Planta Med. 56 (1990) 259
Anti-Platelet Action of GU-7, A 3 -Arylcoumarin Derivative, Purified from Glycyrrhizae Radix Masafo Tawata', Yoshiki Yoda', Kaoru Aida , Hideo Shindo', Hiroshi Sasaki2, Masao Chin2 and Toshiinasa Onaya'3 The Third Department of Internal Medicine, University of Yamanashi Medical School, Tamaho, Yamanashi 409-38, Japan Tsumura Laboratory, Ami-machi, Ibaragi 300-11, Japan 'Address for correspondence 2
Abstract
On the other hand, hyperaggregability of platelets in diabetic patients has also been reported as a cause of chronic diabetic complications (1). So it seems like-
We have purified GU-7, a 3-arylcoumarin
derivative, from glycyrrhizae radix, which is a crude drug of kampo herbal medicines. This was identified to be a new chemical compound and was found to have an anti-platelet action. GU-7 inhibited platelet aggregation, phosphorylation of 40K and 20K dalton proteins, inositol
I ,4,5-trisphosphate production, intraplatelet calcium increase and phosphodiesterase activity in vitro. The data indicate that GU-7 inhibits platelet aggregation by increasing intraplatelet cAMP concentration. We have already reported the existence of aldose reductase inhibitors in some kampo medicines. In addition to the aldose reductase inhibiting action, anti-platelet action may also explain the mechanism by which kampo medicines are effective to diabetic neuropathy.
Key words
3-Arylcoumarin derivative, anti-platelet action, phosphodiesterase inhibitor, kampo medicines.
Introduction Recently, increased poiyoi pathway metaholism and non-enzymatic glycosylation of various proteins have been implicated in the pathogenesis of chronic diabetic complications (1). Several drugs which inhibit aldose reductase, a rate-limiting enzyme in polyol metabolism, are now expected to be used as a possible remedy for the treatment of chronic diabetic complications (2—4). Traditionally in Japan, some of the kampo medicines (traditional oriental herb prescriptions) have long been used for the treatment of subjective symptoms of diabetic neuropathy (5, 6). We have been investigating the
mechanism of actions of these kampo medicines and recently demonstrated the existence of aldose reductase inhibitors in some kampo medicines (7, 8).
ly that normalization of hyperaggregability of platelets in diabetic patients may be beneficial for the prevention or the treatment of chronic diabetic complications. More recently, we have purified GU-7, a 3arylcoumarin derivative, from glycyrrhizae radix, which is widely used as a crude drug of kampo medicines in Japan. This paper describes the anti-platelet action of this cornpound.
Materials and Materials
thods
Boiled water extracts (400 g) of glycyrrhizae radix were applied to Sephadex LI1-20 column and eluted succes-
sively with H,0, 11,0-methanol (1: 1), methanol, and finally acetone-H,0 (1: 1), and six fractions (Frs. A, B, Cs, Cp, D, and F) were obtained as described previously(s). Fr. Cp and Fr. D were retreated with Sephadex L1T-20, MCI gel CHP2Op and eluted with var-
ious solvent systems, and finally seven single compounds have been purified as shown in Fig. 1. These compounds were tentatively named as GUs 1—7 in the order of purification. The identification of the known compounds (GUs 1—6) and the chemical structure of a new compound (Gu-7) were determined by chemical and physical analyses. The molecular weight and structure of GU-7 are shown in Fig. 2.
GU-7 was elucidated to be 3-(3,4-dihydroxyphenyl)- 8 -hydroxymethyl- 8-methyl- 5 -methoxy-6, 7-dihydro-2H, 811-benxo[1 ,2-b;5,4-b']dipyran-2-one.
Aequorin was purchased from Chronolog Japan. Prostaglandin F1 (PGEI) was a generous gift from Ono Pharmaceut-
ical Co., Ltd. Sodtum dodecyl sulfate (SDS), acrylamide, bisacrylamide were purchased from Bin Rad. Adenostne 3',S'-cyclic monophosphate (cAMP), 1 2-O-tetradecanoylphorbol-1 3-acetate (TPA) were purchased from Sigma Chemical Co., Ltd., Thrombin and collagen were purchased from Mochida Pharmaceutical Co., Ltd., and Chronolog Japan, respectively.
X-ray films (X-Omat) were purchased from Kodak. ["PI-Orthophosphate and ['HIcAMP were purchased from Amersham Japan. cAMP assay kits were purchased from Yamasa Co., Ltd.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Received: April 10, 1989
260 PlantczMed. 56 (1990)
Masato Tawata etal.
Boiled water extract of Giycyrrhizae Radix
according to the method of Laernmli (11). The gels were stained, destained and dried, and then finally autoradiography was done.
Sephadex LH —20 COlumn chromatography
Measurements of inositol 1,4,5-trisphos-
H20, H20—methanoi, methanol, acetone—H20
phare (1P3)
[32P]-Labelled platelets were obtained as de-
H20 eluate
scribed above. The experiments were started by the addition ofcollagen. and terminated by the addition of 1 ml of cold methanol at the time indicated, and left on ice for 10mm. One and a half ml of chloroform and 0.5 ml of 2.4 N HC1 were then added, followed by centrifugation at 400xg for 10mm (10). [32P]-1P3 was separated from the aqueous phase according to the method of Berrige (12).
actioj F Fraction Hi supernatant
[tion Cs
precipitate I Fraction
Gu—7
GU-2
GU-3 GlJ-4
Fig. 1 The scheme of the purification of GU-7 from glycyrrhizae radix.
HO H3C
Aequorin was loaded into platelets according to the method of Johnson et a!. (9), and intracellular calcium was determined by a Lumi-aggregometer (AHS, Japan). Intracellular calcium concentrations were calculated according to the specification attached to aequormn.
Determination ofphosphodiesterase activity
— 00 OCH3HO
GU-7
(Mr
Platelet phosphodiesterase activity was deterOH
mined according to the method of Hidaka et al. (13).
Measurement of intraplatelet cAMP concentration
384)
Fig. 2 The molecular structure and the relative molecular mass of GU-7.
Platelet suspensions in the absence or presence of 10 .cg/ml of GU-7 were boiled for 3 mm, followed by centrifuga-
Preparation of washed platelets
tion. cAMP concentration in the supernatant was determined by radioimmunoassay. Statistical significance was assessed by Student's t test.
Blood was obtained from healthy volunteers in
Results
the presence of 1/10 volume of 3.8% citric acid. Platelet rich plasma (PEP) was obtained by centrifugation (150 x g, 15 miii) at room temperature. PGE1 was added to make a final concentration of 1 iM, and then centrifuged again (400 x g, 15 mm) at room temperature. Platelets were suspended in Hepes-Tyrodes buffer (NaC1 129 mM, NaHCO3 8.9 mM, KH2PO4 0.8 mM, MGC12 0.8 mM, glucose
5.6mM, Hepes 10mM, pH 7.4) containing 1 iM of PGE1, and then centrifuged again (400 x g, 15 mm) at room temperature. Platelets
were resuspended in Hepes-Tyrodes buffer, and gel filtered through Sepharose 2B which had been equilibrated with the same buffer (9).
Effect of GU-7 on platelet aggregation rate As shown in Fig. 3, platelet aggregation induced by 0.01 U/ml of thrombin (Fig. 3a) was inhibited progressively as the concentration of GU-7 increased. The effect of GU-7 was observed even at a concentration of 1 .tg/ ml. GU-7 also inhibited platelet aggregation rate induced by collagen (data not shown). Platelet aggregation induced by 50 nM of TPA, however, was not inhibited by GU-7 at a concentration of 20 .tg/ml (Fig. 3b).
The number of platelets was adjusted to 4 x 10/ mm3 by the same buffer, and then diluted by the addition of equal amounts of Hepes-Tyrodes buffer containing 2mM CaCI2 and various concentrations of GU-7. GU-7 was dissolved in ethanol and the
final concentration of ethanol in all experiments was 0.1%. Platelets were exposed to GU-7 for 10mm before the initiation of experiments.
Measurement of platelet aggregation Platelet aggregation was determined by Hematracer (NBS-Japan).
Phosphorylation of platelet proteins Ten ml of PEP were incubated with 1 mCi of[32P1-
Effect of GU-7 on the phosphorylation of
platelet proteins The effects of GU-7 on the phosphorylation of platelet proteins are shown in Fig. 4. GU-7, at a concentration of 20ig/ml, inhibited the phosphorylation of 40 and 20 Kdalton proteins induced by thrombin (Fig. 4A,a). The
effect was observed at 30 seconds after the addition of thrombin. GU-7 also inhibited the phosphorylation of 40 Kdalton protein induced by collagen (data not shown). The effect of GU-7 on 40 Kdalton protein phosphorylation was not observed when platelets were stimulated by TPA (Fig. 4b).
orthophosphate at 37°C for 90mm (10). Then, [32P}-labelled washed platelets were obtained as described above. Ten mm after the pre-exposure to GU-7, the experiments were started by the addition of collagen, thrombin or TPA. and terminated by the addition of an equal amount of SDS sample buffer, followed by boiling for 3 mm. SDS-polyacrylamide gel electrophoresis (PAGE) was done
Effect of GU-7 on 1P3 production Fig. 5 shows the effects of GU-7 on {32P1-1P3
production after collagen stimulation. In the absence of GU-
7, [P]-IP3 began to increase 5 seconds after the exposure
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Measurement of intraplatelet calcium GU-5 GU-6
Planta Med. 56(1990) 261
Aoti-PlalelelAction of GU-7. A 3-Arylcoarnarin Derivative, Parifiedfrorn Glycyrrlzizae Radix
a
thrombin
t
P E1G1 E2E3G3E4G4
MYOSI,
C
G( 1) G(4)
!I11 I V
G(20)
2OK—s.
4
t
20 10
,ct2O
EoH
G:GU-7
GU-7
MWC1 2341234
5
Time (Mm)
—40K
Fig. 3 Platelets were stimulated at the points of arrows. C indicates control and 0 indicates GU-7, with the concentrations indicated in parenthesis in .tglml. The concentrations of thrombin (a) and TPA )b) were 0.01 U/mI and 50 nM, respectively.
—20K
to collagen, reached a maximum at 30 seconds, and then gradually decreased. GU-7 inhibited the increase of [32P11P3 after collagen stimulation.
Effect of GU-7 on intraplatelet calcium
The effect of GU-7 on intraplatelet calcium concentration and on platelet aggregation were shown in Fig. 6. Thrombin, at a concentration of 0.1 U/mi, increased
P El Gi E2G2 E3G3 E4G4
MyosinTTT 40K—.
intraplatelet calcium concentration from 3.16 RM to 4.16 RM. GU-7 at a concentration of 20 .tg/ml reduced the increase of intraplatelet calcium concentration to 3.45 1.tM induced by thrombin. Platelet aggregation rate was slightly retarded at 20 g/ml of GU-7.
ze— —.0
20K—
—
Effect of GU- 7 on platelet
phosphodiesterase activity Fig. 7 shows the effects of GU-7 on platelet phosphodiesterase activity. Platelet phosphodiesterase ac-
tivity was progressively inhibited as the concentration of GU-7 increased. The IC50 was calculated to be 1.05 g/ml. Effect of GU-7 on cAMP concentration in
platelets Table I indicates cAMP concentration in platelets. Intraplatelet cAMP concentration in the absence ofGU-7 was 118.6 12.7 pmoles/105 cells. GU-7, at 10 .tg/ ml, increased intraplatelet cAMP concentration to 190.6 18.3 pmoies/105 cells.
E;EoH
G:GU—7
Fig. 4 Platelets were stimulated either by thrombin (A, a) or TPA )b). Myosin, 40K and 20K indicate phoretic mobility of myosin heavy chain, 40 Kdalton protein and 20 Kdalton protein, respectively. A is protein staining pattern of the SDS-PAGE and a, b are the autoradiographies. Protein staining patterns of )b) are abbreviated. P and MW indicate platelet actomyosin as molecular weight markers. EoH and GU-7 indicate in the absence or in the presence of 20 g/ml of GU7, respectively. C indicates control sample without stimulator. The numbers on the lanes indicate time after stimulation (1:0", 2: 30", 3:60", 4:90"). The concentrations of thrombin and TPA were 0.01 U/mI and 50 nM, respectively.
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30
E:EoH
b
TPA
262 Planta Med. 56(1990)
Masato Tawata et al. Table 1 The effect of GU-7 on cAMP concentration in platelets.
collagen
cAMP content
(pmoles/105 cells)
Control
4-
118.6 190.6
GU-7
E
a.
0 '-4
12.7
18.3'
Cyclic nucleotides were measured after 10 mm exposure to 10 sg/ml of GU.7. The values are expressed as mean SEM from 4 determinations. Significance of difference from control, P < 005.
00, c'J
Discussion
Fig. 5 The ordinate indicates the production of 1P3 and the abscissa indicates time in seconds after stimulation. [32PJ.Iabelled platelets were stimulated by collagen (2 .sg/ml) at the point of arrow and terminated at the time indicated by the addition of cold methanol.
Our experiments have revealed that GU-7 inhibits platelet aggregation, phosphorylation of 40 and 20 Kdalton proteins, [32P1-1P3 production, and intracellular calcium increase. The effect of GU-7 on AlP release could not be observed with the Lumi-aggregometer, because GU7 directly inhibited the luciferin-luciferase system.
21
Twenty Kdalton protein is a substrate of
C
0 Ce
C) a)
myosin light chain kinase, which is calcium and calmodulin dependent (14, 15). Since 1P3 is considered to be a second messenger which mobilizes calcium from microsome (16— 18), it is speculated that GU-7 inhibits myosin light chain kinase, probably by inhibiting the production of 1P3. On the
41
C) C)
I
other hand, since 40 Kdalton protein is reported to be an endogenous substrate of protein kinase C (19), our data suggest that GU-7 also inhibits protein kinase C. Recently, it has become clear that protein kinase C is directly activated
Ca2+
Fig. 6 Thrombin (T, 0.1 U/mI) was added at the point of arrow. Aequorin signals were recorded on the bottom. C indicates control and G indicates 20 .Lg/ml of GU-7. Thrombin increased intraplatelet calcium concentration from 3.16 .tM to 4.16 sM. In the presence of 20 sg/ml of GU-7, maximum concentration of calcium by thrombin was reduced to 3.45 ruM.
by TPA (20, 21). Our observation that 40Kdalton protein phosphorylation and platelet aggregation induced by TPA were not inhibited by GU-7 may suggest that the effect of GU-7 on protein kinase C is an indirect action probably by inhibiting the increase of diacylglycerol, which is co-produced with 1P3 (17, 18).
Our experiments have also revealed that GU-7 inhibits platelet phosphodiesterase activity and increases intraplatelet cAMP concentration. The IC50 of GU-7 on phosphodiesterase activity well agrees with the concent-
ration by which platelets aggregation was inhibited. It has been reported that cAMP inhibits receptor mediated phosphatidylinositol turnover (22). So, we speculate that GU-7
(%)
>
ii
> 0
inhibits phospholipase C activity by increasing intraplatelets cyclic nucleotide concentration. It is not clear whether the whole action of GU-7 on platelets can be
a)
a
Ca
explained only by increasing cyclic nucleotide alone. Hence, experiments are now under way to investigate other possible actions.
a) a)
0
0
Traditionally in Japan, kampo medicines
IC50 1 .05).JgIml 0 0.1
0.5
1
have long been used for the alleviation of subjective 5
10
(j.ig/ml) Fig. 7 The ordinate indicates phosphodiesterase activity and the abscissa indicates the concentration of GU-7 in logarithm.
symptoms of chronic diabetic neuropathy. We have already
reported the existence of aldose reductase inhibitors in kampo medicines (7, 8). In addition to the aldose reductase inhibiting action, anti-platelet action may also explain the mechanism by which the kampo medicines are effective to diabetic neuropathy.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Time (Sec)
Among 7 compounds purified from glycyrrhizae radix, GU-7 has never been reported. The chemical structure has been determined to be a derivative of 3-arylcoumarin, that is, 3-(2 ,4-dihydroxyphenyl)-8-hydroxymethyl-8-methyl-5-methoxy-6,7-dihydro-2H,8H-benzo[1, 2-b;5,4-b']dipyran-2-one.
Anti-Platelet Action of GU-7, A 3-Arylcou,narin Derivative, Purfiedfrom Glycyrrhizae Radix
2
Brownlee, M., Cerami, A. (1981) Ann. Rev. Biochem. 50, 385—432. Beyer-Mears, A., Cruz, E. (1985) Diabetes 34, 15—21. Terashima, H., Hania, K., Yamamoto, R., Tsuboshima, M., Kikkawa, R., Hatanaka, 1., Shigeta, Y. (1984) J. Pharmacol. Experim. Therap. 229, 226—230. Cogan, D. G., Kinoshita, J. H., Kador, P. F., Robison, W. G., Datiles, M. B., Cobo, L. M., Kupfer, C. (1984) Ann. mt. Med. 101,82—91.
Sato, Y., Sakamoto, N., Tomita, A., Shibata, M., Takeuchi, T., 6
8
Harada, N., Kate, K., Takahashi, A., Tsuchida, I. (1984) Rinsho to Kenkyu 61, 2347—2356 (in Japanese). Sakamoto, N., Sato, Y., Goto, Y., Ikeda, Y., Takahashi, A., Yano, S., Takeda, R., Baba, S., Kaneko, T., Mimura, G. (1987) in: Recent trends in management of diabetes mellitus, (Sakamoto, N., Alberti, K. G. M. M., Hotta, N., eds.), Amsterdam, Bxcerpta Med., pp. 552— 554. Aida, K., Shindo, H., Tawata, M., Onaya, T. (1987) Planta Med. 53, 131—135. Aida, K., Tawata, M., Shindo, H., Onaya, T., Sasaki, H., Nishimura, H., Chin, M., Mitsuhashi, H. (1989) Planta Med. 55, 22—26. Johnson, P. C., Ware, J. A., Cliveden, P. B., Smith, M., Dvorak, A. M., Salzman, F. W, (1985) J. Biol. Chem. 260, 2069—2076.
'
18 Agranoff, B. W., Murthy, P., Seguin, E. B. (1983) J. Biol. Chem. 258, 2076— 2078. Laemmli, U. K. (1970) Nature 227, 680—685. 12 Berridge, M. J. (1983) Biochem. J. 212, 849—858. 13 Hidaka, H., Asano, T. (1976) J. Biol. Chem. 251, 7508—7516. 14 Imaoka, T., Lynham, J. A., Haslam, R. J. (1983) J. Biol. Chem. 258, 15 16 17 18
19 20 21
11404—11414. Nishikawa, M., Tanaka, T., Hidaka, H. (1980) Nature 287,863—865. O'Rourke,F.A.,Halenda,S.P.,Zavoico,G.B.,Feinstein,M.B.(1985) J. Biol. Chem. 260, 956—962. Berridge, M. J. (1984) Biochem. J. 220, 345—360. Hokin, L. F.. (1985) Ann. Rev. Biochem. 54, 205—235. Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309—3 17. Castagna,
M., Takai, Y., Kaibuchi, K., Sane, K., Kikkawa, U.,
Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847—7851. Kikkawa, U., Kaibuchi, K., Castagna, M., Yamanishi, J., Sane, K., Tanaka, Y., Miyake, R., Takai, Y., Nishizuka, Y. (1984) Adv. Cyclic Nucleotide and Protein Phosphorylation Research 17, 437—442. Takai, Y., Kikkawa, U., Kaibuchi, K., Nishizuka, Y. (1984) Adv. Cyclic Nucleotide and Protein Phosphorylation Research 18, 119—158. Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
References
Planta Med. 56(1990) 263
Anti-Platelet Action of GU-7, A 3 -Arylcoumarin Derivative, Purified from Glycyrrhizae Radix Masafo Tawata', Yoshiki Yoda', Kaoru Aida , Hideo Shindo', Hiroshi Sasaki2, Masao Chin2 and Toshiinasa Onaya'3 The Third Department of Internal Medicine, University of Yamanashi Medical School, Tamaho, Yamanashi 409-38, Japan Tsumura Laboratory, Ami-machi, Ibaragi 300-11, Japan 'Address for correspondence 2
Abstract
On the other hand, hyperaggregability of platelets in diabetic patients has also been reported as a cause of chronic diabetic complications (1). So it seems like-
We have purified GU-7, a 3-arylcoumarin
derivative, from glycyrrhizae radix, which is a crude drug of kampo herbal medicines. This was identified to be a new chemical compound and was found to have an anti-platelet action. GU-7 inhibited platelet aggregation, phosphorylation of 40K and 20K dalton proteins, inositol
I ,4,5-trisphosphate production, intraplatelet calcium increase and phosphodiesterase activity in vitro. The data indicate that GU-7 inhibits platelet aggregation by increasing intraplatelet cAMP concentration. We have already reported the existence of aldose reductase inhibitors in some kampo medicines. In addition to the aldose reductase inhibiting action, anti-platelet action may also explain the mechanism by which kampo medicines are effective to diabetic neuropathy.
Key words
3-Arylcoumarin derivative, anti-platelet action, phosphodiesterase inhibitor, kampo medicines.
Introduction Recently, increased poiyoi pathway metaholism and non-enzymatic glycosylation of various proteins have been implicated in the pathogenesis of chronic diabetic complications (1). Several drugs which inhibit aldose reductase, a rate-limiting enzyme in polyol metabolism, are now expected to be used as a possible remedy for the treatment of chronic diabetic complications (2—4). Traditionally in Japan, some of the kampo medicines (traditional oriental herb prescriptions) have long been used for the treatment of subjective symptoms of diabetic neuropathy (5, 6). We have been investigating the
mechanism of actions of these kampo medicines and recently demonstrated the existence of aldose reductase inhibitors in some kampo medicines (7, 8).
ly that normalization of hyperaggregability of platelets in diabetic patients may be beneficial for the prevention or the treatment of chronic diabetic complications. More recently, we have purified GU-7, a 3arylcoumarin derivative, from glycyrrhizae radix, which is widely used as a crude drug of kampo medicines in Japan. This paper describes the anti-platelet action of this cornpound.
Materials and Materials
thods
Boiled water extracts (400 g) of glycyrrhizae radix were applied to Sephadex LI1-20 column and eluted succes-
sively with H,0, 11,0-methanol (1: 1), methanol, and finally acetone-H,0 (1: 1), and six fractions (Frs. A, B, Cs, Cp, D, and F) were obtained as described previously(s). Fr. Cp and Fr. D were retreated with Sephadex L1T-20, MCI gel CHP2Op and eluted with var-
ious solvent systems, and finally seven single compounds have been purified as shown in Fig. 1. These compounds were tentatively named as GUs 1—7 in the order of purification. The identification of the known compounds (GUs 1—6) and the chemical structure of a new compound (Gu-7) were determined by chemical and physical analyses. The molecular weight and structure of GU-7 are shown in Fig. 2.
GU-7 was elucidated to be 3-(3,4-dihydroxyphenyl)- 8 -hydroxymethyl- 8-methyl- 5 -methoxy-6, 7-dihydro-2H, 811-benxo[1 ,2-b;5,4-b']dipyran-2-one.
Aequorin was purchased from Chronolog Japan. Prostaglandin F1 (PGEI) was a generous gift from Ono Pharmaceut-
ical Co., Ltd. Sodtum dodecyl sulfate (SDS), acrylamide, bisacrylamide were purchased from Bin Rad. Adenostne 3',S'-cyclic monophosphate (cAMP), 1 2-O-tetradecanoylphorbol-1 3-acetate (TPA) were purchased from Sigma Chemical Co., Ltd., Thrombin and collagen were purchased from Mochida Pharmaceutical Co., Ltd., and Chronolog Japan, respectively.
X-ray films (X-Omat) were purchased from Kodak. ["PI-Orthophosphate and ['HIcAMP were purchased from Amersham Japan. cAMP assay kits were purchased from Yamasa Co., Ltd.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Received: April 10, 1989
260 PlantczMed. 56 (1990)
Masato Tawata etal.
Boiled water extract of Giycyrrhizae Radix
according to the method of Laernmli (11). The gels were stained, destained and dried, and then finally autoradiography was done.
Sephadex LH —20 COlumn chromatography
Measurements of inositol 1,4,5-trisphos-
H20, H20—methanoi, methanol, acetone—H20
phare (1P3)
[32P]-Labelled platelets were obtained as de-
H20 eluate
scribed above. The experiments were started by the addition ofcollagen. and terminated by the addition of 1 ml of cold methanol at the time indicated, and left on ice for 10mm. One and a half ml of chloroform and 0.5 ml of 2.4 N HC1 were then added, followed by centrifugation at 400xg for 10mm (10). [32P]-1P3 was separated from the aqueous phase according to the method of Berrige (12).
actioj F Fraction Hi supernatant
[tion Cs
precipitate I Fraction
Gu—7
GU-2
GU-3 GlJ-4
Fig. 1 The scheme of the purification of GU-7 from glycyrrhizae radix.
HO H3C
Aequorin was loaded into platelets according to the method of Johnson et a!. (9), and intracellular calcium was determined by a Lumi-aggregometer (AHS, Japan). Intracellular calcium concentrations were calculated according to the specification attached to aequormn.
Determination ofphosphodiesterase activity
— 00 OCH3HO
GU-7
(Mr
Platelet phosphodiesterase activity was deterOH
mined according to the method of Hidaka et al. (13).
Measurement of intraplatelet cAMP concentration
384)
Fig. 2 The molecular structure and the relative molecular mass of GU-7.
Platelet suspensions in the absence or presence of 10 .cg/ml of GU-7 were boiled for 3 mm, followed by centrifuga-
Preparation of washed platelets
tion. cAMP concentration in the supernatant was determined by radioimmunoassay. Statistical significance was assessed by Student's t test.
Blood was obtained from healthy volunteers in
Results
the presence of 1/10 volume of 3.8% citric acid. Platelet rich plasma (PEP) was obtained by centrifugation (150 x g, 15 miii) at room temperature. PGE1 was added to make a final concentration of 1 iM, and then centrifuged again (400 x g, 15 mm) at room temperature. Platelets were suspended in Hepes-Tyrodes buffer (NaC1 129 mM, NaHCO3 8.9 mM, KH2PO4 0.8 mM, MGC12 0.8 mM, glucose
5.6mM, Hepes 10mM, pH 7.4) containing 1 iM of PGE1, and then centrifuged again (400 x g, 15 mm) at room temperature. Platelets
were resuspended in Hepes-Tyrodes buffer, and gel filtered through Sepharose 2B which had been equilibrated with the same buffer (9).
Effect of GU-7 on platelet aggregation rate As shown in Fig. 3, platelet aggregation induced by 0.01 U/ml of thrombin (Fig. 3a) was inhibited progressively as the concentration of GU-7 increased. The effect of GU-7 was observed even at a concentration of 1 .tg/ ml. GU-7 also inhibited platelet aggregation rate induced by collagen (data not shown). Platelet aggregation induced by 50 nM of TPA, however, was not inhibited by GU-7 at a concentration of 20 .tg/ml (Fig. 3b).
The number of platelets was adjusted to 4 x 10/ mm3 by the same buffer, and then diluted by the addition of equal amounts of Hepes-Tyrodes buffer containing 2mM CaCI2 and various concentrations of GU-7. GU-7 was dissolved in ethanol and the
final concentration of ethanol in all experiments was 0.1%. Platelets were exposed to GU-7 for 10mm before the initiation of experiments.
Measurement of platelet aggregation Platelet aggregation was determined by Hematracer (NBS-Japan).
Phosphorylation of platelet proteins Ten ml of PEP were incubated with 1 mCi of[32P1-
Effect of GU-7 on the phosphorylation of
platelet proteins The effects of GU-7 on the phosphorylation of platelet proteins are shown in Fig. 4. GU-7, at a concentration of 20ig/ml, inhibited the phosphorylation of 40 and 20 Kdalton proteins induced by thrombin (Fig. 4A,a). The
effect was observed at 30 seconds after the addition of thrombin. GU-7 also inhibited the phosphorylation of 40 Kdalton protein induced by collagen (data not shown). The effect of GU-7 on 40 Kdalton protein phosphorylation was not observed when platelets were stimulated by TPA (Fig. 4b).
orthophosphate at 37°C for 90mm (10). Then, [32P}-labelled washed platelets were obtained as described above. Ten mm after the pre-exposure to GU-7, the experiments were started by the addition of collagen, thrombin or TPA. and terminated by the addition of an equal amount of SDS sample buffer, followed by boiling for 3 mm. SDS-polyacrylamide gel electrophoresis (PAGE) was done
Effect of GU-7 on 1P3 production Fig. 5 shows the effects of GU-7 on {32P1-1P3
production after collagen stimulation. In the absence of GU-
7, [P]-IP3 began to increase 5 seconds after the exposure
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Measurement of intraplatelet calcium GU-5 GU-6
Planta Med. 56(1990) 261
Aoti-PlalelelAction of GU-7. A 3-Arylcoarnarin Derivative, Parifiedfrorn Glycyrrlzizae Radix
a
thrombin
t
P E1G1 E2E3G3E4G4
MYOSI,
C
G( 1) G(4)
!I11 I V
G(20)
2OK—s.
4
t
20 10
,ct2O
EoH
G:GU-7
GU-7
MWC1 2341234
5
Time (Mm)
—40K
Fig. 3 Platelets were stimulated at the points of arrows. C indicates control and 0 indicates GU-7, with the concentrations indicated in parenthesis in .tglml. The concentrations of thrombin (a) and TPA )b) were 0.01 U/mI and 50 nM, respectively.
—20K
to collagen, reached a maximum at 30 seconds, and then gradually decreased. GU-7 inhibited the increase of [32P11P3 after collagen stimulation.
Effect of GU-7 on intraplatelet calcium
The effect of GU-7 on intraplatelet calcium concentration and on platelet aggregation were shown in Fig. 6. Thrombin, at a concentration of 0.1 U/mi, increased
P El Gi E2G2 E3G3 E4G4
MyosinTTT 40K—.
intraplatelet calcium concentration from 3.16 RM to 4.16 RM. GU-7 at a concentration of 20 .tg/ml reduced the increase of intraplatelet calcium concentration to 3.45 1.tM induced by thrombin. Platelet aggregation rate was slightly retarded at 20 g/ml of GU-7.
ze— —.0
20K—
—
Effect of GU- 7 on platelet
phosphodiesterase activity Fig. 7 shows the effects of GU-7 on platelet phosphodiesterase activity. Platelet phosphodiesterase ac-
tivity was progressively inhibited as the concentration of GU-7 increased. The IC50 was calculated to be 1.05 g/ml. Effect of GU-7 on cAMP concentration in
platelets Table I indicates cAMP concentration in platelets. Intraplatelet cAMP concentration in the absence ofGU-7 was 118.6 12.7 pmoles/105 cells. GU-7, at 10 .tg/ ml, increased intraplatelet cAMP concentration to 190.6 18.3 pmoies/105 cells.
E;EoH
G:GU—7
Fig. 4 Platelets were stimulated either by thrombin (A, a) or TPA )b). Myosin, 40K and 20K indicate phoretic mobility of myosin heavy chain, 40 Kdalton protein and 20 Kdalton protein, respectively. A is protein staining pattern of the SDS-PAGE and a, b are the autoradiographies. Protein staining patterns of )b) are abbreviated. P and MW indicate platelet actomyosin as molecular weight markers. EoH and GU-7 indicate in the absence or in the presence of 20 g/ml of GU7, respectively. C indicates control sample without stimulator. The numbers on the lanes indicate time after stimulation (1:0", 2: 30", 3:60", 4:90"). The concentrations of thrombin and TPA were 0.01 U/mI and 50 nM, respectively.
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30
E:EoH
b
TPA
262 Planta Med. 56(1990)
Masato Tawata et al. Table 1 The effect of GU-7 on cAMP concentration in platelets.
collagen
cAMP content
(pmoles/105 cells)
Control
4-
118.6 190.6
GU-7
E
a.
0 '-4
12.7
18.3'
Cyclic nucleotides were measured after 10 mm exposure to 10 sg/ml of GU.7. The values are expressed as mean SEM from 4 determinations. Significance of difference from control, P < 005.
00, c'J
Discussion
Fig. 5 The ordinate indicates the production of 1P3 and the abscissa indicates time in seconds after stimulation. [32PJ.Iabelled platelets were stimulated by collagen (2 .sg/ml) at the point of arrow and terminated at the time indicated by the addition of cold methanol.
Our experiments have revealed that GU-7 inhibits platelet aggregation, phosphorylation of 40 and 20 Kdalton proteins, [32P1-1P3 production, and intracellular calcium increase. The effect of GU-7 on AlP release could not be observed with the Lumi-aggregometer, because GU7 directly inhibited the luciferin-luciferase system.
21
Twenty Kdalton protein is a substrate of
C
0 Ce
C) a)
myosin light chain kinase, which is calcium and calmodulin dependent (14, 15). Since 1P3 is considered to be a second messenger which mobilizes calcium from microsome (16— 18), it is speculated that GU-7 inhibits myosin light chain kinase, probably by inhibiting the production of 1P3. On the
41
C) C)
I
other hand, since 40 Kdalton protein is reported to be an endogenous substrate of protein kinase C (19), our data suggest that GU-7 also inhibits protein kinase C. Recently, it has become clear that protein kinase C is directly activated
Ca2+
Fig. 6 Thrombin (T, 0.1 U/mI) was added at the point of arrow. Aequorin signals were recorded on the bottom. C indicates control and G indicates 20 .Lg/ml of GU-7. Thrombin increased intraplatelet calcium concentration from 3.16 .tM to 4.16 sM. In the presence of 20 sg/ml of GU-7, maximum concentration of calcium by thrombin was reduced to 3.45 ruM.
by TPA (20, 21). Our observation that 40Kdalton protein phosphorylation and platelet aggregation induced by TPA were not inhibited by GU-7 may suggest that the effect of GU-7 on protein kinase C is an indirect action probably by inhibiting the increase of diacylglycerol, which is co-produced with 1P3 (17, 18).
Our experiments have also revealed that GU-7 inhibits platelet phosphodiesterase activity and increases intraplatelet cAMP concentration. The IC50 of GU-7 on phosphodiesterase activity well agrees with the concent-
ration by which platelets aggregation was inhibited. It has been reported that cAMP inhibits receptor mediated phosphatidylinositol turnover (22). So, we speculate that GU-7
(%)
>
ii
> 0
inhibits phospholipase C activity by increasing intraplatelets cyclic nucleotide concentration. It is not clear whether the whole action of GU-7 on platelets can be
a)
a
Ca
explained only by increasing cyclic nucleotide alone. Hence, experiments are now under way to investigate other possible actions.
a) a)
0
0
Traditionally in Japan, kampo medicines
IC50 1 .05).JgIml 0 0.1
0.5
1
have long been used for the alleviation of subjective 5
10
(j.ig/ml) Fig. 7 The ordinate indicates phosphodiesterase activity and the abscissa indicates the concentration of GU-7 in logarithm.
symptoms of chronic diabetic neuropathy. We have already
reported the existence of aldose reductase inhibitors in kampo medicines (7, 8). In addition to the aldose reductase inhibiting action, anti-platelet action may also explain the mechanism by which the kampo medicines are effective to diabetic neuropathy.
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Time (Sec)
Among 7 compounds purified from glycyrrhizae radix, GU-7 has never been reported. The chemical structure has been determined to be a derivative of 3-arylcoumarin, that is, 3-(2 ,4-dihydroxyphenyl)-8-hydroxymethyl-8-methyl-5-methoxy-6,7-dihydro-2H,8H-benzo[1, 2-b;5,4-b']dipyran-2-one.
Anti-Platelet Action of GU-7, A 3-Arylcou,narin Derivative, Purfiedfrom Glycyrrhizae Radix
2
Brownlee, M., Cerami, A. (1981) Ann. Rev. Biochem. 50, 385—432. Beyer-Mears, A., Cruz, E. (1985) Diabetes 34, 15—21. Terashima, H., Hania, K., Yamamoto, R., Tsuboshima, M., Kikkawa, R., Hatanaka, 1., Shigeta, Y. (1984) J. Pharmacol. Experim. Therap. 229, 226—230. Cogan, D. G., Kinoshita, J. H., Kador, P. F., Robison, W. G., Datiles, M. B., Cobo, L. M., Kupfer, C. (1984) Ann. mt. Med. 101,82—91.
Sato, Y., Sakamoto, N., Tomita, A., Shibata, M., Takeuchi, T., 6
8
Harada, N., Kate, K., Takahashi, A., Tsuchida, I. (1984) Rinsho to Kenkyu 61, 2347—2356 (in Japanese). Sakamoto, N., Sato, Y., Goto, Y., Ikeda, Y., Takahashi, A., Yano, S., Takeda, R., Baba, S., Kaneko, T., Mimura, G. (1987) in: Recent trends in management of diabetes mellitus, (Sakamoto, N., Alberti, K. G. M. M., Hotta, N., eds.), Amsterdam, Bxcerpta Med., pp. 552— 554. Aida, K., Shindo, H., Tawata, M., Onaya, T. (1987) Planta Med. 53, 131—135. Aida, K., Tawata, M., Shindo, H., Onaya, T., Sasaki, H., Nishimura, H., Chin, M., Mitsuhashi, H. (1989) Planta Med. 55, 22—26. Johnson, P. C., Ware, J. A., Cliveden, P. B., Smith, M., Dvorak, A. M., Salzman, F. W, (1985) J. Biol. Chem. 260, 2069—2076.
'
18 Agranoff, B. W., Murthy, P., Seguin, E. B. (1983) J. Biol. Chem. 258, 2076— 2078. Laemmli, U. K. (1970) Nature 227, 680—685. 12 Berridge, M. J. (1983) Biochem. J. 212, 849—858. 13 Hidaka, H., Asano, T. (1976) J. Biol. Chem. 251, 7508—7516. 14 Imaoka, T., Lynham, J. A., Haslam, R. J. (1983) J. Biol. Chem. 258, 15 16 17 18
19 20 21
11404—11414. Nishikawa, M., Tanaka, T., Hidaka, H. (1980) Nature 287,863—865. O'Rourke,F.A.,Halenda,S.P.,Zavoico,G.B.,Feinstein,M.B.(1985) J. Biol. Chem. 260, 956—962. Berridge, M. J. (1984) Biochem. J. 220, 345—360. Hokin, L. F.. (1985) Ann. Rev. Biochem. 54, 205—235. Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309—3 17. Castagna,
M., Takai, Y., Kaibuchi, K., Sane, K., Kikkawa, U.,
Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847—7851. Kikkawa, U., Kaibuchi, K., Castagna, M., Yamanishi, J., Sane, K., Tanaka, Y., Miyake, R., Takai, Y., Nishizuka, Y. (1984) Adv. Cyclic Nucleotide and Protein Phosphorylation Research 17, 437—442. Takai, Y., Kikkawa, U., Kaibuchi, K., Nishizuka, Y. (1984) Adv. Cyclic Nucleotide and Protein Phosphorylation Research 18, 119—158. Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
References
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