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 Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.
Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that cell (VSMC VSMC Vascular Smooth Muscle Cell ) 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 microcirculation /mi·cro·cir·cu·la·tion/ (-sir?ku-la´shun) the flow of blood through the fine vessels (arterioles, capillaries, and venules).microcirculato´ry
n. 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 rhizome (rī`zōm) or rootstock, fleshy, creeping underground stem by means of which certain plants propagate themselves. Buds that form at the joints produce new shoots. 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 fractionation /frac·tion·a·tion/ (frak?shun-a´shun)
1. in radiology, division of the total dose of radiation into small doses administered at intervals.
2. 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 alkaloids,
n alkaline phytochemicals that contain nitrogen in a heterocyclic ring structure. They can have powerful pharmacological effects and are more often used in traditional medicine than in herbal treatments. of protoberberine class were studied for their inhibitory activities, berberine berberine /ber·ber·ine/ (bur´bur-en) an alkaloid from species of Berberis and related plants, and from Hydrastis canadensis; 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
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 contractile /con·trac·tile/ (kon-trak´til) able to contract in response to a suitable stimulus.
Capable of contracting or causing contraction, as a tissue. 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 ath·er·o·gen·ic
Initiating, increasing, or accelerating atherogenesis.
atherogenic adjective Referring to the ability to initiate or accelerate atherogenesis—the deposition of atheromas, lipids, and 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 de·dif·fer·en·ti·a·tion
Regression of a specialized cell or tissue to a simpler, more embryonic, unspecialized form. Dedifferentiation may occur before the regeneration of appendages in plants and certain animals and in the 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 cer·e·bro·vas·cu·lar
Relating to the blood supply to the brain, particularly with reference to pathological changes.
pertaining to the blood vessels of the cerebrum or brain. 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 Adv. 1. in other words - otherwise stated; "in other words, we are broke"
put differently , 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
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 Nagoya City University (名古屋市立大学 . 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 The base value in a numbering system. For example, in the decimal numbering system, the radix is 10.
(mathematics) radix - The ratio, R, between the weights of adjacent digits in positional representation of numbers. ; 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 pentobarbital /pen·to·bar·bi·tal/ (pen?to-bahr´bi-tal) a short- to intermediate-acting barbiturate; the sodium salt is used as a hypnotic and sedative, usually presurgery, and as an anticonvulsant. and killed by bleeding. The thoracic and abdominal aortas were excised, cut into 1-mm pieces, and incubated in DMEM DMEM Dulbecco's Modified Eagle's Medium (for cell culture growth)
DMEM Design Manufacture and Engineering Management Department (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 elastase /elas·tase/ (e-las´tas) see pancreatic elastase.
An enzyme found especially in pancreatic juice that catalyzes the hydrolysis of elastin. (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 FCS - Frame Check Sequence (Irvine Scientific Co., Santa Ana, CA, USA), 100 units/ml penicillin, and 100 [micro]g/ml streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other (Gibco, Grand Island, NY, USA). The cells from passages 5-15 were used for the following experiments.
B16 (mouse skin melanoma), 3Y1 (rat embryo fibroblast fibroblast /fi·bro·blast/ (fi´bro-blast)
1. an immature fiber-producing cell of connective tissue capable of differentiating into chondroblast, collagenoblast, or osteoblast.
2. ), dRLh-84 (rat hepatoma hepatoma /hep·a·to·ma/ (hep?ah-to´mah)
1. a tumor of the liver.
2. hepatocellular carcinoma (malignant h.).
n. pl. ), 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 PBS
in full Public Broadcasting Service
Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural, ). 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 DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. 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 MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide
MTT Machine Tool Technology
MTT Microwave Theory and Techniques
MTT Mobile Task Team
MTT Multi-Table Tournament (poker) (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 1. (company) SDS - Scientific Data Systems.
2. (tool) SDS - Schema Definition Set. , the absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .
2. 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 acidified /acid·i·fied/ (ah-sid´i-fid) having been made acid. 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 HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed (YMC YMC Youth Media Council (Oakland, CA)
YMC Yellow-Magenta-Cyan (color model)
YMC Yale Model Congress
YMC Youth Marti Coalition
YMC Yahoo Marketing Communication -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.
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 cytostatic /cy·to·stat·ic/ (sit?ah-stat´ik)
1. suppressing the growth and multiplication of cells.
2. an agent that so acts.
1. 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 dedifferentiation /de·dif·fer·en·ti·a·tion/ (de-dif?er-en?she-a´shun) anaplasia.
Regression of a specialized cell or tissue to a simpler unspecialized form. 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]
"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 quer·ce·tin
A yellow powdered crystalline compound produced synthetically or occurring as a glycoside in the rind and bark of numerous plants, used medicinally to treat abnormal capillary fragility. Also called meletin. (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 Biosynthesis
The synthesis of more complex molecules from simpler ones in cells by a series of reactions mediated by enzymes. The overall economy and survival of the cell is governed by the interplay between the energy gained from the breakdown of compounds 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.
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or red blood cell or red blood corpuscle
Blood cell that carries oxygen from the lungs to the body tissues. deformability deformability /de·form·a·bil·i·ty/ (de-form?ah-bil´it-e) ability of cells to change shape when passing through narrow spaces, such as erythrocytes passing through the microvasculature. in "oketsu" syndrome and its relation to erythrocyte viscoelasticity Viscoelasticity, also known as anelasticity, is the study of materials that exhibit both viscous and elastic characteristics when undergoing deformation. Viscous materials, like honey, resist shear flow and strain linearly with time when a stress is applied. . J. Trad. Med. 13, 156-164.
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H. Tanabe (a), H. Suzuki (a), A. Nagatsu (a), H. Mizukami (a), Y. Ogihara (b), M. Inoue (a,*)
(a) Laboratory of Pharmacognosy pharmacognosy /phar·ma·cog·no·sy/ (fahr?mah-kog´nah-se) the branch of pharmacology dealing with natural drugs and their constituents.
n. , 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: +81 52 836 3415.
E-mail address: email@example.com (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.