Chemical Constituents and Coagulation Activity of Amygdalus persica L. Flowers

Abstract

The ethyl acetate extract of Amygdalus persica L. flowers (family Rosaceae) was fractionated by silica gel and Sephadex LH-20 column chromatography, and, after recrystallization, oleanolic acid (1), ursolic acid (2), quercetin (3), kaempferol (4), hesperetin (5), naringenin (6), and kaempferol-3-O-glucoside (7) were isolated. Their identities were elucidated by spectral techniques and from their physicochemical properties. Compounds 3-7 were identified from A. persica flowers for the first time. All the compounds were evaluated for their procoagulant activity by determining their activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) in vitro. The coagulation activity results showed that oleanolic acid and ursolic acid could significantly prolong APTT and TT; quercetin could significantly shorten PT and TT; kaempferol and hesperetin could significantly shorten APTT, PT, and TT; and naringenin could significantly shorten APTT, PT, and TT and decrease the content of FIB compared with the blank group. All of the above revealed that quercetin, kaempferol, hesperetin and naringenin possessed significant procoagulant activity, while ursolic acid had anticoagulant activity in vitro.

Keywords

chemical constituents coagulation activity quercetin flavonoid

Amygdalus persica L., family Rosaceae, widely distributed in most regions of China, has the effect of reducing diarrhea and defecation, inducing diuresis and removing edema.¹ The “Shennong herbal classic” records that A. persica has the effect of “making people of good color,” and, according to the “Tujing Materia Medica,” A. persica is used as a health food raw material for beauty and constipation treatment, as well as for wine and tea drinks.^2,3

Phytochemical research of A. persica has mainly focused on the extraction and purification technology of polyphenols and the analysis of lipid soluble components.⁴ Wei et al studied by GC-MS the volatile oils of the flower, leaf, stem, and fruit of A. persica extracted by supercritical carbon dioxide.⁵ The volatile oils of the flower and stem showed significant antimicrobial activities with minimum inhibitory concentrations of 0.024-0.188 g/L.

“Chinese Materia Medica” recorded that A. persica was beneficial for water laxation, blood circulation and blood stasis.⁶ On the basis of this, the coagulant activity of the ethyl acetate extract of A. persica was investigated in vitro.

Results

Identification of Compounds

By correlating melting points, ¹H-NMR and ¹³C-NMR spectral, and MS data with literature values, compounds 1-7 were identified as oleanolic acid (1),⁷ ursolic acid (2),⁸ quercetin (3),⁹ kaempferol (4),¹⁰ hesperetin (5),¹¹ naringenin (6),¹² and kaempferol-3-O-glucoside (7).¹³ Compounds 3-7 were identified from A. persica flowers for the first time. The chemical structures of compounds 1-7 are shown in Figure 1.

Figure 1

Chemical structures of compounds 1-7 isolated from A. persica flowers.

Effects on Plasma Coagulation Parameters in Vitro

The results of APTT, PT, TT, and FIB assays in vitro of A. persica flowers are shown in Table 1.

Table 1

Effects of Compounds From A. Persica Flowers on Plasma Coagulation Parameters in Vitro.

Compounds	APTT (s)	PT (s)	TT (s)	FIB (mg/L)
Blank group	15.37 ± 0.06	20.50 ± 0.10	44.90 ± 1.51	85.71 ± 4.70
Yunnan Baiyao	12.07 ± 0.21^***	18.43 ± 0.06^***	48.00 ± 1.77^**	87.71 ± 4.90
Breviscapine	18.57 ± 0.25^***	23.93 ± 0.29^***	46.03 ± 0.42	89.08 ± 4.52
Oleanolic acid	16.10 ± 0.17^{**###△△△}	20.40 ± 0.17	46.63 ± 0.70^*	88.43 ± 3.27
Ursolic acid	16.20 ± 0.35^{**###△△△}	19.70 ± 0.50^*△△	46.93 ± 1.50^*	83.51 ± 6.83
Quercetin	15.67 ± 0.31	19.40 ± 0.10^**△△	36.13 ± 0.15^***	106.92 ± 10.41^**
Kaempferol	14.70 ± 0.44^**△△	18.73 ± 0.86^***	35.37 ± 0.21^***	110.21 ± 8.98^***
Hesperetin	13.57 ± 0.09	18.67 ± 0.07^**###	40.83 ± 0.20^{***###△△△}	95.72 ± 7.73^**△△
Naringenin	12.73 ± 0.35	18.57 ± 0.03^**###	45.6 ± 0.79^***△△△	84.92 ± 0.95
Kaempferol-3-O-glucoside	16.17 ± 0.21^{**###△△△}	20.83 ± 0.15	45.73 ± 0.55	80.74 ± 5.29

Note: Data represent mean ± SD, n = 4.

Compared with blank group, ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Compared with Breviscapine, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001.

Compared with Yunnan Baiyao, ^△ P < 0.05, ^△△ P < 0.01, ^△△△ P < 0.001.

As shown in Table 1, Supplemental Figure S1, compared with the blank group, kaempferol, hesperetin and naringenin could significantly shorten APTT, while oleanolic acid and ursolic acid could significantly prolong APTT, but the effects were lower than that of breviscapine. In Supplemental Figure S2, compared with the blank group, ursolic acid, quercetin, kaempferol, hesperetin, and naringenin could significantly shorten PT, but the effects were lower than those of Yunnan Baiyao. Kaempferol-3-O-glucoside could significantly prolong PT, but there was no difference between kaempferol-3-O-glucoside and breviscapine. Supplemental Figure S3 shows that, in comparison with the blank group, quercetin, kaempferol, hesperetin, and naringenin significantly shorten TT, and the effects were better than that of Yunnan Baiyao. Oleanolic acid and ursolic acid could significantly prolong TT. Supplemental Figure S4 shows that in comparison with the blank group, naringenin and kaempferol-3-O-glucoside significantly decreased the content of FIB, and compared with Yunnan Baiyao, the effect was better than that of the positive control. Quercetin, kaempferol and hesperetin could significantly increase the content of FIB.

Discussion

In clinical tests for blood coagulation, some methods such as APTT, PT, and TT are usually used to test coagulation activity. In the blood coagulation cascade, coagulation activity is related to intrinsic and extrinsic pathways.^14,15 APTT belongs to the intrinsic coagulation pathway, which is related to coagulation factors VIII, IX, XI, and prekallikrein, while PT belongs to the extrinsic coagulation pathway, which is related to coagulation factors I, II, V, VII, and X. TT is a simple screening test for the ﬁbrin polymerization process, which measures the formation time of ﬁbrin from ﬁbrinogen after the addition of known amounts of thrombin to the plasma sample.^16,17 The content of FIB is an important indicator for the detection of cardiovascular diseases. It is mainly synthesized by the liver and can be hydrolyzed to peptides A and B under thrombin, and finally form insoluble fibrin, so as to exert its procoagulant effect.¹⁸ In this study, compared with blank group, oleanolic acid, and ursolic acid could significantly prolong APTT and TT in vitro, which indicated the beneficial effect of them on the intrinsic coagulation pathway. Quercetin could significantly shorten PT and TT, which indicated its beneficial effect on the extrinsic coagulation pathway. Kaempferol and hesperetin significantly shortened APTT, PT, and TT, which indicated their procoagulant effect through intrinsic and extrinsic coagulation pathways. Naringenin could significantly shorten APTT, PT, and TT and decrease the content of FIB in vitro, which indicated its procoagulant effect through intrinsic and extrinsic coagulation pathways, and hindering fibrin formation. This is the first investigation of the procoagulant activity of hesperetin, naringenin and kaempferol-3-O-glucoside with coagulation parameters in vitro.

The previous studies of A. persica flowers were mainly focused on the extraction and purification technology of polyphenols and the analysis of lipid soluble components. In the present study we found, for the first time, that A. persica flowers had a significant coagulation activity. Quercetin, kaempferol, hesperetin, naringenin, and kaempferol-3-O-glucoside have the same 2-phenyl-chromone structure. The results suggest that quercetin, kaempferol, hesperetin, and naringenin possess significant procoagulant activity, but kaempferol-3-O-glucoside has no coagulation activity. The difference in coagulation activity may be related to the C-3 glycosyl group. Hou et al found that the scavenging and antioxidant capacities of flavonoid aglycones were significantly higher than those of flavonoid aglycones in vitro.¹⁹ Wiczkowski et al. studied the bioavailability of quercetin from dietary sources in which it was dispersed in the food matrix. They found that quercetin is more bioavailable than its glucosides in humans.²⁰ Zhang et al. studied the β-glucosidase hydrolysis of flavonoid glycosides in propolis. The contents of many flavonoid aglycones were increased, such as myricetin, quercetin, apigenin, and pinocembrine. The hydrolyzed propolis had stronger antioxidant activity than the original propolis,²¹ and thus deglycosylation of the flavonoids may improve their biological activity.

The 7 compounds isolated from A. persica flowers have a wide range of biological activities.^22,23 Wen et al. showed that naringenin could ameliorate renal function, and had marked protection effects on the kidney in diabetic nephropathy, possibly through inhibiting the activation of TGF-β1/smad and reducing the deposition of ECM proteins.²⁴ Xue et al. showed that hesperitin could inhibit the expression of inflammatory and catabolism genes, and then reduce the chronic inflammation of chondrocytes, thus delaying the degeneration of articular cartilage.²⁵ Quercetin can inhibit the inflammation of mice RAW264.7 cells induced by LPS, whose mechanism may contribute to the regulation of the TLR4/NF-κB signaling pathway.²⁶

In addition, α-glucosidase inhibitors have been used as agents in the treatment of diabetes mellitus type 2 that work by preventing the digestion of carbohydrates such as starch and table sugar. It is known that a number of antidiabetic medicinal plants can be important sources of α-glucosidase inhibitors.²⁷ In this preliminary work, we found that oleanolic acid, ursolic acid, quercetin and kaempferol showed strong α-glucosidase inhibitory activity (IC₅₀ = 4.42 mg·L^-1, 8.86 mg·L^-1, 61.83 mg·L^-1, respectively).^28
-30

The above research provides a theoretical basis for the further study of A. persica.

Materials and Methods

Materials and Reagents

Sodium chloride injection (1707182703), breviscapine injection (20181103, 1), Yunnan Baiyao (2GA1604) and saline sodium citrate were obtained from Kunming Longjin Pharmaceutical Co. LTD (Kunming, Yunnan, China); PT (105317), APTT (112198), TT (121181), and FIB (132120) Assay kits from Shanghai Sun Biotechnology Co. LTD (Shanghai, China); and Sephadex LH-20 from Pharmacia (Burlington, MA, USA). NMR spectra were recorded on a Bruker Avance Am-400 spectrometer.

Plant Materials

A. persica flowers were collected in Anhui Pharmaceutical Zhiyuan Chinese Medicine Co., LTD, (Anhui, China) in March 2015, and identified by Professor Changqin Li of Henan University. A voucher specimen (No.201803027) was deposited in the Institute of Natural Medicine of Huanghe Science and Technology College.

Animals

Male Rex Rabbits from 2.0 to 2.5 kg were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, Henan, China); the animal certificate number was SCXK 2019‐0005. The animals were maintained in a 12 hours light/12 hours dark cycle, at 25 °C and 45 to 65% humidity, and fed with standard rodent diet and water ad libitum.

Samples Extraction and Isolation

The extraction method was similar to that in our previous research.^31,32 Air-dried A. persica flowers (2000 g) were extracted with light petroleum at room temperature (3 times, for 3 days each time) to afford the light petroleum extract. The filtered residues were extracted 3 times, each time for 3 days, with 70% (v/v) ethanol at room temperature. The dry extract was obtained after removing the ethanol. The dry extract (410 g) was suspended in deionized water and then successively partitioned with light petroleum, ethyl acetate, and finally with n-butanol to obtain light petroleum (28 g), ethyl acetate (93 g) and n-butanol extracts (180 g).

The ethyl acetate extract (93 g) was fractionated by silica gel H medium-pressure liquid chromatography by elution with chloroform/methanol (from 100:1 to 7:3, v/v) to obtain 8 fractions: Fr.1~Fr8. Fr.2 (6.5 g) was subjected to silica gel H column chromatography eluting with light petroleum/methylene chloride (from 20:1 to 1:1, v/v), then on silica gel H with methylene chloride/ethyl acetate (10:1), and finally chromatographed on Sephadex LH-20 to give compounds 1 (17.0 mg) and 2 (20.3 mg).

Fr.3 (8.14 g) was purified by silica gel H column chromatography with methylene chloride/methanol (from 30:1 to 5:1, v/v) and further with Sephadex LH-20 (light petroleum/methylene chloride/methanol = 9:9:2, v/v) to yield compound 3 (16.7 mg). Fr.4 (0.86 g) was separated on silica gel H with a stepwise-gradient of light petroleum/ethyl acetate (20:1-1:1, v/v) to obtain 2 fractions: Fr.4.1~Fr.4.2. Fr.4.2 was separated on silica gel H with methylene chloride/methanol (10:1, v/v) and Sephadex LH-20 to give compounds 4 (10.6 mg) and 5 (12.7 mg). Fr.6 (3.40 g) was purified by silica gel H with methylene chloride/methanol (9:1, v/v), and further with Sephadex LH-20 (methylene chloride/methanol = 1:1) to yield 6 (9.3 mg). Fr.7 (9.6 g) was separated on silica gel H with chloroform/ethyl acetate/methanol (20:1:0.5, v/v) and chromatographed on Sephadex LH-20 (methanol) to give compound 7 (8.5 mg).

Coagulation Time Assays in Vitro

Blood samples were drawn from the rabbit’s auricular vein. The method was similar to that used in our previous research.^33
-35 APTT, PT, TT, and FIB were determined.

After collection, the blood was placed in a centrifuge tube with 400 µL 0.109 mol/L sodium citrate to prevent blood clotting. Then serum was separated from the plasma by centrifugation of 3000 rpm at 5 °C for 15 minutes. Basically, serum (100 µL) was mixed with 25 µL of sample, APTT assay reagent (100 µL) was added and incubated for 5 minutes at 37 °C, and then 0.025 mol/L CaCl₂ (100 µL) was added. Clotting times were recorded. For PT assays, serum (100 µL) was mixed with 25 µL of sample and incubated at 37 °C for 3 minutes. For PT assays, reagent (200 µL), which had been maintained at 37 °C for 3 minutes, was added and clotting time was recorded. For TT assays, serum (200 µL) was mixed with 50 µL of sample and incubated at 37 °C for 3 minutes, then TT assay reagent (200 µL) was added, and the clotting time recorded. FIB was determined according to the manufacturer’s recommendations.

In the above tests, blank solvent (dimethyl sulfoxide: Tween 80: normal saline = 2:8:17) was used as negative control group, while breviscapine (13.33 mg/mL) and Yunnan Baiyao (20 mg/mL) were used as positive control groups. All the samples were dissolved in blank solvent, and the concentration of compound was 5 mg/mL.

Statistical Analysis

The results are expressed as arithmetic mean ± standard deviation (SD). Statistical analysis was performed using SPSS19.0 software, and comparison between any 2 groups was evaluated using one-way analysis of variance (One-Way ANOVA). The difference between groups with P < 0.05 was regarded as statistically significant.

Conclusions

In this paper, we investigated the chemical constituents and coagulation activity of A. persica. The present study suggested that quercetin, kaempferol, hesperetin, and naringenin possess significant procoagulant activity, while ursolic acid has anticoagulant activity in vitro. Nevertheless, further study will be required to clarify these additional mechanisms.

Supplemental Material

Online supplementary file 1 - Supplemental material for Chemical Constituents and Coagulation Activity of Amygdalus persica L. Flowers

Supplemental material, Online supplementary file 1, for Chemical Constituents and Coagulation Activity of Amygdalus persica L. Flowers by Juanjuan Zhang, Wei Zhang, Zhenhua Yin, Baocheng Yang and Wenyi Kang in Natural Product Communications

Footnotes

Statement of Human and Animal Rights

All the animal procedures were approved by the Ethical Committee in accordance with “Institute ethical committee guidelines” for Animal Experimentation and Care. Animals were housed in standard cages. The experiment was carried out according to the guidelines of the National Institutes of Health for Care and Use of Laboratory Animals and was approved by the Bioethics Committee of Henan University, Kaifeng, Henan, China.

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.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study received financial support from the Key Research Projects of Colleges and Universities in Henan Province (21B360006) and Zhengzhou Basic Research and Applied Basic Research Special Fund project (ZZSZX202003).

ORCID iD

Wenyi Kang

Supplemental Material

Supplemental material for this article is available online.

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