112 Tech. Bull.. Fac.. Agr.. Kagawa Univ..
O N
THE
SUBERIN I NTHE
O U T E R BARKOF
S O M E JAPANESE T R E E SPECIESKatsumi
HATA,
MuraoSOGO,
ToshioFUKUHARA,*
and MasayasuHOCHI**
The suberin is a constituent of cork cell walls, outer bark. The suberin has been known as being, in its native state, in the form of a polyestolide of hydroxy fatty acids, and can be dissolved by saponification. The elastic properties of cork have been considered to depend on the
presence of suber in.
The suberin content of the outer bark of several species was studied by Sarkov(1). He found in the bark of pine, fir, aspen, oak, and plane trees 2.0-8.3% of suberin, whereas the bark of various birch species contained 19.7-38.8%. The suberin content of the outer bark of cork oak, Ouercus suber, is par titularly high.
The saponification products of suberin of cork oak were investigated by ZETSCHE~~), who isolated phellonic acid, eicosanedicar boxylic acid, phloionic acid, and phloionolic acid from the saponification mixture. Migita et al.(3) studied on the suberin of Abemaki, Quercus variabzlis, produced in Japan, and found that the suberin of this species is composed of almost the same kind fatty acids as those of cork oak suberin. Some years ago, JENSEN(~) found that the countercurrent extraction is a very excellent method for the separation of the fatty acids obtained on the saponification of suberin, and he and his coworkers(5~6) investigated the acids of suberin of cork oak and birch in detail by applying this method.
Studies on the suberin in the outer bark of trees in the use of wood industry are important not only in regard the chemistry of bark constituents but also in connection with the utilization of bark. However, the studies on the suberin in the bark of Japanese trees are practically noneexistent outside the studies on suberin of Q. variabzlzs(3) Accordingly, the suberin contents of outer bark of 15 dicotyledonous and 16 gymnosperm trees were determined by the method of ZETSCHE~~) and the saponification products of suberin in the outer bark of Quercus acutissima, Pinus densiflora, and Ginkgo biloba were investigated in comparison with those of cork oak suberin, by countercurrent extraction method according to the JENSEN'S method(4).
The results of determination of suberin content are shown in Table 1. The outer bark of
Q suber showed particularly high suberin content, 34.0% based on the absolutely dried sample. The bark of Castanea crenata, Q. acutissima, Q serrata, Robinia pseudacacia, and G. bzloba contained 9-14% of suberin, while the suberin contents of the other species were very low. It seemed that the more suberin the bark contains the greater elastic properties it has.
The fatty acids in the saponification mixture of suberin in P densiflora, G bzloba, Q suber, and
Q.
actissima outer barks were studied by the method of JENSEN.(~) The soap mixture was produced by saponifying the extracted and sulfite-treated outer bark with the alcoholic KOH solution It was then freed from betulinol and other neutral organic materials. The soap sample was dissolved in a dilute sodium hydroxide solution and then was hydrochloric acid added toPresent address: *Otsuka Chem Ind Co , Naruto. Tokushima-Ken **Ehime-Ken Paper Making Experiment Station, Kawanoe
Vol.. 20, No.. 2 (1969)
Table 1 Suberin and extractive contents of outer bark..
Samples Ether extracts Dicotyledonous Bakkoyanagi Saliz Bakko Popura Populus nigra Nogurumi Platytar ya strobilatea Kuri Castanea trenata Kor ukugashi Quercus suber Kunugi Quertus atutzsnma Konar a Quertus serrata Keyaki Zelkowa serrata Hantenboku Lirzodendron tultpifera Kusu Cinnamomum Camphora Utsugi Deutna trenata Suzukakenoki Platanus orientalzs Niseakashiya Robinia pseudatatta Sendan Melza Azedarath Nankinhaze Sapium sebifereum Niwatoko
Sambutus Szeboldzana var typzca Gymnosperm Ichyo Gznkgo biloba Inugaya Cephalotazur drupatea Kaya Torreya nuttfera Sugi Cryptornerta japonzta Hinoki Chamaecypart s obtusa Akamatsu
Pinus dennf lora
Alcohol
114 Tech. Bull. Fac.. Agr. Kagawa Univ.
Samples
I
Ether extractsGoyomatsu
Pinus pentaph,y lla Hiba Thujopsis dolabrata Koyamaki Sciadopitys vertitillata Karamatsu Lariz Kaempferi Todomatsu Abies Ma yriana Akaezomatsu Picea Glehnii Byakushin Juniperus chinensis Himarayasugi Cedrrs Libani Senperusekoiya Sequoia .senpavirem Metasekoiya Metasequaia glyptostroboides Suberin
Note : % based on the weight of absolutely dried outer bark samples.
the fatty acids, which were extracted with ether and methylated with diazomethane. T h e methylated mixture was then chsomatogsaphed o n alumina (see Table 2, 4, 6 , a n d 8). T h e mixture of white crystalline substance eluted with ether was subjected t o a renewed chromato- graphy through alumina with different mixtures of petroleum ether and ethyl ether a s eluants
(see Table 3, 5, 7, and 9).
Table 2 Pinus densiflota bark suberin, chromatography-1 of the methyl esters of saponification mixture. (Sample, log)
-
f I I
1
1
EtherI
51
Colorless oilFraction
NO.
//
15-19 Alcohol
White fine crystals (Sample for chromatog. -2)
Yellow oil Eluant
White fine crystals (m. p. 72-74OC) (I) Brown oil I Eluted matter mg Appearance
Vol. 20, No. 2 (1969)
Table 3 Pinus densiflora bark suberin, chromatography-2 of the methyl esters
(Sample, 1 Og, from the fractions No 2-3 of chromatog -1)
Fraction No
Eluant
Petr
..
ether +ether (9+ 1, v./v) N1
coioriess oil Eluted matter mg 367 129 94 36 AppearanceWhite fine crystals in colorless oil@) White fine cr ystals (m p 69-7O0C) (11)
White fine crystals(m.p 74-75OC)(I) Light yellow crystals
1
Total1
70812-16 17-24
Table 4 Ginkgo biloba bark suberin, chromatography-1 of the methyl esters of saponification mixture. (Sample, log)
Petr, ether +ether 14 (1
+
1, v,,/v,) Etheri
25 Fraction No. Brown oil Brown oil1
Eluant Ether // // // AlcoholI
TotalEluted matter
I
AppearanceColorless oil
White cr ystals(Samp1e for chramatog. -2) Yellowish brown oil
White fine crystals in light yellow oil(1) Light yellow oil
Brown oil
Table 5 Ginkgo biloba bark suberin, chromatography2 of the methyl esters (Sample, 0 7g, from the fractions No 2-4 of chromatog. -1)
I ( I
Petr. ether +ether (9+l, v../v.) // // Fraction No Yellow oil
White fine crystals(m.p. 63-66OC)(II) Yellow oil I I I Eluant Total
I
555 11-16 17-24 Eluted matter mg AppearancePetr.. ether +ether 11 (1
+
1, v./v,,)Ether
N
116 Tech. Bull. Fac. Agr.. Kagawa Univ. Table 6 Quertus atutissima bark suberin, chromatography-1 of the methyl esters of saponication
Fraction No.
Eluant Eluted matter mg Ether N // N Alcohol N Total
1
9870 AppearanceWhite fine crystals (Sample for chromatog. -2)
Light yellowish green oil cont a ining fine crystals
Light yellow oil containing fine crystals White fine crystals (m.p. 72-74OC)(I) Yellow wax
Yellow powder
Petr, ether +ether
(9
+
1, v./v..)Table 7 Quertus atutissima bark suberin, chromatography-2 of the methyl esters. (Sample, 1 Og, from the fraction No. 1 of chromatog -1)
1
Petr et; +ether ( l + l , v,/v,) FractionNo
Ether
I
White fine crystals in colorless oil(I1) N
White fine crystals in light yellow oil(I1) White fine crystals(m.p. 69-70°C)(II) White cr ystals(m p 73-75 OC) (I)
Eluant Eluted matter
Light yellow powder
-
-Appearance
1
TotalI
964
I
Table 8 Quertus suber bark suberin, chromatography-1 of the methyl esters of saponification mixture. (Sample, log)
Fraction No,.
Eluant Eluted matter mg Appearance Ether N N Alcohol N Total
White plate crystals (Sample for chromatog.-2) Light yellow wax Yellow powder White fine crystals Yellow wax
Vol. 20, No. 2 (1969) 117 Table 9 Quertus suber bark suberin, chromatography-2 of the methyl esters
(Sample, 1 Og, from the fractions No 1-3 of chromatog -1) Fraction
No.
1
Eluant Eluted matter
Petr, ether +ether (1+1, v./v) // N
1
Ether1
105I
Total1
933 AppearanceWhite fine crystals(m p. 64-65OC)(II) Colorless oil
White wax(m.p 40-40 5OC)(III) Light yellow powder
White fine crystals in yellow oil(1) Light yellow powder
White cr ystals(m p 74-76OC)(I) Light yellow powder
Table 10 Fatty acids isolated in crystalline form from saponification products of suberin.
~ - -
I n this way, crystals of methyl esters of phellonic acid (I), eicosanedicarboxylic acid (11), and a n acid,
CI8
H34 0 4 (111) were isolated from Q. suber bark suberin, and crystals of I and I1 from suberin in the outer bark of Q. acutissima,P,
d e n s i , f l o ~ a , and G. biloba.. T h e corresponding acids were obtained from these methyl esters by saponification with alcoholic alkali. T h e acids obtained in crystalline form from the saponified mixture of suberin in the outer bark of these 4 species are recorded in Table 10.T h e results for
&.
suber bark suberin a r e in accord with the results obtained by ZE'TSCHE(~)and also by JENSEN 4 . However, the suberin of the other 3 species gave only phellonic acid and eicosanedicarboxylic acid. T h e saponification products from Q.. subere bark suberin were composed mainely of white crystallline substances, whereas those from the subesin of Q. a c u t i s s i m a and
G.
biloba contained much oily substances and gave less crystalline substances. P. dens<flora bark suberin gave comparatively more crystalline substances. This indicates that the constituents of suberin differ by species, and also that the materials which have been obtained by applying the suberin determination method of ZE.TSCHE(~) are much variable by species.Phellonic acid(1) Eicosanedicar
-
boxylic acid(I1) Acid, C1 8H3404 (111) Q w t u s Quertus atutissha0
0
Formula
1
Pinus densif lora1
biloba Gtnkgo0
0
0
HOCH2 (CH2) 2 &OOH HOOC (CH2)20COOH0
O
0
O
118 Tech. Bull.. Fac.. Agr. Kagawa Univ. Experimental
1. Determination of suberin.
Five g of finely ground outer bark was successively extracted 2 days with ether, 2 days with alcohol, and 4 x 2 hours with 150 ml of a 5% solution of Na2S03 in water. The purified bark was saponified with 200 ml of a 3% solution of KOH in alcohol for 3 hours. The mixture was filtered through a glass filter. The unsaponified substance was once more boiled with lOOml of alcohol and then filtered through the same filter as above. T h e alcoholic filtrates were combined, and the alcohol was distilled off by vacuum evaporation. T h e residue was dissolved in a small amount of water and the solution was acidified by adding hydrochloric acid. The acidic mixture was shaken with 3 x 100 ml of ether in a separating funnel. The ether was evaporated and the extract was dried and weighed.
2. Preparation of the soap.
Finely ground outer bark(200-500g) was extracted successively 5 days with ether, 5days with alcohol, and 4 x 2 hours with 5% solution of Naz SO3 in water. The purified bark was refluxed in an alcoholic KOH solution. The mixture was filtered and most of the alcohol was distilled off. Small amounts of KOH were added until the solution remained slightly alkaline to phenol- phtalein after boiling. The rest of the alcohol was then evaporated and the residue was dried on a water bath. The soap was extracted with ether by grinding it in small portions to remove betulinol and other extractable substances of the outer bark.
3. Preparation of the methyl esters.
The soap was weighed to contain 10 g of the cork fatty acids and dissolved in distilled water. The solution was acidified to pH 2 with hydrochloric acid and the liberated acids were extracted with ether by shaking in a separating funnel The ether solution was dried with sodium sulfate, cooled to about O°C, and then diazomethane in ether was added with stirring. T h e reaction mixture was allowed to stand overnight and the excess of ether and diazomethane was distilled off,
4. Chr omatogr aphy-1.
The ether solution of the methyl esters was introduced on a column of about 150g of alumina (200-300 mesh), 3.2 cm in diameter The elution was carried out with ether and then with alcohol, taking each 100 ml of eluted solution for every fraction, as presented in Table 2, 4, 6, and 8.
5. Chromatography-2.
About 1 g of white fine crystals which taken in the fractions No. 1-4 of the chromatography
1 was dissolved in a 9 : 1 mixture of petroleum ether (b.p 32-40°C) and ethyl ether and introduced on a column of about 25 g of alumina (200-300 mesh), 2.5 cm in diameter. The course of the elution is shown in Table 3, 5, 7, and 9 Each 50 ml of eluted solution was taken for every fraction.
6. Purification of the methyl esters and the acids.
Crystals of the methyl esters were repeatedly recr ystalized from petroleum ether respectively to a constant melting point. The recrystalized methyl esters were saponified by refluxing 30 minutes with an excess of 0.5 N alcoholic alkali. T h e alcohol was evaporated, the residue
Vol. 20, No. 2 (1969) 119 dissolved in water, the solution acidified, and the acids taken up in ether. The acids were recrystalized repeatedly from ether respectively to a constant melting point. Melting point of the pu~ified substances was respectively 96-96.5"C and 74-76°C for phellonic acid and its methyl ether, 123-124°C and 71-72°C f o ~ eicosanedicarboxylic acid and its methyl ether, and 81-82°C and 40-40.5"C for the acid, C18 H34 O4 and its methyl ether.
Acknowledgement
The authors are grateful to Dr. Waldemar JENSEN, Professor of the Finnish Pulp and Paper Research Institute, Helsinki, Finland for presenting some reprints of his reports of studies on suber in.
This work was presented at the 14th Meeting of the Japan Wood Research Society, held in Tokyo, April, 1964(7).
This investigation has been supported in part by a grant from the Ministry of Education of Japan.
References
(1) SARKOV, V I , KALNINA, V N , SOBETSKIJ, S V : C9 A 341 5273(1940). (2) ZETSCHE, F : Handbuthe der Pflanzen Analyse Bd 111, 224(1932)
(3) MIGITA, N , KANDA. T., FUJINAMI, R : J Japan, Forestry Sot 33, 424(1951) (4) JENSEN, W : Paper and Timber, B, 32, 261(1950)
(5) JENSEN, W , KINENE, P : Ibzd. 35, 407(1953). (6) JENSEN, W., STM MAN, R. : Ib2d. 36, 427(1954)
(7) HATA, K , SOGO, M : Abstracts of Papers 14th Meetzng Japan Wood Researth S o t , 1964, p 73
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