Anti-malarial activity of leaf-extract of hydrangea macrophylla, a common Japanese plant.

To find a new anti-malarial medicine derived from natural resources, we examined the leaves of 13 common Japanese plants in vitro. Among them, a leaf-extract of Hydrangea macrophylla, a common Japanese flower, inhibited the parasitic growth of Plasmodium falciparum. The IC50 of Hydrangea macrophylla leaf extract to Plasmodium falciparum was 0.18 microg/ml. The IC50 to NIH 3T3-3 cells, from a normal mouse cell line, was 7.2 microg/ml. Thus, selective toxicity was 40. For the in vivo test, we inoculated Plasmodium berghei, a rodent malaria parasite, to ddY mice and administered the leaf-extract of Hydrangea macrophylla (3.6 mg/0.2 ml) orally 3 times a day for 3 days. Malaria parasites did not appear in the blood of in the treated mice, but they did appear in the control group on day 3 or 4 after inoculation with the parasites. When leaf extract was administered to 5 mice 2 times a day for 3 days, malaria parasites did not appear in 4 of the mice but did appear in 1 mouse. In addition, the leaf-extract was administered orally 3 times a day for 3 days to Plasmodium berghei infected mice with a parasitemia of 2.7%. In the latter group, malaria parasites disappeared on day 3 after initiating the treatment, but they appeared again after day 5 or 6. Although we could not cure the mice entirely, we confirmed that the Hydrangea macrophylla leaf extract did contain an anti-malarial substance that can be administered orally.

New ferromagnetic bone cement for local hyperthermia.

We have developed a ferromagnetic bone cement as a thermoseed to generate heat by hysteresis loss under an alternate magnetic field. This material resembles bioactive bone cement in composition, with a portion of the bioactive glass ceramic component replaced by magnetite (Fe3O4) powder. The temperature of this thermoseed rises in proportion to the weight ratio of magnetite powder, the volume of the thermoseed, and the intensity of the magnetic field. The heat-generating ability of this thermoseed implanted into rabbit and human cadaver tibiae was investigated by applying a magnetic field with a maximum of 300 Oe and 100 kHz. In this system, it is very easy to increase the temperature of the thermoseed in bone beyond 50 degrees C by adjusting the above-mentioned control factors. When the temperature of the thermoseed in rabbit tibiae was maintained at 50 to 60 degrees C, the temperature at the interface between the bone and muscle (cortical surface) surrounding the material rose to 43 to 45 degrees C; but at a 10-mm distance from the thermoseed in the medullary canal, the temperature did not exceed 40 degrees C. These results demonstrate that ferromagnetic bone cement may be applicable for the hyperthermic treatment of bone tumors.

Time-course alterations of monoamine levels and cerebral blood flow in brain regions after subarachnoid hemorrhage in rats.

To investigate the possible correlation between changes in monoaminergic neuronal activity and cerebral blood flow (CBF) in the same brain region after subarachnoid hemorrhage (SAH), monoamine levels were analyzed by both HPLC-ECD and fluorohistochemistry techniques, and CBF was measured by using colored microspheres. At the second day of SAH, significant and nonsignificant reductions in blood flow were seen in the examined brain regions with a marked increase in CBF appearing in the telencephalon and hypothalamus on the third day. Significant reductions of monoamine levels in most brain regions were also observed on the second day after SAH, whereas norepinephrine (NE) levels in midbrain increased to 1.5 times compared to the normal level. These reductions were sustained until the fourth day of SAH, although at the third day, serotonin (5-HT) and dopamine levels in the hippocampus and 5-HT levels in the cerebelium were significantly elevated. In fluorohistochemical studies, the fluoro-intensities of monoamines, particularly catecholamines, in the midbrain dorsal NE bundle were enhanced at the second day after SAH. These NE neurons originated from the A5 cell group close to the area where homologous blood was applied through the cisterna magna. The results obtained after SAH show an apparent correlation between changes in monoamine levels and CBF in norepinephrine (NE)-rich areas. These results suggest that SAH-induced neuronal dysfunctions, particularly with NE neurons, are caused not only by reductions of blood flow but also by hemorrhage.

[PUVA therapy for cutaneous acute graft-versus-host disease--a case report].

We performed 8-methoxypsolaren and ultraviolet A (PUVA) therapy as a second line treatment for stage +3 cutaneous acute graft-versus-host disease (GVHD) in a 10-year-old girl who had undergone unrelated bone marrow transplantation. Although prednisolone as first line therapy was not effective, the cutaneous lesion began to improve after several times of PUVA treatment and completely disappeared following 11 times of irradiation without additive systemic immunosuppressive therapy. PUVA therapy was considered as a useful treatment for cutaneous acute GVHD.

Influence of a menhaden oil diet on cercarial penetration of Schistosoma mansoni.

The ability of a menhaden oil (MO) diet to influence cercarial penetration into mouse tail skin was evaluated. Male CD-1 mice 4-6 wk old (15.2 g average weight) were fed a 0, 10%, or 20% MO-supplemented diet for 2 wk. After this time mice were infected with either 65 +/- 3 or 145 +/- 3 [35S]methionine/cysteine-labeled cercariae for 1 hr by tail immersion. Twenty-four hours and 7 days later groups of mice were killed and their tail skin removed and autoradiographed. At 24 hr postinfection, mice fed a 20% MO diet had significantly higher cercarial penetration than controls and 10% MO diets (56% +/- 5.2 vs. 44% +/- 2.9, P = 0.02, 1-tailed t-test). After 7 days mice fed a 20% MO diet retained more radioactive foci than controls or 10% MO diets (21% +/- 2.0 vs. 15% +/- 1.3, P = 0.01, 1-tailed t-test).

Neuropsychopharmacological studies of a Ca2+ channel blocker on the modulation of brain Ca2+ mobilization of spontaneously hypertensive rats under mild stress.

The psychotropic effects of a calcium channel blocker (Ca antagonist) were examined in behavioral studies following changes in 45Ca2+ influx in synaptosomal fractions of brain tissues using spontaneously hypertensive rats (SHR). Under a novel circumstance utilizing 85-dB noise, SHR demonstrated hyperactivity and a significant increase in 45Ca2+ uptake into synaptosomal fractions of frontal cortex (FC) and hippocampus. Such hyperactivity may be caused not only be seeking behavior but also by stress-induced anxiety. Such hyperactivity was significantly blocked after 10 days of repeated administration of diazepam (DZP), tandospirone (SM-3997; SM), a 5-HT1A anxiolytic, and nitrendipene (Nit), a Ca antagonist. Moreover, repeated administration of DZP, SM and Nit reduced the maximum binding density of 3H-PN200-110 and reduced the 45Ca2+ uptake in FC of SHR. In hippocampus, midbrain, hypothalamus and striatum, the increased ratio of 45Ca2+ uptake was reduced after repeated administration of Nit or SM. These results suggest that the hyperactivity induced by this novel circumstances was reduced by DZP, SM and Nit and may be attributed to inhibition of voltage-dependent Ca channel activities in FC. In addition, Nit may induce anti-anxiety through the modulation of Ca2+ mobilization in the central nervous system.

[Treatment of iron overload with combination of recombinant human erythropoietin and phlebotomy].

A patient of Coombs negative autoimmune hemolytic anemia was massively transfused of 162 units concentrated red blood cells in 3 months and developed iron overload disease which was confirmed by liver biopsy. Hemolysis was successfully treated with high-dose methyl-prednisolone therapy and splenectomy. To treat iron overload, we administered recombinant human erythropoietin (Epo) in combination with phlebotomy. Total iron removed for 5 months was about 4 g. Thus, combination of Epo and phlebotomy was effective for the treatment of iron overload disease. Furthermore, we compared a degree of clinical effect of subcutaneous administration of Epo with that of intravenous administration in the clinical course and found the former more effective.

Milk of calcium renal stone in a patient with acute promyelocytic leukemia.

A case of milk of calcium renal stone is reported. This is a rare disease in which a suspension of calcium salts is formed within a renal cyst. The pathognomonic sign is a fluid level seen in a standing position and an oval density seen in a supine position. In the present case, the milk of calcium was found to develop in a hydronephrotic kidney during the course of acute promyelocytic leukemia and this condition was suggested to be a complication of infection.

Basic studies on the laboratory assessment of macrofilaricides using Brugia malayi in the jird, Meriones unguiculatus. 1. Longevity and periodicity of microfilaremia.

The longevity and periodicity of microfilaremia were examined in the jird infected with Brugia malayi to be used for assessing the filaricides. Jirds 4 to 6 weeks old were inoculated subcutaneously with 100 to 200 infective larvae of B. malayi. Microfilariae were present in 75 out of 94 jirds observed over 3 years and high microfilaremia, with 10 mf/microliters or higher, developed in 43 out of 75 jirds. Such a high level of microfilaremia was necessary for narrowing the variation of microfilaria counts among the blood samples. The microfilaria negative jirds, 4 months after inoculation, were abandoned, because in those cases where they became patent later the microfilaria density did not reach an appropriate level. The selected jirds were used for experiment from 6 to 15 months after inoculation when most of them revealed the maximal count of microfilariae. The jirds that failed to develop microfilaremia to the level of 10 mf/microliters by 9 months after inoculation were also abandoned because they did not continue the appropriate level of microfilaremia even when they reached this level later. Although a significant periodicity was observed only in of 10 jirds examined by the Aikat and Das method, the peak hour of microfilaria density was observed in most animals in the afternoon and the time was nearly the same in each animal. Therefore, the blood sampling would be performed preferably in the afternoon.

Basic studies on the laboratory assessment of macrofilaricides using Brugia malayi in the jird, Meriones unguiculatus. 2. Establishment and evaluation of a new method of macrofilaricide assessment.

Jird infected subcutaneously with infective stage larvae (L3) of Brugia malayi were evaluated as an animal model for assessing macrofilaricides using a method of observing the change in microfilaria (mf) density but not by recovering adult worms. The animals were treated with a test compound followed by diethylcarbamazine (DEC) at 50 mg/kg for 5 consecutive days for clearing the existing mf from the blood stream. A continuous decrease in mf density was observed when jirds were treated with flubendazole. Nevertheless, slow recovery of mf density was observed in the jirds which were given suramin or Mel W, indicating that mf productivity of female worms was continuing after DEC treatment. The results obtained by monitoring microfilaremia corresponded with those obtained by recovery of adult worms at autopsy, suggesting that the system of L3-induced B. malayi jird model is useful for testing macrofilaricides.

Effect of zonisamide (CI-912) on a synaptic system model.

The effect of the experimental antiepileptic drug zonisamide (1,2-benzisoxazole-3-methanesulfonamide, ZNS) on the trigeminal complex of cats was compared with the effect of established antiepileptic drugs. Intravenous administration of 10-40 mg/kg ZNS significantly depresses descending excitatory mechanisms, as well as segmental and descending inhibitory mechanisms, but has only a minor effect on segmental excitatory mechanisms. This spectrum of activity is similar to that of valproate, and suggests that ZNS should also be a broad-spectrum antiepileptic drug. In agreement with our experimental observations, it has been found that ZNS is effective against complex partial, generalized tonic clonic, and myoclonic seizures. The antiepileptic profile of ZNS in conventional screening tests resembles that of carbamazepine (CBZ) and phenytoin. However, CBZ exacerbates rather than prevents myoclonic seizures. Our experimental model thus provides a more accurate prediction of ZNS's clinical spectrum of activity. The relationship of these findings to the mechanism of action of antiepileptic drugs is discussed.

SEM-EPMA observation of three types of apatite-containing glass-ceramics implanted in bone: the variance of a Ca-P-rich layer.

The progressive changes of a Ca-P-rich layer between bone and three types of apatite-containing glass-ceramics of the same chemical composition: MgO 4.6, CaO 44.9, SiO2 34.2, P2O5 16.3, CaF2 0.5 (in weight ratio) were examined. Plates (15 mm X 10 mm X 2 mm, mirror surface) containing apatite (35 wt%) (designated A-GC), apatite (35 wt%) and wollastonite (40 wt%) (designated A.W-GC), and apatite (20 wt%), wollastonite (55 wt%), and whitlockite (15 wt%) (designated A.W.CP-GC) were prepared. They were implanted into the tibia of mature male rabbits for 5 days, 10 days, 20 days, 30 days, 60 days, 6 months, and 12 months. All three types of glass-ceramics showed direct bonding to the bone 30 days after implantation. It was observed by SEM-EPMA 30 days after implantation that Si and Mg content decreased, P content increased, and Ca content did not change across the reactive zone from the glass-ceramics to bone. The level of P and Si in the A.W.CP-GC changed five days after implantation. In A.W-GC and A-GC, a little change in P and Si levels was observed between 10 and 20 days after implantation. The width of reactive zone was narrowest with A-GC, wider with A.W-GC, and widest with A.W.CP-GC. The dissolution of glass-ceramics stopped 6 months after implantation. This phenomenon shows that the glass-ceramics may be suitable for clinical use.

Suanzaorentang versus diazepam: a controlled double-blind study in anxiety.

The ancient Chinese remedy Suanzaorentang was originally described in Kin-Kue-Yao-Lueh for patients with weakness, irritability and insomnia. In our preliminary observations on suanzaorentang, it seemed to be a promising anxiolytic remedy. A controlled comparative double-blind clinical trial was set up to assess the anxiolytic effect of suanzaorentang. Suanzaorentang (250 mg t.i.d.) and diazepam (2 mg t.i.d.) had almost the same anxiolytic effect. However, suanzaorentang, but not diazepam, improved the psychomotor performance during the daytime. No significant subjective side effects were observed during treatment with suanzaorentang. All laboratory tests, including liver function tests (serum SGOT, SGPT, albumin, globulin, bilirubin), renal function tests (BUN, serum creatinine), electrolyte balances (serum K+, Na+, Cl-, Ca++), serum cholesterol-triglyceride-HDL-c, thyroid function test (serum T4), chest P-A X-ray film, blood-urine-stool routine examinations (BUS routines), were unaltered after one week's administration of the compound.

Suanzaorentang, and anxiolytic Chinese medicine, affects the central adrenergic and serotonergic systems in rats.

Previously, we found that the ancient Chinese remedy of Suanzaorentang was a promising anxiolytic drug (Chen and Hsieh, 1985; Chen and Hsieh, 1985a). We also found that Suanzaorentang decreased the turnover rate of central monoamines and central catecholaminergic activity (Hsieh, et al., 1986). In this study, we found that 5-hydroxytryptophan (5-HTP) induced decrease in locomotor activity was significantly antagonized by Suanzaorentang, p-chlorophenylalanine (p-CPA) induced increase in locomotor activity was significantly inhibited by Suanzaorentang, Suanzaorentang had no significant effects on baclofen, muscimol, aminooxyacetic acid (AOAA) and thiosemicarbazide induced changes in locomotor activity, Suanzaorentang significantly decreased vanillylmandelic acid (VMA) in striatum and hippocampus, homovanillic acid (HVA) in hippocampus and 5-hydroxyindol acetic acid (5-HIAA) in striatum and hypothalamus, Suanzaorentang significantly reversed the alpha-methyltyrosine (alpha-MT) produced decrease in DA concentrations in striatum and hippocampus, and (6) Suanzaorentang significantly reversed the p-CPA produced decrease in 5-HT concentrations on amygdala. These facts implied that Suanzaorentang might decrease the serotonergic activity but have no significant effect on GABAergic activity. The main locus of action might be in the limbic system.

Effects of Suanzaorentang on behavior changes and central monoamines.

Previously, it was found that the ancient Chinese remedy of Suanzaorentang could be a promising anxiolytic drug (Chen and Hsieh, 1985a, Chen and Hsieh, 1985b). To understand the mechanism of the action of Suanzaorentang, the effects of Suanzaorentang on behavior changes and central monoamines and their metabolites were studied in rats. It was found that Suanzaorentang significantly (1) prolonged the period from the onset of clonic to tonic convulsions induced by pentylenetetrazol or picrotoxin, (2) prolonged the sleep duration induced by hexobarbital, (3) reduced locomotor activity, (4) enhanced the hypomotility induced by alpha-MT, (5) reduced the locomotor stimulation produced by levodopa plus benserazide, and (6) reduced central HVA, VMA, and 5-HIAA, but had no significant effects on central DA, NA, and 5-HT. These facts implied that Suanzaorentang decreased the turnover rate of central monoamines and central catecholaminergic activity.

Developmental alterations in maturing rats caused by chronic prenatal and postnatal diazepam treatments.

The post treatment effects of early prenatal, late prenatal, early postnatal or combined prenatal and neonatal treatment with diazepam on the development of pain sensitivity, acoustic startle responsiveness, and benzodiazepine receptors in the cerebral cortex were investigated in rats between 14 and 90 days of age. Tail-flick latency was significantly decreased by combined prenatal and neonatal and by early prenatal diazepam treatment, but not by diazepam during the last half of gestation or during the neonatal period alone. Acoustic startle response was decreased by either late prenatal or neonatal diazepam treatment, but not by early prenatal treatment alone. Density of benzodiazepine receptors in the cortex was increased from postnatal day 1 to 21 by either early or late prenatal diazepam treatment. Neonatal diazepam treatment suppressed cortical benzodiazepine receptor or development until postnatal day 21; thereafter, receptor density increased to significantly higher values than in controls at 90 days of age. The results demonstrate that diazepam can alter development of pain sensitivity by actions during early gestation, startle responsiveness by actions late in pregnancy, and cortical benzodiazepine receptors by actions throughout gestation and the early postnatal period.

Anti-inflammatory effect of diclofenac-sodium ointment (cream) in topical application.

This study was performed to develop a topical ointment of diclofenac-Na which has a potent anti-inflammatory activity by oral administration. At first, research was carried out on the ointment base which influences the external anti-inflammatory effect of the drug. Ointments of diclofenac-Na were prepared with three kinds of bases: lipophilic, emulsion (cream) and gel bases; and their anti-inflammatory effects were compared. The cream was found to have the most potent effect. Therefore, in the next experiment, an optimum concentration of diclofenac-Na in cream was determined comparing the anti-inflammatory effect among the cream preparations containing 0.5, 0.75, 1.0 and 1.5% of the drug. Obvious effects were observed with the cream containing 1.0% and 1.5% of the drug concentration, and there was no significant difference in the anti-inflammatory activities of these two concentrations. Based on these results, the cream preparation containing 1.0% of diclofenac-Na (DF cream) was adopted as the external ointment of the drug. The anti-inflammatory effect of this cream was compared with that of existing anti-inflammatory ointments, i.e., indomethacin gel (IM gel), bufexamac cream (BM cream) and mobilat ointment (ML ointment). DF cream produced obvious inhibition on increased vascular permeability and on acute edema and remarkable suppression of ultraviolet erythema. These activities of DF cream were similar to those of IM gel and more potent than those of BM cream and ML ointment. The inhibitory effect of DF cream on the proliferation of granulation tissue was almost equal to that of ML ointment and more distinguishable than that of IM gel and BM cream. In adjuvant arthritis, DF cream reduced the swelling remarkably in the treated paw and slightly in the untreated paw. The anti-adjuvant activity of DF cream was equal to that of IM gel and more potent than that of BM cream and ML ointment. In pain to pressure stimulation, an analgesic effect was observed in the early stage of DF cream application, and its activity was slightly stronger than that of the other ointments. These results show that DF cream has an obvious anti-inflammatory effect as an external preparation, and the activity is comparable or superior to that of similar existing anti-inflammatory ointments. This cream may be considered as useful in the clinical field as a topical anti-inflammatory preparation.