Abstract

Background: Rokumigan, hachimijiogan and goshajinkigan are traditional Kampo formulations. These formulations are considered to be useful for edema, water disturbance, and urinary problem. Rokumigan is composed of six kinds of crude herbal drugs. Hachimijiogan comprises the same six kinds of herbs and two crude drugs and goshajinkigan includes the same eight herbs of hachimijiogan and two others. We demonstrated that these formulations possess vasodilating actions in rat aorta. Traditionally, rokumigan is thought to be useful for young persons, while hachimijiogan and goshajinkigan are thought to be for elderly persons. In this study, we compare the age-dependent modulation of these Kampo formulations.

Methods: Young wistar rats (10 to 15 week old) and aged rats (35 to 40 week old) were used to investigate the age-dependent actions of these formulations. The isolated aorta ring strips were fixed in Krebs-Henseleit solution. After the contractile response induced by norepinephrine became steady, Kampo formulations are added.

Results: Rokumigan showed potent vasodilatation in young rats. However, the vasodilation - induced by rokumigan was attenuated in aged rats. On the contrary, hachimijiogan - induced vasodilatation in aged rats was to the same extent as that in young rats. Furthermore, compared with young rats, goshajinkigan - induced vasodilatation significantly increased in aged rats. Endothelium removal and L-NAME(NO synthase inhibitor) attenuated rokumigan, hachimijiogan and goshajinkigan - induced vasodilatation in young rats. However, endothelium removal and the pretreatment of L-NAME did not modify rokumigan - induced vasoditation in aged rats. Endothelium removal and L-NAME attenuated hachimijiogan and goshajinkigan - induced vasodilatation in aged rats.

Conclusion: Rokumigan - induced vasodilatation attenuated as age, while the potency of hachimijiogan and goshajinkigan - induced vasodilatations was not changed or increased in aged rats compared with young rats. Our results suggested that the difference of age - related dependency of kampo formulations on vasodilatation was derived from their different dependency to the endothelium.

Keywords

Rokumigan; Hachimijiogan; Goshajinkigan; Vasodilatation; Endothelium

Introduction

Rokumigan, hachimijiogan and goshajinkigan are traditional Japanese herbal (Kampo) formulations. They have been used for the patients suffering from urinary tract symptoms, chronic nephritis and low back pain [1]. The compositions of crude drugs in three kampo formulations are similar, but the number of crude drugs are different. Rokumigan comprises six crude herbal drugs (Rahmanniae Radix, Corni Fuructus, Dioscorea Rhizoma, Alismatis Rhizoma, Poria cocos, and Moutan Cortex). Hachimijiogan comprises the six drugs ofrokumigan and two additional crude drugs (Cinnamomi Cortex and Processi Aconiti Tuber) and goshajinkigan comprise the eight crude drugs of hachimijiogan and two additional crude drugs (Achyranthis Radix and Plantaginis Semen). The exact compositions of crude drugs in three kampo formulations are summarized in Table 1. We have shown the same summary of compositions in previous article entitled “comparative study of vasodilatation induced by rokumigan, hachimijiogan, and goshajinkigan in the Rat aorta” of this journal [2].

In Kampo medicine, rokumigan, hachimijiogan and goshajinkigan are considered to regulate “Jin” functions. The “Jin” has been considered to regulate the water balance of body, urinary track system, hearing system, bone and bone marrow[1,3]. Therefore,“Jin” can be explained as kidney and urinary track system. The failure of “Jin” directly leads to the incidences of edema or dehydration, frequent urination, osteoporosis, and also some dysfunctions related with ageing.

Rokumigan can regulate “Jin” functions, resulting in the improvement of body water balance that is exhausted by inflammatory disorder and unusual heat. Rokumigan is effective for not only children easily to develop a fever, but also adult persons. On the other hand, both hachimijiogan and goshajinkigan have been prescribed traditionally for elderly persons with the symptoms such as dry skin, dry mouth, weakening of legs, benign prostatic hypertrophy and lower back pain [1]. Both drugs have also been well known to improve frequent urination or lower urinary tract symptoms [1,3]. Hachimijiogan relaxes acetylcholine (ACh) - induced contraction of isolated rat bladder strips [4]. Goshajinkigan has been shown to beso effective for overactive bladder (OAB) in male and female patients [5,6]. It has been reported that goshajinkigan regulate bladder activity via sympathetic and parasympathetic nervous systems and suppress C-fiber activation [7,8]. The indications of hachimijiogan and goshajinkigan are similar, but goshajinkigan is relatively useful for more elderly persons suffering from lower back pain and leg pain [1]. Furthermore, the other beneficial effect for elderly people by goshajinkigan has been reported. Sarcopenia is one of the age-related phenomena. Sarcopenia means the phenomenon that muscle mass and strength decrease as age. It has been reported that goshajinkigan protects against sarcopenia promotion in senescence-accelerated mice [9].

Rokumigan, hachimijiogan and goshajinkigan have similar indications; regulating edema and water balance. We ascribe partially these indications to the improvement of blood flow induced by these three Kampo formulations. We infer that these clinical indications are partially derived from the vasodilation induced by these Kampo formulations [2]. And these three Kampo formulations have same mechanisms of vasodilatation such as Ca2+ channel inhibition, β-receptor activation and producing nitric oxide(NO) from endothelium [2].

We have investigated the age-dependent modulation of the vasodilating effects induced by herbal medicine and Kampo formulations. Aging produces various deleterious physiological and histological changes in vascular system such as a plaque formation and wall thickening [10]. Endothelial dysfunction induced by aging is mainly associated with decreased production of NO, resulting in impaired vasodilatation [11]. In aged endothelial cells, NO synthesis decreased [11]. Loss of PI3K/Akt-dependent eNOS phosphorylation is one of the mechanisms explaining the reduction of eNOS activity in old rats [12]. Furthermore, it was reported that age-related change of endothelium-dependent vasodilatation is also involved with reduced bioavailability of NO and response of smooth muscle to NO[11]. Alteration of endothelium-dependent vasodilatation is characterized by reduced agonist-induced vasodilatation. Acetylcholine (Ach) - induced vasodilatation was attenuated in aorta and mesenteric artery of elderly rats due to malfunction of endothelium [13-15]. And sheer stress - induced vasodilatation (which is response of endothelium to shear stress resulting the production of NO) is also impaired by aging [16].

However, we have reported that herbal medicine or Kampo medicine showed the tendency that they exhibit more potent vasodilatation in elder rats than in young rats [17,18].

Ginkgo biloba extract (GBE) is one of the famous herbal medicines [19]. GBE is effective for ischemic brain injury and cerebral disorders with aging. GBE induces dose-dependent vasodilatation [19]. Bilobalide, one of the main constituents in GBE, also exhibits dose-dependent vasodilatation on rat aorta [19]. Bilobalide-induced vasodiltation is attenuated as age develop (5 to 25 week old rats) [17]. Ginkgo biloba extract-induced vasodilatation is also modulated by aging. In 5 to 20 week old rats, GBE-induced vasodilatation (at 0.01 ~ 1 mg/ml) decreased with age. However, in 25 week old rats, GBE - induced vasodilatation (at 0.01 ~ 1 mg/ml) reversely enhanced [17]. The age-dependent attenuation of bilobalide - induced vasodilatation was more severe than that of GBE - induced vasodilatation [17]. The GBE, as a mixture of some constituents, kept vasodilatation potency in elderly rats. However, in 25 week old rats, the vasodilatations - induced by bilobalide were severely attenuated [17]. We infer that the interaction between some constituents in GBE preserve GBE - induced vasodilatations from age - dependent change.

Mokuboito, one of the Kampo formulations, comprises four crude drugs [20]. Mokuboito is useful for edema, dyspnea caused by heart failure [20]. Mokuboito possesses dose - dependent vasodilating actions. Sinomenine acutum is one of the crude herbs in Mokuboito. The extract of Sinomenine acutum also exhibits vasodilatation onrat aorta [18]. Sinomenine is one of the constituents in Mokuboito. This alkaloid is consisted in Sinomenine acutum. Sinomenine has potent vasodilating actions [17,18]. However, sinomenine - induced vasodilating actions severely attenuated in elder rats (35 to 65 week old rats) [18]. The extract of Sinomenin actum was also attenuated in elder rats (35 to 65 week old rats). However, the attenuation of Sinomenine acutum - induced vasodilatation is milder than that of sinomenine [18]. The extract of Sinomenine acutum is a mixture of some constituents. Therefore, Sinomenine acutum is resistant to the influence of age. On the other hand, the vasodilatation induced by Mokuboito did not attenuate in elder rats. Mokuboito comprises four crude drugs and is more resistant to aging than Sinomenine acutum or sinomenine [18]. We infer that the interaction with herbal drugs and the ingredients in Mokuboito enhances vasodilating effects and prevent the age - dependent attenuation of pharmacological responses.

Rokumigan has been traditionally thought to be suitable for young persons, while hachimijiogan and goshajinkigan have been prescribed for elderly persons [1,3]. We infer that the difference of agedependency between Kampo formulations is partially derived from the age - dependent changes of vasodilatations induced by Kampo medicines. Therefore, we compared the vasodilating effects induced by Kampo formulations in young and elder rats and investigate the age - dependent alterations through by using rat aortic ring strips.

Materials and Methods

All experiments were carried out according to the guidelines laid down by the Nara Medical University Animal Welfare Committee and the Doshisha Women’s Collegeanimal Welfare Committee.

Male Wistar rats were used in this study. To investigate the age dependent modulation of pharmacological response, young rats (10 to 15 week old) and aged rats (35 to 40 week old) were used. The rats were anesthetized with ether or isoflurane, and were euthanized by exsanguinations. The thoracic aorta was quickly removed, and was cut into rings of 3 mm in length. The experiments were carried out as reported previously [2,17,18,20]. The vascular rings were suspended between lower and upper triangular - shaped stainless steel stirrups in a jacketed organ chamber filled with 20 ml modified Krebs - Henseleit solution. The lower stirrup was anchored and the upper stirrup was attached to a force - displacement transducer (Nihon Kohden TB-652T, Tokyo, Japan) to record the isometric force. All rings were stretched to generate a resting tension of 1.2 g. The modified Krebs - Henseleit solution was comprised of 118 NaCl, 4.6 KCl, 1.2 MgSO₄, 1.2 KH₂ PO₄, 11.1 glucose, 27.2 NaHCO₃, 0.03 Na₂ - ethylnediaminetetraacetic acid (EGTA), and 1.8 mM CaCl₂. The chamber solution was kept at 37 °C and oxygenated with 95% O₂ and 5% CO₂. After 40 min of resting, 5 µM norepinephrine (NE)was added to the tissue bath. After the contractile response became steady, the drugs were cumulatively administrated into the bath solution. The effects of the drugs were measured 10-20 min after the responses became steady. The relaxation responses were calculated as the percentage decrease compared with the maximal contraction induced by NE. Endothelium was carefully removed by rubbing internal lumen with smooth and thin plastic sticks. The absence of functional endothelium was confirmed by a lack of the relaxant response to acetylcholine (ACh, 1 μM).

Rokumigan (TJ-87), hachimijiogan (TJ-7) and goshajinkigan (TJ-107) are produced by Tsumura Co. (Tokyo, Japan) for clinical use in Japan. They are a spray - dried powder extracted with boiling water of a mixture of medical herbs. The drugs used in this study are supplied by Tsumura Co. The components of three formulations are summarized in Table 1).The Kampo formulations were dissolved with DMSO. And NG-Nitro-L-arginine methyl ester (L-NAME) (Wako Chemical, Osaka, Japan) were used. NaCl, KCl, MgSO₄, KH2PO4,glucose, NaHCO3, CaCl2, and DMSO were also purchased from Wako Chemical. NE (Daiichi Sankyo Co., Tokyo, Japan) was used. All values are represented as the means ± S.E.M. The differences of data in mean values were analyzed by analysis of variance (ANOVA) and a p value of less than 0.05 was considered significant.

Table 1: The compositions of the crude drugs contained in rokumigan, hachimijiogan and goshajinkigan.

Results

Application of NE caused remarkable vasoconstriction of resting aorta. We have reported that rokumigan, hachimijiogan and goshajinkigan caused the vasodilatations in concentration dependent manner in young rats (10 to 15 week old rats) [2].

Rokumigan at 0.03 mg/ml had less vasodilatation (by 0.51 ± 0.25%), butit caused significant vasodilatation at over 0.1 mg/mL; by 69.3 ± 3.9% (n = 8, p < 0.01) at 3 mg/mL in young rats. In aged rats(35 to 40 week old rats), the vasodilatation induced by rokumigan was also observed. At lower concentration, the potency of rokumigan- induced vasodilatations tended to decrease in aged rats, compared with those in young rats. Rokumigan - induced vasodilatations were attenuated significantly in aged rats at 0.1, 0.3 mg/mL (Figure 1A). At 3 mg/mL, rokumigan sustained almost the same potency as young rats. Hachimijiogan at 0.1 - 3 mg/mL also caused the significantvasodilatation in young rats; by 67.0 ± 4.9% (n = 8, p < 0.01) at 3 mg/mL. There are not significant differences in hachimijiogan -induced vasodilatations between young rats and aged rats (Figure 1B).

Figure 1:Age dependent change of vasodilatations induced by rokumigan, hachimijiogan and goshajinkigan.(A) Modulation of rokumigan - induced vasodilatation.(B) Modulation of hachimijiogan - induced vasodilatation. (C) Modulation of goshajinkigan - induced vasodilatation.Symbols are: Open circles: control (young rats) (n = 8) and Close circles: aged rats (n = 8). Values (%) represent mean ± S.E.M.* P < 0.05: significantly different from control.

On the contrary, goshajinkigan induced more potent vasodilatation in aged rats, compared with young rats. Goshajinkigan induced stronger vasodilatations at 1.0 and 3.0 mg/ml in aged rats than those in young rats significantly (Figure 1C). The percent of vasodilatations in young rats was 24.0 ± 4.0 (n = 8) and 58.1 ± 2.9%(n = 8) at 1.0 and 3.0 mg/mL, respectively. Whereas the percent of vasodilatations in aged rats was32.4 ± 4.4 (n = 8, p < 0.05) and 72.8 ± 4.9 % (n = 8, p < 0.05) at 1.0 and 3.0 mg/mL, respectively.

To investigate the endothelium dependency of vasodilatations, L-NAME (100 μM) and endothelium removal were pretreated. In young rats, we have been reported that L-NAME and endothelium removal attenuated kampo formulations-induced vasodilatations in previous reports [2]. However, in aged rats, L-NAME and endotheliumremoval did not attenuate rokumigan-induced vasodilatations at 0.03 - 1.0 mg/mL (Table 2). Only at 3 mg/mL, endothelium removal and L-NAME alter the rokumigan - induced vasodilatation.

Table 2: Controls are vasodilatations in aged rats (35 to 40 week old rats). Values (%) represent Mean ± S.E.M.*: p < 0.05.**: p < 0.01.Symbols of * and ** mean a significance compared with the vasodilating effect of control at each concentration.

We reported that the hachimijiogan - and the goshajinkigan - induced vasodilatations were also reduced by pretreatments with both L-NAME and endothelium removal [2]. The endothelium removal decreased the vasodilatations induced by hachimijiogan at 0.3 - 3.0 mg/mL in young rats [2]. L-NAME also reduced the degree of vasodilatations at 1.0 mg/ml and at 3.0 mg/ml of hachimijiogan in young rats [2]. In aged rats, L-NAME and endothelium - removal also significantly attenuated hachimijiogan - induced vasodilatations at 0.3, 1.0 and 3.0 mg/mL. Endothelium - removal decreased the vasodilatations; 6.8 ± 3.0 % (n = 6, p < 0.05) at 0.3 mg/mL, 16.0 ± 7.2% (n = 6, p < 0.05) at 1.0 mg/mL, 53.3 ± 3.6 % (n = 6, p < 0.05) at 3.0 mg/mL in aged rats.

As well in young rats, the goshajinkigan (3.0 mg/mL) - induced vasodilatation decreased by 43.7 ± 1.3% (n = 8, p < 0.01) in the presence of L-NAME, and by 47.6 ± 3.9 % (n = 8, p < 0.01) under the condition of endothelium-removal [2]. In aged rats, L-NAME and endothelium - removal also significantly attenuated goshajinkigan -induced vasodilatations at 0.3 ~ 3.0 mg/mL (Table 2). In aged rats, endothelium - removal significantly decreased the goshajinkigan - induced vasodilatations; 20.0 ± 5.2 % (n = 6, p < 0.05) at 1.0 mg/ mL, 53.0 ± 3.2 % (n = 6, p < 0.01) at 3.0 mg/mL (Table 2). L-NAME also significantly attenuated goshajinkigan - induced vasodilatationsin aged rats (Table 2). These results indicate that goshajinkigan elicit endothelium - dependent vasodilatation in aged rats.