fact suggests the glycosidicc sugar may be neohesperidose (VIII). The flavonoid pattern of D. wil f ordii is characterized by a large content of glycoside, especially pinocembrin 7-neohesperidoside, and a lesser amounts of flavonol glycosides. The flavonoid pattern of D. wilfordii differs in this point. from that of the four species of the genus Adiantum.

Table 3. Comparison of some properties of pinocembrin 7-rhamnoglucosides reported in literatures and isolated from Dennstaedtia wilfordii.

Acknowledgment

We wish to thank Dr. S. Okada of Osaka Municipal Technical Research Insti- Lute for his kind supply of enzymes and also to Professor K. Kinoshita of Hoshi College of pharmacy for the NMR spectral measurements.

References

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