Effect of the Dried Flowers of Campsis grandiflora on Stagnant Blood Syndrome

Abstract

Dried flower of Campsis grandiflora (Bignoniaceae), known as ryoushouka in Japanese, is a traditional Chinese medicine used to treat stagnant blood, contusion, pruritus, and gynecopathy such as menstrual and menopausal disorders. In the present study, we evaluated the stagnant blood flow (BF) improvement effect of the methanol extract (CG) of dried flowers of C. grandiflora using an in vivo assay, in a continuing effort to improve peripheral circulatory disturbance using natural sources. We used the assay system to monitor a decrease in BF in the tail vein microcirculation of mice subjected to sensitization with hen-egg white lysozyme. Bioassay-guided fractionation of the CG led to the isolation of apigenin (1), acteoside (2), cleroindicin B (3), rengyol (4), and isorengyol (5). Apigenin (1) and acteoside (2) were identified as active compounds as they exhibited significant stagnant BF improvement effect in the peripheral circulation. This study proved the positive effect of ryoushouka against stagnant blood syndrome.

Dried flower of Campsis grandiflora (Bignoniaceae), known as ryoushouka in Japanese, is a traditional Chinese medicine used to treat stagnant blood, contusion, pruritus, and gynecopathy such as menstrual and menopausal disorders. It has been reported to exhibit bioactivities, such as increase blood flow (BF) from the heart, anti-inflammatory effect, and improvement of abnormal lipid metabolism. Several chemical constituents including flavonoids, iridoids, triterpenoids, and phenolic acid derivatives have been identified in the flower of C. grandiflora. ^{1 -6}

Stagnant blood (oketsu in Japanese), considered as a severe important pathological condition in traditional Chinese and Japanese medicine, is one of the leading causes of gynecopathy, poor circulation, and shoulder discomfort. Development of novel drugs to treat oketsu can improve the quality of life of many people suffering from these symptoms. Previously, we reported the application of an in vivo bioassay method to search for novel anti-oketsu drugs. ⁷ The assay system monitors a decrease in BF in the tail vein microcirculation of mice subjected to sensitization with hen-egg white lysozyme (HEL). The decrease in BF is considered to be due to the contraction of peripheral blood vessels and an increase in the viscosity of blood, changing the blood pressure and shortening the blood clotting time. ⁸ The decrease in BF is regulated by various factors, which may be related to stagnant blood, such as nitric oxide, thromboxane (TX) A₂, prostaglandin I₂, and endothelin-1 together with cyclooxygenase (COX)-1 and -2, inducible nitric oxide synthase (iNOS), and constitutive nitric oxide synthase. ^8,9 This assay method had been developed to search for novel preventive allergy substance that monitors BF decrease in the allergy induction phase. Further study of this phenomenon led us to realize a similarity between the BF decrease in the model and the stagnant BF in the oketsu. In addition, not only allergy preventive drug but also Kampo formula (a traditional medicine system practiced in Japan, based on ancient Chinese medicine) and crude drugs clinically used to treat oketsu can recover HEL-induced BF decrease which is used for other purpose showing little effect. Although our primary screening identified drugs to treat stagnant blood syndrome that significantly attenuated HEL-induced BF decrease, we cannot prove a causal connection. ¹⁰ In the present study, we evaluated the stagnant BF improvement effect of the methanol (MeOH) extract (CG) of dried flowers of C. grandiflora and the isolated compounds in the peripheral circulatory system.

As shown in Figure 1(a), the BF in HEL-sensitized mice of the control group gradually and significantly decreased to approximately 80% of that in normal mice on day 9. The oral administration of CG significantly (P < 0.05) attenuated the decrease in BF. However, CG did not affect the decrease in BF in normal mice without HEL sensitization. The blood clotting time in HEL-sensitized mice was significantly reduced to approximately 40% compared with that in the normal control group mice on day 9. ⁸ The administration of CG significantly extended the blood clotting time (Figure 1(b)). Thus, CG can be used to prevent complicated stagnant blood syndrome.

Figure 1

Effect of the MeOH extract (CG) of ryoushouka. (a) Effect on hen-egg white lysozyme induced blood flow decrease. Pretreatment with CG at 0, 3, 6, and 9 days after sensitization. Each value presents the mean ± standard error of 5 mice. *P < 0.05 compared with the control group (Dunnett’s test with Bonferroni). (b) Effect on blood coaggregation. Data present the mean ± standard error of 5 mice. *P < 0.05 compared with the control group (Student’s t-test).

Ethyl acetate (AcOEt) extract of CG significantly attenuated the HEL-induced decrease in BF (Figure 2(a)) and shortened the blood clotting time (Figure 2(b)). Contrarily, the n-butanol (n-BuOH) extract significantly extended the blood clotting time (Figure 2(b)), but it did not increase the BF (Figure 2(a)).

Figure 2

Effect of the fraction extract from CG. (a) Effect on hen-egg white lysozyme induced blood flow decrease. Pretreatment with the extract at 0, 3, 6, and 9 days after sensitization. Each value presents the mean ± standard error of 5 mice. *P < 0.05 compared with the control group (Dunnett’s test with Bonferroni post hoc test). (b) Effect on blood coaggregation. Data present the mean ± standard error of 5 mice. *P < 0.05 compared with the control group (Dunnett’s test).

By bioassay-directed fractionation of CG, compounds 1 and 2 were isolated from the AcOEt extract and compounds 3 to 5 from the n-BuOH extract. These compounds were identified as apigenin (1), acteoside (2), cleroindicin B (3), rengyol (4), and isorengyol (5) by comparing their physical and spectroscopic data with those reported previously. ^{10 -13} To the best of our knowledge, the present study is the first to report compounds 4 and 5 that were isolated from this plant.

Among these compounds, compound 1 at a higher concentration than what was isolated was found to be an active compound in the AcOEt extract of CG. Indeed, we have previously shown that it can significantly inhibit HEL-induced decrease in BF. ¹⁴ In addition, compound 1 has been reported to possess several biological activities to improve BF and blood clotting, such as inhibitory effects on the binding of TXA₂ receptor, signaling pathway related to TXA₂ receptor, expression of iNOS and COX-2, production of NO, aggregation of platelets in vitro, and PAF-induced blood pressure. ^{15 -19} In the present study, the activity of compounds 2 to 5 was tested against HEL-induced BF decrease and whole blood clotting time. As shown in Figure 3, compound 2 significantly improved the BF decrease, as compared with that of the control group after day 7 of HEL sensitization. Compounds 2 to 5 did not affect the BF in normal mice. Compound 2 was isolated from the high polar fraction of the AcOEt extract. However, compound 2 contributes to the activity of both n-BuOH and AcOEt extract, because it is present in the n-BuOH extract at a high concentration. There are previous reports about in vitro activity of 2 on platelet aggregation. ^20,21 However, compound 2 might only inhibit blood clotting in vitro, because compound 2 did not significantly extend HEL-induced shortening of the blood clotting time on day 9 (data not shown). Details of the mode of action of compound 2 for this model are under investigation. There might be other active substances associated with blood clotting in the n-BuOH extract, because compounds 3 to 5 did not extend the blood clotting time (data not shown). These findings showed that apigenin (1) and acteoside (2) were considered as active substances in CG that prevent the decrease in BF.

Figure 3

Effect of compounds 2 to 5 isolated from CG on hen-egg white lysozyme induced blood flow decrease. Pretreatment with the compounds at 0, 3, 6, and 9 days after sensitization. Each value presents the mean ± standard error of 3-5 mice. *P < 0.05 compared with the control group (Dunnett’s test with Bonferroni post hoc test).

Overall, CG improved peripheral circulatory disturbance and increased the blood clotting reaction. Thus, it might be a potential novel drug for stagnant blood. Our findings verify the stagnant blood improvement effect of dried flowers of C. grandiflora using an in vivo assay method in HEL-induced stagnant blood model mice.

Experimental

General Experimental Procedure

The melting point was determined using a Yanagimoto micro melting point apparatus. The IR spectra were recorded on a Shimadzu IR-435 spectrometer and UV absorption spectra on a Shimadzu UV-160A spectrometer. ¹H-NMR (500MHz) and ¹³C-NMR (125MHz) spectra were recorded on a JEOL JNM-ECP 500 spectrometer (TMS as the internal reference). MS was performed on a JMS-700 double-focusing spectrometer with xenon atom of kinetic energy equivalent to 6 kV at ion-accelerating voltage.

Plant Material

The dried petals of C. grandiflora flower were purchased from Matsuura Yakugyo Co., Ltd. (Nagoya, Japan). A voucher specimen is maintained in our laboratory.

Materials

Hen egg-white lysozyme and complete Freund’s adjuvant (CFA) were purchased from Sigma Co., Ltd. (St Louis, MO, United States) and DIFCO (Michigan, United States), respectively.

Animals

Male ddY mice (SPF grade), 5 weeks old, were obtained from Japan SLC, Inc. (Shizuoka, Japan) and housed at 24°C ± 2°C. Food and water were provided ad libitum. All experiments were performed in accordance with the Guidelines for Animal Experiments of Mukogawa Women’s University.

Hen Egg-White lysozyme Sensitization

Immunization with HEL was performed as previously described ⁷ with slight modifications. Male ddY mice (5 weeks old) were sensitized subcutaneously with 50 µg of HEL in 50% CFA on day 0.

Blood Flow Measurement

Subcutaneous BF in the mouse tail was monitored using a contact type Laser Doppler Blood Flow Meter (FLO-C1; Neuroscience, Tokyo, Japan) as previously reported. ⁷ Each mouse was prewarmed for 10 minutes at 36°C before the experiment and was placed on a holder in a measuring chamber maintained at 36°C throughout the experiment. The BF was measured for 10 minutes without anesthetizing the mice. The results were expressed as mean ± standard error (S.E.) of the percent of normal BF in each mouse, measured 1 day before the experiment.

Blood Clotting Time

The whole blood from sensitized mice was sampled within 15 seconds after anesthetizing them with ether on day 9. The natural whole blood clotting time was measured immediately using an Automatic Blood Analyzer (Amelung KC 4A micro, Lemgo, Germany).

Improvement Effect on Stagnant BF

CG, each fraction (100 mg/kg), and each compound (10 mg/kg) were resuspended in water and administered orally to the mice on days 0 (1 hour before sensitization), 3, 6, and 9. The statistical significance was determined in comparison with the HEL-sensitized mice (control group).

Statistical Analysis

All data are expressed as mean ± S.E. All the statistical analyses were conducted using the software GraphPad Prism 7. The results with a P-value of <0.05 were considered statistically significant. In the case of BF, a two-way analysis of variance was used to test statistical differences. When significant differences were identified, the data were further analyzed by Dunnett’s multiple range test coupled with a Bonferroni post hoc test for significant differences between each test group and the control group. In the case of clotting time, the data were analyzed by Student’s t-test or Dunnett’s multiple range test.

Extraction and Isolation

Two kilograms of dried flowers of C. grandiflora were extracted with MeOH at room temperature, and then filtered and evaporated in vacuo to obtain the MeOH extract (CG; yield, 514 g). Further, 285 g of CG was suspended in water and then extracted with AcOEt and n-BuOH to obtain the AcOEt extract (14 g), n-BuOH extract (32 g), and H₂O extract (215 g). The AcOEt extract was subjected to chromatography on a silica gel column using a CHCl₃-MeOH gradient step to obtain 9 fractions (Fr. A I-IX). Fr. A IV (1.79 g), which was eluted with CHCl₃-MeOH (15:1), was subjected to repeated flash chromatography on a silica gel column with a CHCl₃-MeOH gradient step and purified by gel filtration on Sephadex LH-20 using MeOH, to yield compound 1 (1.1 mg). Fr. A IX (836 mg), which was eluted with CHCl₃-MeOH (2:1), was subjected to repeated column chromatography on Sephadex LH-20 using MeOH or ODS column with a H₂O-MeOH gradient step to yield compound 2 (35 mg). The n-BuOH extract was subjected to chromatography on a silica gel column using a CHCl₃-MeOH gradient step to obtain 11 fractions (Fr. B I-XI). Fr. B IV (2.89 g), which was eluted with CHCl₃ H₂O-MeOH (10:1-9:1), was subjected to repeated flash chromatography on a silica gel column with a CHCl₃-MeOH gradient step and purified by gel filtration on Sephadex LH-20 using MeOH, to yield compound 3 (7.6 mg). Fr. B VI (1.85 g), which was eluted with CHCl₃-MeOH (9:1-7:1), was subjected to repeated column chromatography on a silica gel column using a CHCl₃-MeOH gradient step and ODS column with a H₂O-MeOH gradient step to yield compounds 4 (10 mg) and 5 (5.0 mg). Fr. B VII (2.82 g), which was eluted with CHCl₃-MeOH (7:1-5:1), was subjected to repeated column chromatography on a silica gel column using a CHCl₃-MeOH or AcOEt-MeOH gradient step and ODS column with an H₂O-MeOH gradient step to yield compounds 4 (23.6 mg) and 5 (6.0 mg).