Aerial Part Extract of Kummerowia striata as an Anti-Blood Stasis Agent in a Mouse Model of Hen-egg White Lysozyme-Induced Stagnant Blood Flow

Abstract

Background/Objective

Stagnant blood (oketsu in Japanese) is considered a severe pathological condition in traditional Chinese and Japanese medicine. Kummerowia striata is known as a weed in Japan; however, in traditional Chinese medicines, it is used for detoxification and for treating colds, fevers, and bruises. To the best of our knowledge, there is no record of this plant, or its parts, being used as an anti-blood stasis agent. Herein, we demonstrated the effect of the 35% ethanol extract (KS) of the aerial part of K. striata on stagnant blood flow (BF) in the peripheral circulation using an in vivo assay.

Methods

Hen-egg white lysozyme (HEL) was injected into the tail vein of mice to induce stagnant BF, the pathology of which is similar to that of oketsu; then, the changes in BF were observed.

Results

The oral administration of KS (200 mg/kg) significantly improved HEL-induced BF decrease, suggesting that KS can improve blood stasis. Bioassay-guided fractionation of KS resulted in the isolation of 14 flavone and flavonol derivatives. As apigenin (1) and luteolin (2) reportedly have activity in this assay, here, the effects of isovitexin (3), vitexin (5), rhoifolin (12), and vicenin-1 (14) were examined. Compounds 5, 12, and 14 (20 µmol/kg) more effectively inhibited HEL-induced stagnant BF than 1, which was used as a positive control.

Conclusion

The aerial part of K. striata could be useful in the discovery of new leads for the prevention of stagnant blood syndrome.

Keywords

blood stagnation flavonoids apigenin vitexin rhoifolin vicenin-1

Introduction

Stagnant blood (oketsu in Japanese), considered a severe pathological condition in traditional Chinese and Japanese medicine, is an aggravating factor for menopausal disorders, menstrual cramps, sensitivity to cold, and stiff shoulders. Recently, it has been reported to be associated with atopic dermatitis and lifestyle diseases. The development of novel drugs to treat oketsu can improve the quality of life of people suffering from the above conditions.

Previously, we established a mouse model of peripheral blood circulation disorder similar to stagnant blood, and identified anticoagulants using an in vivo assay.^1–3 The blood flow (BF) in the venous microcirculation system of the tail of mice sensitized with hen egg white lysozyme (HEL) gradually and gently decreased without affecting the blood pressure. The decrease in BF is a complex process regulated by various factors, including prostaglandin (PG) I₂, thromboxane (TX) A₂, granulocyte elastase, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX). All these factors contribute to blood stasis-associated inflammatory blood coagulation, platelet aggregation, and blood viscosity.^4,5 The HEL-induced decrease in BF can be considerably improved by Kampo prescriptions (Kamishoyosan, Tokishakuyakusan, Keishibukuryogan, and Tokakujokito) and herbal medicines (Botanpi, Toki, Tounin, and Shakuyaku), which are also commonly used in clinical settings to treat oketsu clinically.¹ Therefore, medicines that inhibit the HEL-induced decrease in BF can improve stagnant blood syndrome.

Kummerowia striata (botanical authority No. 2019-1) belongs to the family Leguminosae and is mainly composed of flavonoids such as luteolin, apigenin, and isovitexin.^6,7 In Japan, K. striata is considered a weed, but in Chinese medicines, it is used for detoxification and for treating colds, fevers, and bruises. However, there is no record of its use as an anti-blood stasis agent. Thus, in a continuing effort to discover new functions of natural products using the in vivo assay developed in our previous study, in this one, we aimed to determine the effect of the aerial part extract of K. striata against stagnant BF.

Results

Effect of KS Extract and its Fractions

As shown in Figure. 1, the BF in the control group gradually, but significantly decreased to approximately 70% of the normal blood flow on day 9. In contrast, in the KS 200 mg/kg group, the decrease in BF was significantly inhibited from day 3. KS improved HEL-induced decrease in BF, suggesting that KS can improve blood stasis.

Figure 1.

Effect of the 35% EtOH extract (KS). Effect on HEL-induced BF decrease. The mice were pretreated with KS at 0, 3, and 6 days after HEL-injection. Each value presents the mean ± SE of five mice. *Results significant at p < 0.05 compared with the control group (Dunnett's test with Bonferroni).

The AcOEt and n-BuOH extracts of KS significantly inhibited the HEL-induced decrease in BF.

Figure 2

Figure 2.

Effect of extracts from KS on HEL-induced BF decrease. The mice were pretreated with the extracts at 0, 3, and 6 days after HEL-injection. Each value presents the mean ± SE of three mice. Dunnett's test was performed between the four groups. *Results significant at p < 0.05 compared with the control group.

The main component of the chloroform (CHCl₃) extract, with a low yield, was identified to be chlorophyll. As we know from our experience that chlorophyll was not active, we did not evaluate the CHCl₃ extract.

Compounds from the AcOEt and n-BuOH Extracts

By bioassay-directed KS fractionation, compounds 1–4 and 6–11 were isolated from the active AcOEt extract and compounds 12–15 from the active n-BuOH extract. By comparing their physical and spectroscopic data with those reported previously,^8–13 these compounds were identified as apigenin (1), luteolin (2), isovitexin (6-C-β-D-glucopyranosylapigenin) (3), apigetrin (apigenin 7-O-β-D-glucoside) (4), 8-C-p-hydroxy-benzylisovitexin (6), 8-C-p-hydroxy-benzylluteolin (7), kaempferol (8), astragalin (kaempferol 3-O-β-D-glucoside) (9), quercetin (10), isoquercitrin (quercetin 3-O-β-D-glucoside) (11), rhoifolin (apigenin 7-O-β-D-neohesperidoside) (12), vicenin-3 (6-C-β-D-glucopyranosyl-8-C-β-D-xylopyranosylapigenin) (13), vicenin-1 (6-C-β-D-xylopyranosyl-8-C-β-D-glucopyranosylapigenin) (14) and apigenin 6-C-β-D-xylopyranosyl-8-C-α-L-arabinoside (15) data are shown in the Supplemental Material）. To the best of our knowledge, compounds 6, 7, 9, and 13–15 have been isolated from Citrullus colocynthis,¹⁴ Thymus hirtus,¹⁵ Viola yedoensis,¹⁶ Glycyrrhiza uralensis,¹⁷ and Pseuderanthemum carruthersii var. atropurpureum.¹⁸ However, this is the first report of their isolation from K. striata.　 [insert K. striata Compounds]

Effect of Compounds of KS

In the present study, the activities of compounds 3, 12, and 14, which were obtained in sufficient amounts, were investigated against HEL-induced BF. Compound 3 is the isomer of vitexin (apigenin 8-C-β-D-glucoside), which is derived from compound 5. Apigenin (1) was used as a positive control. As shown in Figure. 3A and B, glycosides 5, 12, and 14 were more effective than aglycone 1. In contrast, compound 3, the isomer of 5, did not show any effect.

Figure 3.

Effect of compounds on HEL-induced BF decrease. The mice were pretreated with the compounds at 0, 3, and 6 days after HEL-injection. Each value presents the mean ± SE of three mice. For comparison, data for apigenin (1) and control are shown in both graphs. Dunnett's test using Bonferroni was performed between the five groups. *Results significant at p < 0.05 compared with the control group.

Discussion

The decrease in BF in the tail vein microcirculation of mice injected with HEL was reduced by KS, suggesting that it can improve blood stasis. This study demonstrates a new therapeutic function of K. striata.

Using bioassay-directed KS fractionation, compounds 1–4 and 6–11 were isolated from the AcOEt extract and compounds 12–15 from the n-BuOH extract. To our knowledge, the present study is the first to report the isolation of compounds 6, 7, 9, and 13–15 from this plant.

Compounds 5, 12, and 14 significantly improved the decrease in BF 9 days after HEL injection compared with that in the control group. Glycosides 5, 12, and 14 were more effective than aglycone 1. In contrast, compound 3, the isomer of 5, did not show any effect. In the case of C-glycosides, it is suggested that the glucose at position 8 might contribute to their activity.

We have already reported the considerable effects of compounds 1 (the main compound of KS) and 2, which are the active components in the flower petals of Impatiens textori, on peripheral blood circulation disorders in the current model, along with detailed action mechanisms.¹⁹ In contrast, to our knowledge, this is the first report on the activity of 12, which is the O-glycoside of 1, as well as 5 and 14, which are C-glycosides, in this model. Recently, several studies have focused on the absorption mechanisms of flavonoids owing to their various physiological activities. The absorption mechanisms have been suggested to involve flavonoid degradation by bacteria and intestinal mucosal enzymes and transport via transporters; however, no definite conclusion has been reached.^20–24

Therefore, we believe that the present findings regarding the C-glycoside activity of apigenin are of interest.

Compounds 1 and 2 inhibited the activation of macrophages, neutrophils, and eosinophils by inhibiting Toll-like receptors (TLRs) and suppressed the production of inflammatory cytokines and inflammatory mediators such as PGs, TXA₂, and NO, suppressing endothelial cell damage and vascular hyperpermeability in the microcirculatory system. In addition, 1 and 2 suppressed iNOS gene and COX-2 expression induced by HEL administration, and 1 suppressed the induced activity of COX-2. It has also been shown that 1 and 2 suppress platelet aggregation through antagonistic inhibition of the TXA₂ receptor on platelets and further inhibition of signal transduction from the TXA₂ receptor, thereby inhibiting the formation of microthrombi and improving microcirculatory disorders.¹⁹ Compounds 5, 12, and 14, which were found to ameliorate peripheral blood circulation disorders in this model, exert an inhibitory effect on platelet aggregation in vitro.^25–27 In addition, 5 reportedly suppresses iNOS gene expression,²⁸ 5 and 14 inhibit TLRs expression,²⁹ and 5 and 12 inhibit COX-2 expression.^30,31 These results suggest that these apigenin glycosides improve peripheral blood circulation disorders via a mechanism similar to that of 1 and 2, but the details require further investigation. The mode of action of compounds 5, 12, and 14 in this model is under investigation.

In conclusion, to our knowledge, this is the first study to demonstrate the novel function of the aerial part of K. striata as an anti-blood stasis agent using HEL-induced blood stasis model mice.

Material and Methods

General Experimental Procedure

Melting point was determined using a micro-melting point meter (Anatec Yanako, Japan). For ¹H-NMR (500 MHz) and ¹³C-NMR (125 MHz) spectra, the samples were dissolved in either dimethyl sulfoxide (DMSO) or CD₃OD, and the spectra were recorded using a JEOL JNM-ECA 500 spectrometer (JEOL Ltd, Japan) (TMS as the internal reference). MS analysis was performed on a JMS-700 double-focusing spectrometer (JEOL Ltd, Japan) with a xenon atom having a kinetic energy equivalent to 6 kV at an ion-accelerating voltage.

Plant Material

Aerial parts of K. striata were collected from the School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Japan in August 2019 and 2020. The herbarium sample was verified by Professor Kyoko Ishiguro and is stored in the Medical Plant Garden, Mukogawa Women's University. HEL, complete Freund's adjuvant (CFA), and vitexin (5) were purchased from Sigma Co., Ltd (St. Louis, MO, USA), DIFCO (MI, USA), and Cayman Chemical (MI, USA), respectively.

Extraction and Isolation

The soluble compounds of the aerial part of K. striata (2 kg) were extracted using 35% EtOH at room temperature for 3 days and then filtered. The solvent was evaporated in vacuo to obtain the 35% EtOH extract (KS; yield, 152.2 g). After dissolving the extract in water, the water-insoluble precipitate (1.5 g) was removed with CHCl₃. The aqueous solution was then extracted with AcOEt and n-BuOH, and solutions were evaporated in vacuo to obtain the AcOEt extract (5.5 g), n-BuOH extract (18.1 g), and H₂O extract (101.3 g). The AcOEt extract (5.4 g) was subjected to chromatography on a silica gel column using a CHCl₃–MeOH gradient to obtain nine fractions (Fr. A I-IX). Fr. A III (775 mg), which was eluted with CHCl₃–MeOH (30:1), was recrystallized from MeOH to obtain 1 (20 mg). Fr. A IV (919 mg), which was eluted with CHCl₃–MeOH (25:1-23:1), was subjected to repeated flash chromatography on a silica gel column with a CHCl₃–MeOH gradient to obtain ten fractions (Fr. B I-X). Fr. B IV (7.3 mg), which was eluted with CHCl₃–MeOH (50:1), was purified via gel filtration on a Sephadex LH-20 column using MeOH to obtain 8 (1.3 mg) and 10 (1.4 mg). Fr. B IX (510 mg), which was eluted with CHCl₃–MeOH (20:1), was subjected to repeated flash chromatography on a silica gel column with CHCl₃–MeOH (15:1). The eluate was purified via gel filtration on a Sephadex LH-20 using MeOH to obtain 2 (3.8 mg) and 7 (0.4 mg). Fr. A VI (630 mg), which was eluted with CHCl₃–MeOH (18:1-14:1), was subjected to repeated flash chromatography on a silica gel column with CHCl₃–MeOH–H₂O (5:1:0.1). The eluate was purified via gel filtration on a Sephadex LH-20 using MeOH to obtain 4 (1.4 mg) and 9 (1.0 mg).

Fr. A VII (278 mg), which was eluted with CHCl₃–MeOH (12:1-10:1), was subjected to chromatography on a silica gel column using a CHCl₃–MeOH gradient to obtain seven fractions (Fr. C I–VII). Fr. C IV (101 mg), which was eluted with CHCl₃–MeOH (16:1-14:1), was subjected to repeated flash chromatography on a silica gel column with CHCl₃–MeOH–H₂O (5:1:0.1). The eluate was purified via gel filtration on a Sephadex LH-20 column using MeOH, and HPLC (Puresil C18 column, Waters, USA) using CH₃CN–H₂O (30:70) to obtain 11 (1.3 mg). Fr. C VI (58 mg), which was eluted with CHCl₃–MeOH (12:1-10:1), was subjected to column chromatography on a Sephadex LH-20 column using MeOH to obtain 3 (3.9 mg) and 6 (2.4 mg).

The n-BuOH extract (1.5 g) was fractionated by chromatography on a silica gel column using a CHCl₃–MeOH gradient to obtain fourteen fractions (Fr. D I-XIV). Fr.D XII (240 mg), which was eluted with CHCl₃–MeOH (4:1), was purified by gel filtration on a Sephadex LH-20 column using MeOH. The eluate was subjected to chromatography on a Diaion HP-20 column using MeOH–H₂O gradient step, and HPLC using CH₃CN–H₂O (18:82) to obtain 13 (0.5 mg) and 14 (2.4 mg). The n-BuOH extract (2.0 g) was subjected to repeated flash chromatography on a silica gel column (Biotage Sfär Silica HC D, Biotage Japan, Japan) using CHCl₃–MeOH–H₂O (7.5:2.5:0.25). The eluate was purified via gel filtration on a Sephadex LH-20 using MeOH, followed by chromatography on a Diaion HP-20 column using MeOH–H₂O gradient step, and HPLC (Puresil C18 column, Waters, USA) using CH₃CN–H₂O (30:70) to obtain 12 (2.3 mg) and 15 (2.0 mg).

Animals

Four-week-old male ddY mice (SPF grade) were obtained from Japan SLC, Inc. (Shizuoka, Japan) and housed at 24 ± 2 °C. Food and water were provided ad libitum. All animal experiments were performed in accordance with the guidelines for animal experimentation of the Mukogawa Women's University.

Assay Method for Stagnant BF

Mice (5 weeks old) were injected subcutaneously with 50 µg HEL in 50% CFA on day 0. The subcutaneous BF of the tail in each mouse without anesthesia was monitored using a contact-type Laser Doppler Blood Flow Meter (FLO-C1; Neuroscience, Tokyo, Japan) in a measurement chamber maintained at 36 °C, as previously reported.¹ Results are expressed as the mean ± standard error (SE). The percentage of normal BF in each mouse was measured one day before the experiment.

Improvement Effect on Stagnant BF

KS (200 mg/kg), and AcOEt- (10 mg/kg), n-BuOH- (40 mg/kg), and H₂O- (150 mg/kg) extracted frs, and compounds (20 μmol/kg) obtained from KS were re-suspended in water and administered orally to the mice on days 0 (1 h before injection with HEL), 3, and 6. The dosages of the AcOEt-, n-BuOH-, and H₂O-extracted frs were calculated from the amount of compound dissolved in each solvent. Statistical significance was determined by comparison with HEL-injected mice (control group).

Statistical Analysis

All data are expressed as the mean ± standard error (S.E.). All statistical analyses were performed using GraphPad Prism 7. The results with a p-value of < 0.05 were considered statistically significant. Two-way ANOVA was used to test for statistical differences. When significant differences were identified, the data were further analyzed using Dunnett's multiple range test coupled with a Bonferroni post hoc test for significant differences between each test group and the control group. Results with a p-value of < 0.05 were considered statistically significant.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231177128 - Supplemental material for Aerial Part Extract of Kummerowia striata as an Anti-Blood Stasis Agent in a Mouse Model of Hen-egg White Lysozyme-Induced Stagnant Blood Flow

Supplemental material, sj-docx-1-npx-10.1177_1934578X231177128 for Aerial Part Extract of Kummerowia striata as an Anti-Blood Stasis Agent in a Mouse Model of Hen-egg White Lysozyme-Induced Stagnant Blood Flow by Shui Aoki, Hisae Oku, Fumika Kitagawa, Naoki Inoue and Chie Honda in Natural Product Communications

Footnotes

Acknowledgments

This study was partially supported by a Grant-in-Aid for Scientific Research C from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical Approval is not applicable for this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article

Statement of Human and Animal Rights

All experiments on animal were performed in accordance with the guidelines for animal experimentation of the Mukogawa Women's University (FP03-2019-02P). This article does not contain any studies with human subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

ORCID iD

Hisae Oku

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Supplementary Material

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