on the

Studies on the Highly Oxidized Ellagitannins in Medicinal Plants

HIDEYUKI ITO

1999

Studies on the Highly Oxidized Ellagitannins in Medicinal Plants

HIDEYUKI ITO

1999

Studies on the Highly Oxidized Ellagitannins in Medicinal Plants

Introduction

Chapter

I.

1-1.

1-1-1.

1-1-2.

1-2.

1-2-1.

1-2-2.

1-3.

1-2-1.

1-2-2.

1-4.

Contents

Geraniin-Related Ellagitannins

Ellagitannins from Phyllanthus flexuosus Extraction and Isolation

Structures of Phyllanthusiins A- E

Ellagitannins from Geranium thunbergii Extraction and Isolation

Structures of Geraniinic acids 8 and C, and Phyllanthusiin F

Ellagitannins from Acalypha hispida Extraction and Isolation

Structures of Acalyphidins MI, M2 and DI

Geraniin-Related Ellagitannin monomers in Euphorbiaceae and Their Chemotaxonomical Significance

Chapter II. Ellagitannins Having a Gluconic Acid Core

II-I. Ellagitannins from Shepherdia argentea 11-1-1.

II -1-2.

11-2.

11-2-1.

11-2-2.

Extraction and Isolation

Structures of Shephagenins A and 8

Ellagitannins from Elaeagnus umbellata Extraction and Isolation

Structures of Elaeagnatins A-G

5

6

20

28

39

44

44

49

11-3. Taxonomical Significance of Ellagitannins Having A Gluconic Acid Core

11-4. Biogenesis of C-glucosidic Ellagitannin Dimers

Chapter III. Biological Activities of Highly Oxidized Ellagitannins and Related Polyphenols

III-I. Anti-Tumor Activity of Tannins and Related Polyphenols III-I-I. In vitro assay; Inhibitory Effect on Epstein-Barr Virus

Early Antigen Activation

111-1-2. In vivo assay; Inhibitory Effect in Two-Stage Carcinogenesis Assay

111-2. Antibacterial Activity of Tannins and Related Polyphenols against Helicobacter pylori

Concluding Remarks

Experimental

Acknow ledgemen ts

References

List of Compounds

61

62

66

66

71

76

79

94

95

99

Introduction

A large group of polyphenolic compounds, "Tannins", are widely distributed in vegetable kingdom, which are often encountered in our lives, being contained in tea, red wine, fruits, beverages and various medical plants. They are characterized as polyphenols possessing an astringent taste and ability to fonn colored solutions and precipitate with iron and other metals, in addition to binding properties with proteins such as albumin, gelatin and collagen as well as alkaloids. The definition of tannins is proposed to be natural polyphenols with the aoove chemi- cal properties of molecular weights up to 500.¹)

Early studies in tannin chemistry focused mostly on the characterization of components of plant extracts used in the leather industry.2) On the other hand, numerous tannin-rich plants have been used as folk medicines (hemostatics, antidiarrheic, diuretic, antiseptic, etc) and as consumed food and beverages in Asia, Europe, North America and Africa.², 3) However, the chemistry of tannins in medical plants had been little investigated until

1970,

because of diffi- culty in the isolation of intractable, unstable, hardly separable tannins with closely related struc- tures .•

Nevertheless, the number of reports dealing with the isolation and structural elucidation of this class of natural polyphenols from medicinal plants and foods has increased markedly during the last two decades. The remarkable progress in the chemistry of tannins has been mostly based on developments of modern analytical and isolation techniques and various spectroscopies including high-field NMR, CD, and FAB-MS.4)

Generally, tannins are traditionally classified into two large groups, hydrolyzable tannins and condensed tannins.^S) These names are based on their hydrolysis and condensation occurring in the presence of acid or enzyme. The former with structural variation is further sub-divided into gallotannins, ellagitannins and their oxidized metabolites (dehydroellagitannins), C-glucosidic tannins,6) and complex tannins⁷), the last among which are composed of both hydrolyzable tan-

nin and flavan units. Among those various types of hydrolyzable tannins characterized to date, ellagitannins have been attracted a considerable attentions because of their vast structural diver- sity and biological activities specific to structures⁸). Various biological activities including an- tioxidant, antiviral and antitumor activities were noticed and reported for different types of ellagitanni ns.⁹)

Among these ellagitannins having various biological acti vities, the oral administration of geraniin and the geraniin-containing extract from Geranium thunbergii to rats was found to reduce the lipid peroxide concentration in serum and liver, which was raised by feeding the animals with peroxidized corn oil. 10) The levels of serum cholesterol, GOT, and GPT were also lowered. Ellagic acid, which exists in nature as a component unit of ellagitannins, was also found to show a potent anti-carcinogenic activity. I I)

Geraniin is characterized by having a hydrated dehydrohexahydroxydiphenoyl (DHHDP) group in the molecule, and is classified as dehydoellagitannins. The DHHDP group might originate biogenetically from a hexahydroxydiphenoyl (HHDP) group that is a common constituent of ellagitannins. The reactive DHHDP group might be further metabolized into various related acyl groups to lead a large number of mcxlified dehydroellagitannins that show various biologi- cal activities. Chebulagic acid is one of the typical example of modified dehydroellagitannin probably derived from geraniin, and it was reported to exibit an anti-tumor activity and a potent inhibitory effect against DNA-topoisomerase II. Geraniin and related compounds are consid- ered to be metabolized in an animal, and then those metabolites may be activators or indirectly act as mediators. In order to secure positive evidence of a contribution of these ellagitannins to various biological activities, accumulation of much more new findings on structure-activity relationships of this class of tannins would be needed. From this viewpoint, the author tried to search for new tannins focusing on highly oxidized ellagitannins in nature and to estimate their biological acti vity. The plants investigated in this study were (l) Phyllanthus flexuosus, (2) Acalypha hispida belonging to Euphorbiaceae and (3) Geranium thunbergii (Geraniaceae).

On the other hand, although most of these ellagitannins have glucose core as a sugar unit in the molecule, those having a gluconic acid residue as a polyalcohol have rarely been encountered in nature. These ellagitannins have been hitherto found only in four plants

I

Hippophae rhamnoides l2) (Elaeagnaceae), Lagerstroemia speciosa, 13) L. subcostatal³) (Lythraceae) and Punica grana- tum^l4) (Punicaceae)I, but their biological activity are not known at all. Among these plant fami- lies, the author has focused on the elaeagnaceous plants which have little been studied for tannin constituents. Consequently, nine new ellagitannins with highly oxidized structures have been isolated from Shepherdia argentea and Elaeagnus umbel/ata, and their structures have been elucidated on the basis of spectral and chemical evidence. Five among them were ellagitannins based on the gluconic acid core, and the other four were C-glucosidic ellagitannin dimers which are regarded as the metabolites biogenetically produced by an intermolecular oxidative cou- pI i ng between monomers.

This dissertation deals with the investigation on the isolation and structure elucidation of new 17 ellagitannins with highly oxidized structures from the five plant species in three families (Euphorbiaceae, Geraniaceae and Elaeagnaceae). In addition, the biological activities such as in vitro and in vivo anti-tumor promoting effects and antibacterial activity against Helicobacter pylori have also been investigated for the tannins and related polyphenols obtained from the above five plant species.

Chapter I. Geraniin-Related Ellagitannins

Geraniin (1), a yellow crystalline dehydroellagitannin, was originally isolated from an official anti-diarrheic in Japan, Geranium thunbergii (Geraniaceae) 15) and from Acer species (Aceraceae).16) Its wide distribution in Euphorbiaceae as well as Geraniaceae was reported in 1982.¹⁷) Recently, its structure including absol ute configuration was unambiguously substanti- ated by X-ray analysis, 18) which was the first example of chrystallographic analysis of free ellagitannin. A large number of hydrolyzable tannins related with geraniin has revealed its wider distribution in species of Cercidiphyllaceae, 16) Elaeocarpaceae, 19) Melastomataceae,20) Rosaceae,21) Coriariaceae,22) Simaroubaceae, 16) and Betulaceae.23 )

The dehydroellagitannins and their metabolites have been found in the genera Euphorbia, Mallotus, and Macaranga. Among them, chebulagic acid (2) is often found coexisting with a major tannin, geraniin, in those genera, which could be producible by benzylic acid rearrange- ment-like cleavage of cyclohexenetrione ring in the DHHDP group.8) The modified DHHDP group and other analogues would biogenetically originate from chebuloyl group or its equiva- lent. Geraniin is thus regarded as a key compound in the biogenesis of those modified dehydroellagitannins based on the ^I C₄-glucose core.

In order to survey new geraniin-related ellagitannins in nature, geraniin-rich plants

I

Phyllanthus

HO OHHO OH H0-O----O-0H

co co OH

H

731 g /

^{O-C-o-0H 0 -OH}

o _\ 0

,

co co

O~OH~

HO~~~OH

_o

a

\ I

co co

O~Q

^If

~OH

HO -- - OH 0 OH

b

flexuosus, Acalypha hispida (Euphorbiaceae) and Geranium thunbergii (Geraniaceae)

I

have been investigated and several new tannins with highly oxidized structures have been isolated.

1-1. Ellagitannins from Phyllanthusjlexuosus

The plants belonging to the genus Phyllanthus (Euphorbiaceae) consist of approximately 600 species, which are widely distributed throughout tropical and subtropical countries. This genus contains species that have useful medicinal applications?'+) A considerable number of these

species were previously examined and biological active constituents were reported. For ex- ample, the antineoplastic bisabolene glycosides,25) phyllanthoside and phyllanthostatins 1,2 and 3 2-.+,26) were isolated from P. accuminatus. Geraniin and related-polyphenols were isolated

as inhibitors against angiotensin-converting enzyme (ACE) from "Paraparai mi", P. niruri27) which has traditionally been used for the treatment of urolitic disease and as a diuretic in Para-

guay.

Phyllanthus flexuosus (Sieb. et Zucc.) Muell.-Arg. is a common shrub of the Central Japan-

Himalayan geographic region.28) Bergenin and some terpenoids including several new triterpenes and a diterpene were previously isolated from the bark of this plant. 13,29-34) Occurrence of

bergenin, which is structurally related to hydrolyzable tannins, in this species, and the previous finding of a wide distribution of geraniin in Euphorbiaceous plants, prompted to investigate other polyphenols in the leaves of this plant. The author has isolated thirteen polyphenols, including four new ellagitannins (phyllanthusiins A, 8, C and D) with highly oxidized structures and a new related polyphenol (phyllanthusiin E).

1-1-1. Extraction and Isolation

The fresh leaves of P. flexuosus were extracted with ether and acetone to remove triterpene and other lipids. The residual materials were then extracted with aqueous acetone and filtered.

The filtrate was extracted successively with ether, CH₂CI₂ and n-BuOH. The n-8uOH soluble portion was applied to repeated chromatography over Dia-ion HP-20, Toyopearl HW-40, MCI- gel CHP-20P and Sephadex LH-20 to afford five new ellagitannins and a related polyphenol, named phyllanthusiins A (3), B (4), C (5), D (6) and E (7), together with eight known polyphe-

cti-fR ~

9: R=H

9a: R=Me

~Rl-fR

RO~OR

g% ri

^R

H2~ c[nJ8-Q-OR

R

^OR

OR' OR

10: R=R'=H 10a : R=R'=Me

10b: R=Me, R'=H

~H1-fH

f-O~OH

g% OH

Htc(nJ8-<>H

R ^OH

OR' OR

2 12

OH

nols, bergenin (8);~5) brevifolincarboxylic acid (9),36,37) corilagin (10), \5) chebulagic acid (2), 38) repandusinic acid A (11),39) putranjivain A (12)40) and geraniinic acid B (13)41) (The struc- ture of 13 which was first isolated as a new ellagitannin from Geranium thunbergii will be described in section 1-2-2.). The known tannins were identified by direct comparison with au- thentic specimens or by comparison of their physicochemical data with those reported in the literature.

1-1-2. Structures of Phyllanthusiins A (3)-E (7)

The new compounds. except for phyllanthusiin E (7), were shown to be ellagitannins by the characteristic coloration with sodium nitrite and acetic acid reagent⁴²) on thin-layer chroma- tography (TLC).

Structure of Phyllanthusiin A (3)

Phyllanthusiin A (3) was obtained as a pale yellow amorphous powder. The negative fast- atom bombardment mass (FAB-MS) spectrum of 3 showed an (M-HY ion peak at mlz 951, indicating its molecular formula to be C₄,H₂₈0₂^7" The 'H-NMR spectrum of 3 showed a 2H singlet (07.14) and two I H singlets (07.05 and 6.66), ascribable to a galloyl and a HHDP group, respectively. The presence of a fully acylated I C₄ glucopyranose core was demonstrated by the coupling pattern and chemical shifts of the aliphatic proton signals, wHich were assigned by the

'H_'H shift correlation spectroscopy (COSY) spectrum. These sugar proton signals were very similar to those of geraniin (1) (Table I-I). The ¹³C_NMR spectrum of 3 was also closely similar to those of 1, except for the signals due to the DHHDP group in 1, indicating that 3 has a corilagin (10) moiety as a partial structure. In fact, partial hydrolysis of 3 in hot water yielded 10. Phyllanthusiin A was thus assumed to be an ellagitannin in which only the acyl group at 0-

2/0-4 on the glucose core differs from that in 1.

Besides the signals due to the corilagin moiety in the 'H-NMR spectrum of 3, mutually coupled methine 103.46 (br d, 1=7.0 Hz)] and methylene 103.20 (dd, 1=7.0, 16.5 Hz); 0 3.20 (br d, 1= 16.5 Hz)1 protons, and an aromatic proton (07.17, 1 H, s), which were attributable to those of the 0-2/0-4 acyl group, were observed. The ¹³C _NMR spectrum of 3 showed fourteen carbon resonances (four carbonyl carbons, eight ^Sp2 and two ^Sp3 carbons) due to the 0-2/0-4 acyl moi- ety (Table 1-2). Among seven carbonyl carbons in total, two were assignable to a lactone (0

162.2) and a carboxyl (0 169.3) carbon, based on the comparison with the B-ring carbons of

geraniinic acid B (13) (see 1-2-2). The chemical shifts of the A-ring carbon resonances were analogous to those of 13. These NMR features indicated that 3 was the regioisomer of 13 concerning the double bond in the 0-2/0-4 acyl group.

Methylation of 3 with diazomethane afforded a tridecamethylate (3a) that showed mlz 1134 (Mf ion peak in the positive electron impact mass (EI-MS) spectrum. Upon methanolysis with

sodium methoxide in methanol, 3a yielded methyl tri-O-methylgallate (14) and dimethyl hexamethoxydiphenate (IS). However, the product arisen from the 0-2/0-4 acyl moiety was not detected as an outstanding spot on TLC. The linkage mode of the 0-2/0-4 acyl group with

RO ORRO OR RO-Q---Q-OR

rgo ^cQ)

^OR

HC·/ O-C~OR

P1-

^{s 0 -0 , --./ o~ ~ OR}

• _o J

2

0

~

I I

CO CO

,. 7 A ~~ ^OR

3: R=H

3a : R=Me

- C O L O C

OMe

Meooc-Q--OMe OMe 14

MeOOC COOMe

15

Fig. I-I. COLOC Correlations of 3

Tahlc I-I. IH-NMR Spectral Data of 1 and 3-61500 MHz. acetonc-d" + 0,0 (J in Hz)\ La"1 tbal 3 4 5 6 Glucose H-I 6.60 (br 5) 6.60 (br 5) 6.30 (br s) 6.44 (br s) 6.37 (br s) 6.SS (br s) H-2 5.60 (br 5) 5.60 (br 5) 5.42 (br s) 5.32 (br s) 5.54 (br s) 5.S4 (br s) H-3 5.50 (br s) 5.60 (br 5) 5.64 (br s) 6.12 (br s) 5.57 (br s) 5.49 (br s) H-4 5.56 (br s) 5.46 (br s) 5.23 (br s) 5.15 (brs) 5.35 (br 5) 5.41 (br s) H-5 4.81 (m) 4.81 (m) 4.55 (br t, )=8.5) 4.76 (br t, )=8.5) 4.88 (br t, )=7.5) 4.82 (br t, )=7.S) H-6 4.93 (t,)=II) 4.78 (m) 4.79 (t, )= 11.5) 4.75 (t, )= 11.5) 4.83 (t, )= I 0) 4.75 (t, )= 1 0.5) 4.33 (dd, )=8, II) 4.45 (dd, )=6,9) 4.31 (dd, )=8.5, 11.5) 4.32 (dd, J=8.5, 11.5) 4.32 (dd, )=7.5, 10) 4.38 (dd, J=7.5, 10.5) Galloyl H-2,6 7.22 (2H, 5) 7.15 (2H, s) 7.14 (2H, s) 7.14(2H, s) 7.13 (2H, s) 7.15 (2H,s) HHDP H-3 7.13(IH,s) 7.08 (I H, 5) 7.05 (I H, 5) 7.05 (I H, s) 7.06 (l H, s) 7.05 (I H, s) H-3' 6.71 (IH, s) 6.69 (l H, s) 6.66 (I H, 5) 6.58(IH,s) 6.64 (I H, s) 6.63(IH,s) A-ring H-3' 7.25 (l H, 5) 7.28 (1 H, s) 7.17(IH,s) 7.12(lH,s) 7.06 (I H, s) 7.20 (I H, s) B-ring H-I" 5.16(IH,s) 4.72 (I H, d, J= 1.5) 4.S9 (I H, s) 4.89 (I H, d, J= 1.5) H-2" 5.52 (I H, d, )=2) H-3" 6.56 (I H, s) 6.26 (1 H, d, )=1.5) 4.43 (I H, d, )=2) 2.36 (I H, dd, J=6.5, 12) 6.28 (I H, d, J= I.S) 2.20 (l H, t, J= 12) H-4" 3.46 (1 H, br d, )=7) 4.S8 (I H, dd, )=6.5, 12) H-S" 3.20(lH,brdd,J=7,16.5) 3.26(IH,d,)=17) H-7" H-9" a) 400 MHz ~ '--""

8

3:

:r:

.!" 0- '--"" I ~

---- ~

c:: ~ en ::l g. ~ en ~ :3 ~ (") 2- c:: :3 ::l ~ ., ~ ::l ~ ., (") ::r ~ ::l r:ro ~ ~ 0- (b

to

00000000 00~'

-\Ooo__JO\lfl~'-.NN-r:ro o :: : :: :: :: :: :: :: :: o\\o\o\ONlfl~ lflNo\-oo+:-O\ V)VtN~o,o,N o\\o\o\oN~Vt lflNNlfllfl\oN ~~~66NO O\O\'-.N+:-N+:-o\ NN-o\ON\o wN:"'-io'gN~ __J__J~__Jlfloo-..l -N(.;..)__JO\-N Oo~:""~~~~ =--= __J-__J~__JO\ '_.NO\lflO\oo~ Vtioo~:"::"~ N O\'-.NOlflOoo\oN~Vl lflNO'IO\OO__JO'IlflN ~o~oOoioOoio~o

3.09 (I H, br d,)= 16.S) ~ ,

n n n

'l

n n

n~· I I I I I I I ::3 ~ 0:: ~

+:

~ ~

-:00

0'1 +:-'_.N+:---- lfl '_.N 00 lfl '_.N \0 lfl ~~OO~~O'N O'I~'-.N~--N ~:__l:__l:--l~:__l9 NN'-.NO\lfl--

---

O'I+:-'_.N+:--N- lfllfloo~-O__J N~~~N~~ O\lfl~~--­ lflOVl__JN__JO'I NOoVtwOoioOo

---

O'I~'-.N~--­ oo\Olfl__J-__J\O :"::"~VtO:""i.NN O\~'-.N+:--NN ~--J--J__J'-.NOO 006~~~:"":""

nnn'lnnr"J

~~~~~~~ O\~'..;J~ON- 00 lfl 0'1 lfl 00 lfl V"I i.N6~~6~N O'I~(.;..)+:>'ON­ oolflO'llflooVllfl NNVt~NVt6

---

O'I+:>''-.N+:>'ON- OOVlO'llflOOVllfl ~N~~6~N ~ !::

---

O'I~(.;..)+:>.ON- 00 lfl 0'1 lfl 00 Vl lfl ~N~~O~N .s ~ O'I~'-.N+:>.ON­ O'IlflO'llfl__JlflV"l NN~:"::"Ooo,N .s ~

---

O'I~'-.N~ON­ oolflO'lV"l__JVlV"l ~i.N~v,io~i.N .s ~

3.16 (I H, d,)= 17)

:r: 0000~00a

__J 0'1 'J) ~ '_.N N -'ij

---

O'I~'..;J~-N­ O'Ilfl__J~O+:>'__J :...-:...-~~O'OON 0'1 +:>. '..;J +:>. -N - 0'1 v, __J ~ 0 +:>. 0\ :"'-6~NN~io

---

0'1 +:>. '..;J +:>. -N - O'IVl__J~O~O'I i.N6~~~Ooio .!: ~

---

O'I~'-.N~-N­ O'I~__J~O~O\ i.N'!'~~:.,::..~~

---

O\~(.;..)~-N­ +:>'lfl__J~O~O'I ~~~~~iio

---

O'I~'..;J~-N­ O'Ilfl__J+-O~__J ~~00~;..;J~;-.N

C1 nrJnnn~ I I I I I 0 __J+-~...,.J~-~ lfl 0\

---

O\'-.N~-N .p\Olfl-O ~~io6i.N O\'_.N+:>'-N ~\Ov'oo Oo~iooow

---

O\'-.N+:--N +:>'\00\00 ~0,600~

---

0\ '-.N ~ -N +>.\OVlOO ioo,OOo,N O\'_.N~-N ~\OlflOO ~~ioOoN

---

O'I+:>'~-N v,OO\oo ~6:"'-~:"'-

3.46 (I H, d, J=IS.5) 2.95 (I H, d, )=IS.5) 2.17 (3H, 5) C1

n'lnnnrJ~ Q.,V..l:.~N~O

v: ~ O'I__J 0'10\0'1 \0 '..;JNo\'-.N\OO ~~6~iooo O'I__JO\O\__J\O (.,)(.;..)O\NO- ~i.Nio~VtOO O\__J 0\0\ 0'1 \0 +:-'_.NO\ '_.N \0- 6W~N~~ O\__J O'IO\__J \0 ~(.,)__J-O- 00, 00 Vt 0, 00

O'I__JO'IO\O'I\O NNO'INoo- ~ioOVt:""Oo O'I__JO'IO\__J\O '_.N'_.NO\NO- io:....~~~io

-

~::.

-

0;: ~

.,.

VI

;l

0- (b , N ~ ,

z

3: ;;0 C/:J

g [ o

~ S o ^-+,

-

~ ::l Q. ~ I ~ ~ 3:

:r:

N l=S ~ 5' ::l ('1) ~ ~ +

o

~

Il 'g

glucose in 3 was substantiated by 'H_

¹³

C long-range COSY (COLOC) spectrum (JcH=7 Hz), in which H-3' of the A-ring (6 7.17) was correlated with H-2

(6

5.42) of the glucose through three- bond couplings with a common ester carbonyl carbon

(6

165.2). The other long- range correla- tions are shown in Fig. 1-1

.

Based on these data, the structure of phyllanthusiin A was elucidated as

3.

Structure of Phyllanthusiin B (4)

Phyllanthusiin B

(4),

a pale yellow amorphous powder, exhibited an (M-HY ion peak at mlz 969 in the negative FAB

-MS spectrum, to indicate the molecular formula C4IH30028' The H-I

NMR spectrum of 4 showed close similarity to those of 4, except for the B-ring protons. Instead of the B-ring protons of 3, two mutually coupled methine protons 165.52,4.43 (each d,

J=2

Hz)1 and isolated methylene protons Ib 3.26, 3.16 (each d,

J=

17 Hz) I were observed in

4.

Similarly, the aromatic proton signals resembled those of 3 (Table I-I)

.

The

I3

C-NMR spectrum of 4 in aliphatic carbon region showed resonances due to a methylene carbon (643.1), two methine carbons (6 81.6 and 56.7), and a quaternary carbon bearing an oxygen function (677.6) besides glucose signals. Among the seven carbonyl carbon resonances, the signals at 6 172.7 and 171.8

,.

6"

ROOC

:r 3

TCO 7CO 30R

H

g 6 / O -'C~OR

, -0 OR

(J1

^{0 , 0}

"=<

• 3 2

o b

\ I

rco OCT OH1' ~

t-+'" ' A OR :r

4. R=H

4a. R=Me

Me~OOC ^{OH COOMe}

MeOOC H"" r; _ "\ OMe MeOOC" " 0 OMe

H 4b

were assigned to the carboxyl carbons in the 0-2/0-4 acyl moiety as follows. In the COLOC spectrum of

4,

one of the carboxyl carbons at 6 172.7 was correlated with both of methine protons

(6

5.52 and 4.43) through two- and three-bond couplings. The methine proton at 6 4.43 (H-3") was also correlated with C-l' (6 116.8) and C-6' (6 150.8) of the A-ring. Similarly, the other methine proton at

b

5.52 (H-2") exhibited cross peaks with the methine

(b

56.7, C-3"), quaternary (6 77.6, C-4") and A-ring C-6' carbon signals

(b

150.8). Furthermore, methylene protons (C-5") were correlated with quaternary (C-4"), carboxyl (C-6") and ester carbonyl (B

-

ring C-7") carbons (Fig. 1-2).

Methylation of 4 with dimethyl sulfate and potassium carbonate in dry acetone yielded a tridecamethyl derivative

(4a)

which showed the peaks at mlz 1153 (M+Hf and 1175 (M+Naf in the FAB-MS spectrum. The 'H-NMR spectrum of

4a exhibited a 1 H singlet (b

4.43) that disap- peared upon addition of 020. Thus, the presence of an aliphatic tertiary hydroxyl group in

4

was ascertained.

Methanolysis of 4a gave in addition to 14 and 15, a hexamethyl derivative

(4b)

which exhib-

(ppm)

7.0

6.0

50

40

3.0

C(A)-4' C(A)-6\

170 160 150 140 130

C(A)-6'

\

C(S) 4"

C(A)-6'

C(S)-2"j

II I ^I

- t

HO RO HO OH

1--1

B~OB

H I

)-1° fO/'-tf

( '.{:bc,o

-,~o _ ⁰⁸

, -0 I W'>'''H' '08

~ , ^{) 8}

I

,,--:~

?

^o I ,'-, ^J ,:,-,JIO ~CO

CO ~

'- -'1/>-

^(I.

/ , .:1 "\

: HOO ,.I' ,', • 08

C(S)-3" C(8)-5"

'J

H(GI c)-1

>-H(Glc)-3

H(S)-2"

H(Glc )-2 H(Glc) -4

H(GI .,... H(B)-

' H(GI c)-5,6 3"

c)-6

\...

r

H(8)-5"

80 70 60 50 40 (ppm)

. - .... ~ COlOC

Fig. 1-2. COLOC Spectrum of

4

(126 MHz, acetone-d

₆

+020)

ited an (M+Hf

ion

peak at mlz

457

in the FAB-MS spectrum.

The

IH-NMR

spectrum of

4b showed an aromatic proton \67.08 (1 H, s) J, a methylene 162.89, 2.55 (each

1 H, d,

1=

16.5 Hz) I and two methine 165.50,4.31

(each

1 H, d, 1=1.5 Hz)1

signals.

The a-configuration of benzylic proton (H-3") of 4 was determined by nuclear Overhauser enhancement spectroscopy (NOESY) which exhibited a remarkable NOE between the methine pro-

ton

(H-3") and the anomeric

proton of the glucose core (Fig.

1-3)

..

On

the other

hand, the

small

coupling constant (1=2 Hz)

between

the methine protons

(H-2" and

3") in the

^I

H- NMR

spectrum of 4

was indicative of their

trans

relation-

ship, .. B) establishing

the

f)-configuration at H-2". Based on

these data, the structure of phyllanthusiin B was concluded to be represented by

the formula 4, although the absolute con- figuration at C-4" remains undetermined.

Structure of Phyllanthusiin C (5)

- - NOE

Fig

. 1-3. NOE Correlation of 4

The structure

5 of phyllanthusiin C

was determined as follows. The molecular formula was determined to be C40H

30026 based on the negative FAB-MS Imlz 925 (M-HYI

and the elemental

RO OR RO OR

R0-VV-0R

CO CO OR

Hg /

O-C~OR

'2J-?_o--i

^0-\=(

r4

^OR

o 0

\ I

CO CO

HO,,~t)~OR

~rOR

Hd RO 5: R=H

Sa: R=Me

5 ~.--

eOlOe Fig. 1-4. COLOC Correlations of

5

Sa

..... ~ NOE

Fig. 1-5. Stereostructure and NOE Correlations of Sa

analysis. The IH_ and

13C-NMR spectra of

5 indicated the presence a corilagin unit, as shown in Tables I-I and 1-2. The IH-NMR spectrum also exhibited methine proton signal [04.58 (dd, J=6.5, 12 Hz)] coupled with methylene proton signals [02.36 (dd,J=6.5, 12 Hz); 02.20 (t,J=12 Hz)], and an isolated methine proton [04.59 (s)]. The

¹³C-NMR spectrum showed five carbons

[0116.9 and 78.4 (quaternary carbons), 0 75.0 and 63.3 (methine carbons), and 046.6 (methyl- ene carbon)] in aliphatic region, which were analogous to those of the B-ring in 3

and

4. Among these carbons, the signal at 0116.9 can be assigned to a geminal-diol or hemiacetal carbon based on its chemical shift, similar to the corresponding signals of the B-ring in geraniin (la). In addition, the resonances of C-4' and 6' of the A-ring in

5

were similar to those of phyllanthusiin B (4), suggesting the presence of an ether bond at C-6'. The COLOC spectrum (J

CH=8

Hz) of 5 revealed that one of the methylene proton signals (B-ring H-3") showed correlations with all carbons of the B-ring by two- and three-bond couplings. The connectivities among the acyl protons and glucose protons through three-bond couplings with the ester carbonyl carbon reso- nances are illustrated in Fig. 1-4.

Methylation of 5 with diazomethane afforded a dodecamethyl derivative (Sa), whose IH-NMR

spectrum revealed a singlet (0 4.45) and a doublet (0 4.18) that disappeared upon addition of

HO

co co ^OH

H { i 3-

g

- Q - O H

~ OH

o 0

\ to

~

^OH

HO" : (I \\

, ;; 1 \ OH

" ;; 0 OH

Hc5' HO 5

MeO

MeO

co co ^OMe

Hg

/ o-c nOMe

'J-~ -0--1

0 '= {

~ ^OMe

o 0

\ to

~

^OH

HO" : fI \\

, ;; I \ OMe

", : 0 OMe HeY Me6

co co ^OMe

H

pq

²⁰

^{8 /}

^-0

^-c~OMe

^{O'={ OMe}

OH OMe 10a

5a

+

MeOOC Mea OMe

o~M'

o

9a

[

HQ, CooMe CooMe ---', ,H r; '\ OMe

Ha~

COO Me COOMe

- - ... ^6=.Q~

HC{ 0 MeO OMe

Chert 1-1. Methylation of 5

O₂

°,

indicating the presence of a tertiary and a secondary hydroxyl groups. On the other hand, methylation of 5 with dimethyl sulfate and potassium carbonate in dry acetone yielded deca-O- methylcorilagin (10a)44) and methyl tri-O-methylbrevifolincarboxylate (9a). The formation of 9a can be rationalized in terms of a series of reactions including a f3-elimination, keto-enol tautomerizaion and transesterification, as depicted in Chart I-I.

Stereochemistry of the B-ring in 5 was determined by NOESY measurement of 5a as follows.

The anomeric proton of the glucose core showed an NOE correlation with H-I" (64.74) of the B-ring which also showed NOEs with a methoxy signal at 03.41 (C-S"-OMe) and a hydroxyl proton signal at

0

4.18 (C-4"-OH), establishing the a-configurations for all of the functional groups COMe and OH) on the B-ring, as shown in Fig. 1-4. Consequently, phyllanthusiin C was assigned to the structure 5.

Structure of Phyllanthusiin D (6)

Phyllanthusiin 0 (6) was obtained as colorless fine needles. The IH-NMR spectrum of 6 was closely similar to that of geraniin (lb) having five-membered hemiacetal form at the OHHDP group, except for extra signals due to an isolated methylene 16 3.46, 2.96 (each d, 1=IS.S Hz)

I

and a methyl /6 2.17 (s)

I

groups in 6 (Table I-I). As mentioned above, crystalline geraniin readily forms an equilibrated mixture between six-and five-membered hemiacetal forms (la~ Ib) of the OHHOP moiety in an aqueous acetone solution, to give a duplicated signal for each proton in the 'H-NMR spectrum. However, such a phenomenon was not observed for 6. The

'3C _NMR spectrum of 6 also showed close similarity to that of Ib except for extra three carbon resonances (0 206.4, SO.O and 32.0) attributable to a ketonic carbonyl, a methine and a methyl- ene carbons, respectively, as shown in Table 1-2. The COLOC spectrum of 6 indicated that one of the methylene protons (B-ring H-7") was correlated with a, (3-unsaturated ketone carbon through three-bond coupling, indicating that this methylene group located at S" position of the B-ring. Methylation of 6 with dimethyl sulfate and potassium carbonate in dry acetone gave deca-O-methylcorilagin (10a).44) Based on these data, phyllanthusiin 0 was characterized as the condensate of geraniin with acetone, as illustrated in the formula 6, which is consistent with the negative FAB-MS data

Imlz

991 (M-H)l

HO OHHO OH

H0VV-0H

co co ^OH

HOC: 6 /

O -C~OH

p,q

^{5 0 -0 1 0 ~ OH}

4 3 2

o 0

10'60 7'60

3" 3'

o

B""

r; A '\ OH HO ~. "OH

r9H2 0 8"C~H3

6

The structure 6 deduced by the above data was verified by production of a condensate identi- cal with phyllanthusiin

D

upon refluxing

1

in dry acetone containing trifluoroacetic acid for a week. The ROESY spectrum of 6 showed the ROE correlation between the anomeric proton of the glucose and H-I" of the B-ring (04.89), which showed a remarkable correlation with one of the methylene protons (0 2.96, B-ring H-7") (Fig. 1-6). Consequently, the structure of phyllanthusiin D was elucidated as the formula 6.

Phyllanthusiin

D

^{may be} an artifact, since it was isolated from the plant materials soaked in acetone at the initial stage of extraction, and it was most likely to be produced by condensation of geraniin (1) with acetone in a weakly acidic condition. This compound was, however, not

produced when 1 was kept at room temperature for a few weeks in aqueous acetone that is generally employed as the extraction solvent for tannins. Upon refluxing of 1 in an aqueous acetone, compound 1 was slowly decomposed to corilagin (10) after 3 days, without formation of 6, by the normal phase HPLC analysis. On the other hand,

6

was reported to be isolated from

. I f G . h b .. 45)

the aqueous acetone homogenate of cell suspension cu tures 0 eramum t un ergll.

- - ROE

H(8)-9"

H(B)-l"

"hL-~l,

_{L-t\..._}

^j)," J

6.0

!!.!!

!!.o

4.!!

•. 0

3.5 3.0

2.5

~

I

F=

^H(Glc)-l

r-

F F

H(B)-l"

r

I

..

^~.

^~

~'~

L-~-r~,-.-~--~-r~~~~~

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 F2 (PPMI

Fig. 1-6. ROE Spectrum of 6 (500 MHz, acetone-d₆+D₂0)

Therefore, it is likely that phyllanthusiin D is an artefact formed during the extraction proce- dure.

Structure of Phyllanthusiin E (7)

Phyllanthusiin E (7), a brown amorphous powder, showed an (M-Hr ion peak at mlz 291 in the negative FAB-MS spectrum, corresponding to the formula C I3H^gO_g. The IH-NMR spectrum of 7 disclosed a singlet (0 7.38) and two 2H singlets (05.54 and 3.47). The presence of a carboxyl and two lactonic carbonyl (0 173.1, 163.8 and 163.3) carbons, and two methylene (0 68.8 and 32.3) carbons was indicated by the 'H_¹³C heteronuclear shift correlation spectroscopy (HETCOR) spectrum of 7. The remaining carbons were those due to eight Sp2 carbons in the '3C _NMR spectrum. Based on these data and the COLOC spectrum of 7 as shown in Fig. 1-7, phyllanthusiin E was determined as formula 7.

HO

COLOC

Fig. 1-7. COLOC Correlations of 7

1-2. Ellagitannins from

Geranium thunbergii

Geranium thunbergii Sieb et Zucco (Geraniaceae) which is rich in tannins has long been used as a remedy for intestinal disorders in Japan. In the early investigation of the leaf of this plant, crystalline geraniin (1) as a major tannin constituent was isolated, and elucidated its unique structure on the basis of spectroscopic and chemical evidence, 15) which was recently confirmed by X-ray crystallography. 18) Subsequent investigations of the leaf revealed the occurrence of three other dehydroellagitannins, furosinin (16), furosin (17) and didehydrogeraniin (18).46) These tannins are all regarded as the metabolites of geraniin. In addition, a notable ellagitannin, elaeocarpusin (19), which is a condensate of geraniin and ascorbic acid, was also obtained along

with geraniinic acid A (20) from water-soluble portion of the extract.⁴⁷) Further investigation of the polar fraction of the leaf has resulted in the isolation of additional new members of modified dehydroellagitannins named geraniinic acids B (13), C (21) and phyllanthusiin F (22), along with some known tannins.

1-2-1. Extraction and Isolation

A concentrated 70% aqueous acetone homogenate of the dried leaves of G. thunbergii was extracted successively with Et₂0, EtOAc and n-BuOH to give the respective extracts and the water-soluble portion, The water-soluble extract was chromatographed over Dia-ion HP-20 and MCI-gel CHP-20P with aqueous MeOH to give three new ellagitannins, geraniinic acid B (13), geraniinic acid C (21) and phyllanthusiin F (22), along with five known tannins, geraniin (1), corilagin (10), phyllanthusiins B (4), C (5) and E (7).37) The known tannins were identified by direct comparison with authentic specimens.

HO OHHO OH

HO~OH

co co

6

I

^OH

H~~OO-C-o-~

_{;,s'-o~ 0 -} ^OH

["L-('

^OH

OR' OR _HO

~ _~-:>

_COOh

o

A-A' =; , ₇

I I

~

^{COOH co}

HOOC H"" r; "\ ^OH

HOOC"'" -

o ^OH

I ^I

co co

HO·):tj-~OH

HO

~XH

HO

4 5

OH ~H

_ H

r _ ~

^OH

~ O~H

^)...T0H _{CH~H OH}

~ ^O-C~OH

co

ICO

^{co co}

°/-

0

, I I

OH 0 ~

OH

? q

oc co

H k ! - 0 h

? q

oc co co ^I co ^I

O~OH~

HO'~~~OH

O~OH~

HO~~~OH

_o

16

O~-

^{Ij '\ OH}

H O ' -

OH 0 OH

o

OH ~H

r_~ OH~;~HVOH

co co co co

o~

/

^{OH I I}

Hi; /-0, ?-C-Q-OH ~

~

^{0 -OH}

o 0

I \

oc co ^{I I}

O~OH~

HO'~~~OH

_o

co co

O~Q

_{H O '} ^Ij _- ^{'\ OH}

OH 0 OH

18

17

I I

co co

O~-;

_{H O '} ^{Ij '\} _- ^OH

OH 0 OH

HO OH HO OH H 0 - V - V0H

co co OH

¢ /

O-C~OH

H1-?_O-1 0

H "==j

^OH

o 0

HO~0~60

.... 0 ¹⁰ H

b ... .

~H f ~ OH

ItO _ - o HO He) 0 OH

19

HO OHHO OH

HO~OH

yO ^{co OH}

H1 /

O-C-O-~

^OH

R

^o

^I-O.

^{OH 0 - OH}

o I co COOH HO-Q--Q-OH

HO OHHO OH

20

1-2-2. Structures of Geraniinic acids B (13) and C (21), and Phyllanthusiin F (22)

Structures of Geraniinic acids B (13) and C (21)

Geraniinic acid B (13) exhibited an (M+NH4f ion peak at m/z 970 in the electrospray ioniza-

1

tion mass spectrum (ESI-MS), corresponding to the molecular formula C41 H280_27' The H-NMR spectrum of 13 showed a 2H singlet (6 7.19) and two I H singlets (67.06 and 6.64), due to a

galloyl and an HHOP group, respectively. It also showed the signals of an aromatic proton at 6 7.01 (s), a vinyl proton at 6 6.36 (d, J=1 Hz), and two methine protons at 65.33 (d, J=1 Hz) and 65.14 (br s), besides the sugar protons characteristic of the 1 C4-glucopyranose (Table 1-8). The partial hydrolysis of 13 with hot water gave corilagin (10), indicating the presence of corilalgin

RO OR RO OR RO-Q---VOR

CO CO OR

o /

O-C~OR

H1-?

H

_o--1 0- "'==j ^OR

o _I 0 _, CO CO

H ^3'

o B ~ " r; A ~ OR

o F _{H RO} OR ROOC,.

13:R=H 13a : R=Me

RO ORRO OR RO-Q---VOR

CO CO OR

b /

O-C~OR HJ-?_O~ H

o-"'==j ^OR

o 0

co

^CO

O ~ OR

- : d"H A X R

21 : R=H 21.: R=Me

unit as a partial structure of 13. These data suggested that 13 is an analog of 1. In the 13C_NMR spectrum of 13, the resonances due to two ester (or carboxyl) carbonyl carbons (6 171.1 and 161.2) and a methine carbon (6 80.l) signals were observed, instead of the signals attributable to a ketonic carbon (6 191.7), a hemiacetal carbon (696.1) and a geminal-diol carbon (692.4) in the B-ring of 1. From these NMR and MS data, 13 was presumed to have a lactone-carboxylic acid structure in the B-ring of an acyl unit attached to 0-2/0-4.

If 13 has a dihydrocoumarin-type 6-lactone moiety as seen in chebulagic acid (2), 13 should show an IR absorption band at around 1775 cm-I

and a significant upfield shift (ca. 6 ppm) of the C-6' signal relative to C-4' in the 13C_NMR, both of which are observed in 2.48) However, 13 did not exhibit such characterizations, to indicate that hydroxyl group at C-6' in geraniinic acid B does not participate in lactone formation. The COLOC spectrum of 13 showed the methine proton signal at c5 5.14 (B-ring H-2") correlated with two carbonyl carbon resonances at c5 171.1

galloyl-H

1

^HHDP-H

/

^H(A)-3'

'"

H(8)-S" H(Glc)-2

H(8)71311 ./ H(8)-2"

H(Glc)-6 H(Glc)-S H(Glc)-3

H(G IC)-4 "

~ ~

HO

:~~----~"~ , ---~~

I ,

1

,I

I

\

\

\

I I

I I ,/ I "

, ' , , '

"

"

,;

,

I I I I •

I I

! I \ I

, I ,Ii i I J I ,/ I I

, ^"

/1 I ,

I I I I

,! ^{I I}

I , '

, , ,

I I I

"

,

13

--...-,,~~...---"--_~\--''''L~~

,

²¹

i I ^{I t I} J I I I • t I

7.0 6.0 5.0

(ppm)

Fig. 1-8. IH-NMR Spectra of 13 and 21 (500 MHz,

acetone-d

+00)

6 2

Table 1-3. 'H-NMR Spectral Data of 13 and 21

1500 MHz, acetone-d_G + D~O (1 in Hz) I

13 ²¹

Glucose

H-I 6.57 (br s) 6.67 (br s)

H-2 5.32 (br s) 5.27 (br s)

H-3 5.36 (br s) 5.35 (br s)

H-4 5.44 (br s) 5.50 (br s)

H-5 4.61 (br dd, 1=8, 10) 4.73 (br dd, 1=8, 10)

H-6 4.82 (br t, 1= II) 4.91 (brt,l=II)

4.27 (br dd, 1=8, II) 4.25 (br dd, J=8, 11)

Galloyl

7.20 (2H, s)

H-2,6 7.19 (2H, s)

HHDP

H-3 7.06 (I H, s) 7.10(IH,s)

H-3' 6.64 (1 H, s) 6.65 (l H, s)

A-Ring

H-3' 7.01 (tH, s) 6.93 (IH, s)

B-Ring

H-2" 5.14(lH. brs) 5.63 (I H. d. J=6) H-3" 5.33 (tH. d, J=l) 5.17 (I H, dd, J= 1,6) H-5" 6.36 (I H. d. J=l) 6.41 (IH,d,J=I)

(B-ring C-1") and 161.2 (B-ring C-6") through three-bond couplings, indicating an

n,

f3-unsat- urated &-lactone moiety in the B-ring of 13 (Fig. 1-9). The binding modes of the other acyl groups in 13 were also consistent with the long-range correlations in the COLOC spectrum. The NOESY spectrum of 13 showed an NOE between the anomeric proton of the glucose and H-3"

in the B-ring, establishing R-configuration at C-3", An allylic coupling

(J=l

Hz) was observed between H-3" and 5" in the 'H-NMR spectrum of 14 as well as Ib having a five-membered hemiacetal ring in the B-ring, to imply that the angle between C-3" - H-3" and C-5" - H-5" is ca. 90°. The trans-arrangement of H-2" and 3" in this conformation was consistent with their small coupling constants «I Hz).

Atropisomerism of the chiral HHDP was determined to

be

(R)-configuration by a large nega- tive Cotton effect at 225 nm (18\-1.2 x 10

⁵)

in the circular dichroism (CD) spectrum.

⁴⁴)

Fur- thermore, atropisomerism of the HHDP moiety in 13 was confirmed by formation of dimethyl

(R)-hexamethoxydiphenate,15)

[a]D +21

^0,

upon methanolysis of tridecamethyl derivative (13a) of 13. Based on these findings, the Structure of geraniinic acid B was determined as formula 13.

Geraniinic acid C (21) showed a pseudomolecular ion peak at m/z 970 (M+NH4)

⁺

in ESI-MS, corresponding to the molecular formula C41~027 which is identical with that of geraniinic acid B (21). The spectral features (NMR, IR and CD spectra) of 21 showed close similarity to those of 21. The only different point was a large coupling constant (J=6 Hz) between H-2" (6

5.63)

and H-3" (6 5.17) in the

IH-NMR spectrum of

21. The (R)-configuration of C-3" in the B-ring was established by NOE experiment in a similar manner to 21. In addition, the aUylic coupling constant between H-3" and 5" was analogous with th. at of 13 «1 Hz). Thus the conformation of the B-ring at 0-4 should be the same in these two compounds. A significant difference of the chemical shift of H-2" signal between 13 and 21 was interpreted in term of an anisotropic effect

of

the A-ring. The proposed structure 21 for geraniinic acid C was substantiated by its methyla- tion with dimethyl sulfate which afforded an expected tridecamethyl derivative (2la), together with a byproduct, nona-O-methylcorilagin (lOb).

;..".Glc H-4

VVV'

...

- -

... "

13

...

\

\

"---'~COLOC

Fig. 1-9. Stereostructure and COLOC Correlations of the 0-2/0-4 Acyl Moiety in 13

Structure of Phyllanthusiin F (22)

Phyllanthusiin F (22), obtained as an off-white amorphous powder, which was regarded as an analog of phyllanthusiin C (5) based on the following data. The 'H-NMR spectrum of

22~

re- vealed a striking resemblance to those of 5, except for the lack of two I H singlet due to HHDP protons and the upfield shifts of H-3 and H-6 (64.52 and 4.21/3.96) of the glucose core relative to the corresponding signals of 5. The ESI-MS of 22 showed an (M+NH4)+ ion peak at mlz 642, and its molecular formula was determined by high-resolution ESI-MS Imlz 642.1344 (M+NH4)+, calcd for C26H2401S+NH4 642.13061. Thus, 22 was assumed to have a structure lacking 0-3/0- 6 HHDP group in 5. This assumption was substantiated by its hydrolysis with tannase yielding 22 and ellagic acid. The stereochemistry of the B-ring in 22 was established by NOE eXlPeri- ment in an analogous way to that of 5. Consequently, the structure of phyllanthusiin F was determined as 22.

OH

HOHC

O-C~OH

'J-?-~--i

^0-

\=f

A

^OH

o 0

\ I

co co

Ha~OH

,. ~ 0 OH

Hd HO .

22

HO OHHO OH H O { ' { - - 9 - 0 H

co co OH

Hg /

O-C~OH

'2~?_o--i o--w.-

A

^OH

o 0

\ I

co co

Ho.J~)AoH

~rOH

Hd HO

5

Table 1-4. ^I H-NMR Spectral Data of 5 and 22 1500 MHz, acetone-df) + D

20 (J in Hz)

I

Glucose H-I H-2 H-3 H-4 H-5 H-6

Galloyl H-2,6 HHDP

H-3 H-3' A-Ring

H-3' B-Ring

H-I"

H-3"

H-4"

5

6.37 (br s) 5.54 (br s) 5.57 (br s) 5.35 (br s) 4.88 (br t, J=7.5) 4.83 (t, J= 10) 4.32 (dd, 1=7.5, 10)

7.13 (2H, s)

7.06 (I H, s) 6.64(IH,s)

7.06(IH,s)

4.59 (IH, s)

2.36 (IH, dd, 1=6.5,12) 2.20 (lH, t, J=12) 4.58 (I H, dd, J=6.5, 12)

22

6.21 (br s) 5.26 (br s) 4.52 (br s) 4.97 (br s)

4.28 (br dd, 1=6, 7) 4.21 (dd,1=7, II) 3.96 (dd, 1=6, 11)

7.IS(2H,s)

7.01 (I H, s)

4.61 (IH, s)

2.34 (I H, dd, 1=7, 12) 2.17 (IH, br t, 1=12) 4.57 ( I H, dd, 1=7, 12)

1-3. Ellagitannins from Acalypha hispida

Some Acalypha species of Euphorbiaceae have been used as folk medicines for treatment of diarrhea and skin complaints in Southeast Asia. Acalypha hispida Burm. f. widely distributed in Asia is one of them, and its leaves have been used as a remedy for thrush and boils in China and Indonesia.49) Although the medicinal value of these plants is thought to be responsible for their tannin constituents, the poly phenolics in the plants have been little investigated. The author has isolated fifteen polyphenols including new ellagitannin monomers, named acalyphidins

M,

(~~)

and M2 (24), from the leaf extract of this plant. A geraniin dimer, designated as acalyphidin 0, (25), was also isolated as an acetonyl derivative (25a).

1-3-1. Extraction and Isolation

The dried leaves of A. hispida were homogenized in 70% acetone. After concentration of the homogenate, the precipitate deposited was collected by filtration and the concentrated solution was extracted with Et₂0, EtOAc and n-BuOH, successively. The precipitate was subjected to a combination of chromatography over Dia-ion HP-20, Toyopearl HW-40 and/or MCI-gel CHP- 20P, to furnish two new tannins, acalyphidins

M,

(23) and

M2

(24), along with five known tannins, geraniin (1), phyllanthusiin C (5), mallotusinin (26),50) euphorbins A (27) and B (28).51, 52) The n-BuOH extract was similarly chromatographed to give a crude new ellagitannin dimer, acalyphidin D, (25) contaminated with excoecarianin (29),53) in addition to two flavonoids, rutin and kaempferol 3-rutinoside,54) and four tannins, 9,37) 10,15) 29 and euphorbin 0 (30).55) Penta-O-galloyl-~-D-glucose (31),56) furosin (32)15) and repandinin A (33)57) were isolated from

the EtOAc extract in the similar separation procedure. The known tannins were identified by direct comparison with authentic specimens or by comparison of their physicochemical data with those reported in the literature.

HO - " , 'I ~ OH

~ /, -

~ M /

⁵

%~ °

⁰

c: ~~

^{- HOH}

--- ' H

4 3 2

, 0,

R 1 : (1'R)-DHHDP

H

6: (1'R)-Acetonyl-DHHDP

26 (1'R)-DHHDP=

(1'R)-DHHDP

(1'R)-Acetonyl-DHHDP=

32 co I

G~H 2

G 0

G OG

OG

OH

31

H2CO-G

G-O~

_{o O-G}

I ^{G-O o}

a

^{O - G 0 O-G}

G OOC HO HO OH OH

H0-o-0**OH

HO HO CO co

~ ? AH

co n H O H H2

Cb/

0 - G

? tkI 9

(1'R)-DHHDP 27

'r-l:H

^~_lH

H 0 - V V 0H

co co

H2C'O/O-G (1'R)-DHHDP

ft

28