We discovered a phenomenon in which the blood flow in vein microcirculation markedly decreases in response to hen-egg white lysozyme (HEL)-sensitization without any change in blood pressure. Using this blood flow decrease as a guide, we developed an in vivo assay method to search for substances, which can prevent allergies. Antagonists of histamine, serotonin and platelet activating factor (PAF) did not affect the blood flow decrease in response to HEL-sensitization. On the other hand, cyclooxygenase (COX)-1, COX-2, thromboxane (TX) A 2 , endothelin-1 (ET-1), prostacyclin (PGI 2 ) and granulocytic elastase (GE) as well as nitric oxide (NO) from inducible NO synthase (iNOS) were involved in the blood flow decrease. Thus, these substances might injure vascular endothelial cells, and cause a decrease in blood flow in vein microcirculation. Our method can be used to search for preventive agents against allergies involving NO, COX-1, 2 and PGI 2 . This is the first report to applying to an assay method the specific blood flow decrease to occur in the promotion stage of allergy.

Flower buds of Lonicera japonica THUNB. (Caprifoliaceae), one of the most common traditional Chinese medicines, are used to treat various diseases including arthritis, diabetes mellitus, fever, infections, sores and swelling. 1)Pharmacological studies have indicated that the extract of these flower buds have a broad spectrum of biological activities, including antibacterial, anti-inflammatory, antioxidant, antipyretic, antiviral and hepato-protective effects.1,2) A number of chemical constituents including flavonoids, iridoids and saponins have been obtained from this plant. [3][4][5][6][7][8] In our continuing search for allergy-preventive substances from natural sources, 9) we have been using our previously developed in vivo assay method to estimate effects on complex allergies. 10,11) This time, we found that the 35% EtOH extract of flower buds of L. japonica exhibited allergy-preventive activity. Our in vivo assay method monitors the decrease in blood flow (BF) in the tail vein of mice subjected to hen eggwhite lysozyme (HEL) sensitization alone without the HELchallenge as a guide.10) The BF in HEL-sensitized mice (control group) gradually and significantly decreased to about 70% of that in normal mice on day 9. Thus, the induction phase (promotion stage) of allergy caused by xenobiotics can be dynamically and easily measured using BF monitoring. This BF decrease is considered to be due to the contraction of peripheral blood vessels and an increase in blood viscosity, because no relationship with blood pressure was observed. Although anti-HEL immunoglobulin E (IgE) antibody significantly increased after HEL sensitization, there was no significant increase in the number of leukocytes. Thus, a decrease in BF reflects the promoter process of an allergic reaction. The BF decrease is regulated by various factors such as nitric oxide (NO), thromboxane (TX) A 2 , prostacyclin (PGI 2 ), and endothelin (ET)-1, together with granulocytic elastase (GE), cyclooxygenase (COX)-1 and -2, inducible nitric oxide synthase (iNOS), and constitutive nitric oxide synthase (cNOS).10) In addition, the BF decrease occurs via both pathways of iNOS-independent and -dependent responses.11) Therefore this monitoring system should be useful when searching for substances that can prevent complicated inflammatory allergies involving NO, TXA 2 , PGI 2 , ET-1, GE, COX-1, and COX-2. This paper describes the evaluation of the allergy-preventive effects of a 35% EtOH extract (LJ) of flower buds of L. japonica and the following compounds isolated from LJ: chlorogenic acid (1) and three iridoid derivatives, loganin (2), secoxyloganin (3) and sweroside (4).The structure-activity relationships of iridoid derivatives, morroniside (5), geniposide (6), asperuloside (7), aucubin (8) and catalpol (9) were also examined, as iridoid glucosides (2-4) isolated from LJ showed allergy-preventive effects. MATERIALS AND METHODS General Experimental ProceduresMelting points were determined on a Yanagimoto micro-point apparatus. IR spectra were recorded on a Shim...

Xanthorrhoea hastilis R. BR. (Xanthorrhoeaceae), commonly called grass trees, is an Australian native plant genus. A commercially available resin commonly obtained from Xanthorrhoea trees is gum accoroides, but no medical uses have been described for this plant. In our continuing search for allergy-preventive substances from natural sources 1) we found that extracts of X. hastilis exhibited allergy-preventive activity. Known extracts from X. hastilis include benzoic acid, 2) fragrant oil 3) and C-methylated flavonoid, 4) but no detailed chemical study had been reported. In the present study, we isolated from X. hastilis, a new flavanone, 3Ј,5Ј-dihydroxy-7,4Ј-dimethoxyflavanone (1) and two new chalcones, 3,5,2Ј-trihydroxy-4,4Ј-dimethoxychalcone (2) and 5,2Ј-dihydroxy-3,4,4Ј-trimethoxychalcone (3). We report their structures and also the isolation of five known compounds. Finally, we describe the allergy-preventive effects of these compounds isolated from X. hastilis. Results and DiscussionCompound 1 was obtained as colorless needles and its HR-EI-MS showed the [M] ϩ ion at m/z 316.0942 which established the molecular formula as C 17 H 16 O 6 (Calcd 316.0947). The IR spectrum indicated the presence of hydroxy (3500-3200 cm Ϫ1) and conjugated carbonyl (1670 cm Ϫ1 ) groups. The UV spectrum showed l max at 237, 272 and 310 nm, indicating a phenolic nature.1 H-NMR of 1 showed the presence of a methylene (d 2.75, 2.96), an oxymethine (d 5.32), two methoxy groups (d 3.81, 3.84) and five aromatic protons, in which two protons appeared as a singlet at d 6.49, suggesting a symmetrical structure and the other three protons indicated a typical ABX spin system (d 6.54, 6.62, 7.77).13 C-NMR showed the signals for a carbonyl carbon at d 193.1, twelve aromatic carbons between d 102.1 and 168.2, an oxygenated methine at d 80.9, two methoxy groups at d 56.3 and 60.8 and a methylene at d 48.5. These data suggested that 1 is dihydroxy-dimetoxyflavanone. EI-MS showed fragment peaks at m/z 151, 166, which indicated the presence of one methoxy group in the A ring, one methoxy and two hydroxy groups in the B ring.5) The methoxy group of the B ring should be placed at position 4Ј to form the B ring symmetry. The positions of methoxy groups were established at 7 and 4Ј by correlation with the HMBC spectrum ( Table 1). The absolute configuration at C-2 was confirmed by a positive Cotton effect at 331 nm and a negative Cotton effect at 304 nm in the CD spectrum, which is characteristic for the 2S configuration of flavanones. 6)Based on these observations, the new compound 1 was established to be (2S)-3Ј,5Ј-dihydroxy-7,4Ј-dimethoxyflavanone.Compound 2 was isolated as yellow needles. HR-EI-MS showed the [M] ϩ ion at m/z 316.0949 corresponding to the ; 11-68 Koshien, Kyuban-cho, Nishinomiya 663-8179, Japan: and b Gifu Pharmaceutical University; 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan. Received November 22, 2006; accepted January 9, 2007; published online January 12, 2007 Allergy-preventive activity was demonstrated for an ext...

Whole plant of Impatiens textori MIQ. (Balsaminaceae) is an annual herbaceous plant that grows naturally along waterfronts in the mountains in almost every region of Japan. It has been used for detoxication and treatment of carbuncles and contusions in Chinese medicine. 1) However, there has been no report on the antiallergic effects of the flowers of I. textori. In a previous study, we demonstrated the antianaphylactic, antipruritic, and antiplatelet activating factor (PAF) activities of a 35% EtOH extract (IT) of the flowers of I. textori, and isolated apigenin (1), apigenin 7-glucoside (2), luteolin (3), luteolin 7-glucoside (4), quercetin (5), kaempferol (6), and kaempferol 3-glucoside (7). 2,3)In a continuing search for allergy-preventive substances from natural sources, using our previously developed in vivo assay method to estimate complicated allergy, 4) we found that IT exhibited allergy-preventive activity. This in vivo assay method monitors the decrease in blood flow in the tail vein of mice subjected to hen egg-white lysozyme (HEL) sensitization alone without the HEL challenge as a guide.4) The blood flow in HEL-sensitized mice (control group) gradually and significantly decreased to about 70% of that in normal mice on day 9. Thus, the induction phase of allergy caused by xenobiotics can be dynamically and easily measured using blood flow monitoring. This blood flow decrease is considered to be due to the contraction of peripheral blood vessels and increase in blood viscosity, because no relationship with blood pressure was observed. Although anti-HEL immunoglobulin E (IgE) antibody significantly increased after HEL sensitization, there was no significant increase in the number of leukocytes. Thus the decrease in blood flow reflects the promoter process of an allergic reaction. The blood flow decrease is regulated by various factors such as nitric oxide (NO), thromboxane (TX) A 2 , prostacyclin (PGI 2 ), and endothelin (ET)-1, together with granulocytic elastase (GE), cyclooxygenase (COX)-1 and -2, inducible nitric oxide synthase (iNOS), and constitutive nitric oxide synthase (cNOS). 4) Additionally, the blood flow decrease occurs via both pathways of the iNOS-independent and -dependent response.5) Therefore this monitoring system should be useful for searching for preventive substances against complicated allergy involving NO, TXA 2 , PGI 2 , ET-1, GE, COX-1, and COX-2. 4,5) This paper describes the evaluation of the allergy-preventive effects of a 35% EtOH extract (IT) of the flowers of I. textori and compounds (1-7) previously isolated from IT. MATERIALS AND METHODS MaterialsThe 35% EtOH extract (IT) of the flowers of I. textori was used for our present and previous studies. 2,3)Apigenin (1), apigenin 7-glucoside (2), luteolin (3), luteolin 7-glucoside (4), quercetin (5), kaempferol (6), and kaempferol 3-glucoside (7) were isolated from IT as previously reported.2) HEL and complete Freund's adjuvant (CFA) were purchased from Sigma Chemical (St. Louis, MO, U.S.A.), Difco (Detroit, Michigan, U.S.A.)...

Our in vivo assay system was developed to quantitatively estimate mouse hen-egg white lysozyme (HEL)-anaphylaxis, including fatal shock.1) This assay system monitors the decrease in blood pressure 2) or blood flow 3) as anaphylaxis response and makes possible investigation of the dynamics of the anaphylactic response in the same individual animals without killing them. We have also discovered a distinctive phenomenon in which the blood flow of vein microcirculation is markedly decreased by HEL-sensitization alone without the HEL-challenge.4) The blood flow of the HEL-sensitized mice reproducibly decreases to about 65-75% of that of normal mice. Thus, the afferent (promotion) stage of allergy caused by xenobiotics can be dynamically and easily measured by using blood flow monitoring. This blood flow decrease is considered to be due to contraction of peripheral blood vessels and increase in blood viscosity, because no relationship with blood pressure was observed. In this assay system, mice are sensitized with a mixture of HEL and adjuvant. When only HEL or only adjuvant was used, the degree of the blood flow decrease was very small compared with the case of the mixture. Although anti-HEL IgE antibody significantly increased after HEL-sensitization, there was no significant increase in the number of leukocytes. Thus, the decrease of blood flows reflects the promoter process of an allergic reaction by the cooperation action of HEL and adjuvant. Using this blood flow decrease as a guide, we developed an in vivo assay method to search for substances, which can prevent allergies. 5) Various factors are involved in blood flow decrease, including nitric oxide (NO), cyclooxygenase (COX)-1, COX-2, thromboxane (TX) A 2 , endothelin (ET)-1, prostacyclin (PGI 2 ) and granulocytic elastase (GE) from vascular endothelial cells. We also found that the plasma levels of nitrite/nitrate (NO x ), metabolites of NO, together with expression of iNOS protein in the thoracoabdominal aorta synchronously increase with the blood flow decrease in HELsensitized mice. 4)In the present study, we examined how iNOS is involved in the blood flow decrease, using an iNOS KO mouse. The relationships among iNOS and NO, COX-1, COX-2, TXA 2 or ET-1 were also examined. -123) 9) were from Sigma Co., Ltd.; the stable analog of PGI 2 (Beraprost sodium) 10) was from Yamanouchi Pharmaceutical Co., Ltd.; and 3-[4-(1H-imidazol-1-ylmethyl)phenyl] 2-propenoic acid hydrochloride monohydrate (Ozagrel) 11) was from Ono Pharmaceutical Co., Ltd. These agents were dissolved in physiological saline or water. The saline or water solution was administered to mice at a volume of 10 ml/10 g body weight in the case of injection and 100 ml/10 g body weight in the case of oral use. MATERIALS AND METHODS MaterialsAnimals Female iNOS KO mice with a C57BL/6 background (SPF grade), 7-8 week old, were obtained from Jackson Laboratory (Bar Harbor, Maine, U.S.A.). Male ddY mice of 5 weeks (SPF grade) and female WT C57BL/6 mice of 8 weeks old (SPF grade) were obtained from Japan S...