Atta-ur-Rahman (Ed.

) Studies in Natural Products Chemistry, Vol 28

© 2003 Elsevier Science B.V. All rights reserved. 199

CHEMISTRY AND BIOLOGICAL ACTIVITIES OF

ISOPRENYLATED FLAVONOIDS FROM MEDICINAL
PLANTS (MORACEOUS PLANTS AND
GLYCYRRHIZA SPECIES)

TARO NOMURA, TOSHIO FUKAI, and YOSHIO HANO

School of Pharmaceutical ScienceSy Toho University, 2-2-1 Miyama,

Funabashiy Chiba 274-8510, Japan

ABSTRACT: Among a large number of phenolic compounds isolated

from natural source, various isoprenoid-substituted phenolic compounds
have often been found in plants. Moraceous plants and licorice (Glycyrrhiza
species) are rich sources of the isoprenoid-substituted phenolic compounds,
including flavonoids. Some of the Morus flavonoids, such as kuwanons G
and H, have been regarded as optically active Diels-Alder type adducts.
Furthermore, some of the isoprenylated-flavonoids from the moraceous
plants and licorice showed the interesting biological activities. This article
reviews the biological activities of the isoprenylated-flavonoids from the root
barks and/or barks of moraceous plants and from Glycyrrhiza species by
our group. The chemical studies conceming the biological activities of these
compounds are also described briefly.
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I. INTRODUCTION (SURVEY OF ISOPRENYLATED

FLAVONOIDS FROM THE MORACEOUS PLANTS AND
GLYCYRRHIZA SPECIES)

Moraceous plants

Moraceae comprise a large family of sixty genera and nearly 1400

species, including popular species such as Artocarpus, Morus, and Ficus,
which are found in temperate, subtropical, and tropical regions of the
world. Mulberry tree, a typical plant of genus Morus, has been widely
cultivated for its leaves, which serve as indispensable food for silkworm.
In addition, the root bark of the mulberry tree (Mori Cortex, Morus alba
L. and other of genus Morus, "Sang-Bai-Pi" in Chinese, "Sohakuhi" in
Japanese) has been used as a material of traditional Chinese medicine for
an anti-inflammatory, diuretic, antitussive, expectorant, and antipyretic
purposes [1-3]. The earliest written reference to the use of Mori Cortex
is contained in the "Shen Nong Ben Cao Jing" (SNBCJ, shin-no hon-zo
kyo in Japanese), the first Chinese dispensatory whose original
anonymous volumes probably appeared by the end of the third century
[4,5]. In the Chinese book, 365 crude drugs are classified into three
classes (upper: plants with lowest side-effects and nontoxic, useful for
health care; middle: plats that are nontoxic or possess only weak toxicity
in whose use care must be exercised; lower: toxic and only for clinical
use. Mori Cortex is described as belonging to the middle class. The
crude drug is used as a component in traditional Chinese medicinal
prescriptions, such as "Wuhu Tang (Gokotou in Japanese)" and
"Mahuang Lianqiao Chixiaodou Tang (Maou-rensho-shakushozu-tou)",
which are applied clinically as a therapy for bronchitis and for nephritis,
respectively [6]. On the other hand, a few pharmacological studies on
the mulberry tree had demonstrated a hypotensive effect of the extract in
rodents [7,8]. Considering the above and reports described later, it was
suggested that the hypotensive constituents would be a mixture of many
phenolic compounds. Our interests were focused on the phenolic
constituents of the mulberry tree. So, we have studied phenolic
compounds of the mulberry tree and the related plants [8].
About seventy kinds of new phenolic compounds could be isolated
from Japanese cultivated mulberry tree {Morus alba, M. bombycis, and M,
Ihou) and Chinese crude drug "Sang-Bai-Pi" (the root bark of Chinese
mulberry tree). Most of them are isoprenylated flavonoids. Among
them, kuwanon G (1) was the first isolation of the active substance
exhibiting the hypotensive effect firom the Japanese Morus alba root bark
[9]. Furthermore, kuwanon G (1) and its isoprenylated derivative
kuwanon H (2) [10] are considered to be formed through an enzymatic
Diels-Alder type reaction of a chalcone and a dehydrokuwanon C or its
 201

sanggenon A
morusin (3) : R = H
(4): Ri = CH2CH=CMe2. R2 = OH
artonin E (7): R = OH
(4'): Ri = OH, R2 = CH2CH=CMe2

kuwanonG(1):R = H
kuwanon H (2): R = CH2CH=CMe2

OH O

artobiloxanthone (8)

OH
sanggenon C OH 0
(5): Ri = CH2CH=CMe2, R2 = OH sanggenon 0 (6)
cycloartobiloxanthone (9)
(5'): Ri = OH, R2 = CH2CH=CMe2

OH
brosimoneA(13) soroceal (15)

Fig. (1). Structures of compounds 1 - 1 5 from moraceous plants.

202

equivalent, Fig. (1). Since that time, about forty kinds of Diels-Alder
type adducts, structurally similar to that of 1 have been isolated from
Morus species. These Diels-Alder type flavonoids are characteristic
constituents oiMorus species [8,11-14].
Morusin (3), a flavone derivative, isolated from the root bark oi Morus
alba L., as a main isoprenylated flavonoid, has a structure bearing an
isoprenoid moiety at the C-3 position and a 2',4'-dioxygenated pattem in
the B ring [15]. These features are one of the characteristics of the
isoprenylated flavonoids of Morus root bark.
Furthermore, from the Chinese crude drug "Sang-Bai-Pi" purchased in
Japanese market, our group reported a series of isoprenylated flavonoids,
such as sanggenons A (4) [16] and C (5) [17]. Recently, the structure of
sanggenons A and C were revised from 4' and 5' to 4 and 5, respectively,
[18], Fig. (1). Sanggenon C (5) seems to be a Diels-Alder type adduct
of a chalcone derivative and a dehydroprenyl (=3-methyl-1,3-butadienyl)-
phenol having a sanggenon A type partial structure. From the root bark
of one of the Chinese mulberry tree, Morus cathayana, a series of
isoprenylated flavonoids could be isolated [19-21]. Some of the
flavonoids are the sanggenon A type flavanones (SATF), 3-hydroxy-
flavanone having a prenyl (=3-methyl-2-butenyl) group at 2 position and
an ether linkage between C-3 and C-2' positions, such as 4 and 5. Most
SATFs are (27?,55)-flavanones, sanggenons A (4), C (5), L, M (100),
sanggenols F, G, and J, and soroceins D and F [22], but sanggenon O (6)
is (2iS',ii?)-flavanone [23]. The stereochemistry at C-2 and C-3 of
sanggenons B (45), Fig. (6), D (33), E, P (sorocein H), S, sorocein E, and
sanggenols H and I is still unclear. Earlier studies of flavonoids and
stilbenes with one or more isoprenoid groups (prenyl group,
2,2-dimethylpyran ring, geranyl group, famesyl group, etc.) from Morus
species have been summarized in review articles [8,11-13,24-26].
On the other hand, the plants of Artocarpus species distribute over the
tropical and subtropical regions, and have been used as traditional folk
medicine so called "Jamu" in Indonesia against inflammation, malarial
fever and so on. Many kinds of isoprenylated flavonoids have also been
isolated from Artocarpus species by Venkataraman's group and other
several groups [27-30]. Our group also studied the constituents of
Indonesian Artocarpus species, such as A. heterophyllus, A, communis, A.
rigida, A. venenosa (^Paratocarpus venenosa), and A. altilis, a
moraceous plant from Sri Lanka [30-32]. About seventy kinds of
isoprenylated flavonoids have been isolated from these Artocarpus
species. The compounds, except some ones, have a characteristic
structure bearing an isoprenoid side chain at the C-3 position of flavone
skeleton, and the B ring has a 2',4',5'-trioxygenated pattem, such as
artonin E (7) corresponds to 5'-hydroxymorusin [31]. In addition to the
feature, some of the flavone, such as artobiloxanthone (8) and
 203

cycloartobiloxanthone (9) have a unique structure having the C-C linkage

between an isoprenoid side chain at the C-3 position and the 6'-carbon of
the B ring of flavone skeleton. This C-C linkage was considered to be
synthesized biogenetically through a phenol oxidation in the plants
[29,30]. These flavonoids are characteristic constituents oi Artocarpus
species.
Some of the Artocarpus flavonoids, such as artonin I (10), have been
regarded as intermolecular [4+2] cyclo-addition product from the
isoprenyl portion of a dehydroprenylphenol, as a diene, and the a,p-
unsubstituted bond of a chalcone skeleton, as a dienophile. As artonin I
(10) was considered to be formed through the Diels-Alder type reaction
of a chalcone derivative, morachalcone A (11) and artocarpesin (12), the
precursor artocarpesin (12) was added to the Morus alba cell cultures to
produce artonin I (10) [32]. Earlier studies of the isoprenylated phenols
from Artocarpus species have been summarized in review articles
[27,28,30].
Most Diels-Alder type adducts with a dehydroprenylflavonoid and a
chalcone (DADCs) have been isolated from Asian Morus and Artocarpus
species. However, some DADCs have also isolated from American
moraceous plants. From Brosimopsis oblongifolia, a Brazilian
moraceous plant, a series of DADCs, brosimone-family, were isolated.
One of them, brosimone A (13) is a unique adduct, and it is likely to form
through an intra-molecular [4+2] cycloaddition reaction between the
dehydroprenyl moiety at the A' ring, as the diene, and the a,P-double
bond of the chalcone skeleton (A ring-Ca-B ring), as the dienophile, of
brosimone D (14) [33], Fig. (1). From a Brazilian moraceous plant,
Sorocea bonplandii, ketalized Diels-Alder type adducts, such as soroceal
(15), have been isolated [34]. On the other hand, from Paraguayan
moraceous plants, Sorocea bonplandii, our group isolated the similar
ketalized Diels-Alder type adducts along with a unique adducts,
sorocenol B (16) [35], Fig. (2), which may be a derivative induced from
the Diels-Alder type adducts between a chalcone derivative and a de-
hydroprenylated resorcinol through the oxidative reaction.
A series of isoprenylated flavonoids has been isolated from the
following moraceous plants: Brosimopsis oblongifolia, a Brazilian plant
[36], Chinese Cudrania tricuspidata [37], and Taiwanese and Chinese
Cudrania cochinchinensis [38]. Many xanthones with one or two
isoprenoid groups have also been isolated from these Cudrania species
[11]. Latex from the wood of Antiaris toxicaria, a toxic Indonesian
plant, has been used for arrow poison. From the root bark of the plants,
antiarones A (17) and B (18) [39], the first examples of isoprenylated
aurone derivative, have been isolated along with the unique
dihydrochalcone derivatives, antiarones J (19) and K (20) [40], Fig. (2).
These compounds (19 and 20) are biogenetically considered to be formed
204

through a cyclization accompanied by hydration of the isoprenyl group

attached to the B ring of chalcone derivatives such as antiarone E (21)
[41]. Two new cyclomonoterpene-substituted isoflavones, ficusins A
(22) and B were isolated from the Indonesian moraceous plant, Ficus
septica Barm F [42]. Earlier studies of phenolic compounds (flavonoids,
xanthones, benzaldehydes) have been summarized in review articles
[8,11,30].

OH 0

antiarone A (17) antiarone B (18)

sorocenol B (16)

OH O
antiarone J (19): Ri = OH. R2 = CH2CH=CMe2
antiarone K (20): Ri = OMe, R2 = H antiarone E (21)
Fig. (2). Structures of compounds 1 6 - 2 2 from moraceous plants.

Glycyrrhiza species

Licorice (liquorice, kanzoh in Japanese, gancao in Chinese) is the name

applied to the roots and stolons of some Glycyrrhiza species
(Leguminosae or Fabaceae) and has been used by human beings from
ancient times. The genus Glycyrrhiza consists of about 30 species and
chemical studies have so far been carried out on 15 of them. Glycyrrhizic
acid (110) is the major triterpenoid saponin in licorice root and the main
sweetener of the herb. The saponin has been isolated from G. glabra^ G.
uralensis, G. inflata, G. aspera, G. korshinskyi, and G. eurycarpa, and
thus, these plants are generally accepted as licorice. In European
countries, G. glabra is chiefly used as licorice. On the other hand, in
Asian countries, G. glabra, G. uralensis, G. inflata, and G. eurycarpa are
used as licorice. Following extraction, the herb yields the licorice
products of commerce which are used as sweetening agents, flavoring for
American type tobaccos, chewing gums, candies, etc., as a depigmenta-
tion agent in cosmetic, and as pharmaceutical products, e.g., anti-ulcer.
 205

anti-hepatitis medicines, antitussives, etc. Among them the most

important industrial use of the herb is in the production of additives as
flavor and sweetening agents [43].
In SNBCJ, licorice is described as belonging to the upper class and is
recommended for lengthen one's life span, for improving health, for
cures for injury and swelling, and for its detoxification effect. One
hundred ten prescriptions are recorded in the earlier Chinese medicinal
book "Shang Han Lun", where seventy prescriptions include licorice [43].
In the Japanese market, Chinese licorice is classified by its place of
production, e.g., Northeastem licorice (Touhoku kanzoh in Japanese),
Northwestem licorice (Seihoku kanzoh), Xinjiang licorice (Shinkyou
kanzoh), etc. Among these licorice, Northeastem licorice had been
identified as G. uralensis, but the original plants of the others had been
unidentified. We investigated the phenolic constituents of certain
Glycyrrhiza species identified by authorities, and many phenolic
compounds were isolated from these plants [43].
The main phenols of licorice are glycosides of liquiritigenin (100) and
isoliquiritigenin (70), e.g., liquiritin (113), isoliquiritin, liquiritin apioside,
licuraside, etc. [43]. As minor phenolic compounds, many isoprenoid-
substituted flavonoids, chromenes, dihydrophenanthrenes, and dihydro-
stilbenes were isolated from Glycyrrhiza species. Some of them
characterized each plant [43^5]. The 5- and 6-positions of most
flavonoids from European licorice are unsubstituted, but the 5-position of
flavonoids from Chinese licorices is generally substituted with a
methoxyl group or a hydroxy 1 group (the 5-OH of some compounds
forms ether linkage with an isoprenoid group at the 6-position). For
example, the main isoprenoid-substituted flavonoid of G. glabra var.
typica, Russian licorice, is a pyranoisoflavan, glabridin (23). The
5-position of most flavonoids from the plants is unsubstituted, e.g., 23,
glabrene (24), glabrol (25), 3-hydroxyglabrol (26), etc.. Fig. (3).

glabridin (23) giabrene (24) glabrol (25): R = H

3-hydroxyglabrol (26): 3*-(Y,y-dimethylallyl)-
R = OH kievitone (27)

Fig. (3). Structures of compounds 23 - 26 from licorice {Glycyrrhiza glabra).

206

On the other hand, from Chinese and Kyrghiz G. glabra, both

5-unsubstituted flavonoids (e.g., 24) and 5-oxygenated flavonoids, e.g.,
3'-(y,y-diniethylallyl)-kievitone (27), have been isolated. Nevertheless,
5-position of the most flavonoids with one or two isoprenoid groups from
these plants is substituted with a hydroxyl or a methoxyl group. The
main isoprenoid-substituted flavonoid of the Kirghiz licorice is
compound 27, but the isoflavan (23) has not been isolated [46-49],
Isoprenoid-substituted flavonoids isolated from commercial Kyrghiz
licorice and European licorice that was cultivated in Japan were
summarized in Table 1 [49-51].

Table 1, Flavonoids isolated from Kyrgyz and European Glycyrrhiza glabra

Kirghiz [49] European [50,51 ]

3'-(Y,Y-Dimethylallyl)-kievitone (27) +-H-¥

Glisoflavanone (3',6-diprenyl-2',4',5,7-tetrahydroxyisoflavanone) +++ -
Glyasperin A (77) ++
Glyasperin C (61) ++
Glyasperin D (62) +++
Isoderone(DMP;4',3']-5,7-dihydroxyisoflavone)'' +
Semilicoisoflavone B (68) ++
8-(Y,Y-Dimethylallyl)-wighteone (58) ++
Gancaonin G (60) +
Gancaonin H (DMP;4',5']-6-prenyl-3',5,7-trihydroxyisoflavone) ++
1-Methoxyphaseollidin (125) +++
Edudiol (3,9-dihydroxy-l-methoxy-2-prenylpterocarpan) ++
Glabrene (24) ++ +++
Glabridin (23) - ++++
4'-C>-Methylglabridin (123) - ++
Hispaglabridin A (3'-prenylglabridin) - ++-H-
Glabrol (25) - +++
3-Hydroxyglabrol (26)* - +++
Glabrone (DMP;4',3']-2',7-dihydroxyisoflavone) - ++
Medicarpin (3-hydroxy-9-niethoxypterocarpan) - ++++
Shinpterocarpin (DMP;3,4]-9-hydroxypterocarpan) - +++
Euchrenone as (DMP;4',3']-7-hydroxy-8-prenylflavanone) - ++
Glyinflanin K (2DMP;7,8, ;2',3']-isoflavan) - ++
Glyinflanin G (2DMP;4,5, ;4',3']-2',3-dihydroxychalcone) - ++
Kanzonol U (DMP;2',3']-4',6-dihydroxy-2-arylbenzofuran] - ++
Kanzonol V (DMP;2',3']-4',6-dihydroxy5-prenyl-2-arylbenzofuran) - +++
Kanzonol W (DMP;7,8]-2',4'-dihydroxy-3-arylcoumarin) - +++
Kanzonol X (3',8-diprenyl-2',4',7-trihydroxyisoflavan) - +++
Kanzonol Y (3,5'-diprenyl-a,2',4,4'-tetrahydroxy-dihydrochalcone) - +++
Kanzonol Z (DMP;7,8]-3,4'-dihydroxy-3'-prenylflavanone) - +++
3-Hydroxyparatocarpin C** - +++

Yields from dried licorice roots: -H-i-H=more than 0.01%; +-H-=between 0.01 and 0.001%; ++=0.001-0.0001%;
+=1-0.1 ppm. • The compound was obtained from the stolons. ° 2,2-dimethylpyrano[b=DMP. ** Tentative
name used here (DMP;4,5]-3'-prenyl-2',3,4'-trihydroxychalcone).

The difference of the substituents at C-5 is expected that European

and Chinese licorices exhibit different actions in therapeutically use.
For example, 5,6-disubstituted isoflavans do not showed a potency of
 207

anti-HIV activity in vitro, but two isoflavans with no substituent at both

5- and 6-positions obtained from Erythrina lysistemon (Leguminosae)
have the activity as described later [52].
As described the above, moraceous plants and Glycyrrhiza species are
rich sources of isoprenylated phenolic compounds. The phenolic nuclei
having the isoprenoid-derived substituents, e.g., simple isoprene or a
monoterpenoid, vary over a wide range from a simple phenol to
complicated ones. Some of the moraceous plants studied by our group
have been used as traditional herbal medicines in the native countries. It
is interesting to clarify the relationship between the usage and biological
activities of the isoprenylated phenolic compounds. So we studied some
of the biological activities of these compounds. This article reviews the
biological activities of the isoprenylated flavonoids isolated from the
moraceous plants and isoprenoid-substituted phenols (flavonoids,
xanthones, dihydrostilbenes, and dihydrophenanthrenes) from
Glycyrrhiza species by our group and other several groups.

11. HYPOTENSIVE ACTIVITY OF ISOPRENYLATED FLAVO-

NOIDS FROM THE ROOT BARK OF MORUS SPECIES

The first report for the hypotensive effect of the mulberry tree was
presented by Fukutome in 1938, who asserted that oral administration of
the hot water extract of the mulberry tree showed a remarkable
hypotensive effect in rabbits [53]. Ohishi reported the hypotensive
effect of the ethanol extract of mulberry root bark [54]. Suzuki and
Sakuma reported that the hypotensive activity seemed to be due to
phenolic substances, and that the effect disappeared on acetylation [55].
Later, Katayanagi, et aL reported that the ether extract of the root bark
gives to rabbit (6 mg/kg, i.v.) showed a marked hypotensive effect and
that the active constituents seemed to be a mixture of unstable phenolic
compounds [56]. Tanemura ascribed the activity of mulberry root bark
to acetylcholine and its analogous presumably contained in the alcohol
soluble fraction, and that the hypotensive constituents produced a
yellowish-brown precipitate on treatment with Dragendorff reagent [57].
Yamatake, et al reported that n-butanol- and water-soluble fractions of
mulberry root bark had similar effect except for those on the
cardiovascular system. Both fractions showed cathartic, analgesic,
diuretic, antitussive, anti-edema, sedative, anticonvulsant, and
hypotensive actions in mice, rats, guinea pigs and dogs [7]. On the
beginning of our study of mulberry tree, the hypotensive constituents had
not been identified. In view of the reports, we assumed that the
hypotensive compounds of the plant would be a mixture of unstable
208

phenolic compounds and therefore undertook a study of the phenolic

constituents of the root bark of the cultivated mulberry tree.
The root bark of the cultivated mulberry tree was extracted
successively with n-hexane, benzene, and methanol. The methanol
extract, 1-20 mg, showed a dose-dependent decrease in arterial blood
pressure in pentobarbital-anesthetized rabbit, Fig. (4). The extract was
fractionated successively by silica gel column chromatography (C.C.),
polyamide C.C, silica gel preparative (p.) TLC, and p. HPLC leading to
isolated of kuwanons G (1, 0.2% yield) [9] and H (2, 0.13% yield) [10].
The root bark of Moms alba
n-Hexane

Residue Extract
Benzene
Residue Extract
I Methanol C.C, p. TLC
-T
Residue extract Morusin (3), kuwanons C (42), D, E (43), F,
Ethyl acetate soluble portion oxydihydromorusin (46),
I C.C, p. TLC, p. HPLC mulberroftiran A (47)

Kbwanons G (1), H (2), L (44), M (35), albanol B (97)

mulberrofurans C (28), F (29), and G (30)

Fig. (4). Isolation procedure of flavonoids from the root bark of Morus alba.

mmHg

PN n > < l i < M H H H H M M ( H i t i i

kuwanon G 1 mg/kg i.v.

mmHg

tsmmm |ioo
50

iilSliSirJ'^^

10 s
kuwanon H I mg/kg i.v. '

Fig. (5). Effects of kuwanon G (1) and kuwanon H (2) on blood pressure. Electrocardiogram (ECG),
phrenic nerve discharge (PN), and electroencephalogram (EEG) in a gallamine-immobilized rabbit.
 209

Both compounds (1 and 2) almost equally caused decrease of arterial

blood pressure in a dose dependent and reversible manner at the dose of
between 0.1 and 3 mg/kg, i.v. in pentobarbital-anesthetized as well as in
un-anesthetized, gallamine-immobilized rabbits. Fig. (5), [58].
These hypotensive actions of kuwanons G (1) and H (2) were not
modified by atropine or eserine, suggesting the non-cholinergic nature
origin. Furthermore, neither propranol nor diphenhydramine affected
their actions on the arterial blood pressure. Although they produced no
significant change in both electrocardiogram (ECG) and respiration when
administered intravenously in rabbits. The hypotensive effects of kuwa-
non G (1) and H (2) did not accompany with heart rate change [58]. In
pentobarbital-anesthetized pithed dogs, kuwanons G (1) and H (2) also
significantly decrees of femoral arterial blood pressure. These effects
suggested that mechanism of hypotensive effects of kuwanons G (1) and
H (2) mediated through peripheral system.
Mulberrofurans C (28) [59], F (29) [60], and G (30) [60], Fig. (6),
were also isolated as hypotensive components from the mulberry tree.
Mulberrofuran C (28) is considered to be formed by a Diels-Alder type of
enzymatic reaction process of a chalcone derivative and dehydromoracin
C (31) or its equivalent. Furthermore, mulberrofurans F (29) and G (30)
seems to be Diels-Alder type adducts derived from chalcomoracin (32)
and mulberrofuran C (28), respectively, by the intra-molecular ketali-
zation reaction of the carbonyl group with the two adjoining hydroxyl
groups, 3'(5')-OH and 2"-0H. Intravenous injection of mulberrofuran C
(28, 1 mg/kg) produced a significant hypotension (37 mmHg fall) in
rabbit (male, 3.3 kg) anesthetized with pentabarbital sodium (30 mg/kg).
Single intravenous injection of mulberrofurans F (29) and G (30) (both
0.1 mg/kg) caused a marked depressor effect in rabbit by 26 mm Hg and
16 mm Hg, respectively.
On the other hand, in Japan, "Sang-Bai-Pi" (the root bark of Chinese
mulberry tree) imported from China has been used as an herbal medicine,
hence a study of the components of this crude drug purchased in the
Japanese market was undertaken. Its phenolic components are different
from those of Japanese mulberry tree. For example, morusin (3) and
kuwanon G (1) are the main phenolic components of Japanese mulberry
tree, in the case of "Sang-Bai-Pi", these components are minor ones,
while sanggenons A (4) [16], C (5) [17], and D (33) [61] are the main
components [24]. Sanggenons C (5) and D (33) showed the hypotensive
effects as follows: Sanggenon C (5) caused transient decrease in arterial
blood pressure at the doses of 1 mg/kg in pentobarbital-anesthetized
rabbit by 15 mm Hg, while at the doses of 5 mg/kg the compound (5)
caused a transient decrease by 100 mm Hg, which continued for more
210

mulberrofuran C (28): R = H
mulberrofuran F (29): R = CH2CH=CMe2
chalcomoracin (32): R = CH2CH=CMe2
mulberrofuran G (30): R = H

kuwanon E (43)
sanggenon B (45)

Fig. (6). Structures of flavonoids (28 - 44) from moraceous plants.

 211

than one hour by 15 mm Hg [17,62]. Sanggenon D (33) caused a

transient decrease at the dose of 1 mg/kg in pentobarbital and urethane
anesthetized male Wister strain rat by 35 mm Hg, while the compound
(33) caused a decrease by 80 mmHg at the doses of 1 mg^g in
spontaneously hypertensive rat [61,63].

III. ANTI-TUMOR PROMOTING ACTIVITY OF MORUSIN (3)

Cancer chemoprevention is the most important subjects in cancer

research at present and is a new medical strategy for cancer prevention,
which was established by recent understanding of molecular multistage
carcinogenesis in humans. To find nontoxic cancer preventive agents,
Fujiki and his coworker studied natural products derived from marine and
plant sources [64,65]. In 1987, Yoshizawa, et aL reported that (-)-
epigallocatechin gallate (EGCG), which is a main constituent of green tea,
inhibited tumor promotion by teleocidin in mouse skin [66]. In 1988,
Fujita, et aL reported the inhibitory effect of EGCG on carcinogenesis
with 7V-ethyl-A^-nitro-A^-nitrosoguanidine in mouse duodenum [67]. On
the other hand, in the course of our examination the constituents of the
Morus root bark, we found the following novel photo-oxidative
cyclization. When a solution of morusin (3) in chloroform (CHCI3) was
irradiated using high-pressure mercury lamp, morusin hydroperoxide (34),
Fig. (6), was obtained in ca, 80% yield [68]. The reaction did not occur
in the dark and was depend on the solvent; the reaction occurred in low
polar or nonpolar solvent such as CHCI3 and benzene, but not in protic
solvent. The reaction mechanism was suggested as follows [69]:
morusin (3) in the ground state interacts with an oxygen molecular to
form a contact charge transfer complex [3 O2] (CCTC). On
irradiation, the CCTC gives an excited charge transfer state that
presumably leads to reactive species such as free radicals as described in
Fig. (7). Recently, the proof of presence of the CCTC was provided by
laser desorption/ionization time-of-flight mass spectrometry of 3 [70].
The hydroperoxide (34) was also obtained with the oxidation of morusin
(3) with singlet oxygen or radical initiator [71].

HO^^s^^OH
hv
34
•OOH

Fig. (7). Reaction mechanism of photo-oxidative cyclization of morusin (3).

212

This photoreaction and the relative reaction of morusin (3) along with
the anti-tumor promoting activity of EGCG encouraged us to examine the
anti-tumor promoting activities of a series of isoprenylated flavonoids
isolated from Morus species. First we examined the inhibition against
three biochemical effects; the specific binding of ^H-12-O-tetra-
decanolylphorbol-13-acetate (TPA) to mouse particulate fraction, the
activation of Ca^'^-activated phospholipid-dependent protein kinase
(protein kinase C) with teleocidin, and induction of ornithine
decarboxylase (ODC) with teleocidin in mouse skin [72]. Interestingly,
of the eight isoprenylated flavonoids, morusin (3), kuwanons G (1) and M
(35), mulberroforan G (30), and sanggenon D (33) gave similar results in
these biochemical tests as described in Table 2.
Table 2. Effects oi Morus flavonoids on biological and biochemical activities

Inhibiting of Inhibition of Inhibition of

specific [^H]TPA activation of ODC induction
binding protein kinase C (%)
(ED50 jimol/L) (ED50 fimol/L)

Morusin (3) 57 80 43
Kuwanon G (1) 99 40 34
Kuwanon H (2) 100 80 -35
Kuwanon M (35) 85 22 25
Mulberrofuran G (30) 34 46 10
Sanggenon A (4) 62 80 -62
Sanggenon C (5) 48 46 -17
Sanggenon D (33) 60 42 17

100

^ o

Concentration (mol/L) of morusin (3)

Fig. (8). Effects of morusin (3) on specific binding of [^H]TPA to a mouse skin particulate fi-action.
Various concentrations of morusin (•) or TPA (o) were incubated with a particulate fi-action of mouse skin in
the presence of 4 nmol/L [^H]TPA for 2 h at 4°C, and the assay mixture was filtered on glass filter membrane
with acetone cooled in a dry ice-ethanol bath. Non-specific bindings were measured in the presence of
500-fold excess of unlabelled TPA.
 213

Of these five compounds, morusin (3) is the least toxic and can be
isolated as one of the main phenolic compounds from the root bark.
The more detailed data for the above these biochemical tests of
morusin (3) were as follows [73]. As shown in Fig. (8), morusin (3)
caused dose-dependent inhibition of the specific binding pHJTPA to a
mouse skin particulate fraction. The concentration of morusin (3) for
50% inhibition (ED50) was 57 |amol/L, whereas that of unlabelled TPA
was 4 nmol/L.
As morusin (3) was assumed to interact with the phorbol ester receptor,
we examined whether it inhibited the activation of protein kinase C by
teleocidin in vitro [73]. Fig. (9) shows that morusin (3) inhibited the
phosphorylation of histone type III-S by protein kinase C dose-dependent
and that 80 |imol/L morusin caused 50% inhibition.

100 VA

o
50

a
0.

VA 10- 10-*
\o-
Concentration (mol/L) of morusin (3)
Fig. (9). Inhibition by morusin (3) of activation of protein kinase C by teleocidin in vitro.
The assay mixture (0.25 mL) contained 20 jimol/L CaCh, 7.5 |ag of phosphatidylserine, 2.3 (^mol/L teleocidin,
and various concentrations of morusin (3) with 0.05 units of partially purified enzyme. Enzyme activity was
measured as the incorporation of ^^P from [7-^^P]ATP into histone type III-S during incubation for 3 min. at
30^.

Furthermore, we examined the inhibition of the induction of ODC

induction by teleocidin in mouse skin. Application of 11.4 nmol
morusin (3) caused 43% inhibition of the induction of ODC by 11.4 nmol
teleocidin [73]. From the results of these three tests, morusin (3) might
inhibit the tumor-promoting activity of teleocidin on mouse skin.
As shown in Figs. (10) and (11), the percentage of tumor bearing mice
in the group treated with 7,12-dimethylbenz[a]anthracene (DMBA) plus
teleocidin reached 100% by week 15, o in Fig. (10). In contrast, the
onset of tumor formation was delayed 5 weeks by treatment with morusin
(3), • in Fig. (10), and the percentage of tumor-bearing mice in the group
treated with DMBA plus teleocidin and morusin (3) was 60% at week 20.
The average number of tumors per mouse in week 20 was also reduced
from 5.3, o in Fig. (11), to 1.1, • in Fig. (11), by morusin (3) treatment.
214

On the other hand, morusin (3) itself did not show a tumor promoting
activity on mouse skin, x in Figs. (10) and (11). From these results,
morusin (3) is an anti-tumor promoter judging from its ability to inhibit
the short-term effects induced by tumor promoters.

100

to

10 20 10 20
Weeks of promotion Weeks of promotion
Figs. (10) and (11). Inhibition by morusin (3) of tumor promotion by teleocidin in a two-stage
carcinogenesis experiment on mouse skin. Inhibition was achieved by a single application of 100 ^g of
DMBA, and teleocidin (2.5 }ig) and morusin (1 mg) were applied twice a week throughout the experiments.

As mentioned the above, morusin (3), kuwanon G (1), kuwanon M

(35), mulberroforan G (30), and sanggenon D (33) showed inhibitory
effects in the three biochemical tests. The anti-tumor promoting
activities of later four flavonoids with one or two isoprenoid groups have
not been tested in a two-stage carcinogenesis experiments, due to
limitations of their amounts available, but their inhibitory potencies to the
three biochemical tests were almost similar to that of morusin (3).
Furthermore, the twelve isoprenylated flavonoids from the moraceous
plants and two flavonol glycosides (48 and 49) from Epimedium species
(Berberidacaceae) [74] along with quercetin (50) were tested for
inhibitory effects on carcinogenesis by a test for inhibition of specific
binding of [^H]TPA to a mouse skin particulate fraction.
While the other biochemical tests and the inhibition of tumor
promotion of teleocidin in a two-stage carcinogenesis experiment have
not been carried out, due to limitation in their amounts available, some of
isoprenylated flavonoids from the moraceous plants showed the similar
inhibitory potencies to those of morusin (3) and the related compounds,
Figs. (6) and (12), as shown in Table 3.
On the other hand, EGCG and green tea extract are acknowledged
cancer-preventive agents in Japan [75,76]. Natural products with anti-
tumor promotion activity isolated from foodstuff and medicinal plants
have been summarized by Konoshima and his co-worker and Akihisa and
 215

his co-worker [77,78]. Considering these results as well as the results of

biochemical tests and anti-tumor promoting activity of the isoprenylated
flavonoids from the moraceous plants in a two-stage carcinogenesis
experiment with teleocidin, the isoprenylated poly-phenolic compound
seems to be interesting compounds for finding cancer preventive agents
and the more detailed experiments should be carried out.
Table 3. Effects of the isoprenylated flavonoids on inhibition of specific [^H]TPA binding (ID50, ^mol/L)

Kazinol C (36) 80 Kuwanon L (44) 80

Kazinol E (37) 70 Sanggenon B(45) 95
Kazinol F (38) 98 Oxydihydromorusin (46) 95
Kazinol J (39) 90 Mulberroftiran A (47) >100
Kazinol M (40) 100 Ikarisoside A (48) >100
Kazinol N (41) >100 Ikarisoside B (49) >100
Kuwanon C (42) 80 Quercetin (50) >100
Kuwanon E (43) 83

OMe

oxydihydromorusin (46)
ikarisoside A (48): R = Rha
mulberrofuran A (47) ikarisoside B (49): R = Glu(1 ^ 2)Rha

OCH3

OH O

quercetin (50):
Ri = OH,R2=R3 = H
antiarone L (57)
cirsilioi (51):
Ri = H, R2 = 0Me, R3 = Me artonin H (56)

Fig. (12). Structures of flavonoids (46 - 57) from moraceous plants, Epimedium species, and test reagents
(50 and 51).

IV. INHIBITION OF ARTONIN E (7) AND RELATED

COMPOUNDS ON 5-LIPOXYGENASE

Previously, we reported the effects of Morus flavonoids on arachidonate

metabolism in rat platelet homogenates, such as inhibition of 12-hydroxy-
5,8,10-heptadecatrienoic acid (HHT), thromboxane B2, and 12-hydroxy-
5,8,10,14-eicosatetraenoic acid (12-HETE) [79,80]. As described in the
216

introduction, Artocarpus plants (Moraceae) have been used as traditional

medicine in Indonesia for swelling and malarial fever. This usage
seems to be expecting for effect of anti inflammation. As leukotrienes
are known to be chemical mediators of anaphylaxis and inflammation, a
number of compounds have been studied and developed as selective
inhibitors of 5-lipoxygenase, the enzyme initiating leukotriene bio-
synthesis from arachidonic acid. So the inhibitory effect of the
Artocarpus flavonoids against arachidonate 5-lipoxygenase was
examined [81]. Yamamoto, et aL screened various flavonoids, and
found that cirsiliol (51), Fig. (12), potently inhibited 5-lipoxygenase and
proposed two structural factors of the flavonoids for the specific
inhibitory activity, one is catechol type of the B ring and the other is the
presence of an alkyl-like side chain at the C-3 position [82,83]. We had
interesting for the inhibitory effects of a series of Artocarpus flavones on
the 5-lipoxygenase activity.
Seven Artocarpus flavonoids and morusin (3) were tested for their
inhibitory actions on arachidonate-5-lipoxygenase purified from porcine
leukocyte [84]. As shown in Fig. (13), the IC50 values varied depending
on the structural modification of the compound. The compounds having
three hydroxyl groups at positions 2\ 4\ and 5' on the B ring (compounds
7, 8, 52 and 55) were more potent inhibitors. Thus, the vicinal diol
partial structure was important for 5-lipoxygenase inhibition.

OH 0
OH o
artonin A (54)

heterophyllin (52) cycloheterophyllin (53)

Inhibitory effects (IC50 ± SD, N=3, ^imol/L) on

arachidonate 5-lipoxygenase activity 1

Morusin (3) 2.9 ± 0.4

Artonin E (7) 0.36 db 0.03
Artobiloxanthone (8) 0.55 db 0.20
j Cycloartobiloxanthone (9) 1.3 ±0.2
Heterophyllin (52) 0.73 ±0.21 '
OH 0
Cycloheterophyllin (53) 1.6±1.0
artonin B (55) Artonin A (54) 4.3 ± 0.5
Artonin B (55) 1.0 ±0.1

Fig. (13). The inhibitory effect (IC50 ± SD) on arachidonate 5-lipoxygenase activity.

As shown in Fig. (14), 5-lipoxygenase was inhibited depending on the

concentration of artonin E (7), which gave the lowest IC50 (0.36 |Limol/L)
of all the eight compounds. On the other hand, morusin (3), which
 217

lacked the 5'-hydroxyl group of artonin E (7), was a less potent 5-

lipoxygenase inhibitor (IC5o=2.9 |Limol/L). Artonin E (7) was
significantly more potent than cirsiliol (51, Fig. (12), IC5o=1.3 |Limol/L),
which was reported as a 5-lipoxygenase inhibitor. This finding was
consistent with the report that the inhibitory activity of cirsiliol (51) with
5-lipoxygenase was enhanced by introducing a lipophilic alkyl group at
the C-3 position of theflavoneskeleton.
Inhibitory actions of artonin E (7) and morusin (3) on other
mammalian arachidonate oxygenases were examined. Artonin E (7)
inhibited two 12-lipoxygenase from porcine leukocytes and human
platelets, 15-lipoxygenase from rabbit reticulocytes, and fatty acid
cyclooxygenase from bovine vesicular glands (IC5o=2.3, 11, 5.2, and 2.5
|amol/L, respectively). However, IC50 values for these oxygenases were
higher by one order of magnitude than that for 5-lipoxygenase. Morusin
(3) also inhibited these enzymes (except for human platelet 12-
lipoxygenase) with IC50 values of micro molar order as follows: two 12-
lipoxygenase from porcine leukocytes and human platelets, 15-
lipoxygenase from rabbit reticulocytes, and fatty acid cyclooxygenase
from bovine vesicular glands; IC5o=3.4, > 30, 3.3 and 1.6 |imol/l,
respectively. These results indicated that artonin E (7) was a relatively
specific inhibitor for 5-lipoxygenase. Thus, which the selectivity for
5-lipoxygenase was not observed with morusin (3). Significant
differences of IC50 values of artonin E (7) and morusin (3) between
porcine leukocyte 12-lipoxygenase and the human platelet
12-lipoxygenase should be noted since the leukocyte and platelet
12-lipoxygenase were distinct both catalytically and immunologically.

Concentration (|imol/L)
Fig. (14). Dose-dependent inhibition of 5-lipoxygenase by artonin E (7, • ) , morusin (3, o), and cirsiliol (51,
A).

V. INHIBITION OF ARTONIN E (7) AND RELATED

COMPOUNDS ON MOUSE TNF-a RELEASE AND THEIR
CYTOTOXIC ACTIVITIES
218

As described in Chapter III, morusin (3) has been found to be anti-tumor

promoter in a two-stage carcinogenesis experiment with teleocidin.
Considering the similarity of the structures between morusin (3) and
artonin E (7), artonin E (7) was expected to be an anti-tumor promoter.
Furthermore we found a novel photo-oxidative cyclization of artonin E
(7) as follow: photo-reaction of artonin E (7) in CHCI3 containing 4%
ethanol solution with high-pressure mercury lamp produced
artobiloxanthone (8) and cycloartobiloxanthone (9), and the treatment of
artonin E (7) with radical reagent (2,2-diphenyl-l-picrylhydrazyl: DPPH)
resulted in the same products, Fig. (15), [84].

OH 0
(±)-artobiloxanthone (8) (±) -cycloartobitoxanthone (9)
artonin E (7)
8 9

hv, 24 h. CHCI3 34% 3%

DPPH, 24 h, CHCI3 (in the dark) 70% 4%

Fig. (15). Photoreaction of artonin E (7) and the reaction with radical reagent.

As described in Chapter III, we have reported the photo-oxidative

cyclization on morusin (3). These results suggested that the photo-

OH 0 OH 0
(±) -cycloartobiloxanthone (9) (±)-artobiloxanthone (8)

Fig. (16). Plausible mechanism for the formation of artobiloxanthone (8) and cycloartobiloxanthone (9) from
artonin E (7).
 219

oxidative cyciization of artonin E (7) may proceed through phenol

oxidation via the semiquinone radicals described in Fig. (16). This
chemical reactivity and the similarity of the structures between morusin
(3) and artonin E (7) encourage us to examine the anti-tumor promoting
activity of artonin E (7).
Recently, Fujiki, et al. proposed a new tumor promotion mechanism
applicable to human cancer development on the basis of experiment with
okadaic acid. They described that tumor necrosis factor-a (TNF-a)
induced by okadaic acid acts as a mediator of human carcinogenesis [65].
As briefly summarized in Fig. (17), okadaic acid inhibits the action of
protein phosphatase type 1 and 2A, resulting in the accumulation of
phosphorylated protein. Fujiki's group has shown that TNF-a acts as a
timior promoter in BALB/3T3 cell transformation in vitro. The results
of the studies on the okadaic acid class tumor promoters suggest that
inflammatory stimuli or chemical tumor promoters induce TNF-a release
from target tissues, and TNF-a gene expression in the initiated cells.
This released TNF-a acts as a tumor promoter in the autocrine and
paracrine system. According to the assumption that TNF-a is an
endogenous tumor promoter associated with inflammatory potential,
many historical puzzles of tumor promotion, such as its relationship to
inflammation, can be solved. Based on this new tumor-promotion
pathway, inhibition of TNF-a production leads to inhibition of tumor
promotion. Furthermore, recent investigation has revealed that TNF-a
is involved in various diseased, such as rheumatoid arthritis, Crohn's
disease, multiple sclerosis, graft-versus-host disease, HIV, malaria, sepsis,
and cachexia associated with cancer [85-90]. So, specific inhibitions of
TNF-a production will almost certainly be effective not only in cancer
prevention but also in the therapy and prevention of these other diseases.

{jene expression
—1 protein i ^ phosphorylated t - c-fos
okadac acid proteins 1
j — ' phosphatase 1 ojun
NF-KB
ODC
TNF-a - • p
~3
phosporylated
proteins
t
' _
TNF-a — •

V _

Fig. (17). Mechanism of tumor promotion with okadaic acid.

Based on the above descriptions, we examined the inhibitory effect of

the Artocarpus flavonoids on TNF-a release stimulated by okadaic acid
using BALB/3T3 cells. This experiment was carried out in co-operation
with Dr. Fujiki's group (Saitama Cancer Center Research Institute, Japan).
All the compounds tested inhibit the TNF-a release stimulated by
220

okadaic acid at suitable lower concentration. This result suggests that

several Artocarpus flavonoids act as anti-tumor promoter against to the
okadaic acid type promotion. However, the detail mechanism is not
clear at present, Fig. (17). The comparison of the inhibitory effects of
the Artocarpus flavonoids against the TNF-a release (Table 4) and
arachidonate 5-lipoxygenase, Fig. (13), was carried out. Artonin E (7)
was the most potent inhibitor on both tests and the other compounds,
artobiloxanthone (8) and heterophyllin (52), inhibited stronger than
cycloartobiloxanthone (9), cycloheterophyllin (53), and morusin (3).
The compounds showing stronger activity, all have three hydroxyl groups
in the B ring. This characteristic feature might be important factor for
both biological activities [91,92]. It is also noteworthy that the
bioactivities of these flavonoids may reflect the use of Artocarpus species
to the treatment for inflammation and malarial fever in Jamu medicines as
is stated above.
Table 4. Inhibitory effects (IC50, Mmol/L) of six flavonoids for the release of TNF-a from BALB/3T3
cells by treatment of okadaic acid

Morusin (3) 1.76 Artonin E (7) 0.43

Artobiloxanthone (8) 0.94 Cycloartobiloxanthone (9) 1.94
Heterophyllin (52) 0.48 Cycloheterophyllin (53) 7.8

We also examined the cytotoxic activities of the Artocarpus flavonoids,

artonins A (54), B (55), E (7), H (56), heterophyllin (52), and cyclo-
heterophyllin (53), against cancer cells, mouse L-1210 and colon 38.
All compounds tested showed the cytotoxic activities against both cancer
cells (Table 5) [93]. Among them, cytotoxicity of heterophyllin (52),
artonins B (55) and E (7) w^ere stronger than critical drug, l-(2-tetra-
hydrofuryl)-5-fluorouracil (TFFU). While we examined the cytotoxic
activities of three dihydrochalcone derivatives isolated from Antiaris
toxicaria (Moraceae), antiarones J (19), K (20), and L (57), against the
two cancer cells [94]. All the compounds showed the weak cytotoxic
activities against both cancer cells. Artonin E (7) also exhibited

Table 5. Cytotoxic activities (IC50, |ig/mL) of Artocarpus and Antiaris flavonoids against L-12i0 and
Colon 38 cells

L-1210 Colon 38 L-1210 Colon 38

Artonin A (54) 8.8 14.3 Cycloheterophyllin (53) 4.7 4.6

Artonin B (55) 23 1.4 Antiarone J (19) 77.0 70.4
Artonin E (7) 2.2 1.9 Antiarone K (20) 81.3 46.3
Artonin H (56) 8.8 3.5 Antiarone L (57) 80.4 >100
Heterophyllin (52) 2.3 1.3 TFFU* 2.9 3.9

' Positive control.

 221

cytotoxic activities against human oral cells and MT4-cells as shown in

Chapter VII (Table 7).

VI. BOMBESIN RECEPTOR ANTAGONISTS, KUWANONS G

(1) AND H (2), ISOLATED FROM MORUS SPECIES

Bombesin and its mammalian counterparts, gastrin-releasing peptide

(GRP) and neuromedin B (NMB), have been shown to have a wide range
of physiological and pharmacological functions [95]. Ligand-binding
and molecular cloning studies have revealed two pharmacologically
distinct G-protein-coupled receptor subtypes for mammalian bombesin-
like peptides; a GRP-preferring (GRP-R) and an NMB-preferring
bombesin receptor (NMB-R) [96].
A series of observations indicates that the mammalian bombesin-like
peptides may act autocrine growth factors in human small cell lung
carcinoma (SCLC) and other cancers. First, many human SCLC cell
lines have been shown to express bombesin-like peptides [97]. Second,
peptide bombesin receptor antagonists or anti-bombesin antibodies inhibit
SCLC cell growth in vitro and in vivo [98,99]. These data suggested
that the bombesin receptor antagonists might be useful for the treatment
of some kinds of SCLC and other cancers. Because most antagonists
reported thus far are peptides except for CP-70,030 and CP-75,998 (first
synthetic non-peptide antagonists) [100-102], so, Fujimoto's group
(Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan)
screened the four hundred plant extract samples to search for non-peptide
bombesin receptor antagonists. The methanol extract of the under-
ground part of cultivated mulberry tree, Morus bombycis, was found to
potently inhibit [^^^I]GRP binding to Swiss 3T3 cells. Bioassay-directed
fractionation led to the isolation of two known flavone derivatives,
kuwanons G (1) and H (2), which were identified by direct comparison
with the authentic samples [103].
The antagonistic profiles of kuwanons G (1) and H (2) were
characterized from the following results [103]. Kuwanon H (2)
inhibited specific binding of [^^^I]GRP to GRP-referring receptors in
murine Swiss 3T3 fibroblasts with K{ value of 290±50 nmol/L, which is
more potent than that of kuwanon G (1), K\ value=470±60 nmol/L. The
Ki value of 2 was about one order of magnitude more potent than those of
CP-70,030 and CP-75,998, but had no effect on endothelin-1 or
neuropeptide Y binding. While kuwanon H (2) inhibited specific
binding of [^^^I]bombesin to rat esophagus membranes, the Ki value was
about one order of magnitude less potent, Ki value of 2=6,500±2,000,
than that of [^^^I]GRP toSwiss 3T3 cells.
While bombesin (10 ^ mol/L) increased intracellular Ca^"^ levels in
Swiss 3T3 cells, kuwanon H (2, 500 nmol/L) attenuated the bombesin-
222

induced increase in cytosolic free Ca^"^ concentration ([Ca^"^]!) by 60%,

but not bradykinin- or endothelin-1-induced increase in [Ca^"^]}, Fig. (18).

r\ t\
808
r

215 < \ A^ Y1
f t t t t t t t t t t
BK S BK V ET-1 S ET-l
V BOM S BOM

Fig. (18). Effect of kuwanon H (2) on agonist-induced increases in [Ca^^\ in Swiss 3T3 cells. Cells were
stimulated by 10"* mol/L bombesin (BOM), 10"* mol/L endothelin-1 (ET) or 10"* mol/L bradykinin (BK).
Kuwanon H (S, 500 nmol/L at the final concentration) or dimethyl sulfoxide (V) was added 1 min before
stimulation.

In Swiss 3T3 cells, GRP stimulates ["^H]thymidine incorporation in a

concentration-dependent manner. Kuwanon H (2) inhibited GRP-
induced DNA synthesis in Swiss 3T3 cells. The IC50 value was around
100 nmol/L, close to its K, value for [^^^I]GRP binding to Swiss 3T3 cells,
Fig. (19). Kuwanon H (2) demonstrated selectivity toward GRP, as
concentration of 10"^ mol/L uninfluenced basal and 5% serum-induced
[ HJthymidine incorporation. From above results, kuwanon H (2)
appears to be a selective antagonist for GRP-R.

B
o.
35000

B
o
25000

15000

-log (2) mol/L

Fig. (19). Dose-dependent effects of kuwanon H (2) on basal (o) and GRP (10" mol/L)-induced DNA
syntheses in Swiss 3T3 cells (•). Values are the mean ± S.E. for four determinations.

As bombesin family peptides are thought to be autocrine growth

factors for SCLC, the results described above suggested that kuwanon H
 223

(2) might be useful against SCLC. Unfortunately, however, kuwanon H

(2) had no effect on the growth of two human SCLC lines, Lu-134 and
NCI-HI 28.
At the time, kuwanon H (2) was the most potent of non-peptide
bombesin receptor antagonists (NPBRA) that had been reported. Its
affinity might be too low to determine whether the non-peptide antagonist
is effective against human lung cancers. However, kuwanon H (2), and
possibly kuwanon G (1) also, can serve as lead compounds for more
rational drug design in the synthesis of more potent antagonists.
Furthermore, these compounds may be useful tools on the study of
GRP-R. Recently, it was reported that NPBRA, PD 176252, with high
binding affinity which was developed via the application of a peptoid
drug design strategy [104].

VIL EFFECTS OF PHENOLS AGAINST BACILLUS SUBTILIS

(M45) (REC-ASSAY), HUMAN ORAL CELLS, AND
HIV-INFECTED MT-4 CELLS

Rec-assay was developed by Kada et al. for screenings chemical and

enveloped mutagens. Recombination less mutant strain of Bacillus
subtilis (M45) is more sensitive to the cell-killing action of chemical
mutagens, e.g., mytomycin C, A^-nitroso-A/-methylurethane, etc., than the
wild-type bacteria (HI7) [105]. This assay was also useful for pre-
screening of anticancer drugs, such as enediyne-family antibiotics [106].
For the constituents of plants, the assay was modified and used
exclusively for the detection of anti-mutagen compounds [107]. Since
the sensitivity of the rec-assay to chemicals having induction activity of
DNA damage is higher than from other screening technique, such as
Ames test, this method may be useful for pre-screening of anticancer
agents in crude drugs. Furthermore, the antibacterial compounds
against the wild-type strain (HI7) may be expected that these anti-
bacterial compounds have another bioactive potency. We tried the
application of the rec-assay (unmodified) for the detection of bioactive
phenolic compounds obtained from Glycyrrhiza species [51], and spore
rec-assay [108,109] was used for moraceous flavonoids as shown in
Table 7. Sixty-nine Glycyrrhiza phenols out of a total 108 compounds
showed inhibitory activity against the growth of both HI7 and M45
strains. Cytotoxic activities of these antibacterial compounds
{Glycyrrhiza phenols and moraceous phenols) against human oral
squamous cell carcinoma (HSC-2) and human T-lymphoblastoid cell line
MT-4 cells were also shown in Table 7 [110-113] along with other
biological activities reported until the middle of 2002. In the Table,
relatively strong-cytotoxic compounds against HSC-2 (CC5o<25 |ig/mL)
224

that showed no activity against these B. subtilis strains were also shown
in Table 7. On the comparison between these bioactivities of the
phenolic compounds, relationship between the antibacterial activity
against B, subtilis and cytotoxicity against HSC-2 or MT-4 cells was not
found.
Hatano et al. reported antibacterial effect of licorice flavonoids
against methicillin-resistant Staphylococcus aureus (MRSA) [114].
8-(y,Y-Di-methylallyl)-wighteone (58) and 3'-(Y,Y-dimethylallyl)-kievi-
tone (27) showed relatively strong activity against clinically isolated
MRSAs (MIC=8 lag/mL). Licochalcone A (59), gancaonin G (60),
glyasperins C (61) and D (62), glabridin (23), licoricidin (63),
licocoumarone (64), and isoangustone A (65), Fig. (20), showed slightly
weak activity against the bacteria (MIC=16 |ig/mL) [114].

8-(Y,Y-dimethylallyl)-wighteone (58):
Ri = R3 = R4 = H, R2 = CH2CH=CMe2
gancaonin G (60):
Ri = Me, R2 = R3 = R4 = H
isoangustone A (65):
Ri = R2 = H, R3 = 0H,
R4 =CH2CH=CMe2
licochalcone A (59)
^^^
i licocoumarone (64)
glyasperin C (61): Ri = R2 = H
glyasperin D (62): Ri = Me, R2 = H
licoricidin (63):
Ri = H, R2 = CH2CH=CMe2

Fig. (20). Structures of licorice flavonoids (58 - 65).

Table 6. Antimicrobial activity of licorice flavonoids (MIC, ng/mL)

MSSA MSSA MRSA MRSA M luteus E. coli
FDA 209P Smith K3 ST 28 ATCC9341 NIHJ JC-2
Glabridin (23) 12.5 12.5 12.5 12.5 12.5 >100
Glabrene (24) 12.5 12.5 12.5 12.5 25 >100
Licochalcone A (59) 3.13 6.25 6.25 6.25 6.25 >100
Licochalcone B (82) 25 100 100 100 >100 >100
Liquiritigenin (101) >100 >100 >100 >100 >100 >100
Liquiritin (113) >50 >50 >50 >50 >50 >50
Licoisoflavone B (67) 12.5 12.5 12.5 12.5 ND ND
Formononetin (116) >25 >25 >25 >25 >100 >100
Licoricidin (63) 3.13 3.13 6.25 6.25 6.25 >100
Glycyrol (76) >100 >100 >100 >100 >100 >100
Isoglycyrol (117) >25 >25 >25 >25 >100 >100
3-0-Methylglycyrol(118) >16 >16 >16 >16 >16 >16
Vestitol(119) >50 >50 >50 >50 >50 >50
Licoricone (120) 25 >50 >50 >50 >50 >50
Glycyrin (121), >50 >50 >50 >50 >50 >50
Isolicoflavonol (122) 12.5 12.5 25 25 25 >100
GancaonolB(123) >32 >32 >32 >32 >32 >32
Glyasperin D (62) 6.25 6.25 6.25 6.25 12.5 >50
Gancaonin I (126) 3.13 1.56 1.56 3.13 3.13 >50
AMOX 0.1-0.2 0.20 25-50 50 0.025 3.13
MSSA means methicillin-sensitive Staphylococcus aureus. M. and E. mean Micrococcus and Escherichia,
respectively. A positive control used was amoxicillin (AMOX). ND, not determined.
 225

We also screened anti-MRSA flavonoids [115] in the course of the

study of anti'Helicobacter pylori flavonoids from licorice described in
Chapter X. In our screening (Table 6), glabrene (24), licoisoflavone B
(67), and gancaonin I (126) also exhibited anti-MRSA effect. Among
these compounds, 23, 24, 27, 59, 61, 62, 63, 67, and 126 exhibited
relatively high anti-bacterial activities against B. subtilis (HI7), Table 7.

Rec-assay

Seven compounds, licoisoflavanone (66), licoisoflavone B (67),

semilicoisoflavone B (68), gancaonin C (69), isoliquiritigenin (70), 6-
prenyleriodictyol (71), and 8-prenyleriodictyol (72), Fig. (21), showed
positive results in the rec-assay. Isoliquiritigenin (70) was most potent
of the seven compounds (Table 7).
The simple chalcone 70 has distributed widely in Glycyrrhizin plants
as a minor constituent, and its glycosides are mainflavonoidsof licorice
as described in Chapter 1. Recently, Okuyama et al. reported inhibition
effect of the chalcone (70) on azoxymethane-induced murine colon
aberrant crypt foucus formation and carcinogenesis [116].

licoisoflavanone (66)

""OH

6-prenyleriodictyol (71): Ri = CH2CH=CMe2. R2 =H

8-prenyleriodictyol (72): Ri = H, R2 = CH2CH=CMe2
gancaonin C (69) isoliquiritigenin (70)

Fig. (21). Structures o f licorice flavonoids (66 - 72).

Anti-HIV activity

Anti-HIV activity of prenylflavones from mulberry tree, kuwanon H (2),

morusin (3) and its derivatives, was reported by Luo et al. [117]. We
studied the effect of Morus flavones on HIV-1 me infected MT-4 cells,
but no flavone showed anti-HIV activity in our screening system [111].
These discrepant results might be due to multiple acting sites of
226

flavonoids. We also screened anti-HIV flavonoids from moraceous

plants and Glycyrrhiza species, however, only two flavonoids from
Glycyrrhiza species and a 2-arylbenzofuran from Morus species showed
weak anti-HIV activity with selective index (SI=50% cytotoxic
concentration (CC50) for MT-4 cells / 50% effective concentration (EC50)
for HIV-infected MT-4cells): 3-hydroxyglabrol (26, SI=10), kumatake-
nin (73, SI=20), and moracin C (74, SI=12), Figs. (3) and (22), [111].
Manfredi et al. reported the isolation of an anti-HIV diprenylated
bibenzyl from G. lepidota, but its therapeutic index (TI=EC5o/CC5o) was
small [118]. McKee et al. investigated anti-HIV activity of isoprenoid-
substituted isoflavans from Erythrina lysistemon (Leguminosae). They
concluded that both a free 4'-hydroxy1 group and a lack of substituents at
positions C-5 and C-6 are necessary for even minimal in vitro anti-HIV
activity [52].

MeO.

OMe

norartocarpetin (83) wjghteone (84)

Fig. (22). Structures offlavonoids73 - 84 from Glycyrrhiza species and Moraceous plants.
 227

Cytotoxic activity against human oral squamous cell carcinoma

(HSC-2)

Most potent isoprenoid-substituted phenols against HSC-2 cells (CC5o<

10 |ig/mL) were gancaonin R (75), glycyrol (76), glyasperin A (77),
licoricidin (63), antiarone I (78), artonin E (7), broussoflavonols B (79)
and C (80), kazinol B (81), and morusin (3), Table 7. Structure-activity
relationship of isoprenoid-substituted phenols for the cytotoxic activity
against HSC-2 cells could not be clear. Nevertheless, most compounds
having higher cytotoxic activity have two sets of a hydrophilic group
(hydroxyl group) in the vicinity of hydrophobic group (isoprenoid group)
at two different sites on a same plain in the molecule. We also
investigated the cytotoxic activity of these compounds against normal
human gingival fibroblasts (HGF) and compared with activity against
HSC-2 cells. Licochalcone B (82) exhibited significant tumor
selectivity: index of tumor specificity (ITS=CC5o for HGF/CC50 for
HSC-2) was 42 [111]. Norartocarpetin (83) was also showed tumor
selectivity (ITS=11) [99]. ITSs of 23 compounds of 63 isoprenoid-
substituted phenols were 2'-^4 [110-112].
Apoptosis is a normal physiological process that occurs during
embryonic development as well as during the maintenance of tissue
homeostasis. It can be induced by a variety of treatments, such as UV
irradiation, cytotoxic chemotherapy, etc. Cells, which die by apoptosis
usually, suffer similar morphological change, including nuclear
condensation, cytoplasmic blebbing, and DNA fragmentation. Wang et
al. reported induction of apoptosis by apigenin (4',5,7-trihydroxyflavone)
and related flavonoids through cytochrom c release and activation
caspase-9 and caspase-3 in leukaemia HL-60 cells [119]. We found that
glabrol (25) and wighteone (84) induced intemucleosomal DNA
fragmentation in HL-60 cells, but not in HSC-2 cells [111]. This is
consistent with the report by Yanagisawa-Shirota et al.; induction of
intemucleosomal DNA fragmentation depends on the cell types, rather
than apoptosis inducers (ascorbic acid, H2O2, tumor necrosis factor,
etoposide, hyperthermia, and UV irradiation) [120].

VIII. ESTROGEN-LIKE ACTIVITY OF PHENOLS FROM THE

MORACEOUS PLANTS AND GLYCYRRHIZA SPECIES

Traditional Japanese women with their high soy intake, a rich source of
228

Table 7. Inhibitory activity against Bacillus subtilis HI7 and rec-assay (disk division method) of
isoprenoid-substituted phenols (75 |ig/disk), their cytotoxic activities against HSC-2 and MT-4 cells, and
other biological activities

Trivial name Inhibition Rec-assay* HSC-2^ MT-4'' other activity^

forHH" CC50 CC50
(Hg/mL) (Hg/mL)

(Licorice phenols/

Angustone B + + 14 6 AFE, FDC

Bavachalcone -H- - 12 2
(broussochalcone B)
Dehydroglyasperin C -H- - 31 10
3'-(y,y-Dimethylallyl)-
kievitone (27) ++ - 20 10 ABM
8-(Y,Y-Dimethylallyl)-
wighteone (58) + - 22 11 ABM, EBV, LAT
Edudiol +-I- ± ND ND
Echinatin _ ± 45 12 ABE
Formononetin (116) - - 20 100 ABH, APA, EBV, ODD
Gancaonin C (69) + + 125 9
Gancaonin E - - 13 55 ALR, 5LG
Gancaonin G (60) + - 19 28 ABM
Gancaonin H + - 97 14
Gancaonin I (126) ++ - 42 >50 ABH, ABM
Gancaonin O + - 20 55
Gancaonin P ND ND 14 22
Gancaonin Q - - 11 6
Gancaonin R (75) ++ ± 8 18 ALR, ICO, 5LG, NKA,
Gancaonin S -H- ± 10 2 IC0,5LG,NKA
Gancaonin U + ± 12 2 ALR, IOC, 5LG, NKA
Gancaonin V -H- - 34 10
Gancaonin Y + - ND ND
Glabranin ND ND 14 >100 AIA, IPP
(glabranine)
Glabrene (24) -H- ± 12 10 ABE, ABH, ABM, AFE,
AMA, AOA, EAA
Glabridin (23) +++ ± 13 4 ABE, ABH, ABM, AFE,
AOA, EAA, EAB, ETA,
ICO, IMI, PSO, SAE
Glabrol (25) 4-+ ± 18 >100 ABE
Glycycoumarin -f+ - 32 13 ABC, ABE, AOA, CPH
Glycyrin - - 14 11 ABC, ABE, ABH
Glycyrol (76) - - <4 14 ABC, 5LG
(neoglycyrol)
Glyasperin A (77) + - <8 53
Glyasperin B -H- - 46 9
Glyasperin C (61) -H- - 31 34 ABM
Glyasperin D (62) -H- ± 48 >10 ABM, ABH
Glyasperin J ++ ± ND ND
Glyasperin K + - ND ND
Glisoflavanone ++ - 21 46
Glyinflanin A ++ - ND ND
(glycyrdione A)
Glyinflanin B ++ - 31 27
Glyinflanin C ++ - 19 8
(glycyrdione C)
Hispaglabridin A (124) + - 14 >100 ABE, AOA, MOS
3-Hydroxyglabrol (26) -H- - 31 12 ABE
3-Hydroxy-
paratocharpin C^ + - 38 12 ALA
Isoderrone ++ - ND ND
Isoglycyrol(117) - - 16 >100 ABC, ALR
Isoliquiritigenin (70) ++ ++ 22 14 ABE, ATP, EAA, MCC,
MGA, TFV, UFI
 229

Table 7 (continued)

Kanzonol B (81) + ± ND ND SOA

Kanzonol G + - ND ND
Kanzonol H + - 46 >10
Kanzonol P + - ND ND
Kanzonol R ++ - ND ND
Kanzonol S -I-+ - ND ND
Kanzonol U + - ND ND
(glabrocoumarone A)
Kanzonol V + - ND ND
Kanzonol W + - ND ND
Kanzonol X -H- - ND ND
(tenuifolin B)
Kanzonol Y + - ND ND
Kumatakenin (73) — — 375 51 AFE, APV, AVC, SST,
TCD
Licochalcone A (59) -H- — 20 15 ABE, ABH, ABM, ALA,
ATP, CPH, LFP
Licochalcone B (82) - - 4 16 ABE, CPH, LFP
Licoflavonol -H- - 22 13 ABE
Licoisoflavanone (66) ++ + 72 40
Licoisoflavone A + - 55 21 AFE, AOA, TD
(phaseoluteone)
Licoisoflavone B (67) -H- + 43 7 ABM, AFE, TD
Licoricidin (63) -H- ~ 8 15 ABE, ABH, ABM, AOA,
5LG, NKA, LAT
Licoricone (120) + - 45 64
Licorisoflavan A - - 14 47 ABH, BDB
Medicarpin + - 45 53 AOA, FDC, QRI
1-Methoxyphaseollidin (125) -H- - 28 12 ABH, LAT
1 -Methoxyficifolinol + - 11 8
3-(9-Methylgancaonin P ++ - ND ND
4'-(9-Methylglabridin (123) -H- ± ND ND ABE, ABH, AOA, MOS
Naringenin + ± ND ND ARI
Paratocarpin L ++ - 24 14
(macarangaflavanone B)
Pinocembrin -I-+ ± 105 >100 AIA
6-PrenyleriodictyoF (71) ++ + ND ND
S-PrenyleriodictyoF (72) ++ + 35 60
6-Prenylnaringenin (90) ++ - 29 32
Semilicoisoflavone B (68) ++ + 78 22
Shinpterocarpin -H- - ND ND
Sigmoidin A ND ND 20 29 ABE, AFE, ESEC, IMS
Sigmoidin B (99) ++ - 43 26 ABE, AFE, ESEC, IMS
Topazolin + - 19 4
Wighteone (84) ++ - 20 12 ABE, AFE, CTK, FDC,
(erythrinin B) HPE
(moraceous phenols)*
Albanin D _ _ 17 >8
Albanol B (97) ND ND <8 9
Alvaxanthone ND ND 9 ND
Antiarone B (18) ++ - ND ND
Antiarone F ++ - 30 >100
Antiarone G ++ - 13 11
Antiarone H - - 20 >100
Antiarone I (78) -H- - <8 11
Antiarone J (19) ++ - ND ND CTC, CTL
Artobiloxanthone (8) +-I-+ + ND ND 5LG, TAR
(KB-l)
Artonin E (7) -hH- ± <8 4 CTC, CTL, 5LG, RAR
(KB 3)
Broussoflavonol B (79) ND ND <8 >100
Broussoflavonol C (80) - - <8 20
230

Table 7 (c ontinued)

Broussoflavonol E ND ND 12 10
Cudraphenone B ND ND 13 ND
Cudraphenone D ND ND 20
Cycloartobiloxanthone (9) -H- ± ND ND CTC, CTL, 5LG, TAR
Heterophyllin + - ND ND CTC, CTL, 5LG, TAR
Isoalvaxanthone ND ND 14 ND
Kazinol B (81) + - 19 12 ARP,ICO,5LG,NOP
Kazinol E (37) - - <8 9 AOA, ETA, TPA
Kazinol F (38) -H- - 10 9 ETA, TPA
Kazinol N (41) ++ ~ 20 10
Kuwanon C (42) -HH- - 15 44 ABE, AFE, ARP, CTM,
(mulberrin) 5LG, 12LG, NOP, SPB,
TPA
Kuwanon G (1) -H- - 54 66 BRA, FHA, FTB, HPA,
(albanin F, moracein B) ODC, PKC, TPA
Kuwanon M (30) - - 24 7 HPA, ODC, PKC, TPA
Kuwanon R + - ND ND
Moracin C (74) ND ND 17 19 AFA
Morusin (3) ++ - <8 9 ABE, AFE, ANC, AOA,
(mulberrochromene) ARI, ARP, ATP, CTM,
FEA, FHA, FTB, ICO,
5LG, NOP, ODC, PKC,
PSO, SPB, TPA, TAR
Mulberrofiiran B ND ND 23 ND
Mulberrofliran G (25) ND ND <8 2 ARI, FEA, FHA, FTB,
(Albanol A) ODC, PA, PKC, TPA,
Norartocarpetin (83) - - 45 15 ETA, ICO
Oxydihydromorusin (46) ++ + 12 >100 AVR, FEA, FHA, FTB,
(morusinol) SPB, TPA
Sanggenol C ND ND 25 >10
Sanggenol M ND ND 10 ND
Sanggenon A (4) ND ND 23 ND
Sanggenon B (45) -hH- ± 22 ND AFE, ABE, ICO, 5LG,
NOP, TPA
Sanggenon C (5) +++ ± 13 ND ABC, ABE, AFE, FHA,
FTB, HPA, PKC, TPA
Sanggenon M (100) ND ND 21 ND
Sorocein F ND ND 24 ND
"Diameter of inhibition zone (8 mm paper disk was used), +=less than 11 mm, -H-=between 11 and 18 mm,
-H-+=more than 18 mm. Diameter of inhibition zone for kanamicin (10 |ig/disk) is 22 mm.
* Difference in diameter of inhibition zone between Bacillus subtilis M45 (rec: - ) and HI 7 (rec: +), diameter
of inhibition zone on M45 minus that on HI7; -^diameters are same, ±=less than 2 mm, +=between 2 and 5
mm, -H-=more than 5 mm. A positive control is mitomycin C (0.75 ^ig/disk): the difference of inhibition zone
is 6 mm. Inhibition zones of a negative control (kanamicin) were same for the both strains.
^CCso of doxorubicin was 2 |ag/mL.
'^ 50% Cytotoxic concentration against human T-lymphoblastoid cell line MT-4 cells without HIV infection.
" ABC = antibacterial action against a cariogenic bacterium, Streptococcus mutans [121,122];
ABE = antibacterial effect [8,123-132];
ABH = antibacterial effect against Helicobacter pylori [see the text, 126,127],
ABM = antibacterial effect against MRSA [see the text, 114, 115,133];
AFA = anti-feedant activity against silkworm [134];
AFE = antifungal effect [112,134-140];
AIA = anti-inflammatory activity [141,142];
ALA = anti-Leishmania activity [143,144];
ALR = inhibition on aldose reductase [43];
AMA = anti-mutagenic activity [145];
ANE = anti-nociceptive effects in mice [146];
AOA = antioxidant activity [123,125,147-154];
APA = anti-protozoa activity [155];
APV = anti-picomavirus activity [156];
ARI = aromatase inhibitory activity [157];
ARP = inhibition of aggregation of rabbit platelets [158,159];
ATP = anti-tumor promoting activity [see the text, 160,161];
AVC = antiviral activity against Coxsackie virus [162];
 231

AVR = antiviral activity against rhinovirus type 2 [8];

BDB = benzodiazepin-binding stimulator [163];
BRA = bombesin receptor antagonist [see the text];
CPH = inhibition of the cytopathic activity of HIV [164];
CTC = cytotoxic activity against colon 38 [see the text];
CTK = cytotoxic activity against KB cells [165];
CTL = cytotoxic activity against L1210 [see the text, 166];
CTM = cytotoxic activity against mouse macrophage cell line (RAW 264.7) [167];
EAA = estrogen agonist activity [168];
EAB = estrogenic and anti-proliferate properties in human breast cancer cell [169];
EBV = inhibitory effect on TPA-induced Epstein-Barr virus early antigen [170];
ESEC = effect on sea-urchin egg cleavage [171];
ETA = effect on tyrosinase activity [153,172-174];
FDC = feeding deterrents for Costelytra zealandica (white) [135,175];
FEA = inhibition of formation of 12-HET from [l-'*C]arachidonic acid [see the text, 79,80];
FHA = inhibition of formation of 12-HHT from [l-^'^CJarachidonic acid [see the text, 79,80];
FTB = inhibition of formation of thromboxane Bi from [l-''*C]arachidonic acid [79,80];
HPA = hypotensive activity [see the text];
HFE = hepato-protective effect [176];
ICO = inhibition on cyclooxygenase [43,172,177,178];
IFF = inhibition of photo-phosphorylation [179];
IMI = inhibitory effect on melano-genesis and inflammation [172];
IMS = inhibition of macrophage superoxide production [180];
LAT = inhibitory effect on lysoFAF (platelet-activating factor) acetyltransferase [181];
LFF = effect on leukotriene formation in human polymorpho-nuclear neutrophils [182];
5LG = inhibition on 5-lipoxygenase [43,177],
12LG = inhibition on 12-lipoxygenase [177];
MCC = inhibition effect on murine colon carcinogenesis [116];
MGA = mutagenic activity [183];
MOS = protection of mitocondrial fractions against oxidative stresses [184];
NKA = inhibition on Na^ K^-ATPase [43];
NOP = inhibitory activity on NO production from lipopolysaccharide-induced nitric oxide (NO) production
from mouse macrophage cell line (RAW 264.7) [167];
ODC = inhibitory activity on induction of ODC [see the text];
ODD = reducing of endogenous oxidative DNA damage [185];
PKC = inhibition of activation of protein kinase C [see the text];
PSO == inhibitory effect on production of superoxide anions [172];
QRI = quinone reductase-inducing activity [186];
SAE = synergistic anti-oxidative effect with lycopene [187];
SPB = substrate for PCB-degrading bacterium, Burkholderia sp. [188];
SOA = stimulation of superoxide anion generation in rat neutrophils [189,190];
SST = suppression of SOS-inducing activity of Trp-P-l {umu test) [191];
TAR = inhibitory effect on TNF-a release [see the text];
TCD = inhibitory activity on TNF-a-induced cell death in mouse hepatocytes [192];
TFV = tube formation from vascular endothelial cells of rats [193];
TPA = inhibiting of ^H-TFA binding [see the text];
UFI = preventive effect on ulcer formation induced by severe necrotizing agents in rats [194].
/Eriodictyole, liquiritigenin (101), sophoraflavanone B (86, AFA), isobavachin (94), kanzonol Z, euchrenone
as, ovaliflavanone B, prenyllicoflavone A, glepidotin A, 3,3'-di-0-methylquercetin, 3,4'-di-0-methylquercetin,
paratocarpins B and C, glyinflanin G, and licuraside exhibited no effect against both M45 and HI7 strains.
^ No name: names in the table are tentative name used here.
* Antiarones A and K, brosimone L, cudraflavanone A, and cyclohetrophyllin (53) showed no effect against
both M45 and HI 7 strains. These data were obtained with spore rec-assay (38 ^ig of sample/disk).
ND means not determined. The structures of all phenolic compounds isolated from Gfycyrrhiza species until
1996 were reviewed in the reference [43], new compounds from the plants since the time to 2000 were
summarized in the proceeding [44].

plant-derived estrogens (phytoestrogens [195]), have a low incidence of

breast cancer and few menopausal symptoms. This has led to the
hypothesis that at the menopause phytoestrogens might act as natural
selective estrogen receptor modulators, tweaking estrogenic responses in
the cardiovascular system, bone, and brain, but dampening responses in
232

the breast and uterus. The finding that the soy-derived phytoestrogen
genistein (85), Fig. (23), preferentially binds to the form of the estrogen
receptor found mainly in the cardiovascular system lends some credence
to that belief [196]. It is expected by recent many evidence that phyto-
estrogens exert beneficent actions to chronic diseases, e.g., heartattacks
and other cardiovascular problems, osteoporosis, Alzheimer's disease, etc.
[197]. Nevertheless, the isoflavonoids and Ugnans bind w^ith low^
affinity to estrogen receptors, and thus, it is also suggested that they may
induce production of sex hormone binding globulin in the liver and in this
way influence sex hormone metabolism and biological effects [198].
Recently, Cooke et al. reported that genistein (85) decreased mouse
thymocyte numbers and doubled apoptosis, indicating that the mechanism
of the genistein effect on loss of thymocytes is caused in part by
increased apoptosis [199]. In addition, genistein (85) produced
suppression of humoral immunity. These data indicate that use of soy-
based infant formulas and soy/isoflavone supplements has aroused
concern: genistein (85) and daidzein (102) may be capable of producing
thymic and immune abnormalities. Therefore, the screening of phyto-
estrogen from medicinal plants may be important.

genistein ( 8 5 ) : R = OH
daidzein ( 1 0 2 ) : R = H OH 0
sophorafiavanone B ( 8 6 ) : R = OH licoflavone C (87): R^ = R2 = H
isobavachin ( 9 4 ) : R = H 8-prenylquercetin (88): R i = R2 = OH
noranhydroicaritin (93): Ri = OH, R2 = H

lupiwighteone (89)
6-prenylnaringenin ( 9 0 ) : R = H
17p-estradiol(92)
lonchocarpd A ( 9 1 ) : R = CH2CH=CMe2

Fig. (23). Structures of phytoestrogens (85, 87, 88, 93, and 102) and related compounds having no
estrogenic effect (89-91).

Numerous phytoestrogens with a diversity of structures have now been

recognized [168,169,200-210]. Akiyama et al. reported that
sophorafiavanone B (86), Fig. (23), isolated from a Thai crude drug,
Anaxagorea luzonensis (Annonaceae), showed about two-fold higher
affinity for the bovine uterine estrogen receptor than that of genistein (85),
Table 8 [197]. They also reported that synthetic 8-prenylated flavonoids,
licoflavone C (87) and 8-prenylquercetin (88), also exhibited the binding
 233

affinity but their binding affinities were weaker than that of 86. In the
report [197], it was also reported that a synthetic 6-prenylated isoflavone,
lupiwighteone (89), and 6-prenylated and 6,8-diprenylated flavanones, 6-
prenylnaringenin (90) and lonchocarpol A (91), Fig. (23), did not exhibit
the binding affinity against the receptor. This study indicated that the
position of the steric hydrophobic bulky group (prenyl group) is
important for the binding affinity of prenylated flavonoids.
We examined whether other types of phenols with two or more
aromatic rings bind to the estrogen receptor [45,211]. About one
hundred phenols with isoprenoid groups or without side chains from
moraceous plants and Glycyrrhiza species and synthetic flavonoids were
evaluated with the estradiol receptor-binding assay [204]. Among them,
13 compoimds exhibited weak binding affinities (1C50<1 |ig/mL) in
which three compounds were isolated from moraceous plants (95, 97, and
100), and six were constituents oi Glycyrrhiza species (24, 75, 76, 94, 99,
and 101), Fig. (24).

HOJ

tetrahydrogiabrene (96) * bH
alband B (97)

OH

sigmoidin B (99): Ri = OH, R2 = CH2CH=CMe2 sanggenon M (100)

8-geranylapigenin (98) liquiritigenin (101): Ri = R2 = H

Fig. (24). Estrogenic compounds from moraceous plants (95, 97, and 100) and Glycyrrhiza species (99 and
101) and synthetic estrogenic flavonoids with a isoprenoid group (96 and 98).

Relative binding affinities of these compounds against 17p-estradiol

(92) (RBA=[IC5o of 92 (nmol/L)] / [IC50 of test sample (nmol/L)]) values
are shown in Table 8. The affinity of gancaonin R (75) was stronger
than those of genistein (85, RBA=0.004) and daidzein (102,
RBA=0.00035) [205]. The affinities of the other 12 compounds were
similar to those of the isoflavones (85 and 102) in dietary foods.
Recently, Vaya et al. reported binding affinities of glabrene (24),
isoliquiritigenin (70), and glabridin (23) for human estrogen receptor
[168].
234

Table 8. Relative binding affinities of phenolic compounds for the bovine uterine estrogen receptor

Compound RBA Sources

Gancaonin R (75) 0.016 G. uralensis (aerial part)

Noranhydroicaritin (93) 0.0064 synthesis
8-Prenylquercetin (88) 0.0057 synthesis
Isobavachin (94) 0.0054 G. pallidiflora (root)
Isobavachalcone (95) 0.0045 M cathayana (root)
Glabrene (24) 0.0022 G. glabra (root)
Tetrahydroglabrene (96) 0.0016 synthesis
Glycyrol (76) 0.0012 G. uralensis (underground part), G. aspera (root)
Albanol B (97) 0.0011 M alba (root bark)
8-Geranylapigenin (98) 0.00095 synthesis
Sigmoidin B (99) 0.00077 G. uralensis (aerial part), G. eurycarpa (aerial part)
Sanggenon M (100) 0.00048 M cathayana (root bark)
Liquiritigenin (101) 0.00038 Glycyrrhiza species (root and aerial part)

Compound RBA Sources [ref.]

Sophoraflavanone B (86) 0.0071 Anaxagorea luzonensis [197]
Sophoraflavanone B (86) 0.0091 synthesis [197]
Licoflavone C (87) 0.0063 synthesis [197]
8-Prenylquercetin (88) 0.0015 synthesis [197]
Hinokiresinol 0.00083 Chamaecyparis obtusa [209]
Nyasol (c/5-hinokiresinol) 0.00017 Anemarrhena asphodeloides [209]
(3i?)-nyasol 0.0010 Anemarrhena asphodeloides [209]
(35)-nyasol 0.0067 Anemarrhena asphodeloides [209]
Neoflavone dimer T (0.028)* Pistacia chinensis [210]
Neoflavone dimer 2° (0.0028)* Pistacia chinensis [210]
Dihydrochalcone* 0.0010 Dracaena loureiri [204]
Homoisoflavone*" 0.0024 Draceana loureiri [204]
10-Hydroxycoronaridine (0.003)* Tabernaemontana penduliflora [208]
Coronaridin (0.0005)* (not reported) [208]

" 3,3"-Dimer of 3,4-dihydro-4-(4'-hydroxyphenyl)-7-hydroxycoumarin. * No data of the positive control (92)

was reported. * 4,4'-Dihydroxy-2,6-dimethoxydhihydrochalcone. *" 5,7-Dihydroxy-3-(4-hydroxybenzyl)-4-
chromone.

On the molecular modeling examination of gancaonin R (75), the

prenyl groups did not lie into the lipophilic pocket of estrogen receptor
that is illustrated in Fig. (27). These groups existed near the B and C
rings of 17p-estradiol (92) as shown in Fig. (25). On the molecular
models, the volume of 17p-estradiol (92) was 266.9 A^ (based on van der
Waals radius of atoms) and that of compound 75 was 374.5 A^, and the
distance between 3-0- and 17-0- of 92 was 10.9 A and 4'-0- and 3(5)-0-
of 75 was 11.0 A. The overlapping volume of these two models of 75
and 92 was 204.2 A l
We also studied the structure-activity relationships with computation
modeling of these estrogenic phenols (Table 8) and non-estrogenic
phenols [45]. For the examination of molecular modeling of the iso-
prenoid-substituted phenols, we made a space model of estrogen receptor
(SMER) with an imaginary compound (103), Fig. (26), that built up with
 235

estrogenic steroids (104-108) having higher binding affinities than that of

17p.estradiol(92)[212].

Fig. (25). Molecular models of gancaonin R (75, ball and stick) and 17p-estradiol (92, stick): overlay of B
ring of 75 and A ring of 92. These molecular models were minimized with KfM2, and then calculated with
Mopac6.

104
SMERmodd(103)

106 107 108

Fig. (26). Structures of an imaginary compound (103) that built up with estrogenic steroids (104 - 108)
having higher binding affmities than that of 17p-estradiol (92).

The SMER, Fig. (27), is similar model to estrogen receptor excluded

volume (RExV) postulated by Kym et al. [212]. The volume of the
SMER was 424.7 A^. Most flavonoid skeletons (A-C rings) lay into the
SMER, however, the prenyl (geranyl) groups existed out of the SMER as
shown in Fig. (27).
By our examination of the modeling as illustrated in Fig. (27), it was
indicated that orientations of these estrogenic compounds in tiie binding
site of the estrogen receptor relative to 17P-estradiol (92) depend on tiieir
236

SMER

A ring of 76 and 103

Prenyl group of 76 Lipophilic pocket

Fig. (27). Overlapping of the SMER and molecular model of glycyrol (76).

skeletal structures as shown in Fig. (28). The binding sites of 17p-

estradiol in estrogen receptor-a and -(5 have been determined by X-ray
crystallographic study [213,214]. In Fig. (28), the amino acid residues
beside the phenols mean hydrogen-bonding binding site of the estrogen
receptor-a [213]. The orientations of these prenylated phenols against
binding site of estrogen receptor-p [214] may be similar to against
estrogen receptor-a. Fig. (28). The binding sites of albanol B (97), a 2-
arylbenzofuran derivative w^ith three additional aromatic rings, in
estrogen receptor-a may be different from those of prenylated phenols.
Fig. (28): the binding sites of 97 may be similar to those of raloxifene.
Asp 351, Glu 353, Arg 394, and His 524 [213], but the binding of 97 is
not completely because of lacking of the binding to His 524, and the A, B,
and C rings of 97 may lie in the lipophilic pocket of the receptor as
shown in Fig. (29). A and E rings of sanggenon M (100) may also
locate in the lipophilic pocket. Fig. (29).
From the molecular modeling analyses of isoprenoid-substituted
phenols that did not showed binding affinity for the estrogen receptor, it
was indicated that the binding sites at C-3 and C-17 of 17p-estradiol are
rigid, but the lipophilic pocket near C-4~C-7 is flexible.

IX. AKll'HELICOBACTER PYLORI ACTIVITY OF LICORICE

FLAVONOIDS

Helicobacter pylori is a bacterium that lives in the human stomach and

duodenum. The bacterium is generally recognized as one of the
etiological agents of peptic ulcer. Therefore, it is generally accepted
that ulcer patients with H. pylori infection require treatment with
antimicrobial agents in addition to anti-secretory drugs, whether on first
 237

[Glu 3531
H----[His 524]
[Glu 353]

[H2O]-

IH2O] [Arg 394]

17p-estradioi(92)
[Arg394] isoflavones (Ri = O. A^-^ : 85 (R2 = OH), 102 (R2 = H)
lsoflav-3-ene (R, = R2 = H, A^"*): 24
Isoflavan (Ri = H2. R2 = H, no A^'^= ^•^): 96

[Glu 3531 H - - . [His 524]

....-[His 524]
[Glu 353].,

[H2O]'''
[H2O]-
[Arg 394]
dihydrostilbene :75
[Arg 394]
coumestan: 76

[Asp 351],^
[His 524]
[Glu 353]»

[H2O]- [Glu 353],

[Arg 394] [H2O]-'

[Arg 394]

flavones (R = H, A^^): 87,98

flavonols (R = OH. A^^): 88,93 albanol B (97)
flavanones (R = H, no A^'^): 86,94,99,101

[His 524]
[Glu 353]

[H2O]'' /
[Arg 394]
[Arg 394] isobavachalcone (95)
sanggenon M (100)

Fig. (28). Orientations that flavonoids and a dihydrostilbene (75) may adopt in the binding site of the
estrogen receptor relative to 17p-eatradiol (92): the amino acid residues mean binding site of estrogen
receptor-a. A-D mean the positions of the A-D rings of 17p-estradiol but not the rings of these phenols.

presentation with the illness or on recurrence [215]. On the other hand,

gastric cancer is one of the most frequent cancers on a worMwide and the
leading cause of death from cancer. Since the discovery of H. pylori
[216,217], an association between the bacterium and gastric cancer has
238

A ring

Ertng

(SMERandlOO)

(SMERand97)

Fig. (29). Overiapping of the SMER (stick) and molecular model of albanol B (97, ball and stick) and
sanggenon M (100, ball and stick).

been suspected. Descriptive epidemiological data indicate that gastric

cancer occxirs morefrequentlyin some populations that have higher rates
of H. pylori infection. Furtheraiore, rates of both K pylori infection
and gastric cancer are correlated inversely with socioeconomic status and
increase as a ftmction of age and/or intake of dietary salt [218,219].
Recently, Uemura et al. reported the first evidence of the association
between K pylori and gastric cancer with a long-term study [220].
They studied a large group of Japanese patients with duodenal ulcer,
gastric hyperplasia, or non-ulcer dyspepsia. During the study, gastric
cancer developed in 2.9% of the patients infected with K pylori but in
none of the uninfected patients.
However, most people with chronic H. pylori infection have no
symptoms of peptic ulcer or gastric cancer, which raises questions
regarding preventive agents against these diseases; infected individuals
without disease symptoms may be protected by anti-bacterial compounds
in the diet and/or medicinal plants used frequently [221-239]. Many
anti-//. pylori agents with a diversity of structures have been isolated
from plant sources [240-252]. However, their antibacterial activities
against the bacterium in stomach are unclear [253] as the bacterium in
the narrow interface between the gastric epithelial cell surface and the
overlying mucus gel [215,217]. Among these antibacterial compounds,
flavonoids could be expected to show anti-//. pylori effects in vivo,
because kaempferol (109) exhibited antibacterial action in K pylori-
infected Mongolian gerbils [243]. As the biological activities of
 239

flavonoids are generally weak, the phenolic compounds may act as

bacterial suppressors in the stomach.
Most elderly Japanese favor Kampo-medicines (traditional Chinese
medicines modified in Japan) rather than synthetic medicines.
Generally, traditional Chinese medicines consist of mixtures of crude
drugs and require extraction with boiling water for lengthy periods.
Sasaki et al. reported that aqueous solutions of some kinds of licorice
saponins solubilize water-insoluble substances such as a-tocopherol and
oleanolic acid [254]. The solubilizing effect of the saponins is expected
to result in licorice extract containing lipophilic compounds such as the
isoprenoid-substituted flavonoids. Licorice extract is frequently used in
Japanese over-the-counter (OTC) drugs that can be purchased without a
doctor's prescription, e.g. stomachic, cough medicines etc. [255].
Therefore, we studied anti-K pylori activities of the flavonoids from
licorice [256].
Licorice is for the most part derived from Glycyrrhiza glabra, G.
uralensis and G. inflata [43]. G. glabra is used worldwide but the other
two species are mainly consumed in Asian countries as described in
Chapter 1. The common constituents of these licorices are triterpenoid
saponins, glycyrrhizic acid (110) and licorice-saponin G2 (112), Fig. (30),
a flavanone, liquiritigenin (101), Fig. (24), its 4'-0-glucoside, liquiritin
(113), and an isoflavone, formononetin (116). These saponins (110 and
112) exhibited no anti-//. pylori activity and the aglycone of 110,
glycyrrhetic acid (111), showed weak activity (Table 9) as reported
previously [257]. The flavanone glucoside (113) also exhibited no
inhibitory activity against the bacterium, and its aglycone (101) showed
weak activity (Table 9). The main flavanone of the licorices is
compound 113 but the aglycone (101) is a minor component in these
plants. Kim et al. reported the anti-//. pylori activity of the metabolites
of poncirin (114) from Poncirus trifoliate (Rutaceae) by human intestinal
bacteria [246]. They speculated that one of the metabolites,
isosakuranetin (115), contributes to the prevention of gastritis to some
degree because of its anti-if. pylori activity (MIC=10 |ig/mL against H,
pylori ATCC 43504, 5x10^ cfu). In the case of licorice, the intestinal
bacteria would be expected to hydrolyze these glycosides (110 and 113).
However, it may be difficult that these hydrolysates (111 and 101) to be
transferred from the intestine to the stomach. Formononetin (116) is
frequently isolated from leguminous plants. The compound exhibited
weak anti-//. pylori activity when bacterial concentration was 2x10^ cfu.
The compound also showed weak antibacterial activity against a
clarithromycin (CLAR, a macrocyclic antibiotic)-resistant strain, GP98,
at higher bacterial concentration (2x10^ cfu) but not against
CLAR-sensitive strains (Table 9). Strain GP98 is also an amoxicillin
(AMOX, a penicillin class antibiotic)-resistant strain. The compound
240

may be a bacteriostatic agent for these CLAR (AMOX)-sensitive strains.

Nevertheless, the isoflavone (116) is a minor constituent of licorice.
These observations suggested that anti-//. pylori agents in licorice are
isoprenoid-substituted flavonoids

OOH H00<

= A

OH 0
kaempferoi (109)

glycyrrtiizic acid (110): Ri = A, R2 = CH3

glycyrrtietlc acid (111): Ri = H, R2 = CH3
licorice-saponin G2 (112): Ri = A, R2 = CH2OH

a OCHa

OH 0
OMe
liquiritin (113): R = p-D-gtucopyranosyl poncirin(114):
R = 2-0-a-L-rhamnopyranosyl-
p-D-glucopyranosyl
isosakuranetin (115): R = H

Fig. (30). Structures of compounds 103 - 109.

We selected eight flavonoids from licorices (G. glabra^ G, inflata,

and G, uralensis) to test for anti-//. pylori activity (Table 9). A
pyranoisoflavan, glabridin (23), and a pyranoisoflav-3-ene, glabrene (24),
are characteristic flavonoids of European G. glabra as described in
Chapter 1. These compounds exhibited weak anti-//. pylori activity
against these four strains. The characteristic flavonoids of G. inflata are
licochalcones A (59) and B (82). Compound 82 did not exhibit anti-^.
pylori activity. However, compound 59 showed weak bioactivity. The
characteristic flavonoids of G. uralensis are an isoflavan with two prenyl
groups, licoricidin (63), a prenylated coumestan, glycyrol (76), a
coumestan with a dihydropyran ring, isoglycyrol (117), and a pyrano-
isoflavone, licoisoflavone B (67). These compounds were also isolated
from G aspera but this licorice is commercially unimportant because of
its small plant size [43]. No anti-//. pylori activity of the coumestans
(76 and 117) could be detected against the strains examined, but the
isoflavan (63) and the isoflavone (67) exhibited antibacterial activity.
The inhibitory activity of 67 against the growth of CLAR and AMOX-
resistant strain GP98 is of considerable value: its minimum inhibitory
concentration (MIC) was 3.13 |ig/mL against 2x10^ cfu of this strain.
Nevertheless, this compound is a minor component of licorice, and thus
 241

the main anti-//. pylori agent of G. uralensis may be licoricidin (63).

Tabic 9. AnXi'Helicobacter pylori activities (MIC, ng/mL) of the characteristic compounds of licorices
(Glycyrrhiza glabra, G. inflata, and G. uralensis)

ATCC 43504 ATCC 43526 ZLM 1007 GP98 (cfii)'* source

Glycyrrhizic acid (110) >100 >100 >100 >100 (a)

>100 >100 >100 >100 (b)
Glycyrrhetic acid (111) 50 50 50 50 (a)
25 25 25 25 (b)
Licorice-saponin G2 (112) >100 >100 >100 >100 (a)
>100 >100 >100 >100 (b)
Liquiritigenin (101) 50 50 50 50 (a)
50 50 50 50 (b)
Liquiritin (113) >50 >50 >50 >50 (a)
>50 >50 >50 >50 (b)
Formononetin (116) >100 >100 >100 12.5 (a)
12.5 12.5 12.5 12.5 (b)
Glabridin (23) 12.5 12.5 25 12.5 (a) G. glabra
12.5 12.5 12.5 12.5 (b)
Glabrene (24) 12.5 12.5 12.5 12.5 (a) G. glabra
12.5 12.5 12.5 12.5 (b)
Licochalcone A (59) 25 25 25 25 (a) G. inflata
25 25 12.5 12.5 (b)
Licochalcone B (82) >50 >50 >50 >50 (a) G. inflata
>50 >50 >50 >50 (b)
Licoricidin (63) 12.5 12.5 6.25 12.5 (a) G. uralensis
12.5 12.5 6.25 6.25 (b)
Licoisoflavone B (67) 6.25 6.25 6.25 6.25 (a) G. uralensis
6.25 6.25 6.25 3.13 (b)
Glycyrol (76) >50 >50 >50 >50 (a) G. uralensis
>50 >50 >50 >50 (b)
Isoglycyrol (117) >100 >100 >100 >100 (a) G. uralensis
>100 >100 >100 >100 (b)
AMOX** 0.05 0.05 0.05 0.20 (a)
0.025 0.025 0.025; 0.10 (b)
* (a): 2x10^ colony forming units (=cfti), (b): 2x10^ cfii. ** Positive control; amoxicillin (=AMOX).
ATCC 43504, ATCC 43526, and ZLM 1007 are CLAR-sensitive strains.

Next, we attempted to isolate further flavonoids exhibiting anti-^.

pylori activity from the extract of G. uralensis. In 1967, Takagi and
Ishii reported that one of the flavonoid-rich fractions of G. uralensis
(FMIOO), which also included about 15% glycyrrhizic acid (110), is
effective in prevention of digestive gastric ulcer by suppressing gastric
secretion [258,259]. The fraction was developed as an anti-ulcer drug
and ten similar medicines containing licorice extract have been also
supplied as prescribed drugs for treatment of gastric ulcer, duodenal ulcer,
and gastritis [255]. Our study of FMIOO showed that the medicine
exhibited anti-/f. pylori activity but did not contain licoricidin (63),
which is the main isoprenoid-substituted flavonoid in G. uralensis
[259,260] and exhibited anti-//. pylori activity as described above. The
other antibacterial agent 67 was not detected in FMIOO on TLC analysis.
The above investigations indicated strongly that licorice extract contains
some anti-//. pylori flavonoids.
242

H3CO.

3-0-methylglycyrol (118)

OH 0

glycyrin (121) isdicofiavonol (122)

6,8-diprenylorobol (124) 1-methoxyphaseollidin (125)

HO^ ^..^ ^ 0 ^

OH O _ . ^ ^
0CH3

gancaonin I (126) gancaond C (127) dihydroisoflavone A (128)

OCH3

4'-0-methylglabriclin (129) hispagiabridin A (130) shinflavanone(131)

Fig. (31). Structures of compounds 117-128 isolated from the active fractions of the methanol extract of
G. uralensis and compounds 129 -131 from the dichloromethane extract of G. glabra (Russian licorice).

The isolation of flavonoids from the methanol extract of G. uralensis

was carried out under non-basic conditions, because some flavonoids
isomerize under basic conditions, e.g. racemization of flavanones and
isoflavanones, ring-open reaction of flavanones etc. Bioactive fractions
were separated by some chromatographic methods and each step was
monitored with anti-//. pylori activity with the paper disk method.
Eighteen compounds were isolated from these bioactive fractions and
 243

their anti-H. pylori activities were shown in Table 10. The MICs of the
growth of H. pylori of vestitol (119), licoricone (120), 1-methoxy-
phaseollidin (125), and gancaonol C (127), Fig. (31), were similar to that
of licoricidin (63). The activities of the other flavonoids were weak and
similar to those of glycyrrhetic acid (111) and liquiritigenin (101). All
the compounds investigated here had weaker anti-//. pylori activity;
however, these compounds may be chemopreventive agent agents the H.
pylori infection. Furthermore, these compounds may be bacteriostatic
agents for the bacteria in the stomach and prevent peptic ulcer or gastric
cancer disease in H. pylori-mfQCtcd people. However, further pharma-
cological and clinical studies including the antibacterial effect in liquid
medium are required for confirmation of this hypothesis.
Imakiire et al. also reported antibacterial activities of compound 23,
4'-0-methylglabridin (129), hispaglabridin A (130), glabrol (25) and
shinflavanone (131), Fig. (31), from the lipophilic extract of Russian
licorice, G. glabra; Maruzen P-TH® that is a material of medicines and
cosmetics [126,127].

Table 10. Anti-Helicobacter pylori activities (MIC, |ig/mL) of the flavonoids from Glycyrrhiza uralensis

ATCC 43504 ATCC 43526 ZLM 1007 ZLM 1200 GP98 (cfu)*

Glyasperin D (62) 25 25 12.5 25 12.5 (a)

25 25 12.5 25 6.25 (b)
3-0-Methylglycyrol (118) >16 >16 >16 >16 >16 (a)
>16 >16 >16 >16 >16 (b)
Vestitol (119) 12.5 12.5 12.5 12.5 12.5 (a)
12.5 12.5 12.5 12.5 6.25 (b)
Licoricone (120) 12.5 12.5 12.5 25 12.5 (a)
12.5 12.5 12.5 12.5 12.5 (b)
Glycyrin (121) 50 50 50 50 25 (a)
50 50 25 50 25 (b)
Isolicoflavonol (122) 50 25 25 50 25 (a)
25 25 25 25 12.5 (b)
Gancaonol B (123) >32 32 32 32 16 (a)
32 16 32 16 16 (b)
6,8-Diprenylorobol (124) >50 >50 50 >50 50 (a)
50 50 50 50 50 (b)
l-MethoxyphaseoUidin (125) 16 16 16 16 16 (a)
16 8 16 16 8 (b)
Gancaonin I (126) 50 50 50 >50 50 (a)
50 50 50 50 50 (b)
Gancaonol C (127) 16 16 32 32 16 (a)
16 8 16 16 16 (b)
Dihydrolicoiso- >25 >25 25 >25 25 (a)
flavoneA(128)^ >25 >25 25 >25 25 (b)
CLAR** 0.025 0.0125 < 0.0063 0.0125 50 (a)
0.0125 < 0.0063 < 0.0063 < 0.0063 12.5 (b)
AMOX** 0.05 0.05 0.05 0.025 0.2 (a)
0.025 0.025 0.025 0.125 0.1 (b)

* (a): 2x10^ cfu, (b): 2x10^ cfu. ^ Tentative name used here.
** Positive control; clarithromycin (=CLAR) and amoxicillin (=AMOX).
244

Protonpump inhibitor-based triple therapy is now the most

commonly accepted eradication regimen for peptic ulcer patients with H.
pylori infection. However, CLAR resistance is an increasing problem
as its use has become more common in recent years [234,261]. It is
interesting that licorice flavonoids exhibited anti-//. pylori activity
against not only CLAR and AMOX-sensitive strains but also CLAR and
AMOX-resistant strain CP98: Although licorice has been used as a crude
drug in Japan from more than 1200 years [262], these strains have not
developed resistance to the licorice flavonoids. These compounds may
be useful as lead compounds in the development of a new class of anti-//.
pylori agents.

X. EFFECTS OF ISOPRENYLATED FLAVONOIDS FROM

MORUS SPECIES ON TESTOSTERONE 5a-REDUCTASE

In Japan, the extracts of mulberry tree have been used for promotion of
hair growth and prevention of baldness [263]. Testosterone
5a-reductase catalyses the reduction of testosterone to its active form,
5a-dihydrotestosterone (5a-DHT). 5a-DHT has been implicated in
certain androgen-dependent conditions such as benign prostatic
hyperplasia, acne, and male pattern boldness [264]. And 5a-reductase
activity is high in situ. Inhibitions of 5a-reductase may be usefuU for
the treatment of these diseases. Therefore we studied on 5a-reductase
inhibitory activity of some isoprenylated flavonoids isolated from the
root bark of Japanese mulberry tree [265]. Table 11 shows the
5a-reductase inhibitory activity of flavonoids isolated from the root bark
of Morus species. Most of the flavonoids had inhibitory activities
against 5a-redactase, and showed the activity in the range of 10^ - lO"''
mol/L. Kuwanon E (43) had the most potent activity of these
compounds and its IC50 value is 6.9x10"^ mol/L, while kuwanon G (1)
had no effect at 10"^ mol/L. Fig. (32) shows the effects of kuwanon E
(43) on the Lineweaver-Burk plots of rat prostate 5a-reductase activity
using testosterone as a substrate. The addition of 3x10"^ mol/L
kuwanon E (43) produced a parallel shift indicating un-competitive
inhibitor. And the apparent K\ value is 7.6x 10~^ mol/L.
Enzyme kinetic studies of inhibitor are very important for
considering as a therapeutic agent. It is interesting to note that
isoprenoid-substituted flavonoids having non-steroidal structures are
potent un-competitive inhibitors of 5a-reductase. So, it would be
expected that the isoprenoid-substituted flavonoid derivertive would be
an interesting lead compounds for testosterone 5a-reductase inhibitor.
 245

Table 11. Effects of Morus flavonoids on testosterone 5a-reductase

Inhibition (%)* IC50 (mol/L)

Morusin (3) 59.6

Oxydihydromorusin (46) 35.6
Kuwanon C (42) 63.0 8.2x10"^
Kuwanon E (43) 94.0 6.9x10"'
Kuwanon G (1) 0
Kuwanon H (2) 100 1.8x10"^
Kuwanon L (44) 53.0 4.4x10"^
Mulberrofiiran A (47) 24.2
Mulberrofuran G (30) 37.0
' Final concentration at 100 |imol/L.

lA'estosterone (1/10^ mol/L)

Fig. (32). Lineweaver-Burk plots of inhibition of prostatic 5a-reductase by kuwanon E (43). The assay
was carried out at varied concentration of [4-*'*C]testosterone in the absence (o) or in the presence of 0.3
Hmol/L kuwanon E (•).

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