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
morusin (3) : R = H artonin E (7): R = OH
sanggenon A (4): Ri = CH2CH=CMe2. R2 = OH (4'): Ri = OH, R2 = CH2CH=CMe2
kuwanonG(1):R = H kuwanon H (2): R = CH2CH=CMe2
sanggenon C (5): Ri = CH2CH=CMe2, R2 = OH (5'): Ri = OH, R2 = CH2CH=CMe2
OH
sanggenon 0 (6)
OH O
artobiloxanthone (8)
OH 0
cycloartobiloxanthone (9)
OH
brosimoneA(13) soroceal (15)
Fig. (1). Structures of compounds 1 -15 from moraceous plants.
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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
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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].
sorocenol B (16)
OH 0
antiarone A (17) antiarone B (18)
OH O
antiarone E (21)
Fig. (2). Structures of compounds 1 6 - 2 2 from moraceous plants.
antiarone J (19): Ri = OH. R2 = CH2CH=CMe2 antiarone K (20): Ri = OMe, R2 = H
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 depigmentation agent in cosmetic, and as pharmaceutical products, e.g., anti-ulcer.
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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): R = OH
3*-(Y,y-dimethylallyl)-kievitone (27)
Fig. (3). Structures of compounds 23 - 26 from licorice {Glycyrrhiza glabra).
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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
Benzene
Residue
I Methanol
Residue -T
Extract
extract Ethyl acetate soluble portion
I C.C, p. TLC, p. HPLC
Extract
C.C, p. TLC
Morusin (3), kuwanons C (42), D, E (43), F, oxydihydromorusin (46), 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
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mulberrofuran C (28): R = H chalcomoracin (32): R = CH2CH=CMe2
mulberrofuran F (29): R = CH2CH=CMe2 mulberrofuran G (30): R = H
kuwanon E (43) sanggenon B (45)
Fig. (6). Structures of flavonoids (28 - 44) from moraceous plants.
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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
•OOH 34
Fig. (7). Reaction mechanism of photo-oxidative cyclization of morusin (3).
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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
Morusin (3) Kuwanon G (1) Kuwanon H (2) Kuwanon M (35) Mulberrofuran G (30) Sanggenon A (4) Sanggenon C (5) Sanggenon D (33)
Inhibiting of specific [^H]TPA binding (ED50 jimol/L)
57 99
100 85 34 62 48 60
Inhibition of activation of protein kinase C (ED50 fimol/L)
80 40 80 22 46 80 46 42
Inhibition of ODC induction (%)
43 34
-35 25 10
-62 -17
17
^ o
o
100
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.
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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.
o
100
50
a 0.
VA
VA 10- \o- 10-*
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 Weeks of promotion
10 20 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 antitumor 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) Kazinol E (37) Kazinol F (38) Kazinol J (39) Kazinol M (40) Kazinol N (41) Kuwanon C (42) Kuwanon E (43)
80 70 98 90
100 >100
80 83
Kuwanon L (44) Sanggenon B(45) Oxydihydromorusin (46) Mulberroftiran A (47) Ikarisoside A (48) Ikarisoside B (49) Quercetin (50)
80 95 95
>100 >100 >100 >100
oxydihydromorusin (46)
OMe
mulberrofuran A (47)
ikarisoside A (48): R = Rha ikarisoside B (49): R = Glu(1 2)Rha
OH O
quercetin (50): Ri = OH,R2=R3 = H cirsilioi (51): Ri = H, R2 = 0Me, R3 = Me artonin H (56)
OCH3
antiarone L (57)
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 biosynthesis 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.
j Cycloartobiloxanthone (9) Heterophyllin (52) Cycloheterophyllin (53) Artonin A (54) Artonin B (55)
2.9 ± 0.4 0.36 db 0.03 0.55 db 0.20 1.3 ±0.2 0.73 ±0.21 ' 1.6±1.0 4.3 ± 0.5 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 the flavone skeleton.
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].
artonin E (7)
hv, 24 h. CHCI3 DPPH, 24 h, CHCI3 (in the dark)
(±)-artobiloxanthone (8)
8 9
OH 0 (±) -cycloartobitoxanthone (9)
34% 70%
3% 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 (±) -cycloartobiloxanthone (9)
OH 0
(±)-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.
okadac acid
TNF-a - • ~ 3
—1 protein i ^ j — ' phosphatase 1
p V _
phosphorylated t proteins 1
phosporylated t proteins ' _
{ jene expression - c-fos
ojun NF-KB
ODC
TNF-a — •
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
Artonin E (7) 0.43 Cycloartobiloxanthone (9) 1.94 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 cycloheterophyllin (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
Artonin A (54) Artonin B (55) Artonin E (7) Artonin H (56) Heterophyllin (52)
L-1210
8.8 23 2.2 8.8 2.3
Colon 38
14.3 1.4 1.9 3.5 1.3
Cycloheterophyllin (53) Antiarone J (19) Antiarone K (20) Antiarone L (57) TFFU*
L-1210
4.7 77.0 81.3 80.4
2.9
Colon 38
4.6 70.4 46.3
>100
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 underground 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).
808
215
r\
< \
r
A^
t\
Y1 f t t t V BOM S BOM
t BK
t t S BK
t t t t V ET-1 S ET-l
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.
B o
35000
25000
15000
-log (2) mol/L
Fig. (19). Dose-dependent effects of kuwanon H (2) on basal (o) and GRP (10" syntheses in Swiss 3T3 cells (•). Values are the mean ± S.E. for four determinations.
mol/L)-induced DNA
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 antibacterial 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): licochalcone A (59) 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 i licocoumarone (64)
Fig. (20). Structures of licorice flavonoids (58 - 65).
glyasperin C (61): Ri = R2 = H glyasperin D (62): Ri = Me, R2 = H licoricidin (63): Ri = H, R2 = CH2CH=CMe2
Table 6. Antimicrobial activity of licorice flavonoids (MIC, ng/mL)
Glabridin (23) Glabrene (24) Licochalcone A (59) Licochalcone B (82) Liquiritigenin (101) Liquiritin (113) Licoisoflavone B (67) Formononetin (116) Licoricidin (63) Glycyrol (76) Isoglycyrol (117) 3-0-Methylglycyrol(118) Vestitol(119) Licoricone (120) Glycyrin (121), Isolicoflavonol (122) GancaonolB(123) Glyasperin D (62) Gancaonin I (126) AMOX
MSSA FDA 209P
12.5 12.5 3.13
25 >100 >50
12.5 >25
3.13 >100
>25 >16 >50
25 >50
12.5 >32
6.25 3.13
0.1-0.2
MSSA Smith
12.5 12.5 6.25
100 >100 >50
12.5 >25
3.13 >100 >25 >16 >50 >50 >50
12.5 >32
6.25 1.56 0.20
MRSA K3
12.5 12.5 6.25
100 >100 >50
12.5 >25
6.25 >100 >25 >16 >50 >50 >50
25 >32
6.25 1.56
2 5 - 5 0
MRSA ST 28
12.5 12.5 6.25
100 >100 >50
12.5 >25
6.25 >100 >25 >16 >50 >50 >50
25 >32
6.25 3.13
50
M luteus ATCC9341
12.5 25
6.25 >100 >100
>50 ND
>100 6.25
>100 >100 >16 >50 >50 >50
25 >32
12.5 3.13 0.025
E. coli NIHJ JC-2
>100 >100 >100 >100 >100 >50 ND
>100 >100 >100 >100 >16 >50 >50 >50
>100 >32 >50 >50
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 main flavonoids of 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
gancaonin C (69) isoliquiritigenin (70)
Fig. (21). Structures of licorice flavonoids (66 - 72).
6-prenyleriodictyol (71): Ri = CH2CH=CMe2. R2 =H 8-prenyleriodictyol (72): Ri = H, R2 = CH2CH=CMe2
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'-hydroxy 1 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 of flavonoids 73 - 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
(Licorice phenols/
Angustone B Bavachalcone (broussochalcone B) Dehydroglyasperin C 3'-(y,y-Dimethylallyl)-kievitone (27) 8-(Y,Y-Dimethylallyl)-wighteone (58) Edudiol Echinatin Formononetin (116) Gancaonin C (69) Gancaonin E Gancaonin G (60) Gancaonin H Gancaonin I (126) Gancaonin O Gancaonin P Gancaonin Q Gancaonin R (75) Gancaonin S Gancaonin U Gancaonin V Gancaonin Y Glabranin (glabranine) Glabrene (24)
Glabridin (23)
Glabrol (25) Glycycoumarin Glycyrin Glycyrol (76) (neoglycyrol) Glyasperin A (77) Glyasperin B Glyasperin C (61) Glyasperin D (62) Glyasperin J Glyasperin K Glisoflavanone Glyinflanin A (glycyrdione A) Glyinflanin B Glyinflanin C (glycyrdione C) Hispaglabridin A (124) 3-Hydroxyglabrol (26) 3-Hydroxy-paratocharpin C Isoderrone Isoglycyrol(117) Isoliquiritigenin (70)
Kanzonol B (81) Kanzonol G Kanzonol H Kanzonol P Kanzonol R Kanzonol S Kanzonol U (glabrocoumarone A) Kanzonol V Kanzonol W Kanzonol X (tenuifolin B) Kanzonol Y Kumatakenin (73)
Licochalcone A (59)
Licochalcone B (82) Licoflavonol Licoisoflavanone (66) Licoisoflavone A (phaseoluteone) Licoisoflavone B (67) Licoricidin (63)
Licoricone (120) Licorisoflavan A Medicarpin 1-Methoxyphaseollidin (125) 1 -Methoxyficifolinol 3-(9-Methylgancaonin P 4'-(9-Methylglabridin (123) Naringenin Paratocarpin L (macarangaflavanone B) Pinocembrin 6-PrenyleriodictyoF (71) S-PrenyleriodictyoF (72) 6-Prenylnaringenin (90) Semilicoisoflavone B (68) Shinpterocarpin Sigmoidin A Sigmoidin B (99) Topazolin Wighteone (84) (erythrinin B)
(moraceous phenols)*
Albanin D Albanol B (97) Alvaxanthone Antiarone B (18) Antiarone F Antiarone G Antiarone H Antiarone I (78) Antiarone J (19) Artobiloxanthone (8) (KB-l) Artonin E (7) (KB 3) Broussoflavonol B (79) Broussoflavonol C (80)
Broussoflavonol E Cudraphenone B Cudraphenone D Cycloartobiloxanthone (9) Heterophyllin Isoalvaxanthone Kazinol B (81) Kazinol E (37) Kazinol F (38) Kazinol N (41) Kuwanon C (42) (mulberrin)
Kuwanon G (1) (albanin F, moracein B) Kuwanon M (30) Kuwanon R Moracin C (74) Morusin (3) (mulberrochromene)
Mulberrofiiran B Mulberrofliran G (25) (Albanol A) Norartocarpetin (83) Oxydihydromorusin (46) (morusinol) Sanggenol C Sanggenol M Sanggenon A (4) Sanggenon B (45)
"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 phytoestrogens 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 phytoestrogen from medicinal plants may be important.
genistein (85): R = OH daidzein (102): R = H
sophorafiavanone B (86): R = OH isobavachin (94): R = H
OH 0
licoflavone C (87): R = R2 = H 8-prenylquercetin (88): Ri = R2 = OH noranhydroicaritin (93): Ri = OH, R2 = H
lupiwighteone (89) 6-prenylnaringenin (90): R = H lonchocarpd A (91): R = CH2CH=CMe2 17p-estradiol(92)
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)
8-geranylapigenin (98) sigmoidin B (99): Ri = OH, R2 = CH2CH=CMe2 liquiritigenin (101): Ri = R2 = H
OH
sanggenon M (100)
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
Gancaonin R (75) Noranhydroicaritin (93) 8-Prenylquercetin (88) Isobavachin (94) Isobavachalcone (95) Glabrene (24) Tetrahydroglabrene (96) Glycyrol (76) Albanol B (97) 8-Geranylapigenin (98) Sigmoidin B (99) Sanggenon M (100) Liquiritigenin (101)
Compound
Sophoraflavanone B (86) Sophoraflavanone B (86) Licoflavone C (87) 8-Prenylquercetin (88) Hinokiresinol Nyasol (c/5-hinokiresinol) (3i?)-nyasol (35)-nyasol Neoflavone dimer T Neoflavone dimer 2° Dihydrochalcone* Homoisoflavone*" 10-Hydroxycoronaridine Coronaridin
G. uralensis (aerial part) synthesis synthesis G. pallidiflora (root) M cathayana (root) G. glabra (root) synthesis G. uralensis (underground part), G. aspera (root) M alba (root bark) synthesis G. uralensis (aerial part), G. eurycarpa (aerial part) M cathayana (root bark) Glycyrrhiza species (root and aerial part)
" 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 Al
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
A ring of 76 and 103
Prenyl group of 76
SMER
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 353] H----[His 524] [Glu 3531
[H2O]-
IH2O]
[Arg394] 17p-estradioi(92)
[Arg 394]
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
[H2O]'''
H - - . [His 524]
[Arg 394]
dihydrostilbene :75
[Glu 353].,
[H2O]-
[Arg 394]
....-[His 524]
coumestan: 76
[Glu 353]»
[H2O]-
[His 524]
[Arg 394]
flavones (R = H, A^^): 87,98
flavonols (R = OH. A^^): 88,93
flavanones (R = H, no A^'^): 86,94,99,101
[Asp 351],^
[Glu 353],
[H2O]-'
[Arg 394]
albanol B (97)
[Glu 353]
[H2O]'' /
[Arg 394] isobavachalcone (95)
[His 524]
[Arg 394]
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 more frequently in 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
OH 0
kaempferoi (109)
liquiritin (113): R = p-D-gtucopyranosyl
OOH H00<
= A
glycyrrtiizic acid (110): Ri = A, R2 = CH3 glycyrrtietlc acid (111): Ri = H, R2 = CH3 licorice-saponin G2 (112): Ri = A, R2 = CH2OH
OH 0
aOCHa
poncirin(114): R = 2-0-a-L-rhamnopyranosyl-
p-D-glucopyranosyl isosakuranetin (115): R = H
OMe
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)
* (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.
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
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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 pharmacological 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
* (a): 2x10^ cfu, (b): 2x10^ cfu. ^ Tentative name used here. ** Positive control; clarithromycin (=CLAR) and amoxicillin (=AMOX).
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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
Morusin (3) Oxydihydromorusin (46) Kuwanon C (42) Kuwanon E (43) Kuwanon G (1) Kuwanon H (2) Kuwanon L (44) Mulberrofiiran A (47) Mulberrofuran G (30)
Inhibition (%)*
59.6 35.6 63.0 94.0 0
100 53.0 24.2 37.0
IC50 (mol/L)
8.2x10"^ 6.9x10"'
1.8x10"^ 4.4x10"^
' 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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