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Vasorelaxant effects of forsythide isolated from the leaves of forsythia viridissima on NE-induced aortal contraction.


Forsythide (F1) isolated from the leaves of Forsythia viridissima (Oleaceae) showed vasorelaxant effects on norepinephrine (NE)-induced contraction of rat aorta with or without endothelium. This compound did not affect contraction induced by high concentration potassium (60 m M [K.sup.+]) and phorbol 12,13-diacetate, but inhibited NE-induced contraction in the presence of nicardipine. These results demonstrated the inhibitory effects of F1 on NE-induced vasocontraction presumably due to decrease of calcium influx from extracellular area, which was induced by NE.

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Keywords: Forsythia viridissima; Forsythide; Iridoid; Vasorelaxant activity; ROC


Forsythia viridissima and F. suspensa are listed in Japanese Pharmacopoeia as the original plants of Forsythiae fructus. Earlier studies revealed that F. viridissima constituents demonstrate a broad spectrum of medicinal properties including anti-inflammatory, diuretic and anti-hypertensive activities (Nishibe, 2002). Moreover, constituents such as lignans, pheny-lethanoids, and flavonoids were previously isolated from these species (Tokar and Klimek, 2005). In our earlier studies, two vasorelaxing constituents, forsythiaside from F. suspensa and acetoside (F2, Fig. 1) from F. viridissima, were isolated and elucidated their vasorelaxing activities via receptor-operated [Ca.sup.2+] -channels (ROCs) inhibitor (Wong et al., 2001; Iizuka and Nagai, 2005; Iizuka et al., 2005). In the present study, we isolated another active constituent, forsythide (Fl, Fig. 1), an iridoid derivative from the leaves of F. viridissima, and demonstrated vasorelaxing activities of forsythide on isolated rat aorta, compared with F2 as a positive control.


Materials and methods

Plant material

F. viridissima was identified by Dr. Nishibe at Health Science University of Hokkaido and dried leaves of F. viridissima were generously donated by Dr. Deyama in Yomeishu Seizo Co., Ltd. (Japan).


NE, nicardipine, ethyleneglycol-bis-([beta]-aminoethyl ether)-tetraacetic acid (EGTA), phorbol 12, 13-diaceta-te(PDA), and acetylcholine chloride (Ach) were all purchased from Sigma Chemical (St. Louis, MO, USA).


Melting points were determined on a Yanagimoto microscopic melting point apparatus and are uncorrected (Yanagimoto, Japan). [.sup.1]H (500 MHz) -and [.sub.13] (125 MHz) -NMR spectra were measured with a JEOL JNM-LA 500 spectrometer (JEOL, Japan), while chemical shifts are given on the 3 scale (ppm) with tetramethylsilane (TMS) as an internal standard.

Extraction and isolation

The extraction, isolation and confirmation of Fl and F2 were described in previous studies (Iizuka and Nagai, 2005; Iizuka et al., 2005). The purity of Fl and F2 were more than 98% based on HPLC analysis. The preparatory HPLC system as follows: pump, LC-6AD (Shimadzu, Japan); detector, S-310A model-2 (Soma, Japan) at 254 nm; column, YMC ODS-AQ 250 x 20 mm i.d. (YMC Co. Ltd., Japan); mobile phase, 1% formic acid:MeCN (88:18-88:12-94:6). NMR-spectra and m.p. of Fl were identical with those in the literature (Damtoft et al., 1994; Wu et al., 2004).

Isolation of rat aortic strips

These animal experimental studies were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, and under the supervision of the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Male Wistar rats (Tokyo Laboratory Animals Science) weighing 240-340 g were housed in a room maintained at 23 [+ or -] 1[degrees]C with a 12 h light/dark cycle (lights on 8:00 am to 8:00 pm). Food and water were available ad libitum. After 7-10 days of habituation, rats were killed by exsanguination from the carotid arteries under anesthesia. A section of the thoracic aorta between the aortic arch and the diaphragm was removed and placed in oxygenated, modified Krebs-Henseleit buffer (KHB: NaCl 118.0mM, KCl 4.7 mM, [NaHCO.sub.3] 25.0mM, [CaCl.sub.2] 1.8 mM, [NaH.sub.2][Po.sub.4] 1.2 mM, MgS04 1.2mM, and glucose 11.0mM). The aorta was cleanedand cut in ring preparations 3 mm in length or cut into helical strips 3 mm in width and 20 mm in length. To detach the endothelium, endothelial cells on each strip were removed by gentle rubbing of the endothelial surface with a disposable cotton applicator.

The tissue was placed in a well-oxygenated (95% [O.sub.2], 5% [CO.sub.2]) bath of KHB 10 ml at 37 [degrees]C with the ringed aorta connected to a tissue holder and to a force-displacement transducer (Nihon Kohden, Japan, TB-61 IT). The tissue was equilibrated for 60min under a resting tension of 1.0g. During this time, the KHB solution in the tissue bath was replaced every 20min at 37 [degrees]C.

Experimental protocol

Experiments were performed according to method as described in our previous studies (Nagai et al., 1996; Iizuka and Nagai, 2005; Iizuka et al., 2005, 2006). After equilibration, each aortic ring was contracted by treatment with NE 3 x [10.sup.-7]M. The presence of functional endothelial cells was confirmed by demonstrating relaxation with response to Ach [10.sup.-5] M, and aortic rings in which 80% relaxation occurred was considered as tissue with endothelium. The endothelial cells removed by rubbing were confirmed by observing the loss of Ach-induced relaxation. When the NE-induced contractions reached a plateau, each sample was added cumulatively. When NE-induced contractions in the presence of Fl were examined, thoracic aortic strips were exposed to Fl ([10.sup.-4]M) for 1 h and then NE was added to the afore-mentioned bath. For examination of the contraction in depolarized muscle, normal KHS in the tissue bath was replaced to KHS containing high concentration [K.sup+] (NaCl 62.7 mM, KC1 60.0 mM, [NaHCO.sub.3] 25 mM, [CaCl.sub.2] 1.8 mM, [NaH.sub.2][PO.sub.4] 1.2mM, [MgSO.sub.4] 1.2 mM, and glucose 11.0 mM). When the high [K.sup.+] (60.0 mM)-induced contraction reached a plateau, Fl ([10.sup.-4]M) were added. Then, relaxant effects were calculated against the contraction. For examination of [Ca.sup.2+]-induced contractions in the presence of NE, the aortic rings were exposed to Fl (4 x [10.sup.-5] M) or F2 ([10.sup.-5]M, as s positive control) in [Ca.sup.2+]-free KHS containing 0.01 mM EGTA for 1 h, followed by the addition of nicardipine [10.sup;.-6] M and NE [10.sup.-6]M. Next, [Cas.up.2+] ([10.sup.-5]-[10s.up.-3]M) were added cumulatively to the bath. When PDA-induced contractions in the presence of Fl were examined, thoracic aortic strips were exposed to Fl (4 x [10.sup.-5] M) for 1 h and then PDA [10.sup6]M was added to the afore-mentioned bath. Compounds Fl and F2 were dissolved in DMSO and diluted with saline. The final concentration of DMSO in the organ bath was less than 0.1%, and did not show any effect on contraction or relaxation. All other drugs used were dissolved in saline.

Statistical analysis

The results are expressed as mean [+ or -] S.E. Statistical evaluation of the data was performed using Bonferroni-type multiple t-test ( javastat/JavaStat-j.htm). P values of less than 5% were considered significant.


Effect of Fl on NE-induced contraction

When NE 3x [10.sup.-7]M was applied to thoracic aortic rings with endothelium after maximum response was achieved, we added Fl at [10.sup.-4] M and observed slow vasorelaxant actions. These vasorelaxant effects of Fl ([10.sup.-6], 3 x [10.sup.-6], [10.sup.-5], 3 x [10.sup.-5] and [10.sup.-4] M) were shown in a concentration-dependent manner (13.2%, 15.3%, 22.8%, 43.6% and 90.9% relaxant, n = 3). Such relaxant actions were also seen in the sample of aortic rings without endothelium (data not shown), suggesting that the inhibitory effects of Fl on NE-induced contraction of aortic rings are not dependent on the presence of endothelium. On the other hand, Fl did not affect the basal tension of aortic rings (data not shown).

Effect of Fl on NE-induced contraction (pre-administration)

We also investigated NE-induced contractions with the addition of Fl to aortic rings. We applied NE 3 x [10.sup.-7]M to contract aortic rings 60 min after adding Fl at [10.sup.-4]M or F2 at 3x [10.sup.-5]M (Fig. 2). The NE-induced contractions were divided into two phases.


The first contraction as phasic phase is characterized by an initial rapid and transient contraction. The second phase as tonic phase is identified as the maintained contraction, exhibiting a long-lasting contraction. As shown in Fig. 2, Fl did not inhibit the phasic contraction, while the tonic contraction was evidently inhibited by Fl. Similarly, F2 at [10.sup.-5]M inhibited tonic contraction only.

Effect of Fl on high [K.sup.+]-induced contraction

The aortic rings depolarized with isotonic high [K.sup.+] (60 mM) caused contractions with the [Ca.sup.2+] influx via voltage-dependent [Ca.sup.2+] channels (VDCs). The contractions of depolarized aorta were not affected by treatment of Fl at [10.sup.-4]M (data not shown).

Effect of Fl on [Ca.sup.2+] -induced contraction of NE-stimulated muscles

The NE [10.sup.-6] M-induced contraction of the aortic rings in the presence of [10.sup.-6]M nicardipine in [Ca.sup.2+] -free KHS occurred in [Ca.sup.2+] ([10.sup.-5] to [10.sup.-3]M) concentration-dependent manner presumably due to [[Ca.sup.2+]], via receptor-operated [Ca.sup.2+] channels (ROCs). Compound Fl inhibited these contractions at 4 x [10.sup.-5] M (Fig. 3), suggesting that Fl exerts inhibitory effects on [[Ca.sup.2+] ],-. Compound F2, which reported as a ROCs inhibitor (Wong et al., 2001), showed inhibitory effect on these contractions.


Effect of Fl on PDA-induced contractions of rat aortic strips

Protein kinase C was involved in the maintenance of the agonists-induced contraction in rat aorta and it regulates a [Ca.sup.2+] sensitivity of smooth muscle (Rasmus-sen et al., 1984; Weber et al., 1995). Although PDA, a protein kinase C activator, was known to induce a slowly development contraction, the PDA ([10.sup.-6]M) -induced contraction was not inhibited by pretreatment with Fl (4 x [0.sup.-5]M) for 1 h. Thus, compound Fl may not affect regulation of [Ca.sup.2+] sensitivity of smooth muscle in agonists-induced contraction (data not shown).


The vasorelaxant effects of Fl were examined in NE-treated isolated rat thoracic aorta. Compound Fl relaxed NE-induced vasocontraction in a concentration-dependent manner. And Fl inhibited only the tonic contraction in characteristically as with F2 (Fig. 2). It is generally accepted that NE-induced tonic phase contraction is mediated via [Ca.sup.2+] influx through ROCs and VDCs (Meisheri et al., 1981). In many cases, vasoconstrictor-induced contraction is largely mediated by [Ca.sup.2+]-influx so that inhibitors of [Ca.sup.2+]-influx cause vasorelaxant effect. As described above, no effect of Fl was observed on high [K.sup.+] (60 mM) depolarized contractions, while Fl inhibited [Ca.sup.2+]-induced contraction of the aortic rings pre-incubated with NE in the presence of nicardipine, just like way of F2 known as ROCs inhibitor (Wong et al., 2001; Iizuka et al., 2005).

The above results demonstrate that Fl relaxes NE-induced vasocontraction; it indicates that the vasorelaxant effect of Fl may be due to the inhibition of [Ca.sup.2+]-influx through ROCs similar to that of F2, but not dependent on VDCs and regulation of [Ca.sup.2+]-sensitivity of smooth muscle.

The effects of several iridoid derivatives on smooth muscle were reported before (Chung et al., 1980; Sarg et al., 1990; Yamahara et al, 1991; Breschi et al., 1992; Ghisalberti, 1998; Schapoval et al., 1998; Rojas et al., 2000), but few reports directed to the effects on vascular smooth muscle (Breschi et al., 1992). As previously reported, we confirmed vasorelaxant effects of F2 isolated from the leaves of F. viridissima (Iizuka et al., 2005). Therefore, it is suggestive that Fl contributes to the vasorelaxant effects of the F. viridissima in association with F2.

In conclusion, forsythide (Fl) inhibited NE-induced vasocontractions, which mechanism of action might be in part attributable to the involvement of [Ca.sup.2+]-influx through ROCs.


The authors are grateful to Dr. Nishibe (Health Science University of Hokkaido) and Dr. Deyama (Yomeishu Seizo Co., Ltd.) for the valuable advice and kindly supplying the plant material, respectively.


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* Corresponding author at: Laboratory of Pharmacognosy, Yokohama College of Pharmacy, Matano-cho 601, Tozuka-ku, Yokohama, Kanagawa 245-0066, Japan. Tel.: +81 45859 1300; fax: +8145859 1301.

E-mail address: (T. Iizuka).

T. Iizuka (a), (b), * H. Sakai (b), H. Moriyama (c), N. Suto (a), M. Nagai (d), D. Bagchi (e)

(a) Laboratory of Pharmacognosy, Yokohama College of Pharmacy, Matano-cho 601, Tozuka-ku, Yokohama,

Kanagawa 245-0066, Japan

(b) Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ehara, Shinagawa-ku, Tokyo 142-8501, Japan

(c) Laboratory of Pharmcotherapeutics, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen,

Machida, Tokyo 194-8543, Japan

(d) Ohu University School of Pharmaceutical Sciences, Misumido 31-1, Tomita-machi, Koriyama 963-8611, Japan

(e) Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA

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Title Annotation:SHORT COMMUNICATION; norepinephrine
Author:Iizuka, T.; Sakai, H.; Moriyama, H.; Suto, N.; Nagai, M.; Bagchi, D.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9JAPA
Date:Apr 1, 2009
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