Functional constituents in the peel and seed of camu-camu (Myrciaria dubia)

氏

名

金

嶋

泰

学位（専攻分野の名称）

博 士（生物産業学）

学 位 記 番 号

甲 第 683 号

学 位 授 与 の 日 付

平成 27 年 3 月 17 日

学 位 論 文 題 目

Functional constituents in the peel and seed of

camu-camu (Myrciaria dubia)

論 文 審 査 委 員

主査 教

授・薬 学 博 士

西 澤

信

教

授・農 学 博 士

戸 枝 一 喜

教

授・農 学 博 士

中 川 純 一

論 文 内 容 の 要 旨

The fruits of camu-camu (Myrciaria dubia) are

harvested in the rain forest of the upper Amazon in

Peru and Brazil. Camu-camu is famous for its high

vitamin C content (more than 2000mg/100g) compared

with those of other tropical fruits such as acerola and

passion fruit. Therefore, the fruit has been attracted

attention of researchers, and many studies were

re-ported on the constituents of camu-camu ; carotenoids,

anthocyanins, pulp in juice and volatile compounds.

Because the fruit is difficult to keep fresh, its juice has

been produced in the harvest area, and residual

substances, peel and seed, are discarded as industrial

wastes. Recently, many processed foods using

camu-camu juice such as canned and bottled juice and

vinegar are commercially available in advanced

countries, and the cultivation area expanded from

Peru to neighboring country, Brazil, Venezuela, and

Colombia. According to the increase of camu-camu

juice production, the residual peel and seed were also

increased. The amount of residual peel and seed is

nearly 40% of weight of fresh fruit. The pulp in the

fruit has been used for ingredient of functional foods,

but the residual peel and seed have not yet been

utilized. The utilization of the residues must be

beneficial for the total industry of camu-camu,

in-cluding juice production and cultivation. Therefore,

we have been studying the functional constituents in

the peel and seed of camu-camu fruits, and our

in-vestigation resulted in the isolation of C-glycosidic

ellagitannins along with condensed tannin with

oxidant constituents, and acylphloroglucinols as

anti-microbial constituents.

1． The functions of the extracts of camu-camu

peel and seed

As the preliminary studies, the n-hexane extracts

and 50% acetone extracts were prepared from the peel

and seed of camu-camu. The n-hexane extracts

showed antimicrobial activities against

Staphylo-coccus aureus. On the other hand, the 50% acetone

extracts were demonstrated to contain large amount of

polyphenols by Folin-Ciocalteu method. The contents

of polyphenols in the extracts of the peel and seed were

150.0mg/g and 404.5mg/g, respectively, and the

con-tent in the seed are more than four times higher than

that of acerola juice residue (94.6mg/g). Moreover,

vanillin-HCl assay indicated that the content of

densed tannin was about 20% of total polyphenol

con-tent. The 50% acetone extracts and their fractions

obtained by Sephadex LH-20 chromatography

exhib-ited antioxidant activities by 1,

1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay. The

ac-tivity was shown to be related to the polyphenol

con-tents of the extracts and fractions.

2． Antioxidant activities of C-glycosidic

ellagi-tannins in the peel and seed of camu-camu

Six C-glycosidic ellagitannins, grandinin (1),

vesca-lagin (2), castavesca-lagin (3), methylvescavesca-lagin (4),

sta-chyurin (5), and casuarinin (6) were isolated from the

50% acetone extracts of the peel and seed by repeated

chromatographic purifications such as medium

pres-sure reversed phase chromatography and preparative

high performance liquid chromatography (HPLC).

Among these tannins, 3 and 2 were main constituents

─ 4 ─

and were consisted 10% and 5% of total polyphenol

content in 50% acetone extracts of the peel and seed,

and HPLC analysis indicated that the peel and seed of

camu-camu contains same C-glucosidic ellagitannins.

These C-glycosidic ellagitannins exhibited strong

antioxidant activities by DPPH and 2,2′

-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays

(single electron transfer assays). Compound 5 and 6

are diasteromeric isomers of C1 position of

-glucose

with the same substituents, two

hexahydroxydipheno-yl (HHDP) groups and a gallohexahydroxydipheno-yl group. The activities

of 5 in single electron transfer assays were stronger

than 6, and the activities of 2 and 3 were weaker than 5

and 6, respectively, but the relation between the

activities of 2 and 3 was the same in the case of 5 and 6.

These tannins (2 and 3) are also diasteromeric isomers

of C1 position of

-glucose with the same substituents,

a nonahydroxyterphenoyl (NHTP) group, a HHDP

group and a galloyl group. These results indicated

that the antioxidant activities the C-glycosidic

ellagi-tannins are affected by the flexibility around C3, C4

and C5 positions of

-glucose and the conformation of

the hydroxyl group at C1 position of

-glucose. These

tannins also showed antioxidant activities by oxygen

radial absorbance capacity (ORAC) assay, a hydrogen

atom transfer assay, but the order of strength of the

activities was not the same as those of single electron

transfer assays, thus the activities by ORAC method

were assumed to be affected other structural factors of

the tannins than single electron transfer assays.

These C-glycosidic ellagitannins were isolated from

many plants mainly belonging to Fagaceae,

Myr-taceae, Juglandaceae, Casuarinaceae, Stachyuraceae,

Betulaceae, Punicaceae, Lythraceae, Combretaceae,

Melastomataceae, and Trapaceae, and in most cases,

these tannins were isolated from leaves, bark, wood,

and branches. However, camu-camu is the first case

in which these tannins were isolated from the peel and

seed of a plant belonging to Myrtaceae.

3． Condensed tannins in the peel and seed of

camu-camu

Condensed tannin in the 50% acetone extracts of the

peel and seed was purified by Sephadex LH-20

chro-matography. Condensed tannins were composed of

catechin 3-ols connected by C-C bond, thus their

structure must be estimated by thiol degradeation.

The condensed tannin was degraded with

2-sul-fanylethanol in 0.2M HCl, and the products were

isolated by MCI gel CHP-20P chromatography and

preparative HPLC. The structures of these

com-pounds were characterized as (-)-epigallocatechin

3Ogallate (7, EGCG), ()4b(2hydroxyethylsulfanyl)

-EGCG (8) and (-)-4b-(2-hydroxyethylsulfanyl) -

epi-catechin 3-O-gallate (9). According to these results,

the condensed tannin in the peel and seed of

camu-camu was supposed to be composed of EGCG and

(-)-epicatechin 3-O-gallate (ECG), and the C4 positions of

EGCG and ECG were involve in one side of C-C bond,

but another position of C-C bond have not yet been

determined, because dimeric degradation products

(10) was not characterized. In the chromatogram of

the degradation products, EGCG was main component

of the condensed tannin. The end unit of condensed

tannin chain was supposed to be EGCG, as ECG was

not detected in the chromatogram.

4． Acylphloroglucinols as antimicrobial

con-stituents in the peel and seed of camu-camu

As antimicrobial constituents in the n-hexane

ex-tracts, two compounds (11 and 12) were isolated from

the peel of camu-camu and other two (13 and 14) from

the seed. Counter-current partition was effective for

the isolation of these compounds, because presence of

large amounts of neutral lipid disturbed the

fractiona-tion by preparative HPLC and thin layer

chromatogra-phy (TLC). Structure elucidation of these compound

by

_H-

_{C- and 2D-nuclear magnetic resonance (NMR),}

and mass spectra revealed these compounds as

acyl-phloroglucinols.

Compound 11 was characterized as rhodomyrtone

(6,8-dihydroxy-2,2,4,4-tetramethyl-7-(2-methyl-

1-oxo-butyl) -9-(2-methylpropyl) -4, 9-dihydro-1H-xanthene-1,

3(2H) -dione) isolated from Rhodomyrtus tomentosa,

There was also reported rhodomuytosone B, an isomer

of rhodomyrtone, but the structure was confirmed by

comparison of their spectral data and NOESY

spec-trum.

Compound 12 was characterized as 6,8-dihydroxy-2,2,

4, 4-tetramethyl-7-(2-methyl-1-oxopropyl) -

9-(2-meth-─ 5 9-(2-meth-─

ylpropyl) -4, 9-dihydro-1H-xanthene-1, 3(2H) -dione. It

is a new acylphlorogulcinol, and is named as

myrciar-one A, having 2-methyl-1-oxopropyl group at C7

position instead of 2-methyl-1-oxobutyl group of 11.

Compound 13 was characterized as

isomyrtucommu-lone B,

2,2,4,4tetramethyl7(2methyl1oxopropyl)9(1methylethyl)4, 9dihydro1Hxanthene1, 3 (2H)

-dione. Isomyrtucommulone B was reported as the

degradation product of myrtucommulone A, a trimeric

acylphloroglucinol. Thus, it is the first time that

iso-myrtucommulone B was isolated from natural

re-source.

Compound 14 was characterized as 2, 2, 4,

4-tetramethyl-7-(1-methyl-1-oxobutyl) -9-(1-methylethyl)

-4,9-dihydro-1H-xanthene-1,3(2H)-dione. It is another

new acylphloroglucinol, and is named as myrciarone

B, having 1-methyl-1-oxobutyl group instead of

2-methyl-1-oxopropyl group of 12.

These acylphloroglucinols exhibited antimicrobial

activities against mainly to Gram-positive bacteria

and the MIC values (0.78-6.25mg/ml) were equal or

lower than those of kanamycin (1.56-6.25mg/ml).

Acylphloroglucinols were isolated mainly from plants

of Hypericaceae and Myrtaceae, and various biological

activities, such as antimicrobial, antivirus,

anti-inflammatory activities were reported. Among many

phloroglucinols reported, rhodomyrtone has been

attracted attention because of its antimicrobial

activi-ties against meticillin-resistant Staphylococcus aureus

and vancomycin-resistant Enterococcal strains.

5． Other functional properties of camu-camu.

Inhibitory effects of 50% acetone extract of the seed

and its fractions against a-amylase and pancreatic

lipase were tested. The fractions containing the

con-densed tannin exhibited inhibitory effects on

a-amylase, especially 100% acetone Fr. was more than 7

times stronger effect compared with that of positive

control, acarbose. The difference in the inhibitory

effects was recognized among fractions, precise

inves-tigation must be needed to clarify the effects of the

condensed tannin against a-amylase activity. The

100% acetone Fr. also inhibited on pancreatic lipase,

and the inhibitory effect was more potent compared

with that of positive control, EGCG. Moreover,

C-glycosidic ellagitannins, 2 and 3, showed inhibitory

effects against pancreatic lipase. Effects of the

con-stituents in the 50% acetone extract on digestive

en-zymes may lead to development of functional foods

using residual parts of camu-camu juice production.

Deodorant effects of the 50% acetone extracts were

also tested using n-valeric acid, isovaleric acid and

caprylic acid, as specific constituents in sweat.

Sig-nificant deodorant effect against n-valeric acid was

recognized. As condensed tannin, such as persimmon

tannin, is known to have deodorant effect. The

con-densed tannin in camu-camu may have deodorant

effects and may applied for cosmetic goods.

In conclusion, six C-glycosidic ellagitannins were

isolated as responsible constituents of antioxidant

ac-tivities of the peel and seed of camu-camu, along with

the characterization of component units of the

con-densed tannin. Four acylphloroglucinols were

iso-lated as responsible constituents of antimicrobial

ac-tivities of the peel and seed of camu-camu and two

acylphloroglucinols were new compounds. Beside

antioxidant and microbial activities, many n-hexane

and 50% acetone extracts were revealed to have many

functional effects, thus utilization of the peel and seed

of camu-camu will be expected as functional foods,

cosmetics and medicines.

審 査 報 告 概 要

本論文は，南米ペルー原産のフトモモ科植物であるカ

ムカムの果皮と種子に含まれている機能性成分の探索と

抗酸化性，抗菌性に関する研究である。カムカムは東農

大メルカードが深く関与している植物で，現地の換金作

物として発展が期待されている。カムカム果実は傷みや

すく現地で果汁を生産しているが，果皮と種子が廃棄物

として破棄されており，これの有効利用法が確立すれ

ば，カムカム産業の安定的発展が期待できる。そこで，

カムカム果皮と種子の機能性成分の探索研究を行った。

機能性成分として，抗酸化性を有するタンニンとして

─ 6 ─

2 つのカテゴリーに属する化合物を探索し，C-配糖体エ

ラジタンニンと縮合型タンニンの存在を明らかにした。

単離した 6 種の C-配糖体エラジタンニンは DPPH，

ABTS および ORAC 法による抗酸化性試験で活性を示

した。また，そのうち 4 種は類縁化合物で，抗酸化性の

メカニズムの違いによる構造と相関関係を議論した。こ

れまで，C-配糖体型エラジタンニンは葉や材から単離

されているが，果皮や種子から単離されたのは初めてで

ある。一方，縮合型タンニンはエピガロカテキンガレー

トとエピカテキンガレートから構成されていることが解

かり，a-amylase や膵リパーゼ活性を阻害することを

明らかにした。

抗菌性成分としては，Rhodomyrtone など 4 種のアシ

ル フ ロ ロ グ ル シ ノ ー ル 類 を 単 離・構 造 決 定 し た。

Rhodomyrtone 以外の化合物のうち 1 つは天然から初

めて単離された化合物で，他の 2 つは新規化合物であっ

た。Rhodomyrtone は MRSA，VRE などに抗菌活性を

示すことが報告されており，カムカム種子からこれらが

単離されたことは興味深い。

以上のように，本論文は廃棄物であるカムカム果皮と

種子から多くの機能性成分を単離構造決定し，カムカム

果皮と種子の機能性食品，香粧品および医薬品としての

利用に道を開く重要な研究論文である。

よって，審査員一同は博士（生物産業学）の学位を授

与する価値があると判断した。

─ 7 ─