137
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity of Polymorphonuclear Leucocytes Kaoru Kinoshita , Kaoru Morikawa2, Masahiko Fujita2, and Shinsaku Natori " 1 Meiji College of Pharmacy, Yato-cho, Tanashi-shi, Tokyo 188, Japan
National Institute of Public Health, Shirokanedai-4-chome, Minato-ku, Tokyo 108, Japan Address for correspondence Received: March 5, 1991
The inhibitory effects of 151 natural prod-
ucts, representing most of the frequently occurring types, on the cytotoxicity to MM2 tumor cells of polymorphonuclear leucocytes (PMN) induced by TAK, a polysac-
charide immunomodulator, were examined. Forty-two compounds inhibited the TAK-induced activation of PMN. Among them, some naturally occurring quinones and various alkaloids (nicotine, Cinchona alkaloids, isoquinoline alkaloids such as cepharanthine, and indole alkaloids such as ajmaline) exhibited potent inhibitory effects. Using the inhibition assay for monitoring, the extracts of Hydrangea Dulcis folium, Scopoliae rhizoma, Cinchona cortex, Magnoliae cortex, Stephania tuber, and Rauwolfia radix were analysed to characterize the active constituents. Key words
Cytotoxicity, inhibition of TAK-induced activation, polymorphonuclear leucocytes, secondary metabolites, quinones, isoquinoline alkaloids, indole alkaloids.
dered to exhibit anti-inflammatory effects. In this study, we investigated the effects of natural products or herbal drug constituents on the PMN activation by TAK using a PMN cytotoxicity assay system. The results for 151 compounds, covering most of the frequently occurring types of natural products, are presented in this paper. Compounds such as
quinones, isocoumarins, and several types of alkaloids showed inhibitory effects to the activation of PMN. Thus, ex-
tracts of herbal drugs containing these compounds were examined and the active principles were characterized to evaluate the usefulness of the assay for the crude extracts of herbal drugs.
Materials and Methods Mice Inbred male C3H/He mice were purchased from Shizuoka Experimental Animal Farm (Shizuoka, Japan) and were used at 8 to 10 weeks of age.
Tumor cells
MM2, a transplantable ascites tumor from a spontaneous mammary carcinoma in a C3H/He mouse, was used as a target of effector cells (4).
PMN
Introduction Polymorphonuclear leucocytes (PMN) are the most abundant effector cells in the circulation that kill invading microorganisms, and may also be active in killing tumor cells. In previous papers, one of the authors (K. M.)
and his colleagues reported that some antitumor immunomodulators such as TAK, a linear fl-i, 3-D-glucan from
Alcaligenes faecalis var. rnyxogenes IFO 13140, induced potent tumoricidal activity of PMN in vitro, and showed that
hydrogen peroxide is a direct cytolytic mediator in the cytotoxicity (1, 2). Although PMN are important components in the surveillance and protection systems for a broad spectrum of host defenses, they also give rise to an inappropriate response, hypersensitivity reactions. In inflam-
matory processes, undesirable tissue damage can be caused by attracted activated PMN (3).
A volume of 2 ml of 8% sodium casein in 0.9% NaC1 solution (saline) was injected into the peritoneal cavity of C3H/He mice; 6 h later, the peritoneal exudate was harvested in RPMI 1640 (Nissul Seiyaku Co., Tokyo, Japan) supplemented with penicillin (100 units/ml; Banyu Pharmaceutical Co., Tokyo, Japan) and streptomycin (100 g/ml; Meiji Seika Co., Tokyo, Japan). The peritoneal cells (about 95% PMN) were harvested by centrifugation at 300 x g for 5mm and washed 3 times by resuspension in RPMI 1640. Finally, they were suspended in RPMI 1640 containing 5% heat-inactivated FCS (Flow Laboratories, North Ryde, Australia).
Cytolytic assay Cytolysis was assayed as described previously (5). Briefly, MM2 tumor cells (2 x 106 cells/ml) were labeled with Na251Cr04 (100tCi/ml) in RPMI 1640 with 10% heat-inactivated FCS for 1.5 hand then washed 4 times. Peritoneal cells obtained 6 h after injection of 2 ml of 8 % casein solution into mice and target cells (usually 5 x io cells) and the sample solutions with or without TAK were mixed in 96 wells of flat-bottomed microplates (Nunc,
OK-4000 Roskilde, Denmark). The mixtures were incubated in
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Many traditional herbal drugs are consi-
Abstract
138 Planta Med. 58(1992)
Cytolysis was calculated as: Experiment count — control count x 100. /oofcytolysis = . Maximum releasable count — control count
Measurement of direct H202 scavenger action of inhibitory compounds Samples, at various concentrations, were preincubated with 1 x i0 M H202 in 200 yl of RPMI containing heat-inactivated FCS for 2 hat 37°C under 5% CO2 in air. After centrifugation, the supernatant was mixed with [51Cr]-labelled MM2 tumor cells and the mixtures were incubated for 16h at 37°C under 5% CO2 in air. The residual cytolytic activity was measured by the same method as used in the cytolytic assay.
Reagents TAK, a linear ji-i ,3-glucan from Alcaligenes faecalis var. myxogenes IFO 13140, was kindly provided by Takeda Chemical Industries Ltd. (Osaka, Japan). Sodium casein from milk was a product of Tokyo Kasei Kogyo (Tokyo, Japan).
RPMI medium was from Nissui Seiyaku Co. (Tokyo, Japan). Herbal
drugs were purchased from Kinokuniya Kanyakkyoku (Tokyo, Japan).
Physical data and chromatography The 1H- and 13C-NMR spectra were recorded using a JEOL GX-400 spectrometer in CDC13 with tetramethylsilane as an internal standard. Kieselgel 60 F254 (Merck) precoated plates
were employed for thin-layer chromatography (TLC). Column chromatography was carried out on 70—230 mesh silica gel (Merck). HPLC was performed using a Waters M45J pump with an Oyo Bunko Uvilog-5111A UV detector.
Isolation of the active constituents from Hydrangea Dulcisfolium [leaves of Hydrangea macrophylla Seringe var. thunbergii Makino (Saxfragaceae)] (6) The crude drug (lOOg) was extracted with 70% methanol and the benzene-soluble part of the extract (12.8 g) was chromatographed through silica gel. The fractions containing phyllodulcin and hydrangenol were pooled separately and the com-
bined fractions were further purified by recrystallization from CH2C12 and by HPLC (Nucleosil 50-5). Phyllodulcin was thus obtained (180mg) showed m.p. 127—130°C; [a]0: + 7.1°, as well as hydrangenol(SOmg), m.p. 185—187°C; [a]0: +18.7°. Each ofthe extracts and fractions was assayed for the inhibitory activity but all the samples up to the stage of chromatographic fractions exhibited direct toxicity to the target cells at around 100 jig/ml and the inhibition of the TAK-induced activation of PMN was not observable.
Isolation of the active constituents from Scopoliae rhizoma [rhizomes of Scopoliajaponica Maxim. (Solanaceae)] The rhizomes (bOg) were extracted with 70% MeOH and the lyophilized extract (20.6 g) was dissolved in 2% H25O4. The solution was filtered and the filtrate was washed with ether, basified, and extracted with CHC13. The extract (0.55 g) was chromatographed on a column of silica gel. Fractions containing scopolamine were combined and subjected to HPLC on a Nucleosil
50-5 column to give scopolamine and hyoscyamine. The extract and most of the eluate fractions showed cytotoxicity to the target cells at around 300 yg/ml and inhibitory effects on the activation of PMN were not observable.
Isolation of the active constituents from Cinchonae cortex[bark of Cinchona ledgeriana Moeus et Trimen (Rubiaceae)] The bark (500 g) was extracted with 6% HC1 under warming and the filtrate was basified and extracted with CHC15-ether (3 : 9). The organic layer was dried and evaporated. The residue (30.3 g) was dissolved in CHCI5 and the insoluble part
(containing cinchonine and cinchmnidine) was removed. The chloroform solution was subjected to silica gel column chromatography employing a gradient system of CHC15-MeOH as the developer. The eluates were divided into twelve fractions on the basis of TLC examination. The fractions 6—10 contain the major alkaloids, quinine, qumnidine, cinchonmne, and cmnchonidine. The TLC and the bioassay showed the presence of other alkaloids and active constituents in fractions 5 and 10. Fraction 5 was subjected to HPLC on a column of Develosil 60-5 with hexane-CHC15-MeOH-Et2NH (70:6 : 2 :0.3) as
the developer. From the main fraction, colorless needles (9mg) of
m.p. 148—156°C, [a]0:+87,6° (EIOH), MS m/z: 312 (Mt, C19H54N202) were obtained. The physical data (liv, Ill, iH and isC NMR) suggested that the compound might be quinamine (7, 8) and
this was confirmed by comparison of the spectral data with data provided by Professor Verpoorte. Fraction 10 [eluted by CHC15-MeOH (1: 1)] was subjected to HPLC on a Develosil 60-5 column with hexane-CHC13-
MeOH-Et2NH (66: 27 : 6: 1) as the developer. Two alkaloids, I (184mg) and 11(72mg), were obtained, each as a pale yellow amorphous powder. Compound I showed UV A I!2' nm: 224, 280,
300, 314; IRvcm: 3400,1620,1510,1450; 12C-NMR(CDCI3)O ppm: 29.0, 30.0, 35.8, 38.4, 42.7, 46.0, 51.6, 70.0, 116.4, 117.4, 123.1, 125.5, 126.3, 129.0, 130.0, 137.2, 148.0, 150.2, 151.4. nm: 237, 312; lB vcm-': 3400, Compound II showed UV A 1640, 1570, 1530; iSCNMR (CDC13) 6 ppm: 28.5, 28.9, 33.9, 42.6,
43.3, 45.5, 50.5, 63.3, 112.1, 117.3, 119.6, 120.5, 121.1, 126.5, 128.4,132.9,136.1,137.2, 192.7.While ourworkwasinprogress, two alkaloids, [1S,3'R,4'R]-3-(3-ethenyl-4-piperidinyl)-1 -(4quinolinyl)- 1-propanol (cinchonicinol) and [3'R,4'S]-2-[2-(3ethenyl-4-piperidinyl)acetyl]-1H-indole-3-ethanol (cinchonamin-
one), have been isolated from Cinchonae cortex as monoamine oxidase inhibitors (9). Direct comparison showed I and II to be identical with the above compounds, respectively.
Isolation of the active constituents in Magnoliae cortex [bark of Magnolia obovata Thunb. (Magnoliaceae)J The bark (100 g) was extracted with methanol and the extract was treated with 3% AcOH. The acetic acid solution was partitioned with diethyl ether and, after being made alkaline,
the aqueous layer was again extracted with diethyl ether. The aqueous layer containing quaternary bases was neutralized and lyophilized, and the residue was treated with MeOH to remove inorganic salts. The methanol fraction containing quaternary bases such as magnocurarine (10) showed inhibition of the activation.
Isolation of the active constituents from Stephania cephalantha Hayata (Menispermaceae) The powdered root tuber (100 g) was extracted with MeOH and the extract (11.3 g) was dissolved in 3% HC1 under warming. The solution, after being washed with diethyl ether, was
basified by ammonia and extracted with diethyl ether (total basic fraction). The diethyl ether layer was partitioned with 5% NaOH and the diethyl ether layer afforded a non-phenolic base fraction
(235 mg) containing cepharanthine, isotetrandrine, and cycleanine (identified by TLC). The alkaline layer, after the addition
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0.2 ml of RPMI 1640 with 5% heat-inactivated FCS for 16 hat 37°C
under 5% CO2 in air. The plates were centrifuged at 450 x g for 10 mm, and the radioactivity of the supernatant was measured.
Kaora Kinoshita et at
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity ofPolymorphonuclear Leucocytes
>,
0
100
100
80
80
60
60
Planta Med. 58(1992) 139
Fig. 1 Effects of coumarin, mompain, atropine, and quinine on TAK-induced PMN cytotoxicity. PMN (6 x 10) and {51Cr]-labelled MM2 target cells (5 x 10) were incubated at 37°C for 16 h
with TAK (100 ffg/ml) (•) orwithoutTAK (0). Compounds were added at the indicated concentrations. After incubation, the radioactivity of the supernatant was determined. Values are means for duplicate determinations.
0 4, 40
' 40
20
20
0
0
-/, 0
Coumarin (jig/mi)
2.5
10
40
160
Mompain (jig/mi)
>
0 4,
100
80
80
60
,m 60 0
0>,
40
40
20
20
0
0
0 '" 25
50 100200400
0
Atropine (pg/mi)
6.25
12.5
25
50
Quinine (pg/mi)
of ammonium chloride, was extracted with diethyl ether to afford
the phenolic bases such as cepharanoline, berbamine, and homoaromoline (155 mg)(l1).
Isolation of the active constituents in Rauwol,fiae radix [root ofRauwolfia serpentina Benth. (Apocynaceae)J The powdered drug (500 g) was extracted with 70% MeOH and the extract was evaporated and lyophilized. The residual powder was extracted with CHC13 after alkaline treatment with ammonia and the chloroform layer was partitioned with 0.5 N H2S04 to remove strong bases, including ajmaline, which were recovered by extraction with chloroform after basification. The original chloroform layer was concentrated, diethyl ether was added, and the organic solution was extracted with 2 % citric acid solution. The acidic layer was made alkaline and extracted with CHC13 to recover weak bases such as reserpine and rescinamine (12).
Results Inhibitory effects of natural products on the PMN cytotoxicity
As shown in the previous paper (1), PMN in the presence of TAK are capable of destroying MM2 target cells. The cytolysis is almost 100% at E : T ratios of 3: 1 to 50: 1 in the presence of TAK at more than 6.3 ig/ml. All the experiments in this study were done at an E :T ratio of 12: 1 with TAK at 100 jig/mI. The dose-response curves of some of the active compounds are shown in Fig. 1. Table 1 summarizes the inhibitory effect of the 151 natural products. The compounds indicated by (—) showed neither toxicity to
the target tumor cells nor inhibition of the activation of PMN by TAK. The compounds shown by the letter (t) were toxic to the target MM2 tumor cells directly and the inhibitory effects were not observable. None of these tested compounds
caused the activation of PMN, as TAK and other immunomodulators did (1, 2). Forty-two compounds, listed in Table 1 with their inhibitory concentrations, inhibited the PMN cytotoxicity. The chemical structures of the fifteen
compounds exhibiting activity at concentrations of less than 5Opg/ml are shown in Fig. 2.
Among the six derivatives related to primary metabolites, sorbic acid was inhibitory. Ellagic acid, a
component of tannins, and podophyllotoxin, a lignan known as an antitumor agent, were inhibitory among the
sixteen phenolic compounds of shikimate origin. Usnic acid was the only inhibitory compound among six phenolics of the polyketide type. Two coumarins, coumarin and 6,7-di-
methylesculetin, and two isocoumarins, hydrangenol and phyllodulcin, showed the inhibition though not so strong. Among the seventeen naturally ocurring quinones tested, several compounds such as alizarin, ardisiaquinone A, embelin, helicobasidin, and mompain, showed inhibition of the TAK-induced PMN activation at 8—50 jig/mI concentrations, but the other half of the group of compounds showed
direct toxicity to the target cells and the inhibitory effects were not observable. No obvious effects were observed among isoprenoids, i.e. eleven monoterpenes, three sesquiterpenes, nine diterpenes and their glycosides, fourteen
triterpenes and saponins, and nine steroids, except for
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——'—II
100
140 Planta Med. 58(1992)
Kaoru Kinoshita et al.
Sourc&
Compound
Table 1 Inhibitory effects of plant secondary ID50 )jLg/ml)
metabolites on TM-induced cytotoxicity of PMN.
I) Compounds related to primary metabolites (a) Fatty acid derivatives agaricic acid lichesterinic acid protolichesterinic acid sorbic acid
(b) Sugarderivat/ve sinigrin (c) Amino acid den vative kainicacid
Fomeslaricis(13) Iichens(14) lichens (14)
c
66
c
t
c
ii) Phenolic compounds
iii) Isoprenoids (a) Monoterpenes aucubin borneol
(+)-camphor catalpol gardenoside gentiopicroside
c c c C
c c
— —
— — —
t 62 —
310 — — — —
— — — — —
53 182
65 —
500 100
t t t —
t —
15.8
t
15.2 7.8
t
10
t
—
t t
56 —
I — —
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(a) Phenol/cs derived from shikimate c amygdalin arbutin c caffeic acid c c chiorogenic acid c cinnamaldehyde c coniferin curcumin Curcumalonga(15) c 26-dimethoxyphenol c ellagic acid c ferulic acid c podophyllotoxin safrole c c salicin c syringic acid vanillic acid c vanillin c (b) Phenol/cs derived from acetate-malonate asterric acid Oosporasulphurea-ochracea (16) C griseofulvin lecanoric acid Iichens(14) p-orsellic acid Iichens(14) sulochrin Oospora sulphurea-ochracea (16) usnicacid lichens(14) (c) Coumarins and isocoumarins c coumarin c 6,7-dimethylesculetin esculin c hydra ngenol Hydrangea macrophylla var. thunbergiic Hydrangea macrophylla var. thunbergii' phyllodulcin (d) Flavonoids c astragalin baicalein c c baicalin c hesperidin c hematoxylin kaempferol 3-rutinoside Leonurussibiricus (17) (e) Qu/nones alizarin c alkannin c Ardisiasp. (18) ardisiaquinoneA embelin Ardisiasp.(18) emodin Preussiasp.(19) helicobasidin Hel/cobasidiummompa (20) C 2-hydroxynaphthoquinone islandicin Pen/c/Ilium islandicum (19) isodiospyrin Diospyrosspp.(21) juglone Diospyrosspp.(21) Pen/c/Ilium islandicum (19) luteoskyrin mompain Helicobasidium mompa (20) c sennosideA c shikonin Pen/c/Ilium islaridicum (19) skyrin tanshinone II Salvia m/ltiorrhiza c ubiquinone 9
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity of Polymorphonuclear Leucocytes Source
Compound
(—(-menthol nero!
perillalehyde
thymo! (b) Sesqu/terpenes picrotoxin ptaquiloside santonin (C) Diterpenes abietic acid
andrographolide gibberellin rebaudioside A rebaudioside B rebaudioside 0 rebaudioside E rubusoside stevioside (d) Triterpenes and saponins bauerenol dihydrolanosterol eburicoic acid entadasaponin II entadasaponin II! entadasaponin IV
c c c c c
C
Table 1 (continued). ID50 (pg/mI)
t — —
t —
—
Pteridium apuilinum (22) c c
Andrographispaniculatae
238
t
c
—
Stevia rebaudiana Stevia rebaudiand Stevia rebaudiana Stevia rebaudiana
—
Rubussp!
—
Stevia rebaudiana
—
Ardisiasieboldii(23( fungi of Polyporaceae (24) fungi of Po!yporaceae (24)
Entadasp. Entadasp.0
— — —
500 — — —
—
Entadasp. Panaxginseng
—
ginsenoside Rd
ginsenosideR
Panaxginsen
—
glycyrrhetic acid glycyrrhizin hederagenin lanosterol oleanolic acid pachymic acid (e) Sterols
cholesterol cholic acid deoxycholic acid digitonin digitoxin digitoxigenin diosgenin ergosterol /3-sitosterol
G/ycyrrh/zaglabra0
G/ycyrrhizag/abra Hedera tob/eri5 fungi of Polyporaceae (24)
Glycyrrhizag/abra fungi of Polyporaceae (24)
t —
— —
—
190
c c c c c c
—
c c c
—
t — —
t t
iv) Alkaloids and other nitrogen compounds (a) Tropane alkaloids
atropine scopolamine tropine
c c c
180
308 230
(b) Quinoline alkaloids
cinchonicinol cinchonidine cinchonine quinidine quinine (c) Isoquinoline alkaloids
berbamine berberine cepharanthine cepharanoline coclaurine cycleanine emetine homoaromoline isotetrandrine magnocurarine papaverine sinomenine tubocurarine trilobine (d) lndole alkaloids ajmaline
Cinchona ledger/anach
70
c c c c
53
Menispermaceae plants
33
c Menispermaceae plants Menispermaceae plants' Menispermaceae plants Menispermaceae plants
c Menispermaceae plants' Menispermaceae plants' Menispermaceae plants'
c Menispermaceae plants'
44 37 25
—
8 — —
57
t
10 38 8.7 52
400
c
—
Menispermaceae plants
—
c
38
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limonene
Planta Med. 58(1992) 141
142 Planta Med. 58(1992) Compound
Kaoru Kinoshita et a1.
Sourcea
I
cinchonaminone ergotamine quinamine reserpine strychinine vinbiastine vincrystine (e) Other alkaloids arecoline coichicine nicotine pilocarpine phalloidine physostigmine sparteine veratramin (f) Other nitrogen compounds caffeine chlorophyll a
ID5, (JtWnl)b
Table 1 (continued).
36 84
Cinchona ledgeriana c
Cinchona ledge riana' c C
c c c c c c c
340 2.2
575
C C
c
243
c
130
C
amphotericin B benzylpenicillin cefalexin chloramphenicol daunomycin erythromycin josamycin kanamycin kasugamycin oxytetracycline
c C
c C
c c
50 365
C C C
c
c = commercial products from Wako Pure Chemicals, Ltd. or Tokyo Kasel Kogyo Co., Ltd. = Inhibitory effects were not observable due to the cytotoxicity to the target cells. — = Compounds showing neither inhibition of activation of PMN nor cytotoxicity to the target cells. See text. Unpublished data of our laboratory. Provided by Dr. M. Kuroyanagi (University of Shizuoka(. Provided by Prof. 0. Tanaka (Hiroshima University). Provided by Prof. S. Shibata (University of Tokyo, retired). Provided by Dr. T. Naro (University of Shizuaka(. Provided by Dr. M. Akasu (Kaken Drug Co., Ltd.). Provided by Prof. R. Verpoorte (State University of Leiden).
three (santonin, bauerenol, and pachymic acid) which showed weak inhibition.
It might be possible that some of the tested compounds showed a selective toxic potential to PMN. How-
chonae cortex, quinine, quinidine, cinchonine, cm-
ever, the inhibitory effects of these compounds were unlikely to be due to the direct cytotoxicity to PMN, since the inhibitory effects were observed at nontoxic concentrations or, at least, in thirty times lower concentrations than those toxic to the target cells. Therefore, the inhibitory effects on
chonidine, and cinchonicinol, also showed the inhibition.
PMN is due not to dying PMN but to some inhibitory
Eight compounds among the fourteen isoquinoline al-
mechanism for the production of active oxygen from PMN.
Various alkaloids showed potent inhibition.
Three tropane alkaloids, atropine, scopolamine, and
tropine, were inhibitory. Five quinoline alkaloids of Gin-
kaloids, berbamine, cepharanthine, cycleanine, homoaromoline, isotetrandrine, magnocurarine, papaverine, and
sinomenine, showed the inhibition. However, no clear structure-activity relationship was observed among the al-
kaloids. Among the eight indole alkaloids tested, cmchonaminone from Cinchona, ajmaline from Rauwoijia, and ergotamine from ergot showed the inhibition. Among the other compounds tested, eight alkaloids of other types, two other nitrogen compounds and ten antibiotics, nicotine showed the highest activity (ID50 2.2zg/ml). Other alkaloids, arecoline, pilocarpine, and veratramine, caffeine, and two antibiotics, erythromycin and josamycin, were weakly inhibitory.
Examination of the positive compounds as hydrogen peroxide scavengers In the TAK-induced PMN cytotoxicity, hydrogen peroxide is a direct cytotoxic mediator (1, 2). A possible mechanism of the inhibitory effect of the positive compounds is direct hydrogen peroxide scavenging. Thus, the scavenging activity was examined by the method described
above. Among the compounds showing the inhibition, naturally occurring quinones such as ardisiaquinone A, embelin, and mompain, which have o-hydroxyquinone structures and were quite reactive to oxidizing agents,
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v) Antibiotics
Planta Med. 58(1992) 143
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity ofPolymorphonuclear Leucocytes
H3C CH3
0
OH3C '
OH HO
(CH2)10—CH3
0 alizarin
HO
H3C (OH 0
heticobasidin
embe [in
ardisiaquinone A
H3
R1
R1
R2
R3
magnocurarine
R'
cepharanthine
—H quinine H3CO HO H HO ---H quinidine H3cO H cinchonine H H HO ---H
isotetrandrine
homoaromotine
Oci OH
H3C
0
II
H 2C
cinchonamine
berb amine
ajmat me
nicotine
Fig.2 Structures of the compounds exhibiting the inhibitory activity with ID50 value of less than 50ig/ml.
proved to be H202 scavengers at 20—100 g/ml, while other compounds such as ajmaline, arecoline, atropine, cmchonidine, coumarin, homoaromoline, magnocurarine, nicotine, pachymic acid, papaverine, podophyllotoxin, scopolamine, sorbic acid, tropine, and usnic acid were found not to be H202 scavengers.
activation were not observable until the final stage of the separation due to the toxicities exhibited by the fractions.
The total alkaloid fraction from Cinchona cortex showed inhibition of the same order of magnitude (ID50 53uglml) as the four quinoline alkaloids, quinine, quinidine, cinchonine, and cinchonidine. Besides these
compounds, three alkaloids quinamine (7, 8), cm-
Isolation of the active constituents
chonicinol, and cinchonaminone (9), were isolated from the
from herbal drugs To learn the applicability of the assay for the monitoring of crude extracts and in separation procedures, six drugs were analyzed with the assay to characterize the active constituents. In the case of Hydrangea Dulcis folium con-
taining isocoumarins, hydrangenol, and phyllodulcin, and Scopolia rhizome containing the tropane alkaloids, hyoscyamine and scopolamine, the inhibitory effects on the PMN
fractions exhibiting the inhibitory effects. The first compound did not show any effects, but the other two compounds isolated recently from the drug as monoamine oxidase inhibitors, showed inhibitory effects as shown in Table 1.
Several isoquinoline alkaloids showed the inhibitory effects. Thus two crude drugs, Magnolia cortex
and the roots of Stephania cephalantha, containing isoquinoline alkaloids, were analyzed. In the former, the
quaternary base fraction containing magnodurarine, Table 2 Inhibitory activity of constituents of Step/ian/a cepha/antha. Part
MeOH extract Total base Non-phenolic base Phenolic base
proved to be inhibitory. In the latter, non-phenolic bases such as cepharanthine, isotetrandrine, and cycleanine, and
lD50g/ml) 140 20 19 23
Substance
Cepharanthine Isotetrandrine Cycleanine Homoaromoline Berbamine Cepharanoline
lD50IygIml)
8
phenolic bases such as berbamine and homoaromoline proved to be the inhibitory constituents (Table 2).
38 57 10 33 —
The inhibitory constituent in Rauwolfia radix, containing indole alkaloids, was proved to exist in the strong base fraction and ajmaline but not reserpine and rescinamine proved to be the active constituent as shown in
Fig.3.
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3C
144 Planta Mcd. 58(1992)
Kaoru Kinos/zita et aL
induces the same potent PMN cytotoxicity as TAK, with hydrogen peroxide as a direct cytotoxic mediator (26). The effective alkaloids also inhibited the PMA-induced cytotoxicity (data not shown). PMAis thought to act through the acti-
100
It) U,
>,
60
C
4.) >.)
LI
40
20
0
0
6.25 12.5 25
50
100 200
Constituents of Rauwoifiae Radix (pg/mi) Fig.3 Effects of constituents of Rauwolfiae radix on TAK-induced PMN cytotoxicity. In the presence of TAK (100 yg/mI), the indicated concentrations of MeOH ext. (•), strong base fr. (A), weak base fr. (A), ajmaline and reserpine (LI) were added to the cytotoxicity assay mixture containing PMN (6 x 10) and [51Cr]-labelled MM2 target cells (5 x 1O). After incubation for 16 h, the radioactivity of the supernatant was determined. Values are means of duplicate determinations.
Discussion Among 151 natural products tested, covering a wide range of structural varieties and biosynthetic origins, the 42 compounds shown in Table I inhibited the activation of PMN by TAK. All of the compounds exhibiting po-
tent inhibitory activities (IDso: less than 50 yg/ml) are quinones or alkaloids. The modes of action of these two groups are different; most of the quinone derivatives showed direct H202 scavenging action, while the alkaloids showed the inhibition by different mechanism(s).
Several herbal drug constituents have been shown to exhibit anti-inflammatory activities. A naphthoquinone, shikonin, and an isoquinoline alkaloid, berberine, showing inhibitory activities in this study, are known to be anti-inflammatory agents. Inhibitory activities of cepharanthine, the isoquinoline alkaloid from Step/ian/a cepharantha, on the production of active oxygen by PMN and on PMA-induced inflammation have been known (M. Akasu, personal communication). Nicotine showed a remarkable inhibitory effect in this study. Sasagawa et al. (25) examined the effect of nicotine on the functions of human PMN. They reported that nicotine had no effect on migration but inhibited both lysosomal enzyme release and O
production from PMN and they found that the effect of nicotine was not altered by cholinergic antagonists and agonists. They suggested that nicotine acts directly on PMN through the nicotine-specific receptors. The inhibitory mechanism of these effective alkaloids has not been analyzed in this study. However, we examined the effect on PMN cytotoxicity induced by a different type of stimulus; phorbol myristate acetate (PMA). PMA
The assay that we used for screening and monitoring of the inhibitory compounds was not entirely satisfactory. As shown in the cases of Hydrangea Dulcis folium and Scopoliae rhizome, when the crude drugs contained cytotoxic compounds and the inhibitory activities were not so strong, the activities were not observable until the final stages of the separation. However, as in the cases of Rauwolfiae radix, Cinchonae cortex, and the root of Stephania cepharantha, when the drugs did not contain cytotoxic compounds, the method was found to be effective for monitoring systems of the separation. In the case of alkaloids, the method seems in general to be applicable after separation of the total alkaloidal fraction.
Acknowledgements The authors indebted to Professors S. Shibata, 0. Tanaka, and R. Verpoorte, and Drs. M. Akasu, T. Noro, and M. Kuroyanagi for providing samples and spectral data. The authors thank Mr. I. Takahashi and Misses J. Gonoi, H. Kohsaka, N. Ogi, andY. Ono and Mrs. C. Wakabayashi for their help in a part of this work.
References 1
Morikawa, K., Takeda, fi., Yamazaki. M., Mizuno, D. (1985) Cancer Res. 45. 1496—1451. Morikawa, K., Kamegaya, S., Yamazaki, M., Mizuno, D. (1985) Cancer Res. 45, 3482—3486. Roit, I. M., Brostoff, J., Male, D. K. (1989) in: Immunology, 2nd edn., pp. 19.1—22.10, Blackwell Scientific Publications, Oxford. " Nishtoka, K., Inc. B. F., Kawana, T., Takeucht, S. (1969) lot. J. Cancer 4, 139—149. Yamazaki, M., Shinoda, H., Mizuno, D. (1975) Gann 66, 489—497. 6 Kaneko, H., Fujimori, M., Matsushita, H., Noguchi, M. (1973) Nogei KagakuKaishi 10,605—609: C. A. 80, 93153 (1974). 2
8
° 12
13 14 15 16
17 18
Goutarel, R., Janot, M.-M., Prelog, V., Taylor, W. I. (1950) Helv. Chim.Acta33, 150—169. Mulder-Krieger, Th., Verpoorte, R., de Water, A., van Gessel, M., van Oeveren, B. C. J. A., Svendsen, A. B. (1982) Planta Med. 46, 19—24. Mitsui, N., Noro, T., Kuroyanagi, M., Miyase, T., Umemura, K., Ueno, A. (1989) Chem. Pharm. Bull. 37, 363—366. Tomtta, M., Inubushi, Y., Yamagata, M. (1951) Yakugaku Zasshi 71, 1069—1075; C. A. 46, 5059 (1952). Tomita, M., Sawada, T., Kozuka, M., Takeuchi, M., Akasu, M. (1969) Yakugaku Zasshi 89, 1678—1681; C. A. 72, 107846 (1970). Japanese Pharmacopoeia, IX Edition (1976) p. D-215. Yokoyama, A., Natori, S., Aoshima, K. (1975) Phytochemistry 14, 487—497. Asahina, Y., Shibata, 5. (1954) Chemistry of Lichen Substances, Japan Society of Promotion of Science, Tokyo. Kuroyanagi, M., Natori, 5. (1970) Yakugaku Zasshi 90, 1467—1470; C. A. 74, 61612 (1971). Natoni, S., Nishikawa, H. (1962) Chem. Pharm. Bull. 10, 117—124. Kuroda, K., Kinoshita, K., Natort, S., Wang, C.-E., Yano, S.,
Watanabe, K. (1991) Chem. Pharm. Bull., in press. Ogawa, H., Natori, S. (1968) Chem. Pharm. Bull. 16, 1709—1720.
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vation of protein kinase C (27). On the other hand, in the case of TAK, there may be a receptor mediated Ca2-dependent step prior to activation of protein kinase C (26). Therefore, it is presumed that these alkaloids could impair the NADPH oxidase system in the plasma membrane of PMN. Further studies are needed on the inhibitory mechanism to characterize the effect of the active constituents.
80
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity ofPolymorphonuclear Leucocytes
24 Yokoyama, A., Natori, S., Aoshima, K. (1975) Phytochemistry 14,
20
386. Natori, S., Inouye, Y., Nishikawa, H. (1967) Chem. Pharm. Bull. 15,
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22
380— 390. Tezuka, M., Takahashi, C., Kuroyanagi, M., Satake, M., Yoshihira,
26
22 23
K., Natori, S. (1973) Phytochemistry 12, 175—183. Saito, K., Nagao, T., Matoba, M., Koyama, K., Natori, S., Murakami, T., Saiki, Y. (1989) Phytochemistry 28, 1605—1611. Ogawa, H., Natori, S. (1968) Chem. Pharm. Bull. 16, 1709—1720.
487—497.
27
Sasagawa, S., Suzuki, K., Sakatani, T., Fujikura, T. (1985) J. Leukocyte Biology 37, 493—502. Morikawa, K., Noguchi, T., Yamazaki, M., Mizuno, D. (1986) Cancer Res. 46,66—70.
Castagna,
M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U.,
Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847—7851.
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19 Natori, S., Sam, F., Udagawa, S. (1965)Chem. Pharm. Bull. 13, 385—
Planta Med. 58 (1992) 145
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity of Polymorphonuclear Leucocytes Kaoru Kinoshita , Kaoru Morikawa2, Masahiko Fujita2, and Shinsaku Natori " 1 Meiji College of Pharmacy, Yato-cho, Tanashi-shi, Tokyo 188, Japan
National Institute of Public Health, Shirokanedai-4-chome, Minato-ku, Tokyo 108, Japan Address for correspondence Received: March 5, 1991
The inhibitory effects of 151 natural prod-
ucts, representing most of the frequently occurring types, on the cytotoxicity to MM2 tumor cells of polymorphonuclear leucocytes (PMN) induced by TAK, a polysac-
charide immunomodulator, were examined. Forty-two compounds inhibited the TAK-induced activation of PMN. Among them, some naturally occurring quinones and various alkaloids (nicotine, Cinchona alkaloids, isoquinoline alkaloids such as cepharanthine, and indole alkaloids such as ajmaline) exhibited potent inhibitory effects. Using the inhibition assay for monitoring, the extracts of Hydrangea Dulcis folium, Scopoliae rhizoma, Cinchona cortex, Magnoliae cortex, Stephania tuber, and Rauwolfia radix were analysed to characterize the active constituents. Key words
Cytotoxicity, inhibition of TAK-induced activation, polymorphonuclear leucocytes, secondary metabolites, quinones, isoquinoline alkaloids, indole alkaloids.
dered to exhibit anti-inflammatory effects. In this study, we investigated the effects of natural products or herbal drug constituents on the PMN activation by TAK using a PMN cytotoxicity assay system. The results for 151 compounds, covering most of the frequently occurring types of natural products, are presented in this paper. Compounds such as
quinones, isocoumarins, and several types of alkaloids showed inhibitory effects to the activation of PMN. Thus, ex-
tracts of herbal drugs containing these compounds were examined and the active principles were characterized to evaluate the usefulness of the assay for the crude extracts of herbal drugs.
Materials and Methods Mice Inbred male C3H/He mice were purchased from Shizuoka Experimental Animal Farm (Shizuoka, Japan) and were used at 8 to 10 weeks of age.
Tumor cells
MM2, a transplantable ascites tumor from a spontaneous mammary carcinoma in a C3H/He mouse, was used as a target of effector cells (4).
PMN
Introduction Polymorphonuclear leucocytes (PMN) are the most abundant effector cells in the circulation that kill invading microorganisms, and may also be active in killing tumor cells. In previous papers, one of the authors (K. M.)
and his colleagues reported that some antitumor immunomodulators such as TAK, a linear fl-i, 3-D-glucan from
Alcaligenes faecalis var. rnyxogenes IFO 13140, induced potent tumoricidal activity of PMN in vitro, and showed that
hydrogen peroxide is a direct cytolytic mediator in the cytotoxicity (1, 2). Although PMN are important components in the surveillance and protection systems for a broad spectrum of host defenses, they also give rise to an inappropriate response, hypersensitivity reactions. In inflam-
matory processes, undesirable tissue damage can be caused by attracted activated PMN (3).
A volume of 2 ml of 8% sodium casein in 0.9% NaC1 solution (saline) was injected into the peritoneal cavity of C3H/He mice; 6 h later, the peritoneal exudate was harvested in RPMI 1640 (Nissul Seiyaku Co., Tokyo, Japan) supplemented with penicillin (100 units/ml; Banyu Pharmaceutical Co., Tokyo, Japan) and streptomycin (100 g/ml; Meiji Seika Co., Tokyo, Japan). The peritoneal cells (about 95% PMN) were harvested by centrifugation at 300 x g for 5mm and washed 3 times by resuspension in RPMI 1640. Finally, they were suspended in RPMI 1640 containing 5% heat-inactivated FCS (Flow Laboratories, North Ryde, Australia).
Cytolytic assay Cytolysis was assayed as described previously (5). Briefly, MM2 tumor cells (2 x 106 cells/ml) were labeled with Na251Cr04 (100tCi/ml) in RPMI 1640 with 10% heat-inactivated FCS for 1.5 hand then washed 4 times. Peritoneal cells obtained 6 h after injection of 2 ml of 8 % casein solution into mice and target cells (usually 5 x io cells) and the sample solutions with or without TAK were mixed in 96 wells of flat-bottomed microplates (Nunc,
OK-4000 Roskilde, Denmark). The mixtures were incubated in
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Many traditional herbal drugs are consi-
Abstract
138 Planta Med. 58(1992)
Cytolysis was calculated as: Experiment count — control count x 100. /oofcytolysis = . Maximum releasable count — control count
Measurement of direct H202 scavenger action of inhibitory compounds Samples, at various concentrations, were preincubated with 1 x i0 M H202 in 200 yl of RPMI containing heat-inactivated FCS for 2 hat 37°C under 5% CO2 in air. After centrifugation, the supernatant was mixed with [51Cr]-labelled MM2 tumor cells and the mixtures were incubated for 16h at 37°C under 5% CO2 in air. The residual cytolytic activity was measured by the same method as used in the cytolytic assay.
Reagents TAK, a linear ji-i ,3-glucan from Alcaligenes faecalis var. myxogenes IFO 13140, was kindly provided by Takeda Chemical Industries Ltd. (Osaka, Japan). Sodium casein from milk was a product of Tokyo Kasei Kogyo (Tokyo, Japan).
RPMI medium was from Nissui Seiyaku Co. (Tokyo, Japan). Herbal
drugs were purchased from Kinokuniya Kanyakkyoku (Tokyo, Japan).
Physical data and chromatography The 1H- and 13C-NMR spectra were recorded using a JEOL GX-400 spectrometer in CDC13 with tetramethylsilane as an internal standard. Kieselgel 60 F254 (Merck) precoated plates
were employed for thin-layer chromatography (TLC). Column chromatography was carried out on 70—230 mesh silica gel (Merck). HPLC was performed using a Waters M45J pump with an Oyo Bunko Uvilog-5111A UV detector.
Isolation of the active constituents from Hydrangea Dulcisfolium [leaves of Hydrangea macrophylla Seringe var. thunbergii Makino (Saxfragaceae)] (6) The crude drug (lOOg) was extracted with 70% methanol and the benzene-soluble part of the extract (12.8 g) was chromatographed through silica gel. The fractions containing phyllodulcin and hydrangenol were pooled separately and the com-
bined fractions were further purified by recrystallization from CH2C12 and by HPLC (Nucleosil 50-5). Phyllodulcin was thus obtained (180mg) showed m.p. 127—130°C; [a]0: + 7.1°, as well as hydrangenol(SOmg), m.p. 185—187°C; [a]0: +18.7°. Each ofthe extracts and fractions was assayed for the inhibitory activity but all the samples up to the stage of chromatographic fractions exhibited direct toxicity to the target cells at around 100 jig/ml and the inhibition of the TAK-induced activation of PMN was not observable.
Isolation of the active constituents from Scopoliae rhizoma [rhizomes of Scopoliajaponica Maxim. (Solanaceae)] The rhizomes (bOg) were extracted with 70% MeOH and the lyophilized extract (20.6 g) was dissolved in 2% H25O4. The solution was filtered and the filtrate was washed with ether, basified, and extracted with CHC13. The extract (0.55 g) was chromatographed on a column of silica gel. Fractions containing scopolamine were combined and subjected to HPLC on a Nucleosil
50-5 column to give scopolamine and hyoscyamine. The extract and most of the eluate fractions showed cytotoxicity to the target cells at around 300 yg/ml and inhibitory effects on the activation of PMN were not observable.
Isolation of the active constituents from Cinchonae cortex[bark of Cinchona ledgeriana Moeus et Trimen (Rubiaceae)] The bark (500 g) was extracted with 6% HC1 under warming and the filtrate was basified and extracted with CHC15-ether (3 : 9). The organic layer was dried and evaporated. The residue (30.3 g) was dissolved in CHCI5 and the insoluble part
(containing cinchonine and cinchmnidine) was removed. The chloroform solution was subjected to silica gel column chromatography employing a gradient system of CHC15-MeOH as the developer. The eluates were divided into twelve fractions on the basis of TLC examination. The fractions 6—10 contain the major alkaloids, quinine, qumnidine, cinchonmne, and cmnchonidine. The TLC and the bioassay showed the presence of other alkaloids and active constituents in fractions 5 and 10. Fraction 5 was subjected to HPLC on a column of Develosil 60-5 with hexane-CHC15-MeOH-Et2NH (70:6 : 2 :0.3) as
the developer. From the main fraction, colorless needles (9mg) of
m.p. 148—156°C, [a]0:+87,6° (EIOH), MS m/z: 312 (Mt, C19H54N202) were obtained. The physical data (liv, Ill, iH and isC NMR) suggested that the compound might be quinamine (7, 8) and
this was confirmed by comparison of the spectral data with data provided by Professor Verpoorte. Fraction 10 [eluted by CHC15-MeOH (1: 1)] was subjected to HPLC on a Develosil 60-5 column with hexane-CHC13-
MeOH-Et2NH (66: 27 : 6: 1) as the developer. Two alkaloids, I (184mg) and 11(72mg), were obtained, each as a pale yellow amorphous powder. Compound I showed UV A I!2' nm: 224, 280,
300, 314; IRvcm: 3400,1620,1510,1450; 12C-NMR(CDCI3)O ppm: 29.0, 30.0, 35.8, 38.4, 42.7, 46.0, 51.6, 70.0, 116.4, 117.4, 123.1, 125.5, 126.3, 129.0, 130.0, 137.2, 148.0, 150.2, 151.4. nm: 237, 312; lB vcm-': 3400, Compound II showed UV A 1640, 1570, 1530; iSCNMR (CDC13) 6 ppm: 28.5, 28.9, 33.9, 42.6,
43.3, 45.5, 50.5, 63.3, 112.1, 117.3, 119.6, 120.5, 121.1, 126.5, 128.4,132.9,136.1,137.2, 192.7.While ourworkwasinprogress, two alkaloids, [1S,3'R,4'R]-3-(3-ethenyl-4-piperidinyl)-1 -(4quinolinyl)- 1-propanol (cinchonicinol) and [3'R,4'S]-2-[2-(3ethenyl-4-piperidinyl)acetyl]-1H-indole-3-ethanol (cinchonamin-
one), have been isolated from Cinchonae cortex as monoamine oxidase inhibitors (9). Direct comparison showed I and II to be identical with the above compounds, respectively.
Isolation of the active constituents in Magnoliae cortex [bark of Magnolia obovata Thunb. (Magnoliaceae)J The bark (100 g) was extracted with methanol and the extract was treated with 3% AcOH. The acetic acid solution was partitioned with diethyl ether and, after being made alkaline,
the aqueous layer was again extracted with diethyl ether. The aqueous layer containing quaternary bases was neutralized and lyophilized, and the residue was treated with MeOH to remove inorganic salts. The methanol fraction containing quaternary bases such as magnocurarine (10) showed inhibition of the activation.
Isolation of the active constituents from Stephania cephalantha Hayata (Menispermaceae) The powdered root tuber (100 g) was extracted with MeOH and the extract (11.3 g) was dissolved in 3% HC1 under warming. The solution, after being washed with diethyl ether, was
basified by ammonia and extracted with diethyl ether (total basic fraction). The diethyl ether layer was partitioned with 5% NaOH and the diethyl ether layer afforded a non-phenolic base fraction
(235 mg) containing cepharanthine, isotetrandrine, and cycleanine (identified by TLC). The alkaline layer, after the addition
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0.2 ml of RPMI 1640 with 5% heat-inactivated FCS for 16 hat 37°C
under 5% CO2 in air. The plates were centrifuged at 450 x g for 10 mm, and the radioactivity of the supernatant was measured.
Kaora Kinoshita et at
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity ofPolymorphonuclear Leucocytes
>,
0
100
100
80
80
60
60
Planta Med. 58(1992) 139
Fig. 1 Effects of coumarin, mompain, atropine, and quinine on TAK-induced PMN cytotoxicity. PMN (6 x 10) and {51Cr]-labelled MM2 target cells (5 x 10) were incubated at 37°C for 16 h
with TAK (100 ffg/ml) (•) orwithoutTAK (0). Compounds were added at the indicated concentrations. After incubation, the radioactivity of the supernatant was determined. Values are means for duplicate determinations.
0 4, 40
' 40
20
20
0
0
-/, 0
Coumarin (jig/mi)
2.5
10
40
160
Mompain (jig/mi)
>
0 4,
100
80
80
60
,m 60 0
0>,
40
40
20
20
0
0
0 '" 25
50 100200400
0
Atropine (pg/mi)
6.25
12.5
25
50
Quinine (pg/mi)
of ammonium chloride, was extracted with diethyl ether to afford
the phenolic bases such as cepharanoline, berbamine, and homoaromoline (155 mg)(l1).
Isolation of the active constituents in Rauwol,fiae radix [root ofRauwolfia serpentina Benth. (Apocynaceae)J The powdered drug (500 g) was extracted with 70% MeOH and the extract was evaporated and lyophilized. The residual powder was extracted with CHC13 after alkaline treatment with ammonia and the chloroform layer was partitioned with 0.5 N H2S04 to remove strong bases, including ajmaline, which were recovered by extraction with chloroform after basification. The original chloroform layer was concentrated, diethyl ether was added, and the organic solution was extracted with 2 % citric acid solution. The acidic layer was made alkaline and extracted with CHC13 to recover weak bases such as reserpine and rescinamine (12).
Results Inhibitory effects of natural products on the PMN cytotoxicity
As shown in the previous paper (1), PMN in the presence of TAK are capable of destroying MM2 target cells. The cytolysis is almost 100% at E : T ratios of 3: 1 to 50: 1 in the presence of TAK at more than 6.3 ig/ml. All the experiments in this study were done at an E :T ratio of 12: 1 with TAK at 100 jig/mI. The dose-response curves of some of the active compounds are shown in Fig. 1. Table 1 summarizes the inhibitory effect of the 151 natural products. The compounds indicated by (—) showed neither toxicity to
the target tumor cells nor inhibition of the activation of PMN by TAK. The compounds shown by the letter (t) were toxic to the target MM2 tumor cells directly and the inhibitory effects were not observable. None of these tested compounds
caused the activation of PMN, as TAK and other immunomodulators did (1, 2). Forty-two compounds, listed in Table 1 with their inhibitory concentrations, inhibited the PMN cytotoxicity. The chemical structures of the fifteen
compounds exhibiting activity at concentrations of less than 5Opg/ml are shown in Fig. 2.
Among the six derivatives related to primary metabolites, sorbic acid was inhibitory. Ellagic acid, a
component of tannins, and podophyllotoxin, a lignan known as an antitumor agent, were inhibitory among the
sixteen phenolic compounds of shikimate origin. Usnic acid was the only inhibitory compound among six phenolics of the polyketide type. Two coumarins, coumarin and 6,7-di-
methylesculetin, and two isocoumarins, hydrangenol and phyllodulcin, showed the inhibition though not so strong. Among the seventeen naturally ocurring quinones tested, several compounds such as alizarin, ardisiaquinone A, embelin, helicobasidin, and mompain, showed inhibition of the TAK-induced PMN activation at 8—50 jig/mI concentrations, but the other half of the group of compounds showed
direct toxicity to the target cells and the inhibitory effects were not observable. No obvious effects were observed among isoprenoids, i.e. eleven monoterpenes, three sesquiterpenes, nine diterpenes and their glycosides, fourteen
triterpenes and saponins, and nine steroids, except for
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——'—II
100
140 Planta Med. 58(1992)
Kaoru Kinoshita et al.
Sourc&
Compound
Table 1 Inhibitory effects of plant secondary ID50 )jLg/ml)
metabolites on TM-induced cytotoxicity of PMN.
I) Compounds related to primary metabolites (a) Fatty acid derivatives agaricic acid lichesterinic acid protolichesterinic acid sorbic acid
(b) Sugarderivat/ve sinigrin (c) Amino acid den vative kainicacid
Fomeslaricis(13) Iichens(14) lichens (14)
c
66
c
t
c
ii) Phenolic compounds
iii) Isoprenoids (a) Monoterpenes aucubin borneol
(+)-camphor catalpol gardenoside gentiopicroside
c c c C
c c
— —
— — —
t 62 —
310 — — — —
— — — — —
53 182
65 —
500 100
t t t —
t —
15.8
t
15.2 7.8
t
10
t
—
t t
56 —
I — —
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(a) Phenol/cs derived from shikimate c amygdalin arbutin c caffeic acid c c chiorogenic acid c cinnamaldehyde c coniferin curcumin Curcumalonga(15) c 26-dimethoxyphenol c ellagic acid c ferulic acid c podophyllotoxin safrole c c salicin c syringic acid vanillic acid c vanillin c (b) Phenol/cs derived from acetate-malonate asterric acid Oosporasulphurea-ochracea (16) C griseofulvin lecanoric acid Iichens(14) p-orsellic acid Iichens(14) sulochrin Oospora sulphurea-ochracea (16) usnicacid lichens(14) (c) Coumarins and isocoumarins c coumarin c 6,7-dimethylesculetin esculin c hydra ngenol Hydrangea macrophylla var. thunbergiic Hydrangea macrophylla var. thunbergii' phyllodulcin (d) Flavonoids c astragalin baicalein c c baicalin c hesperidin c hematoxylin kaempferol 3-rutinoside Leonurussibiricus (17) (e) Qu/nones alizarin c alkannin c Ardisiasp. (18) ardisiaquinoneA embelin Ardisiasp.(18) emodin Preussiasp.(19) helicobasidin Hel/cobasidiummompa (20) C 2-hydroxynaphthoquinone islandicin Pen/c/Ilium islandicum (19) isodiospyrin Diospyrosspp.(21) juglone Diospyrosspp.(21) Pen/c/Ilium islandicum (19) luteoskyrin mompain Helicobasidium mompa (20) c sennosideA c shikonin Pen/c/Ilium islaridicum (19) skyrin tanshinone II Salvia m/ltiorrhiza c ubiquinone 9
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity of Polymorphonuclear Leucocytes Source
Compound
(—(-menthol nero!
perillalehyde
thymo! (b) Sesqu/terpenes picrotoxin ptaquiloside santonin (C) Diterpenes abietic acid
andrographolide gibberellin rebaudioside A rebaudioside B rebaudioside 0 rebaudioside E rubusoside stevioside (d) Triterpenes and saponins bauerenol dihydrolanosterol eburicoic acid entadasaponin II entadasaponin II! entadasaponin IV
c c c c c
C
Table 1 (continued). ID50 (pg/mI)
t — —
t —
—
Pteridium apuilinum (22) c c
Andrographispaniculatae
238
t
c
—
Stevia rebaudiana Stevia rebaudiand Stevia rebaudiana Stevia rebaudiana
—
Rubussp!
—
Stevia rebaudiana
—
Ardisiasieboldii(23( fungi of Polyporaceae (24) fungi of Po!yporaceae (24)
Entadasp. Entadasp.0
— — —
500 — — —
—
Entadasp. Panaxginseng
—
ginsenoside Rd
ginsenosideR
Panaxginsen
—
glycyrrhetic acid glycyrrhizin hederagenin lanosterol oleanolic acid pachymic acid (e) Sterols
cholesterol cholic acid deoxycholic acid digitonin digitoxin digitoxigenin diosgenin ergosterol /3-sitosterol
G/ycyrrh/zaglabra0
G/ycyrrhizag/abra Hedera tob/eri5 fungi of Polyporaceae (24)
Glycyrrhizag/abra fungi of Polyporaceae (24)
t —
— —
—
190
c c c c c c
—
c c c
—
t — —
t t
iv) Alkaloids and other nitrogen compounds (a) Tropane alkaloids
atropine scopolamine tropine
c c c
180
308 230
(b) Quinoline alkaloids
cinchonicinol cinchonidine cinchonine quinidine quinine (c) Isoquinoline alkaloids
berbamine berberine cepharanthine cepharanoline coclaurine cycleanine emetine homoaromoline isotetrandrine magnocurarine papaverine sinomenine tubocurarine trilobine (d) lndole alkaloids ajmaline
Cinchona ledger/anach
70
c c c c
53
Menispermaceae plants
33
c Menispermaceae plants Menispermaceae plants' Menispermaceae plants Menispermaceae plants
c Menispermaceae plants' Menispermaceae plants' Menispermaceae plants'
c Menispermaceae plants'
44 37 25
—
8 — —
57
t
10 38 8.7 52
400
c
—
Menispermaceae plants
—
c
38
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limonene
Planta Med. 58(1992) 141
142 Planta Med. 58(1992) Compound
Kaoru Kinoshita et a1.
Sourcea
I
cinchonaminone ergotamine quinamine reserpine strychinine vinbiastine vincrystine (e) Other alkaloids arecoline coichicine nicotine pilocarpine phalloidine physostigmine sparteine veratramin (f) Other nitrogen compounds caffeine chlorophyll a
ID5, (JtWnl)b
Table 1 (continued).
36 84
Cinchona ledgeriana c
Cinchona ledge riana' c C
c c c c c c c
340 2.2
575
C C
c
243
c
130
C
amphotericin B benzylpenicillin cefalexin chloramphenicol daunomycin erythromycin josamycin kanamycin kasugamycin oxytetracycline
c C
c C
c c
50 365
C C C
c
c = commercial products from Wako Pure Chemicals, Ltd. or Tokyo Kasel Kogyo Co., Ltd. = Inhibitory effects were not observable due to the cytotoxicity to the target cells. — = Compounds showing neither inhibition of activation of PMN nor cytotoxicity to the target cells. See text. Unpublished data of our laboratory. Provided by Dr. M. Kuroyanagi (University of Shizuoka(. Provided by Prof. 0. Tanaka (Hiroshima University). Provided by Prof. S. Shibata (University of Tokyo, retired). Provided by Dr. T. Naro (University of Shizuaka(. Provided by Dr. M. Akasu (Kaken Drug Co., Ltd.). Provided by Prof. R. Verpoorte (State University of Leiden).
three (santonin, bauerenol, and pachymic acid) which showed weak inhibition.
It might be possible that some of the tested compounds showed a selective toxic potential to PMN. How-
chonae cortex, quinine, quinidine, cinchonine, cm-
ever, the inhibitory effects of these compounds were unlikely to be due to the direct cytotoxicity to PMN, since the inhibitory effects were observed at nontoxic concentrations or, at least, in thirty times lower concentrations than those toxic to the target cells. Therefore, the inhibitory effects on
chonidine, and cinchonicinol, also showed the inhibition.
PMN is due not to dying PMN but to some inhibitory
Eight compounds among the fourteen isoquinoline al-
mechanism for the production of active oxygen from PMN.
Various alkaloids showed potent inhibition.
Three tropane alkaloids, atropine, scopolamine, and
tropine, were inhibitory. Five quinoline alkaloids of Gin-
kaloids, berbamine, cepharanthine, cycleanine, homoaromoline, isotetrandrine, magnocurarine, papaverine, and
sinomenine, showed the inhibition. However, no clear structure-activity relationship was observed among the al-
kaloids. Among the eight indole alkaloids tested, cmchonaminone from Cinchona, ajmaline from Rauwoijia, and ergotamine from ergot showed the inhibition. Among the other compounds tested, eight alkaloids of other types, two other nitrogen compounds and ten antibiotics, nicotine showed the highest activity (ID50 2.2zg/ml). Other alkaloids, arecoline, pilocarpine, and veratramine, caffeine, and two antibiotics, erythromycin and josamycin, were weakly inhibitory.
Examination of the positive compounds as hydrogen peroxide scavengers In the TAK-induced PMN cytotoxicity, hydrogen peroxide is a direct cytotoxic mediator (1, 2). A possible mechanism of the inhibitory effect of the positive compounds is direct hydrogen peroxide scavenging. Thus, the scavenging activity was examined by the method described
above. Among the compounds showing the inhibition, naturally occurring quinones such as ardisiaquinone A, embelin, and mompain, which have o-hydroxyquinone structures and were quite reactive to oxidizing agents,
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v) Antibiotics
Planta Med. 58(1992) 143
Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity ofPolymorphonuclear Leucocytes
H3C CH3
0
OH3C '
OH HO
(CH2)10—CH3
0 alizarin
HO
H3C (OH 0
heticobasidin
embe [in
ardisiaquinone A
H3
R1
R1
R2
R3
magnocurarine
R'
cepharanthine
—H quinine H3CO HO H HO ---H quinidine H3cO H cinchonine H H HO ---H
isotetrandrine
homoaromotine
Oci OH
H3C
0
II
H 2C
cinchonamine
berb amine
ajmat me
nicotine
Fig.2 Structures of the compounds exhibiting the inhibitory activity with ID50 value of less than 50ig/ml.
proved to be H202 scavengers at 20—100 g/ml, while other compounds such as ajmaline, arecoline, atropine, cmchonidine, coumarin, homoaromoline, magnocurarine, nicotine, pachymic acid, papaverine, podophyllotoxin, scopolamine, sorbic acid, tropine, and usnic acid were found not to be H202 scavengers.
activation were not observable until the final stage of the separation due to the toxicities exhibited by the fractions.
The total alkaloid fraction from Cinchona cortex showed inhibition of the same order of magnitude (ID50 53uglml) as the four quinoline alkaloids, quinine, quinidine, cinchonine, and cinchonidine. Besides these
compounds, three alkaloids quinamine (7, 8), cm-
Isolation of the active constituents
chonicinol, and cinchonaminone (9), were isolated from the
from herbal drugs To learn the applicability of the assay for the monitoring of crude extracts and in separation procedures, six drugs were analyzed with the assay to characterize the active constituents. In the case of Hydrangea Dulcis folium con-
taining isocoumarins, hydrangenol, and phyllodulcin, and Scopolia rhizome containing the tropane alkaloids, hyoscyamine and scopolamine, the inhibitory effects on the PMN
fractions exhibiting the inhibitory effects. The first compound did not show any effects, but the other two compounds isolated recently from the drug as monoamine oxidase inhibitors, showed inhibitory effects as shown in Table 1.
Several isoquinoline alkaloids showed the inhibitory effects. Thus two crude drugs, Magnolia cortex
and the roots of Stephania cephalantha, containing isoquinoline alkaloids, were analyzed. In the former, the
quaternary base fraction containing magnodurarine, Table 2 Inhibitory activity of constituents of Step/ian/a cepha/antha. Part
MeOH extract Total base Non-phenolic base Phenolic base
proved to be inhibitory. In the latter, non-phenolic bases such as cepharanthine, isotetrandrine, and cycleanine, and
lD50g/ml) 140 20 19 23
Substance
Cepharanthine Isotetrandrine Cycleanine Homoaromoline Berbamine Cepharanoline
lD50IygIml)
8
phenolic bases such as berbamine and homoaromoline proved to be the inhibitory constituents (Table 2).
38 57 10 33 —
The inhibitory constituent in Rauwolfia radix, containing indole alkaloids, was proved to exist in the strong base fraction and ajmaline but not reserpine and rescinamine proved to be the active constituent as shown in
Fig.3.
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3C
144 Planta Mcd. 58(1992)
Kaoru Kinos/zita et aL
induces the same potent PMN cytotoxicity as TAK, with hydrogen peroxide as a direct cytotoxic mediator (26). The effective alkaloids also inhibited the PMA-induced cytotoxicity (data not shown). PMAis thought to act through the acti-
100
It) U,
>,
60
C
4.) >.)
LI
40
20
0
0
6.25 12.5 25
50
100 200
Constituents of Rauwoifiae Radix (pg/mi) Fig.3 Effects of constituents of Rauwolfiae radix on TAK-induced PMN cytotoxicity. In the presence of TAK (100 yg/mI), the indicated concentrations of MeOH ext. (•), strong base fr. (A), weak base fr. (A), ajmaline and reserpine (LI) were added to the cytotoxicity assay mixture containing PMN (6 x 10) and [51Cr]-labelled MM2 target cells (5 x 1O). After incubation for 16 h, the radioactivity of the supernatant was determined. Values are means of duplicate determinations.
Discussion Among 151 natural products tested, covering a wide range of structural varieties and biosynthetic origins, the 42 compounds shown in Table I inhibited the activation of PMN by TAK. All of the compounds exhibiting po-
tent inhibitory activities (IDso: less than 50 yg/ml) are quinones or alkaloids. The modes of action of these two groups are different; most of the quinone derivatives showed direct H202 scavenging action, while the alkaloids showed the inhibition by different mechanism(s).
Several herbal drug constituents have been shown to exhibit anti-inflammatory activities. A naphthoquinone, shikonin, and an isoquinoline alkaloid, berberine, showing inhibitory activities in this study, are known to be anti-inflammatory agents. Inhibitory activities of cepharanthine, the isoquinoline alkaloid from Step/ian/a cepharantha, on the production of active oxygen by PMN and on PMA-induced inflammation have been known (M. Akasu, personal communication). Nicotine showed a remarkable inhibitory effect in this study. Sasagawa et al. (25) examined the effect of nicotine on the functions of human PMN. They reported that nicotine had no effect on migration but inhibited both lysosomal enzyme release and O
production from PMN and they found that the effect of nicotine was not altered by cholinergic antagonists and agonists. They suggested that nicotine acts directly on PMN through the nicotine-specific receptors. The inhibitory mechanism of these effective alkaloids has not been analyzed in this study. However, we examined the effect on PMN cytotoxicity induced by a different type of stimulus; phorbol myristate acetate (PMA). PMA
The assay that we used for screening and monitoring of the inhibitory compounds was not entirely satisfactory. As shown in the cases of Hydrangea Dulcis folium and Scopoliae rhizome, when the crude drugs contained cytotoxic compounds and the inhibitory activities were not so strong, the activities were not observable until the final stages of the separation. However, as in the cases of Rauwolfiae radix, Cinchonae cortex, and the root of Stephania cepharantha, when the drugs did not contain cytotoxic compounds, the method was found to be effective for monitoring systems of the separation. In the case of alkaloids, the method seems in general to be applicable after separation of the total alkaloidal fraction.
Acknowledgements The authors indebted to Professors S. Shibata, 0. Tanaka, and R. Verpoorte, and Drs. M. Akasu, T. Noro, and M. Kuroyanagi for providing samples and spectral data. The authors thank Mr. I. Takahashi and Misses J. Gonoi, H. Kohsaka, N. Ogi, andY. Ono and Mrs. C. Wakabayashi for their help in a part of this work.
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Inhibitory Effects of Plant Secondary Metabolites on Cytotoxic Activity ofPolymorphonuclear Leucocytes
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