Selective inhibition of vascular smooth muscle cell proliferation by coptisine isolated from Coptis rhizoma, one of the crude drugs composing Kampo medicines Unsei-in.

Abstract

Acceleration of vascular smooth muscle cell (VSMC) proliferation is closely linked to the pathogenesis of vascular diseases. We, therefore, focused on traditional Japanese herbal medicines (Kampo medicines) used to ameliorate the impairment of microcirculation or blood stasis and screened them for their ability to inhibit rat VSMC proliferation. Among them, Unsei-in was found to effectively suppress VSMC proliferation, and Coptis rhizome was the responsible constituent crude drug. The extract of Coptis rhizome inhibited VSMC proliferation with the G[I.sub.50] value of 4.4 [micro]g/ml, which was much lower than those against the proliferation of 3Y1, dRLh-84, B16, and HeLa cells. The Coptis rhizome extract inhibited the progression of VSMC arrested at [G.sub.0]/[G.sub.1] phase from [G.sub.0]/[G.sub.1] to S phase, but not that of 3Y1 cells. Biological assay-guided fractionation revealed that an alkaloid of Coptis rhizome, coptisine, was the active ingredient in selectively preventing VSMC proliferation with G[I.sub.50] of 3.3 [micro]M (1.2 [micro]g/ml). When the structurally-related isoquinoline alkaloids of protoberberine class were studied for their inhibitory activities, berberine decreased the VSMC proliferation with G[I.sub.50] of 95.1 [micro]M (35.4 [micro]g/ml), about 30 times higher concentration than coptisine, while palmatine failed to show any activity. This study provides evidence that coptisine, an ingredient of Unsei-in, prevents VSMC proliferation selectively at lower concentrations compared with various cells or other structurally related alkaloids.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Coptisine; Coptis rhizoma; Kampo medicine; Vascular smooth muscle cell; Growth inhibition

Introduction

The abnormal and excessive proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathology of coronary artery atherosclerosis, restenosis following angioplasty, and hypertension. VSMC proliferation and differentiation are intricately regulated by a complex array of local environmental impetuses including growth factors, contractile agonists, inflammatory stimuli, and mechanical stresses (Ross, 1999). Although medial VSMCs are normally highly specialized for contraction, they retain the ability to modulate their phenotype to a more proliferative synthetic state. Retention of this plasticity is important in that it permits processes such as vessel repair after injury, but it may also be detrimental since it increases susceptibility to atherogenic risk factors (Mack et al., 2000). In atherosclerosis, the change in medial VSMCs from the contractile to the synthetic state is so far the most widely accepted mechanism of neointimal formation. Then, selective inhibition of the excessive proliferation of VSMC is considered to have the potential to protect from vascular disorders as well as dedifferentiate VSMC from the synthetic to the contractile state and induce apoptosis.

There is a pathophysiological concept called "oketsu," blood stasis or stagnant syndrome, in traditional Japanese herbal medicine (Kampo medicine) (Yakazu, 1975). This pathological state refers to a state of insufficient blood circulation and blood stasis. Patients suffering from "oketsu" syndrome reveal impaired microcirculation often accompanied by cerebrovascular disorders including atherosclerosis. It is well known that blood flow is influenced not only by blood components but also by the functions of heart and blood vessels, which are further controlled by the nervous system and some hormones. Therefore, it is conceivable that some Kampo medicines used to improve the "oketsu" syndrome would regulate VSMC function to restore or maintain normal vascular tone in addition to improving blood properties (Terasawa et al., 1986; Kohta et al., 1992). Kampo medicines have long been used and have never been reported as having severe side effects in suitable applications. In other words, Kampo medicines are a promising medicinal resource.

Therefore, the present study was designed to examine the possibility that VSMC proliferation-modifying compounds exist in Kampo medicines, to identify any such compounds in order to develop a novel therapeutic agent for vascular disorders, to clarify the mechanism by which Kampo medicines are used to ameliorate the "oketsu" syndrome, and to regulate vascular functions.

Materials and methods

Animals

Male Sprague-Dawley rats at 9 weeks of age were purchased from Nippon Crea Co., Ltd. (Shizuoka, Japan), and were housed in a temperature-controlled room (at 23[+ or -]1[degrees]C), with lighting from 6 a.m. to 6 p.m., under specific-pathogen-free conditions, at the Laboratory Animal Center of Nagoya City University . Medicinal crude drugs were obtained from Tsumura Co., Ltd. (Ibaragi, Japan).

Preparation of Kampo medicines and hot-water extract of crude drugs

Kampo medicines (dose per person per day) and each crude drug extract were prepared as follows. The crude drugs which are the components of Unsei-in shown in Table 1 were weighed, added to 700 ml water, decocted for 1 h, and then concentrated to 300 ml. These decoctions were lyophilized to give 9.32 g of powdered extract. The yields of the other Kampo medicines were as follows: 6.21 g, Kami-shoyo-san; 2.94 g, Keishi-bukuryo-gan; 4.43 g, Otsuji-to; 5.78 g, Toki-shakuya-ku-san; 6.70 g, Chiketsu-kyo-ippo; 1.94g, Choyo-to; 6.70 g, Daio-botampi-to; 11.41 Kyuki-kyogai-to; 6.54g, Sessyo-in; 7.46g, Shimotu-to; 7.71 g, Sokei-kakketsu-to; 4.33 g, Tokaku-joki-to; 8.21 g, Toki-shi-gyaku-ka-goshuyu-shokyo-to; 8.07 g, Unkei-to; and 5.16 g, Zokumei-to. The extract of each crude drug composing Unsei-in was also prepared from the same amount of crude drug contained in Unsei-in as described above, and their respective yields were: 2.41 g, Angelicae radix; 1.55 g, Paeoniae radix; 1.54 g, Cnidii rhizoma; 3.03 g, Rehmanniae radix; 2.00 g, Scutellariae radix; 0.59 g, Gardeniae fructus; 0.45 g, Coptidis rhizoma; and 0.44 g, Phellodendri cortex.

Rat vascular smooth muscle cell isolation and culture

Male Sprague-Dawley rats (10 weeks of age) were anesthetized by intraperitoneal injection with pentobarbital and killed by bleeding. The thoracic and abdominal aortas were excised, cut into 1-mm pieces, and incubated in DMEM (Irvine Scientific Co., Santa Ana, CA, USA) containing 600 U/ml collagenase-I (Nitta Gelatin Inc., Osaka, Japan) for 30min at 37[degrees]C to remove the endothelial cells. Then, aortas were washed with DMEM and dispersed in DMEM containing 500 U/ml colla-genase-I, 20 U/ml elastase (Wako, Osaka, Japan), and 0.5 mg/ml trypsin inhibitor (Sigma, St. Louis, MO, USA). Following 2h incubation at 37[degrees]C, smooth muscle cells were harvested and maintained in DMEM supplemented with 10% FCS (Irvine Scientific Co., Santa Ana, CA, USA), 100 units/ml penicillin, and 100 [micro]g/ml streptomycin (Gibco, Grand Island, NY, USA). The cells from passages 5-15 were used for the following experiments.

Cell culture

B16 (mouse skin melanoma), 3Y1 (rat embryo fibroblast), dRLh-84 (rat hepatoma), and HeLa (human carcinoma) were obtained from Health Science Research Resources Bank (Sennan, Osaka, Japan). These cells were maintained in MEM-Eagle's salt medium (Irvine Scientific Co., Santa Ana, CA, USA), supplemented with 10% heat-inactivated FCS, 50 U/ml penicillin, and 50 [micro]g/ml streptomycin. Each cell was maintained at 37[degrees]C in a humidified atmosphere of 5% C[O.sub.2] in air.

Cell cycle analysis

For cell synchronization at [G.sub.0]/[G.sub.1] phase, VSMC and 3Y1 at a density of 6 x [10.sup.3] cells/[cm.sup.2] were cultured in DMEM and MEM supplemented with 10% FCS for 24 h, respectively. Then, VSMC and 3Y1 were incubated in DMEM with high glucose in the absence of FCS for 96h, and in MEM supplemented with 0.5% FCS for 36 h, respectively. To examine the effect of medicinal crude drug extracts, cultures were growth arrested as described above, followed by incubation in a medium supplemented with 10% FCS containing the extracts at the varying concentrations. At the indicated time points after release, cells were harvested by trypsinization and washed with phosphate-buffered saline (PBS). The cells were fixed with ice-cold 70% methanol for 30 min, washed with PBS, and then treated with 1 ml of 1 mg/ml RNase A in 1 mM Tris-HCl, pH 7.4/1.5 mM NaCl at 37[degrees]C for 30 min. Cells were harvested by centrifugation at 400g for 5 min, and stained with 250 [micro]l of nuclear staining solution (10 mg propidium iodide, 0.1 mg trisodium citrate, and 0.03 ml Triton X-100 were dissolved in 100 ml [H.sub.2]O) at room temperature for 30 min in the dark. After adding 750 [micro]l PBS, the DNA content in each cell cycle phase was determined by FACSort, using an analytic software, ModFit (Becton Dickinson, San Jose, CA, USA).

Assay of cell proliferation

Rat VSMCs and the other cells were plated at the density of 1.0 x [10.sup.4] cells/well into 24-well plates and cultured for 24 h, respectively. Then, Kampo medicines, crude drug extracts, or isolated compounds were added into cultures and incubated for 72 or 48 h. After exchanging with fresh medium, 500 [micro]l of MTT (1 mg/ml in DMEM for VSMC; 200 [micro]g/ml in MEM for the other cells) was added to each well and incubated for another 4h. Following the solubilization of the formed formazan with SDS, the absorbance at 590 nm was measured and also the cell growth percentage; GP was calculated according to the following formula: GP (>0%) = [(absorbance of cells treated with samples)-(absorbance at 0 time)]/[(absorbance of control cells)-(absorbance at 0 time)], GP (-100~0%) = [(absorbance of cells treated with samples)-(absorbance at 0 time)]/(absorbance at 0 time).

Isolation of coptisine

The hot-water extract of Coptis rhizome (10 g) was dissolved in 500 ml [H.sub.2]O and then acidified by HC1 (25 ml). Water phase was washed with AcOEt (300 ml times). The pH of the water phase was raised to 10 by addition of N[H.sub.3] aqueous and was extracted by AcOEt. The organic phase was evaporated to give the total alkaloid (110 mg). The alkaloid fraction was subjected to Si[O.sub.2] column chromatography (CH[Cl.sub.3]-MeOH = 4:1 [right arrow] MeOH), and the obtained active fraction was then separated by HPLC (YMC -pack ODS-AG, 0.1% formic acid aq.-MeOH = 3:7) to give coptisine (4.0 mg) as active compound.

Analysis of coptisine and berberine contents with HPLC

The coptisine and berberine contents in the hot-water extract of Coptis rhizome were determined with HPLC as follows. The HPLC system included a PU-980 pump and MD-2010 Plus detector. The HPLC system and detectors were controlled and results calculated by the Jasco Borwin HPLC software. A 50 [micro]l sample was injected on a Cosmosil 5C18-AR column (4.6 mm i.d. x 250 mm) (Nacalai Tesque Co., Kyoto, Japan) at room temperature with a flow rate set at 0.5 ml [min.sup.-1]. The mobile phase consisted of phosphate buffer (pH2.1; 0.1 M)/methanol (80/20; v/v). The coptisine and berberine contents were 10.3% and 26.4% of the extract, respectively.

Results

We first screened 16 Kampo medicines (the names of which are described in Materials and methods), which are used to ameliorate the "oketsu" syndrome, by their respective ability to inhibit VSMC proliferation (Fig. 1). Among them, Unsei-in effectively suppressed VSMC proliferation with a 50% growth-inhibitory concentration (G[I.sub.50]) of 65.5 [micro]g/ml, and did not show cytotoxicity at the highest concentration of 500 [micro]g/ml. This result indicated that Unsei-in possessed a cytostatic effect on VSMC without cytotoxicity. However, Otsuji-to also suppressed VSMC proliferation, but showed cytotoxicity at the concentration of 500 [micro]g/ml. The other 14 Kampo medicines revealed only marginal effects on VSMC proliferation even at the high concentation of 500 [micro]g/ml (the data of representaive Kampo medicines are shown in Fig. 1). Therefore, we next examined the antiproliferative effect of the eight crude drugs composing Unsei-in. Results shown in Table 2 demonstrated that the Coptis rhizome extract showed a cytostatic activity, and its G[I.sub.50] value was 4.4 [micro]g/ml. Scutellaria root extract inhibited VSMC proliferation at relatively low concentrations, but induced cytotoxicity at a concentration of 125 [micro]g/ml (data not shown). Phellodendron bark and Paeonia root extracts also reduced VSMC proliferation, although much less than Coptis rhizome. No such inhibitory effect was found with the other four crude drugs. When the specificity of the antiproliferative effect was investigated, the Coptis rhizome extract demonstrated weak inhibitory activities against 3Y1, and dRLh-84 cells with G[I.sub.50] of around 200 [micro]g/ml, and against B16 and HeLa cells with G[I.sub.50] of 57.3 and 90.3 [micro]g/ml (Table 2). This result suggested that the inhibitory effect of Coptis rhizome extract was considerably selective for VSMC. In contrast, the extracts of Scutellaria root, Phellodendron bark, and Paeonia root also exhibited moderate cell-proliferation inhibitory activity against all cells tested, but did not show prominent selectivity. Therefore, the effect of the Coptis rhizome extract on the progression of cell cycle was evaluated using VSMC synchronized at [G.sub.0]/[G.sub.1] phase by serum deprivation. In the control VSMC, the addition of the medium containing 10% FCS obviously induced the S and [G.sub.2]/M phases 21 h after the addition. In contrast, the addition of Coptis rhizome extract dose-dependently inhibited the progression and completely abolished the release from [G.sub.0]/[G.sub.1] at 200 [micro]g/ml (Fig. 2A). Interestingly, when the effect of Coptis rhizome extract on the cell cycle progression was studied using rat fibroblast, 3Y1 cell, the extract failed to show any cell cycle inhibitory activity (Fig. 2B). This result coincided with that of the antiproliferative activity shown by Coptis rhizome extract. To distinguish between the inhibition of cell proliferation and dedifferentiation from the synthetic to the contractile state, mRNA expressions of 1-caldesmon, a marker of synthetic VSMC, and h-caldesmon, a marker of contractile VSMC, were determined by semi-quantitative RT-PCR. Although the data were not shown, the growth-arrested VSMC after Coptis rhizome extract treatment expressed only 1-caldesmon mRNA. This result indicated that the growth inhibition was due to merely the interruption of proliferation, but not the dedifferentiation from the synthetic to the contractile state of VSMC.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

We then attempted to isolate the active ingredients from the Coptis rhizome extract by bioassay-guided fractionation in which the antiproliferative activity against VSMC was used as the index of bioassay. First, total alkaloids were collected from the Coptis rhizome extract according to the conventional method. The antiproliferative activity was efficiently recovered in the alkaloid fraction (data not shown). The obtained fraction was next subjected to silica gel column chromatography and HPLC (YMC-pack ODS-AG) to give coptisine as the compound responsible for an antiproliferative activity. The structure was confirmed on the basis of [.sup.1.H]-, and [.sup.13.C]-NMR and mass spectral data. Then, its activity was compared with those of the structurally-related isoquinoline alkaloids, berberine and palmatine, which are also contained in Coptis rhizome. As shown in Fig. 3, coptisine effectively suppressed VSMC proliferation with the G[I.sub.50] value of 3.3 [micro]M (1.2 [micro]g/ml), whereas palmatine failed to show such an activity and berberine showed only a weak activity with the G[I.sub.50] value of 95.1 [micro]M (35.4 [micro]g/ml). These activities were confirmed using commercially available coptisine, berberine, and palmatine. Furthermore, when the specificity of its antiproliferative effect was assessed, coptisine was able to suppress the proliferation of the other cells, although at a G[I.sub.50] value at least 10-fold higher than for VSMC (Figs. 4A and B). In contrast, berberine inhibited the proliferation of all cells tested at relatively high G[I.sub.50] values without the specificity for VSMC. Analysis of coptisine and berberine contents in the hot-water extract of Coptis rhizome revealed coptisine and berberine contents of 10.3% and 26.4%, respectively (Fig. 5). When considering the relationship between the potency of activity (G[I.sub.50]: 3.3 [micro]M for coptisine, 95.1 [micro]M for berberine) and the alkaloid content in the hot-water extract of Coptis rhizome, the selective antiproliferative activity in the extract can be ascribed to coptisine.

[FIGURE 3 OMITTED]

Discussion

"Oketsu", blood stasis or stagnant blood syndrome, is one of the few pathological conditions that is suitable for treatment by Kampo medicine. Patients with "oketsu" syndrome demonstrate impairment of microcirculation and hemorheological abnormalities (Terasawa et al., 1986; Hikiami et al., 1996). Currently many Kampo medicines are administered to patients under clinical settings, despite the lack of knowledge of the drug mechanism. Among the 16 Kampo medicines used to ameliorate "oketsu" syndrome, we have demonstrated here that Unsei-in has antiproliferative activity against VSMC. Additionally, we isolated coptisine as the compound responsible for the antiproliferative activity from the extract of Coptis rhizome, one of the crude drugs composing Unsei-in. This result suggests that Unsei-in may regulate vascular tone by suppressing abnormal VSMC proliferation.

To date, a number of biological activities have been reported for Coptis rhizoma, a crude drug that is frequently used in Kampo medicines: anticancer activity against esophageal and other cancer cells (Iizuka et al., 2000a; Colombo et al., 2001; Lin et al., 2004), antibacterial activity (Hu et al., 2000), anti-ulcer activity (Takase et al., 1989), antidiarrhoeal activity (Kase et al., 1999), and anticachectic activity (Iizuka et al., 2000b). Screenings for active compounds in Coptis rhizome have identified berberine as the active compound in most cases. In contrast, there are only a few reports on the biological activities of coptisine (Hattori et al., 1992; Tanaka et al., 1993; Colombo et al., 2001; Hirano et al., 2001; Ro et al., 2001; Lin et al., 2004). Here we report that VSMC proliferation is more highly sensitive to coptisine (G[I.sub.50]: 3.3 [micro]M) compared to the other cells (G[I.sub.50]: 35~> 140 [micro]M). In addition, the selective inhibition of VSMC proliferation by coptisine was confirmed by the finding that coptisine blocked the release from the [G.sub.0]/[G.sub.1] phase of cell progression in VSMC, but not 3Y1 cells. Although compounds with antiproliferative activity specific to VSMC have never been reported, some natural compounds that suppress VSMC proliferation have been identified: fangchinoline (Zhan et al., 2003), ajoene (Ferri et al., 2003), senkyunolide (Kobayashi et al., 1992), and quercetin (Alcocer et al., 2002). That is, selective antiproliferative activity against VSMC is one of the most potent and novel activities among the inherent biological activities of coptisine. However, our data did not provide evidence to explain the mechanisms underlying the selective inhibition of VSMC proliferation by coptisine. To date, berberine and palmatine are known to interact with DNA (Nandi et al., 1990; Cushman et al., 1979) and relax arterial contraction by reducing the intracellular [Ca.sup.2+] concentration (Chang et al., 1999; Ko et al., 2000). These findings suggest that the selective antiproliferative activity of coptisine is based on the activities of DNA binding and decreasing intracellular [Ca.sup.2+] levels, both of which might be regulated by coptisine in slightly different ways than berberine and palmatine. In addition, the possibility exists that coptisine targets the signaling pathway specific to VSMC proliferation. However, further detailed study is required to clarify the selectivity shown by coptisine.

[FIGURE 5 OMITTED]

Coptisine is an isoquinoline alkaloid with a structure that is very similar to those of berberine and palmatine. When the relationship between structure and antiproliferative activity against VSMC was examined, the inhibitory activity of berberine was much less potent than that of coptisine (G[I.sub.50] value was about one thirtieth), while palmatine failed to show any inhibitory activity against VSMC proliferation. Thus, these three alkaloids possess differing potency in their antiproliferative activities, despite the similarities in their structures. In fact, hydrophobicity increases with increasing number of carbon atoms in the order of coptisine, berberine, and palmatine. This fact suggests that an increase in hydrophobicity could lead to a decrease in inhibitory activity. By contrast, a study to assess inhibitory effects of isoquinoline alkaloids on dopamine biosynthesis in PC12 cells showed that berberine and palmatine reduced dopamine content at a concentration of 20 [micro]M, whereas coptisine failed to do so (Shin et al., 2000). Taken together, the biological activity of these three alkaloids is unlikely to be completely explained by their hydrophobicity. Rather, the characteristic structures of each alkaloid appears to be crucial to their respective activities.

In conclusion, we identified a compound with antiproliferative activity against VSMC in Coptis rhizome, one of the crude drugs used in Unsei-in. The clinical observation of the ability of Unsei-in to ameliorate insufficient blood circulation and blood stasis may be due in part to the suppression of VSMC proliferation, which leads to the regulation of vascular tone. In addition, the finding that coptisine is a selective antiproliferative compound against VSMC paves the way for a novel therapeutic agent to treat the diseases involving abnormal vascular remodeling.

References

Alcocer, F., Whitley, D., Salazar-Gonzalez, J.F., Jordan, W.D., Sellers, M.T., Eckhoff, D.E., Suzuki, K., Macrae, C., Bland, K.I., 2002. Quercetin inhibits human vascular smooth muscle cell proliferation and migration. Surgery 131, 198-204.

Chang, Y.L., Usami, S., Hsieh, M.T., Jiang, M.J., 1999. Effects of palmatine on isometric force and intracellular calcium levels of arterial smooth muscle. Life Sci. 64, 597-606.

Colombo, M.L., Bugatti, C., Mossa, A., Pescalli, N., Piazzoni, L., Pezzoni, G., Menta, E., Spinelli, S., Johnson, F., Gupta, R.C., Dasaradhi, L., 2001. Cytotoxicity evaluation of natural coptisine and synthesis of coptisine from berberine. Farmaco 56, 403-409.

Cushman, M., Dekow, F.W., Jacobsen, L.B., 1979. Conformations, DNA binding parameters, and antileukemic activity of certain cytotoxic protoberberine alkaloids. J. Med. Chem. 22, 331-333.

Ferri, N., Yokoyama, K., Sadilek, M., Paoletti, R., Apitz-Castro, R., Gelb, M.H., Corsini, A., 2003. Ajoene, a garlic compound, inhibits protein prenylation and arterial smooth muscle cell proliferation. Br. J. Pharmacol. 138, 811-818.

Hattori, T., Furuta, K., Nagao, T., Nagamatsu, T., Ito, M., Suzuki, Y., 1992. Studies on the antinephritic effect of plant components (4): Reduction of protein excretion by berberine and coptisine in rats with original-type anti-GBM nephritis. Jpn. J. Pharmacol. 59, 159-169.

Hikiami, H., Kohta, K., Sekiya, N., Shimada, Y., Itoh, T., Terasawa, K., 1996. Erythrocyte deformability in "oketsu" syndrome and its relation to erythrocyte viscoelasticity. J. Trad. Med. 13, 156-164.

Hirano, H., Osawa, E., Yamaoka, Y., Yokoi, T., 2001. Gastric-mucous membrane protection activity of coptisine derivatives. Biol. Pharm. Bull. 24, 1277-1281.

Hu, J.P., Takahashi, N., Yamada, T., 2000. Coptidis rhizoma inhibits growth and proteases of oral bacteria. Oral Dis. 6, 297-302.

Iizuka, N., Miyamoto, K., Okita, K., Tangoku, A., Hayashi, H., Yosino, S., Abe, T., Morioka, T., Hazama, S., Oka, M., 2000a. Inhibitory effect of Coptidis rhizoma and berberine on the proliferation of human esophageal cancer cell lines. Cancer Lett. 148, 19-25.

Iizuka, N., Miyamoto, K., Hazama, S., Yoshino, S., Yoshimura, K., Okita, K., Fukumoto, T., Yamamoto, S., Tangoku, A., Oka, M., 2000b. Anticachectic effects of Coptidis rhizoma, an anti-inflammatory herb, on esophageal cancer cells that produce interleukin 6. Cancer Lett. 158, 35-41.

Kase, Y., Saitoh, K., Makino, B., Hashimoto, K., Ishige, A., Komatsu, Y., 1999. Relationship between the antidiarrhoeal effects of Hange-Shashin-To and its active components. Phytother. Res. 13, 468-473.

Ko, W.H., Yao, X.Q., Lau, C.W., Law, W.I., Chen, Z.Y., Kwok, W., Ho, K., Huang, Y., 2000. Vasorelaxant and antiproliferative effects of berberine. Eur. J. Pharmacol. 399, 187-196.

Kobayashi, S., Mimura, Y., Notoya, K., Kimura, I., Kimura, M., 1992. Antiproliferative effects of the traditional Chinese medicine Shimotsu-to, its component cnidium rhizoma and derived compounds on primary cultures of mouse aorta smooth muscle cells. Jpn. J. Pharmcol. 60, 397-401.

Kohta, K., Hiyama, Y., Terasawa, K., Hamazaki, T., Itoh, T., Tosa, H., 1992. Hemorrheological studies of "oketsu" syndrome--Erythrocyte aggregation in "oketsu" syndrome. J. Med. Pharm. Soc. WAKAN-YAKU 9, 221-228.

Lin, C.C., Ng, L.T., Hsu, F.F., Shieh, D.E., Chiang, L.C., 2004. Cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and their major constituents (berberine, coptisine, and icariin) on hepatoma and leukaemia cell growth. Clin. Exp. Pharmacol. Physiol. 31, 65-69.

Mack, C.P., Thompson, M.M., Lawrenz-Smith, S., Owens, G.K., 2000. Smooth muscle [alpha]-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex. Circ. Res. 86, 221-232.

Nandi, R., Debnath, D., Maiti, M., 1990. Interactions of berberine with poly(A) and tRNA. Biochim. Biophys. Acta 1049, 339-342.

Ro, J.S., Lee, S.S., Lee, K.S., Lee, M.K., 2001. Inhibition of type A monoamine oxidase by coptisine in mouse brain. Life Sci. 70, 639-645.

Ross, R., 1999. Atherosclerosis--an inflammatory disease. N. Engl. J. Med. 340, 115-126.

Shin, J.S., Kim, E.I., Kai, M., Lee, M.K., 2000. Inhibition of dopamine biosynthesis by protoberberine alkaloids in PC12 cells. Neurochem. Res. 25, 363-368.

Takase, H., Imanishi, K., Miura, O., Yumioka, E., Watanabe, H., 1989. Features of the anti-ulcer effects of Oren-gedokuto (a traditional Chinese medicine) and its component herb drugs. Jpn. J. Pharmacol. 49, 301-308.

Tanaka, T., Metori, K., Mineo, S., Hirotani, M., Furuya, T., Kobayashi, S., 1993. Inhibitory effects of berberine-type alkaloids on elastase. Planta Med. 59, 200-202.

Terasawa, K., Toriizuka, K., Tosa, H., Ueno, M., Hayashi, T., Shimizu, M., 1986. Rheological studies on "oketsu" syndrome I. The blood viscosity and diagnostic criteria. J. Med. Pharm. Soc. WAKAN-YAKU 3, 98-104.

Yakazu, D., 1975. Considerations on Oketsu (stagnation of disordered blood), one of the cardinal pathogenetic factors in oriental medicine. J. Japan Soc. Oriental Med. 25, 167-187.

Zhan, Y.H., Fang, L.H., Ku, B.S., 2003. Fangchinoline inhibits rat aortic vascular smooth muscle cell proliferation and cell cycle progression through inhibition of ERK 1/2 activation and c-fos expression. Biochem. Pharmacol. 66, 1853-1860.

H. Tanabe (a), H. Suzuki (a), A. Nagatsu (a), H. Mizukami (a), Y. Ogihara (b), M. Inoue (a,*)

(a) Laboratory of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan

(b) Laboratory of Kampo Medicine, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan

*Corresponding author. Tel./fax: [phone].

E-mail address: [e-mail] (M. Inoue).

Table 1. Constitutive crude drugs of Unsei-in

Crude drugs      Plant name                   Part used  Composition (g)

Angelicae radix  Angelica acutiloba KITAGAWA  Root       4.0
Paeoniae radix   Paeania lactiflora PALLAS    Root       4.0
Cnidii rhizoma   Cnidiun officinale MAKINO    Rhizome    4.0
Rehmanniae       Rehmannia glutinosa          Root       4.0
radix            LIBOSCHITS var.
                 purpurea MAKINO
Scutellariae     Scutellaria baicalensis      Root       2.0
radix            GEORGI
Gardeniae        Gardenia jasminoides ELLIS   Fruits     2.0
fructus
Phellodendri     Phellodendron amurense       Bark       1.5
cortex           RUPRECHT
Coptidis         Captis japanica MAKINO       Rhizome    1.5
rhizoma

Table 2. G[I.sub.50] values of eight crude drugs component of Unsei-in
against proliferation of VSMC and various cells

                 VSMC     3Y1      dRLh-84  B16      HeLa

Angelicae r.     > 625.0  > 625.0  > 625.0  > 625.0  > 625.0
Paeoniae r.        172.7    336.7  > 625.0    298.1  > 625.0
Cnidii rh.       > 625.0  > 625.0  > 625.0  > 625.0  > 625.0
Rehmanniae r.    > 625.0  > 625.0  > 625.0  > 625.0  > 625.0
Scutellariae r.     26.8     90.0     69.4     48.8    103.2
Gardeniae f.     > 625.0  > 625.0  > 625.0  > 625.0  > 625.0
Coptidis rh.         4.4    220.1    229.5     57.3     90.3
Phellodendri c.     45.9    258.6    469.7    175.9    182.6

Antiproliferative effects of crude drugs were determined as described in
Materials and methods. G[I.sub.50] values are represented as
[micro]g/ml.

(B)

           3Y1    dRLh-84  B16     Hela

coptisine  >140   >140       91.4    34.7
berberine  >134   >134       58.3    64.1
palmatine  >129   >129     >129    >129

([micro]M)

Fig. 4. Specificity of antiproliferative effect of coptisine, berberine,
and palmatine. (A) Effects of three alkaloids on proliferation of
various cells were determined as described in Materials and methods.
Closed circle: 3Y1; open square: dRLh-84; closed square: B16; open
triangle: HeLa. Each value represents means [+ or -] SE of four wells
(SEs were so small that most of the SEs hid behind the symbols.). (B)
G[I.sub.50] values ([micro]M) were calculated from the data in Fig. 4A.