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Inhibitory effects of Choto-san (Diao-teng-san), and hooks and stems of Uncaria sinensis on free radical-induced lysis of rat red blood cells.

Summary

The present study is designed to test our hypothesis that the ingestion of Uncaria sinensis (US), the main medicinal plant of Choto-san (Diao-teng-san, CS), would protect red blood cell (RBC) membrane from free radical-induced oxidation if polyphenolics in US could be absorbed and circulated in blood. When incubated with RBC suspension, Choto-san extract (CSE) and Uncaria sinensis extract (USE) exhibited strong protection for RBC membrane against hemolysis induced by 2,2-azo-bis (2-amidinopropane) dihydrochioride (AAPH), an azo free-radical initiator. The inhibitory effect was dose-dependent at concentrations of 50 to 1000 [micro]g/mL. Ingestion of 200 mg of USE was associated with a significant decrease in susceptibility of RBC to hemolysis in rats. Furthermore, caffeic acid, an antioxidative hydroxycinnamic acid, was identified in rat plasma after administration of URE.

Key words: Choto-san, Uncaria sinensis, erythrocyte, caffeic acid, free radical, membrane oxidation

Introduction

It has become clear that Choto-san (Diao-teng-san, CS) is an essential and effective crude drug in the treatment of vascular dementia (Shimada et al., 1994; Terasawa et al., 1997). Moreover, Choto-san was shown to improve hemorrheological factors, i.e., erythrocyte and leukocyte deformability (Yang et al., 1999).

Recent data revealed that Uncaria sinensis (US), the main medicinal plant of Choto-san, had a vasodilator effect, whose mechanism consisted of both endothelium-independent relaxation with [Ca.sup.2+] channel blocking effect (Goto et al., 1999) and endothelium-dependent relaxation with nitric oxide (Goto et al., 2000). Polyphenolic substances contained in URE are considered as the active components responsible for these mechanisms.

Reactive oxygen species are thought to be involved in the damage of biomembrane during ischemia, inflammation and aging. It was shown that low vitamin E in red blood cell (RBC) membrane was associated with increased susceptibility to hemolysis (Delmas-Beauvieus et al., 1995).

This paper reports the role of CS and US an antioxidative point of view. We hypothesize that CS and US strengthen the protection of RBC membrane against free radical-induced oxidative damage. Therefore, the present study is designed to examine the effect on RBC hemolysis induced by an azo free-radical initiator after a gavage dose of Uncaria sinensis extract (USE) in rats.

Materials and Methods

Preparation of Drugs

Choto-san extract (CSE) and extract of Choto-san without Uncaria sinensis (CS-US.E): One hundred g and 90.3 g of mixtures of the pharmacons shown in the Table 1, were each suspended in 500 mL of distilled water and boiled for 40 min. Then, 300 mL of infusion solutions were formed and centrifuged at 12,000 rpm for 30 min, filtered and lyophilized to yield 11.0 g of CSE and 10.2 g of CS-US.E, respectively. All the components of CS were purchased from Tochimoto Pharmaceuticals (Osaka, Japan).

Uncaria sinensis extract (USE): USE was prepared from the hooks and stems of Uncaria sinensis purchased commercially (Chinese origin, Tochimoto Pharmaceuticals). The extract was obtained by boiling the hooks in water for 50 minutes and then freeze-drying into a resultant powder. We got 8.0 g of extract from 100 g of raw material.

In our study, the powdered extract was dissolved in phosphate buffered saline (PBS, pH = 7.4) for animal experiments.

Other reagents: Caffeic acid was purchased from Tokyo Kasei (Tokyo, Japan). Procyanidin B-1, procyanidin B-2, epicatechin and catechine were kindly provided by Tsumura & Co. (Tokyo, Japan).

HPLC analysis of USE

The HPLC system consisted of a SD-8022 degassa, CCPM-II pump, CO-8020 column oven and PD-8020 detector (Tosoh, Tokyo, Japan). 15 [micro]L of USE was analyzed and polyphenols (caffeic acid, procyanidin B-1, procyanidin B-2, epicatechin and catechin) were monitored by HPLC. HPLC was performed using a column (TSK-gel ODS-80TM, 150 x 4.6 mm I.D., Tosoh) with the mobile phase consisting of 0.02 M [KH.sub.2][PO.sub.4]-[H.sub.3][PO.sub.4] (pH 2.4) -MeOH (75:25) at a flow rate of 0.8 mL/min. The column temperature was 37 [degrees]C. The detector wavelength was 270 nm. All solvents of analytical and HPLC grade were obtained from Wako (Osaka, Japan). Fig. 1 shows the HPLC chart of the phenolic fractions from USE.

In vitro study of anti-hemolytic activity of CSE, CS-US.E, USE and caffeic acid

Male Wistar rats (350 g) were fed commercial diet (type CE-2, CLEA Japan Inc., Tokyo, Japan) for one week. Blood obtained from rats by cardiac puncture was collected into heparinized tubes. RBC was separated from plasma by centrifugation at 1500 g for 20 min. Crude RBC was then washed three times with 5 volumes of PBS. Packed RBC was thereafter suspended in 4 volumes of PBS solution. Oxidative hemolysis in RBC induced by 2,2 -azo-bis (2-amidinopropane) dihydrochloride (AAPH) has been extensively studied as a model for peroxidative damage in biomembrane (Miki et al., 1987; Sugiyama et al., 1993; Zhang et al., 1997). In the present study, the method of Zhang et al. was used to determine the hemolysis of RBC mediated by AAPH. The addition of AAPH (a peroxyl radical initiator) to the suspension of RBC causes oxidation of lipids and proteins in cell membrane and thereby induces hemolysis. It is known that AAPH-induced hemolysis in RBC is a function of incubation time and is proportional to the concentration of fre e radicals. The inhibitory effect on RBC hemolysis is also proportional to the concentration of antioxidants in the incubation mixture. Two mL of RBC suspension was mixed with 2 mL of PBS solution containing various amounts of CSE, CS-US.E, and USE (10-1000 [micro]g/mL) and caffeic acid (1-50 [micro]g/mL), respectively. Two mL of 200 mM AAPH in PBS solution was then added to the mixture. The incubation mixture was shaken gently in a water bath at 37 [degrees]C for 3 hours. After incubation, 8 mL of PBS solution was added to the reaction mixture, followed by centrifugation at 1000 g for 10 min. The absorbance (A) of the supernatant at 540 nm was recorded with a UV-visible recording spectrophotometer (UV-265FS, Shimadzu, Kyoto, Japan). Percentage inhibition was calculated by the following equation:

% Inhibition = [[A.sub.AAPH] - [A.sub.CS]]/[A.sub.AAPH]

where [A.sub.CS] is the absorbance of the sample containing CSE, CS-US.E, USE, and caffeic acid, and [A.sub.AAPH] is the absorbance of the control sample containing no CSE and USE.

In vivo study of anti-hemolytic activity of USE

Male Wistar rats (350 g) were randomly divided into two groups. One group was gavage-dosed with 1 mL of distilled water containing 200 mg of USE, while the control group was gavage-dosed with 1 mL of distilled water only. 30 min after administration, blood from the heart was collected in heparinized tubes. RBC was separated from plasma by centrifugation at 1500 g for 20 min. After removal of white blood cells (WBCs) and platelets (PLTs), the remaining RBC was mixed with the volume of plasma therefrom. Two mL of the reconstituted blood (without WBCs and PLTs) was then used for the hemolysis assay by adding 2 mL of AAPH solution and 2 mL of PBS followed by incubation at 37 [degrees]C for 2, 3 and 4 hours, respectively. As mentioned above, 8 mL of PBS solution was added to the incubation mixture followed by centrifugation at 1000 g for 10 min. The absorbance (A) of the supernatant at 540 nm was measured. The percentage inhibition of a gavage dose of USE was calculated in the same way as in the in vitro study:

% Inhibition = [[A.sub.CTL] - [A.sub.USE]]/[A.sub.CTL]

where [A.sub.USE] is the absorbance of the reconstituted blood obtained from rats administered USE, and [A.sub.CTL] is the absorbance of the reconstituted blood from control rats.

To investigate the absorption and circulation of polyphenols after a gavage dose of 200 mg USE or distilled water, the rats were killed at 30 min. Blood was collected from the aorta, and 1 mL of plasma was extracted twice with 1 mL of ethyl acetate. 278 [micro]L of 100 [micro]M caffeic acid was added to 1 mL of plasma from control and USE rat (final concentration of caffeic acid: 100 [micro]g/mL) followed by extraction in a similar manner. The ethyl acetate was then removed under a gentle stream of nitrogen. The residues were dissolved in 50 [micro]L of pure water and subjected to HPLC analysis as described above in the section for HPLC analysis of USE.

Statistical analysis

Data are reported as mean values [+ or -] standard deviation (S.D.). One-way and two-way repeated-measures ANOVA followed by Fisher's PLSD were used for statistical analysis. A p value < 0.05 was regarded as significant.

Results

The results of the in vitro tests are showed in Figures 2-4. CSE and CS-US.E inhibited the hemolysis of rat RBC due to AAPH-induced peroxy-radicals in a dose-dependent manner. This inhibitory effect was more noticeable in CSE than CS-US.E, and the difference was significant (Fig. 2). USE inhibited AAPH-induced hemolysis in a dose-dependent manner (Fig. 3). Moreover, caffeic acid strongly inhibited RBC hemolysis dose-dependently (Fig. 4).

The results of the in vivo tests are presented in Figure 5. RBC in reconstituted blood (without white blood cells and platelets) obtained from USE rats was more resistant to AAPH-induced hemolysis than that obtained from control rats (Fig. 5).

Figure 6 shows the result of the HPLC analyses of rat plasma. There was a peak in the plasma of USE rats, but not in control rats (arrow in Fig. 6c). The peak was heightened by the addition of caffeic acid to plasma of USE rats (Fig. 6d). Furthermore, plasma of control rats with caffeic acid showed the same peak as USE rats (arrow in Fig. 6e). These findings suggested that caffeic acid included in URE was absorbed from the alimentary tract and circulated through the body.

Discussion

The biomembrane may be most susceptible to free radical attack due to its content of polyunsaturated fatty acids. The present study revealed that US, the chief medicinal plant of CS, can protect these polyunsaturated fatty acids from oxidation in the membrane of RBC incubated with AAPH not only in vitro but also in viva. Previously, it was reported that CS improved erythrocyte deformability in patients with asymptomatic cerebral infarction (Yang et al., 1999). So far, the reason why CS has favorable effects on the microcirculation has not been determined. The direct protection of RBC membrane from free radical attack as observed in the present study would provide an important pathophysiological basis for making use of the helpful hemorrheological effects of CS and US.

Although we could not identify procyanidin B-1, procyanidin B-2, epicatechin and catechine in rat plasma after USE administration, this study indicated that caffeic acid could be absorbed and circulate in blood. It was reported that caffeic acid formed a transient chelating complex with cupric ions, coupled with its free radical scavenging properties, thereby accounting for its inhibitory activity of LDL oxidation (Nardini et al., 1995; Brown et al., 1998). Furthermore, it was suggested that caffeic acid inhibited carcinogenesis and increased the antioxidant defense system in vivo (Tanaka et al., 1993; Nardini et al., 1997). It has been clarified that when rats are orally administered 200 mg of caffeic acid, almost all of the caffeic acid metabolites found in rat plasma are in the form of glucronide, sulfate, and sulfate/glucronide conjugates of caffeic acid or its methylated compound, ferulic acid (Azuma et al., 2000). The metabolism of caffeic acid has been reported (Rechner et al., 2001; Moridani et al., 2001). Moreover, Azuma et al. assumed that ingested caffeic acid, absorbed from the alimentary tract, may be metabolized through the same pathway as that proposed for flavonoids (Piskula et al., 1998), so the presence of nonconjugated caffeic acid might be due to overdosing of the animals. Rats were prescribed 200 mg of USE in the present study, meaning that the caffeic acid contained in the USE administrated was less than 20 mg, a dosage level that did not seem to be overdosing. So, we supposed that the absorption of crude drugs might be different from that of pure compounds. The present results, together with those previously reported in the literature, suggest that caffeic acid and its metabolites which are supplied by USE administration at least create a sound antioxidant defense system and favorable hemorrheological conditions.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]
Table 1. Composition of Choto-san.

Herbal Medicine Choto-san Choto-san
 without URE

Gypsum Fibrosum 16.0 g 16.0 g
Chrysanthemi Flos 9.7 g 9.7 g
Uncariae Uncis cum Ramulus 9.7 g 0 g
Aurantii nobilis Pericarpium 9.7 g 9.7 g
Ginseng Radix 9.7 g 9.7 g
Ophiopogonis Tuber 9.7 g 9.7 g
Ledebouriellae Radix 9.7 g 9.7 g
Hoelen 9.7 g 9.7 g
Pinelliae Tuber 9.7 g 9.7 g
Glycyrrhizae Radix 3.2 g 3.2 g
Zingiberis Rhizoma 3.2 g 3.2 g

Total 100.0g 90.3g


Acknowledgments

This work was supported by a Health Sciences Research Grant for Comprehensive Research on Aging and Health from the Japanese Ministry of Health, Labour and Welfare.

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Abbreviations:

AAPH -- 2,2,-azo-bis (2-amidinopropane) dihydrochloride; CS -- Choto-san; CSE -- Choto-san extract; US -- Uncaria sinensis: USE -- Uncaria sinensis extract; CS-US.E -- extract of Choto-san without Uncaria sinensis

N. Sekiya (1), Y. Shimada (1), N. Shibahara (2), S. Takagi (1), K. Yokoyama (1), Y, Kasahara (1), I. Sakakibara (3), and K. Terasawa (1)

(1.) Department of Japanese Oriental Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama Japan

(2.) Department of Kampo Diagnostics, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan

(3.) Kampo & Pharmacognosy Laboratory, Tsumura & Co., Amimachi, Ibaraki, Japan

Address

N. Sekiya, Department of Japanese Oriental Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan Tel: ++81-76-434-7393; Fax ++81-76-434-0366; e-mail: s114@ms.toyama-mpu.ac.jp
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Author:Sekiya, N.; Shimada, Y.; Shibahara, N.; Takagi, S.; Yokoyama, K.; Kasahara, Y.; Sakakibara, I.; Tera
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Oct 1, 2002
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