The search for anti-allergic substances from natural products has attracted much interest in recent years. Previously, we developed an in vivo assay system for quantitatively estimating mouse anaphylaxis, including fatal shock.1) The method of monitoring blood pressure in unanesthetized mice enabled us to study the dynamics of the anaphylactic response in the same individual animals without killing them.
2)Hence, we investigated the participation of nitric oxide in mouse anaphylactic hypotension, 3) and the anti-anaphylactic effects of a 35% ethanol extract (IB) of white petals of Impatiens balsamina L., from which were isolated anti-anaphylactic compounds. 4,5) Some of the antianaphylactic mechanisms of IB were also characterized using a blood pressure monitoring method, with administration of exogenous histamine 6) or platelet-activating factor (PAF).
7)The mortality rate due to the shock of Hen egg-white lysozyme (HEL)-anaphylaxis is very high. 1) Ohtsuka et al. 8) observed a decrease in the blood flow 30 min after an oral challenge with antigen in mouse intestine. This observation suggested that not only hypotension but also a decrease in the blood flow is involved in severe HEL-anaphylaxis, and thus led us to quantitatively determine anaphylaxis by blood flow. This paper describes the monitoring of blood flow as a reliable and useful method for quantitating murine anaphylaxis and evaluating the effects of I. balsamina. The results with currently used anti-allergic agents, diphenhydramine hy-disodium cromoglycate (DSCG) 10) and rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxy-propyl 2-thiazoliethyl phosphate 11) are also reported.
MATERIALS AND METHODS
AnimalsMale ddY mice (SPF grade), 5 weeks old, were obtained from Japan SLC (Shizuoka, Japan) and housed at 24Ϯ2°C. Food and water were available ad libitum.Materials DPH was purchased from Nakalai Tesque; Ketanserin was from Funakoshi Co., Ltd.; DSCG was from Molecular Biology Resources, Inc.; CV-3988 was from Biomol Research Laboratories, Inc. These agents were dissolved in 10 ml saline/10 g body weight.Plant Materials and Extraction I. balsamina L. was planted in our medicinal plant garden and the white flowers were collected in August. Fresh flowers (2.5 kg) were freezedried (200 g), extracted with cold 35% EtOH for 24 h and concentrated in vacuo, giving a residue (designated IB) of 8.47 g. IB was dissolved in 10 ml saline/10 g body weight before use in biological experiments.Isolation of kaempferol 3-rutinoside (1) and lawsone (2-hydroxy-1,4-naphthoquinone) (2) from IB was done as previously reported.4) These compounds were dissolved in 100 ml saline/10 g body weight.HEL Sensitization and Challenge Immunization with HEL was performed as previously described.2) Male ddY mice, 5 weeks of age, were sensitized intraperitoneally (i.p.) on day 0 with 50 mg of HEL (Sigma, 6 times recrystallized) emulsified in Freund's complete adjuvant (DIFCO). On day 9, each mouse was challenged intravenously (i.v.) with 100 mg of HEL in 30 ml saline.Blood Flow Measurement Sub...