Comparative efficacy of Keishi-bukuryo-gan and Pentoxifylline on RBC deformability in patients with "oketsu" syndrome.

Summary

Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is one of the prescriptions in Japanese traditional medicine for improving the "oketsu" syndrome, so-called blood stasis syndrome. "Oketsu" syndrome is an important pathological conception in Japanese traditional medicine and often accompanies cerebro-vascular disorders. Previously, we were able to reveal a deterioration of RBC (Red blood cell) deformability and viscoelasticity in patients with "oketsu" syndrome. The purpose of the present study was to evaluate whether KBG has an effect on RBC deformability in comparison with pentoxifylline (PXF). The subjects were 30 male patients with multiple lacunar infarctions. Eighteen patients (44-79 yrs, mean [+ or -] SD, 66.1 [+ or -] 10.7 yrs) were treated with 12 g of KBG daily for 4 weeks (KBG group). Twelve patients (59-78 yrs, 70.7 [+ or -] 6.4 yrs) were treated with 300 mg of PXF daily for 4 weeks (PXF group). Based on the "oketsu" score, the patients of each group were divided into two subgroups, a non-"oketsu" group ("oketsu" score 20 points or less) and an "oketsu" group ("oketsu" score 21 points or higher). KBG had significant effects on RBC deformability as evaluated by filtration method. KBG also significantly increased intracellular ATP content, as did PXF. Moreover, KBG was more effective for patients with a more severe "oketsu" state. However, PXF was effective only in patients with "oketsu" syndrome, who might have deteriorated RBC deformability. In conclusion, the effect of KBG on RBC deformability was by no means inferior to PXF.

Key words: Keishi-bukuryo-gan, Pentoxifylline, Kampo medicine, RBC deformability, hemorheology

* Introduction

"Oketsu", blood stasis syndrome, is one of the pathological concepts unique to Japanese traditional medicine. In Japanese traditional medicine, "ketsu" ("Xue" or blood), means human red body fluid containing life energy ("ki" or "Qi"), that circulates in the body. "Oketsu" is a pathological state induced by circulatory failure of this "ketsu". The "oketsu" state was evaluated by the criteria presented by Terasawa et al. (Terasawa et al., 1989) in Japan. We previously reported that most of the patients with multiple cerebral infarction were complicated with "oketsu" syndrome (Hikiami et al., 1996a). These diagnostic criteria are popularly called the "oketsu" score (Table 1). Patients with "oketsu" syndrome revealed impairment of microcirculation on the basis of observiation of the blood flow of their bulbar conjunctiva by means of a video-microscope system (Terasawa et al., 1988), and they presented with elevated blood viscosity. (Terasawa et al., 1986) and increased RBC aggregability (Kohta et al., 1992). Recently, we established that RBC deformability and viscoelasticity were impaired in patients with "oketsu" syndrome (Hikiami et al., 1996b).

Keishi-bukuryo-gan (KBG: Gui-Zhi-Fu-Ling-Wan), one of the prescriptions in Japanese traditional medicine for improving the "oketsu" syndrome, exerts some favorable pharmacological effects on improving a conjunctival microcirculation (Itoh et al., 1988), inhibiting a platelet aggregation, suppressing the production of Thromboxane A2 in platelets, and decreasing blood viscosity (Tosa et al., 1987) and RBC aggregability (Kohta et al., 1993). Clinically, KBG has a favorable effect on several symptoms of patients with cerebro-spinal vascular disorder (Tosa et al., 1989).

Several drugs such as pentoxifylline (PXF) were developed for improving hemorheological factors, as hemorheological abnormalities were involved in cerebrovascular diseases. PXF is one of the most important drugs for improving the deterioration of erythrocyte deformability.

The present study was designed to elucidate the influence of KBG on RBC deformability in patients with multiple lacunar infarction. In addition, we compared KBG with PXF, which has been generally considered to have some efficacy on RBC deformability.

* Subjects and Methods

Subjects

Thirty male patients (range 44-79 yrs, mean [+ or -] SD, 67.6 [+ or -] 9.6 yrs) with multiple lacunar infarctions who visited the Department of Japanese Oriental (Kampo) Medicine, Toyama Medical and Pharmaceutical University Hospital, were examined. All patients in this series were diagnosed with multiple lacunar infarctions by neurological examination and magnetic resonance imaging, and were in the chronic phase. Eighteen patients (range 44-79 yrs, mean [+ or -] SD 66.1 [+ or -] 10.7 yrs) were treated with 4 g of KBG three times dally for 4 weeks (KBG group).

KBG consists of five medicinal plants. One KBG tablet weighs 2 g and consists of honey (1 g), Cinnamomi Cortex (0.2 g, Cinnamomum cassia Blume), Paeoniae Radix (0.2 g, Paeonia lactiflora Pallas), Persicae Semen (0.2 g, Prunus persica Batsch), Hoelen (0.2 g, Poria cocos Wolf) and Moutan Radix (0.2 g, Paeonia frutricosa Andrews). Twelve patients (59-78 yrs, 70.7 [+ or -] 6.4 yrs) were treated with 100 mg of PXF three times daily for 4 weeks (PXF group). Based on the "oketsu" score, the patients of each group were divided into two subgroups, a non-"oketsu" group ("oketsu" score 20 points or less) and "oketsu" group ("oketsu" score 21 points or higher). The "oketsu" score was determined by two of the authors, both specialists in Japanese traditional medicine. "Oketsu" score, RBC deformability, and intracellular ATP content were measured at 0 (immediately before administration) and 4 weeks after administration. All patients completed an informed consent form prior to the study.

Blood samples

To determine RBC deformability and intracellular ATP content, 7 ml of blood, anticoagulated in EDTA-2Na (1.5 mg/ml), and 5 ml of heparinized blood were withdrawn from the cubital vein. Every blood sample was taken in the morning after overnight fasting and examined within four hours after sampling.

Measurement of RBC deformability

The apparatus for measurement of RBC deformability was prepared according to Reid's filtration method (Reid et al., 1976), with certain modifications. After high-speed centrifugation, plasma and buffy coat were removed. The remaining packed RBCs were washed three times with isotonic phosphate buffer (PBS) (pH = 7.4, 295 mOsm/kg). Washed RBCs, aspirated from the middle of the packed RBC column, were resuspended in isotonic PBS to a final concentration of 15%. RBC deformability was evaluated by RBC filtration time, which was determined by measuring their ability to pass through a 5 [micro]m pore falter (Nucleopore, Costar Co., Ltd., USA) under constant -10 cm [H.sub.2]O pressure. In each filtration experiment, the time required for 400 [micro]l of the 15% RBC suspension to pass through the filter was determined. The deformability index was calculated as the average of eight repeated tests. A fresh filter was used for each measurement, and all measurements were performed at 25 [degrees]C.

Hematocrit (microhematocrit, 12,500 x g for 10 min., in duplicate), total plasma protein, albumin, total plasma cholesterol, triglyceride, high density lipoprotein (HDL) cholesterol and fasting plasma glucose were measured. The cell count of RBCs and hemoglobin concentration were also determined (Auto cell counter Celltac MEK-4500; Nihon Kohden, Co., Ltd., Tokyo, Japan) to determine mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC).

Measurement of intracellular ATP content

After plasma and buffy coat were removed from 5 ml heparinized blood, the remaining packed RBCs were washed three times with isotonic PBS at 4 [degrees]C. ATP in the packed RBCs was extracted by Mosior's method (Mosior et al., 1990a). By using an ATP assay kit (LL-100-1, Toyo Ink Co., Ltd., Tokyo, Japan), luminescence was caused by reacting the extracted ATP on luciferase (firefly's luminescent enzyme). The degree of luminescence was measured with a luminescence reader (BLR-301, Aloka Co., Ltd., Tokyo, Japan).

Statistical analysis

Comparisons of the laboratory data between the non-"oketsu" subgroup and "oketsu" subgroup in the KBG and PXF groups were performed with the use of the Mann-Whitney test. Comparisons of the variables before and after treatment of KBG and PXF were performed with the use of two-tailed Wilcoxon signed-rank test. Analyses of independence were performed by chi-square test. Differences with a p value less than 0.05 were considered significant.

Analysis of Keishi-bukuryo-gan by three-dimensional HPLC 

Keishi-bukuryo-gan was extracted with 20 ml of methanol under ultrasonication for 30 min. The solution was filtered with membrane filter (0.45 [micro]m) and then submitted for HPLC analysis. HPLC equipment was controlled with a SLC-10A (Shimadzu, Tokyo, Japan) using a TSK-GEL ODS-80TS column (4.6 [phi] x 250 mm), eluting with solvents (A) 0.05 M AcONH4 (pH 3.6), (B) CH3CN. A linear gradient of 100% A and 0% B changing over 60 min to 0% A and 100% B was used. The flow rate was controlled with LC 10 AD pump at 1.0 mL/min. The eluate from the column was monitored and three-dimensional data were processed by SPD-M10A diode array detector. All assigned peaks were identified by co-injection test with authentic samples and compared by UV spectral data.

* Results

Comparison of laboratory data between the non-"oketsu" and "oketsu" subgroups

From the standpoint of "oketsu" score, the KBG group was classified into two subgroups, the non-"oketsu" group (KBG-NO, n = 7, 62.0 [+ or -] 10.7 yrs) and the "oketsu" group (KBG-O, n = 11, 67.7 [+ or -] 10.5 yrs). The PXF group was also divided into two subgroups, the non-"oketsu" group (PXF-NO, n = 6, 68.5 [+ or -] 5.4 yrs) and the "oketsu" group (PXF-O, n = 6, 72.8 [+ or -] 7.1 yrs). There was no statistical significance in any laboratory data (hematocrit, MCV, MCHC, total plasma protein, albumin, total plasma cholesterol, HDL-cholesterol, triglyceride and fasting plasma glucose) among each group (Table 2).

Changes in "oketsu" score (Fig. 1)

[FIGURE 1 OMITTED]

The mean ([+ or -] SEM) "oketsu" score in the KBG group significantly decreased from 28.3 [+ or -] 2.5 points to 20.7 [+ or -] 1.8 points after the administration of KBG (p < 0.05). In terms of severity of the "oketsu" state, the "oketsu" score of the KBG-O group significantly decreased from 34.4 [+ or -] 2.0 points to 23.7 [+ or -] 1.9 points after 4 weeks (p < 0.05), but that of the KBG-NO did not show a significant change (from 12.7 [+ or -] 2.8 points to 12.9 [+ or -] 2.7 points). On the other hand, there was no significant change in "oketsu" score in the PXF group. However, "oketsu" score of PXF-O group was significantly decreased from 41.6 [+ or -] 3.8 points to 29.7 [+ or -] 3.7 points after 4weeks (p < 0.05).

Changes in RBC deformability (Fig. 2)

[FIGURE 2 OMITTED]

The mean RBC filtration time in the KBG group significantly decreased from 12.9 [+ or -] 0.3 to 12.0 [+ or -] 0.3 msecs after 4 weeks (p < 0.05). RBC filtration time of the KBG-O group significantly decreased from 13.3 [+ or -] 0.3 to 12.2 [+ or -] 0.4 msecs after 4 weeks (p < 0.05), but not significantly changed in the KBG-NO group (from 11.8 [+ or -] 0.7 to 11.3 [+ or -] 0.5 msecs). On the other hand, there was no significant change of RBC filtration time in the PXF group. However, RBC filtration time of the PXF-O group was significantly decreased from 14.7 [+ or -] 0.5 to 13.0 [+ or -] 0.7 msecs after 4weeks (p < 0.05).

Changes in intracellular ATP content (Fig. 3)

[FIGURE 3 OMITTED]

The mean intracellular ATP content in the KBG group significantly increased from 4.8 [+ or -] 0.5 to 6.8 [+ or -] 1.3 x [10.sup.-9] M after 4 weeks (p < 0.05). The intracellular ATP content of the KBG-O group significantly increased from 4.3 [+ or -] 0.6 to 6.6 [+ or -] 1.0 x [10.sup.-9] M after 4 weeks (p < 0.05), but did not significantly change in the KBG-NO group (from 6.4 [+ or -] 1.2 to 7.3 [+ or -] 1.8 x [10.sup.-9] M). On the other hand, there was no significant change in intracellular ATP content in the PXF group. However, in the PXF-O group intracellular ATP was significantly increased from 2.9 [+ or -] 0.7 to 4.4 [+ or -] 1.5 x [10.sup.-9] M after 4 weeks (p < 0.05).

Three-dimensional HPLC profile of Keishi-bukuryo-gan

The three-dimensional HPLC profile of Keishi-bukuryo-gan is shown in Fig. 4.

[FIGURE 4 OMITTED]

* Discussion

KBG was first mentioned "Kinki-Yohryaku" (Jin- Gui-Yao-Lue), a classic medical work of Chinese medical science written in the 3rd century A.D. (Liao, 1994). This prescription is one of the most popular prescriptions for improving the "oketsu" syndrome in Japanese traditional medicine, and has the effect of improving hemorheological parameters such as blood viscosity and RBC aggregability. In a previous report., by the use of a cetrifugation method, KBG was observed to improve RBC deformability of the rat (Oda et al., 1984). We previously reported that the severity of the "oketsu" state was correlated with RBC deformability, and deterioration of RBC deformability in the "oketsu" state was assumed to be caused by decreased RBC membrane viscoelasticity due to lower intracellular ATP concentration (Hikiami et al., 1996b). These findings led us to study whether kampo treatment improves RBC deformability in the "oketsu" state or not.

The present study is the first to elucidate the influence of KBG on RBC deformability in patients with multiple lacunar infarctions, as well as to evaluate hemorheological abnormality from the view point of "oketsu" syndrome. It was revealed that KBG improved RBC deformability according to a decreasing "oketsu" score. In other words, patients with a severe "oketsu" state had a greater degree of deterioration of RBC deformability than those without the "oketsu" syndrome.

The RBC membrane is composed of a lipid bilayer consisting of free cholesterol, phospholipid and a cytoskeletal protein network (i.e., spectrin, actin, etc.) (Shiga et al., 1990), and RBC deformability depends on these structures. ATP plays many important roles in the RBC membrane, and especially in its viscoelasticity. Stability of the lipid bilayer is maintained by ATP-dependent aminophospholipid translocase (Middlekoop et al., 1989). Therefore, lipid mobility is influenced by the intracellular ATP concentration (Mosior et al., 1990b). Changes in the cytoskeletal protein structure result from a lowering of intracellular [Ca.sup.++] concentration due to a reduction in intracellular ATP concentration (Rendell, 1992). Moreover, because the strength of cytoskeletal protein bundling is also changed by the phosphorylation of cytoskeletal proteins (i.e., spectrin) that is induced by ATP, the cytoskeletal architecture is influenced by the intracellular ATP concentration (Backman, 1988.). It was reported that PXF improved RBC deformability by increasing the ATP content in RBC (Stefanovich, 1975; Palek, et al. 1977).

In this study, KBG seemed to improve RBC deformability by activating ATP metabolism, as did PXF, since the intracellular ATP content increased after the KBG treatment. Our results suggested that KBG may in fact not be inferior to PXF on the effect for improving RBC deformability.

PXF is generally considered to be a drug that improves RBC deformability (Muller, 1978, Chyzy et al., 1987, Ott et al., 1988). Nevertheless, it has also been reported that this drug has no effect on RBC deformability (Cummings et al., 1992). Further, the opinion has also been expressed that RBC deformability of the subjects in the latter report was within normal range before the PXF treatment, so there might not have been any effect with PXF (Martinez et al., 1994). Thus, needless to say, the effect of PXF on RBC deformability is still a matter of some controversy. In the present study, PXF was not effective on RBC deformability for all the subjects, but it was significantly effective for the patients with the severe "oketsu" state and the deterioration of RBC deformability. KBG is effective for the RBC deformability of patients with "oketsu" syndrome, and its effect dose not take second place to PXF. KBG had greater efficacy on RBC deformability for those patients with the "oketsu" state than for those without. Prescriptions of oriental medicine that include Japanese traditional medicine are restricted to a suitable pathology under the system of oriental diagnosis. Therefore, these results suggest that the diagnosis of "oketsu" state plays an important part in the determination of those patients who have an affinity for KBG in advance.

Table 1. Diagnostic criteria for "oketsu" syndrome.

symptoms                                  score

                                          male   female

dark shade around the eyes                10     10
pigmentation over the face                 2      2
rough skin                                 2      5
purple discoloration of lips               2      2
purple discoloration of gums              10      5
purple discoloration of tongue            10     10
telangiectasis, vascular spider            5      5
susceptibility to subcutaneous bleeding    2     10
redness of palms, palmar erythema          2      5
tenderness of left narvel region           5      5
tenderness of right narvel region         10     10
tenderness under the narvel region         5      5
tenderness of iliocoecal region            5      2
tenderness of hypochondrial region         5      2
tenderness of sigmoid region               5      5

hemorrhoids                               10      5
dysmenorrhea                                     10

Evaluation:
20 points and less: non-"oketsu" state.
21 points and above: mildly affected "oketsu" state.
40 points and above: severely affected "oketsu" state.

Proposed by the research group, supported by the Ministry of
Science and Technology of Japan.

Table 2. Comparison of the laboratory data between the non-"oketsu"
subgroup and "oketsu" subgroup in each groups.

                                  KBG group

                                  KBG-NO
                                  group
                                  (n = 7)

Age                                62.0 [+ or -] 10.7
"Oketsu" score                     12.7 [+ or -] 7.4

Hematocrit         (%)             43.7 [+ or -] 3.8
Mean               ([micro]        95.2 [+ or -] 3.2
  corpuscular        [m.sup.3])
  (MCV) volume
Mean               (%)             33.9 [+ or -] 1.1
  corpuscular
  hemoglobin
(MCHC)
  concentration
Total protein      (g/dl)           7.3 [+ or -] 0.4
Albumin            (g/dl)           4.5 [+ or -] 0.2
Total              (mg/dl)        209.3 [+ or -] 43.4
  cholesterol
Triglyceride       (mg/dl)        133.2 [+ or -] 89.1
HDL-cholesterol    (mg/dl)         51.4 [+ or -] 14.4
Fasting            (mg/dl)         99.6 [+ or -] 8.1
  plasma glucose

                                  KBG group

                                  KBG-O
                                  group
                                  (n = 11)

Age                                67.7 [+ or -] 10.5
"Oketsu" score                     34.4 [+ or -] 8.3 *

Hematocrit         (%)             44.0 [+ or -] 3.7
Mean               ([micro]        97.3 [+ or -] 2.5
  corpuscular        [m.sup.3])
  (MCV) volume
Mean               (%)             32.9 [+ or -] 0.2
  corpuscular
  hemoglobin
(MCHC)
  concentration
Total protein      (g/dl)           7.4 [+ or -] 0.5
Albumin            (g/dl)           4.3 [+ or -] 0.3
Total              (mg/dl)        195.6 [+ or -] 37.8
  cholesterol
Triglyceride       (mg/dl)        134.4 [+ or -] 78.7
HDL-cholesterol    (mg/dl)         47.9 [+ or -] 16.9
Fasting            (mg/dl)         99.3 [+ or -] 11.8
  plasma glucose

                                  PXF group

                                  PXF-NO
                                  group
                                  (n = 6)

Age                                68.5 [+ or -] 5.4
"Oketsu" score                     17.7 [+ or -] 7.5

Hematocrit         (%)             44.2 [+ or -] 3.2
Mean               ([micro]        96.1 [+ or -] 6.4
  corpuscular        [m.sup.3])
  (MCV) volume
Mean               (%)             33.5 [+ or -] 0.9
  corpuscular
  hemoglobin
(MCHC)
  concentration
Total protein      (g/dl)           7.4 [+ or -] 0.6
Albumin            (g/dl)           4.3 [+ or -] 0.3
Total              (mg/dl)        173.0 [+ or -] 32.7
  cholesterol
Triglyceride       (mg/dl)        123.8 [+ or -] 66.5
HDL-cholesterol    (mg/dl)         55.5 [+ or -] 23.3
Fasting            (mg/dl)        106.2 [+ or -] 12.5
  plasma glucose

                                  PXF group

                                  PXF-O
                                  group
                                  (n = 6)

Age                                72.8 [+ or -] 7.1
"Oketsu" score                     41.6 [+ or -] 9.2 **

Hematocrit         (%)             43.9 [+ or -] 3.2
Mean               ([micro]        99.4 [+ or -] 6.5
  corpuscular        [m.sup.3])
  (MCV) volume
Mean               (%)             33.4 [+ or -] 1.7
  corpuscular
  hemoglobin
(MCHC)
  concentration
Total protein      (g/dl)           7.6 [+ or -] 0.5
Albumin            (g/dl)           4.4 [+ or -] 0.2
Total              (mg/dl)        197.8 [+ or -] 42.2
  cholesterol
Triglyceride       (mg/dl)        117.3 [+ or -] 81.1
HDL-cholesterol    (mg/dl)         57.0 [+ or -] 20.4
Fasting            (mg/dl)        104.2 [+ or -] 11.1
  plasma glucose

The values are expressed as the mean [+ or -] S.D.
Sttatistical analysis was done by Mann-Whitney test.

* p < 0.05 vs KBG-NO group; ** p < 0.01 vs. PXF-NO group

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* Address

Hiroaki Hikiami, Department of Japanese Oriental (Kampo) Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan

Tel.: ++81-76-434-7393; Fax: ++81-76-434-0366; e-mail: [e-mail]

H. Hikiami (1), H. Goto (2), N. Sekiya (1), N. Hattori (3), I. Sakakibara (3), Y. Shimada (1), and K. Terasawa (1)

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

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

(3) Department of Phytochemistry, Tsumura Co., Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki, Japan