Platelet Anti-Aggregation Activities of Compounds from Cinnamomum cassia

Abstract

Cinnamomum cassia is a well-known traditional medicine for improvement of blood circulation. An extract of this plant showed both platelet anti-aggregation and blood anti-coagulation effects in preliminary testing. Among the 13 compounds obtained from this plant, eugenol (2), amygdalactone (4), cinnamic alcohol (5), 2-hydroxycinnamaldehyde (7), 2-methoxycinnamaldehyde (8), and coniferaldehyde (9) showed 1.5–73-fold greater inhibitory effects than acetylsalicylic acid (ASA) on arachidonic acid (AA)-induced aggregation (50% inhibitory concentration [IC₅₀] = 3.8, 5.16, 31.2, 40.0, 16.9, and 0.82 μM, respectively, vs. 60.3 μM) and 6.3–730-fold stronger effect than ASA on U46619 (a thromboxane A₂ mimic)-induced aggregation (IC₅₀ = 3.51, 33.9, 31.0, 51.3, 14.6, and 0.44 μM, respectively, vs. 321 μM). The other compounds, coumarin (3), cinnamaldehyde (6), cinnamic acid (10), icariside DC (11), and dihydrocinnacasside (12), also inhibited (2.5 to four times greater than ASA) U46619-induced aggregation. In addition, compounds 2, 4, 5, 6, 7, 8, and 9 were 1.3–87 times more effective than ASA against epinephrine-induced aggregation (IC₅₀ = 1.86, 1.10, 37.7, 25.0, 16.8, 15.3, and 0.57 μM, respectively, vs. 50.0 μM). However, the 13 compounds were only very mildly effective against blood coagulation, if at all. In conclusion, compounds 2, 4, 8, and 9 showed stronger inhibitory potencies than others on AA-, U46619-, and epinephrine-induced platelet aggregation. Eugenol (2) and coniferaldehyde (9) were the two of the most active anti-platelet constituents of C. cassia.

Introduction

P latelets play a pivotal role in the hemostatic process that occurs after an injury of the blood vessels as well as having a role in the pathogenesis of thromboses resulting from improperly regulated hemostatic stimuli in the blood following the formation of a platelet or a hemostatic plug. The process of platelet activation is initiated by stimulating multiple receptors with specific agonists and the associated receptor-mediated signaling pathways.^1,2 Activation of the blood coagulation system also has a significant role in hemostatic plug formation.^3,4 An activated coagulation system, which consists of intrinsic and extrinsic pathways, sequentially reacts to form thrombin and fibrin. Thus, anti-platelet or anticoagulant compounds can be therapeutic for various thrombotic circulatory diseases. The process of anti-platelet or anticoagulation activity is initiated by the inhibition of one or multiple receptors with specific antagonists.⁵ However, the current widely used anti-platelet and anticoagulant agents such as aspirin, warfarin, etc., have considerable limitations because of their associated adverse effects.^6,7

The genus Cinnamomum, which belongs to the Lauraceae family, comprises over 250 aromatic evergreen trees distributed mostly in Asia. Cinnamomum cassia Blume is distributed in southern China as well as in Laos and Vietnam. The bark and twigs of this plant have long been used as a source of aromatic spices worldwide. In addition, those plant parts are used as a traditional Chinese herbal medicine for alleviation of fever, inflammation, chronic bronchitis, and induction of perspiration and to improve blood circulation.^8,9 Chemical and pharmacological investigations on C. cassia have resulted in the isolation of several compounds such as cinnamaldehyde, cinnamic acid, coumarins, diterpenoids, and polyphenols,^10,11 which possess a wide variety of activities, including antifungal, antipyretic, antioxidant, and antimicrobial.^12

–15 However, most of the pharmacological investigations of the chemical constituents of this plant have been limited to the effects of its essential oil and the oil's major component cinnamaldehyde, which has shown bactericidal,¹⁶ sedative,¹⁷ hypotensive,¹⁸ peripheral vasodilation,¹⁸ and platelet anti-aggregation^19,20 effects.

In the course of our continuing screening program, a 70% ethanol (EtOH) extract of the bark and twigs of C. cassia was found to have platelet anti-aggregation and blood anticoagulation effects. Solvent fractionation of the methanol extract and repeated column chromatography yielded 13 known compounds with sufficient quantities to proceed with the evaluation of their effects on platelet aggregation and blood coagulation.

Materials and Methods

Instruments and reagents

The detailed procedures of extraction and isolation of the compounds were previously described by the present authors.¹¹ The structures of the 13 compounds (Fig. 1) obtained from the aforementioned extract were identified by direct comparison of the one and two-dimensional ¹H- and ¹³C-nuclear magnetic resonance, mass, and other physical and spectroscopic data with previously reported data: 4-hydroxybenzoic acid (1),²¹ eugenol (2),²² coumarin (3),²³ amygdalactone (4),²⁴ cinnamic alcohol (5),¹¹ cinnamaldehyde (6),¹¹ 2-hydroxycinnamaldehyde (7),¹¹ 2-methoxycinnamaldehyde (8),¹¹ coniferaldehyde (9),¹¹ cinnamic acid (10),¹¹ icariside DC (11),¹¹ dihydrocinnacasside (12),¹¹ and lyoniresinol 3α-O-β-d-glucopyranoside (13).²⁵

FIG. 1.

Structure of the compounds from C. cassia.

Platelet counts were determined on a hematology analyzer (Excell™ 18, Drew Scientific Inc., Dallas, TX, USA), and the degree of platelet aggregation was determined by using a four-channel platelet aggregometer (model 490-X, Chrono-Log Corp., Havertown, PA, USA) connected to a personal computer. Blood coagulation time was detected by a fibrometer (US/BBL^® Fibrosystem^®, Fisher Scientific, Pittsburgh, PA, USA). Wright's stain was purchased from YD Diagnostics (Gyeonggi-do, Republic of Korea). Collagen was purchased from Chrono-Log Corp. Dimethyl sulfoxide, ADP, arachidonic acid (AA) (sodium salt), U46619 (9,11-dieoxy-11α,9α-methanoepoxyprostaglandin F2α) (a thromboxane A₂ [TXA₂] mimetic), and acetylsalicylic acid (ASA) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Activated partial thromboplastin time (aPTT) (STA^®-PTT Automate 5), prothrombin time (PT) (Neoplastin^® Cl Plus), and thrombin time (TT) (STA^®-Thrombin) test kits, STA-Coag Control (N)+(P), and STA CaCl₂ were purchased from Diagnostica-STAGO (Asnieres, France). Thromboxane B₂ (TXB₂) enzyme immunoassay kits were purchased from GE Healthcare (Little Chalfont, UK).

Animals

The rats (Sprague-Dawley) (weighing 252 ± 20 g) used in this study were purchased from Orient Bio (Seoul, Republic of Korea). They were fed with a diet of animal feed and tap water and were housed at 23 ± 0.5°C and 60% relative humidity in a 12-hour light–dark cycle in accordance with the Guide for the Care and Use of Laboratory Animals ²⁶ by Seoul National University, Seoul. The animal experiments were conducted under an animal ordinance license issued by the Institute of Laboratory Animal Resources of Seoul National University and performed according to the guidelines of the Seoul National University Institutional Animal Care and Use Committee (approval number SNU050502-13).

Preparation of rat platelet-rich plasma

After surgery, blood was collected from rat hearts using a syringe containing 0.1 volume of 2.2% sodium citrate. The blood was centrifuged at 200 g for 10 minutes at room temperature to obtain the supernatant platelet-rich plasma (PRP). The PRP was then diluted with platelet-poor plasma obtained after centrifugation of the initial centrifuged residue at 1,500 g for 20 minutes. Physiological saline was added to the diluted PRP to adjust the final platelet concentration to 4.0–4.5 × 10⁸ platelets/mL with the aid of a platelet counter.

Smear screening test

To 160 μL of adjusted PRP, 20 μL of test sample solution (dissolved in 10% EtOH to give a final EtOH concentration of 1%) or vehicle was added. After incubation for 2 minutes at 37°C, 20 μL of an aggregation-inducing agent (ADP [2–5 μM], collagen [2–5 μg/mL], or AA [0.2 mM]) was added. The mixture was agitated vigorously for 10 seconds, and the incubation at 37°C was continued for 4 more minutes. Thin smears were prepared on slide glasses and dried quickly in the air. The slides were stained with Wright's stain, washed, and dried. The smears were examined under an ordinary light microscope using an oil immersion objective lens (×1,000). The smears were graded as follows: (—), no aggregation; (±), slight aggregation of platelets; (+), less aggregation than with an inducing agent; and (++), as much aggregation as with an inducing agent.²⁷

Platelet aggregation (turbidimetric method)

Platelet aggregation responses were monitored using a turbidimetric method with an optical aggregometer.²⁸ The degree of platelet aggregation was determined after the final addition of an aggregating agent and was standardized by assuming that platelet-poor plasma (500 μL) represented 100% light transmission and that PRP represented 0% light transmission. Adjusted PRP was equilibrated at 37°C for 3 minutes prior to the initiation of each experiment. Five microliters of sample or vehicle (final dimethyl sulfoxide concentration, 0.7%) was added, and an aggregation-inducing agent (ADP [2–5 μM] or collagen [2–5 μg/mL]) was added at 30 seconds. Platelet aggregations induced by AA (40–60 μM), U46619 (1–5 μM), or epinephrine (1–5 μM) were measured in the presence of the threshold concentration of collagen (1.0–1.5 μg/mL), which was added 30 seconds before the addition of the agent. The anti-aggregating effect of each compound was expressed as an IC₅₀ value, the concentration of a compound causing a 50% inhibitory effect.²⁹

Measurement of TXB₂

Rat blood was drawn into syringes containing one-sixth volume of a mixture of 1.25 g of sodium citrate, 0.7 g of citric acid, and 1 g of glucose in 50 mL and then centrifuged at 200 g for 10 minutes at room temperature to obtain the PRP. Apyrase (1 U/mL) was added to the PRP, which was then centrifuged at 220 g for 10 minutes at room temperature. The platelet pellet obtained was suspended in Tyrode's buffer (138 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 3 mM NaH₂PO₄, 5.5 mM glucose, 10 mM HEPES [pH 7.4], and 0.2% bovine serum albumin) containing 0.01 U/mL apyrase, and the final platelet number was adjusted to 4.0–4.5 × 10⁸ platelets/mL with the aid of a platelet counter. The adjusted washed platelet suspension was equilibrated at 37°C for 2 minutes prior to the initiation of each experiment. Five microliters of a sample solution or vehicle was added to the suspension, an aggregation-inducing agent, AA (100 μM), was added at 30 seconds, and the reaction was continued for 10 minutes. The reaction mixture was placed in ice to terminate the reaction and was then stored at −20°C until the TXB₂ assay was conducted. The concentration of TXB₂ was analyzed by enzyme immunoassay kit according to the manufacturer's (GE Healthcare's) instructions.

Measurement of blood coagulation time

Human plasma, obtained from the Blood Bank, Seoul National University Hospital, was stored in a freezer at −80°C. An appropriate amount of plasma was thawed at room temperature and immediately used in the PT, TT, and aPTT assays. The plasma (200 μL) was preincubated with 3 μL of a sample solution or vehicle at 37°C for 2 minutes. Prewarmed Neoplastin Cl Plus (PT reagent) or STA-Thrombin (TT reagent) was added, and the time until coagulation (PT time or TT time, respectively) was measured by the fibrometer. STA-PTT Automate 5 (aPTT reagent) was added to the preincubated plasma, as described above, and incubation was continued at 37°C for 3 additional minutes. Coagulation was initiated by addition of CaCl₂ (25 mM) to the plasma, and the time to coagulation (aPTT time) was measured by fibrometer. All data were standardized to the clotting time of the control plasma. The presented PT, TT, and aPTT data were obtained from the average of a minimum of three tests.

Statistical analysis

The IC₅₀ values were determined from the Regression Wizard and from the Sigma Plot Equation Library (both from Systat Software, Chicago, IL, USA). At minimum, three experiments were performed for each test. Values of R (correlation coefficient) up to 0.9 were in all test variables. The data were presented as mean ± SD values and were analyzed by Student's t test (Sigmastat; Systat Software) to determine whether the compound mean was significantly different from the control mean. P values below .05 were considered significant.

Results

Platelet anti-aggregatory activity

The 70% EtOH extract of C. cassia strongly inhibited AA-induced platelet aggregation, whereas the effects were very mild on either ADP- or collagen-induced aggregation as shown in Table 1. Among the 13 compounds isolated from the methanol extract of this plant, compounds 2, 4, 5, 7, 8, and 9 showed 1.5–73 times higher inhibitory effects than ASA on AA-induced aggregation (IC₅₀ = 3.8, 5.16, 31.2, 40.0, 16.9, and 0.82 μM, respectively, vs. 60.3 μM) and 6.3–730-fold stronger effect than ASA on U46619 (a TXA₂ mimetic)-induced aggregation (IC₅₀ = 3.51, 33.9, 31.0, 51.3, 14.6, and 0.44 μM, respectively, vs. 321 μM) as shown in Table 2. The other compounds—3, 6, 10, 11, and 12—were also inhibitory (2.5 to four times that of ASA) against U46619-induced aggregation. In addition, compounds 2, 4, 5, 6, 7, 8, and 9 were 1.3–87-fold more effective than ASA on epinephrine-induced aggregation (IC₅₀ = 1.86, 1.10, 37.7, 25.0, 16.8, 15.3, and 0.57 μM, respectively, vs. 50.0 μM). Against collagen-induced platelet aggregation, compounds 9 and 12 showed higher inhibitory effects than ASA (IC₅₀ = 21.1 and 210 μM, respectively, vs. 420 μM), whereas the other compounds had negligible effects. However, all of the tested compounds, including ASA, at 300 μM, produced only negligible effects on ADP-induced platelet aggregation (data not shown).

Table 1.

Effects of the 70% Ethanol Extract of C. cassia on Platelet Aggregation

	Aggregating agent ^a
	Collagen	ADP	AA
PRP (without agents)	−	−	−
PRP (with agents)	++	++	++
ASA^b	−(±)	−(±)	−
70% EtOH extract^c	+±	+±	±

After the induction of platelet aggregation, thin smears were prepared on glass. The degrees of aggregation were examined under a microscope.

ADP, 3 μM; collagen, 30 μg/mL; arachidonic acid (AA), 0.2 mM.

Acetylsalicylic acid (ASA), 500 or 1 mg/mL.

Sample concentration, 2 mg/mL.

EtOH, ethanol; PRP, platelet-rich plasma.

Table 2.

Effects of the Compounds from C. cassia on Platelet Aggregation

	IC₅₀ (μM)
Compound	AA	U46619	Epinephrine	Collagen
ASA	60.3	321	50.0	420
4-Hydroxybenzoic acid (1)	≥300	≥300	≥300	≥300
Eugenol (2)	3.80	3.51	1.86	≥300
Coumarin (3)	256	123	58.1	≥300
Amygdalactone (4)	5.16	33.9	1.10	≥300
Cinnamic alcohol (5)	31.2	31.0	37.7	≥300
Cinnamaldehyde (6)	187	79.9	25.0	≥300
2-Hydroxycinnamaldehyde (7)	40.0	51.3	16.8	≥300
2-Methoxycinnamaldehyde (8)	16.9	14.6	15.3	≥300
Coniferaldehyde (9)	0.82	0.44	0.57	21.1
Cinnamic acid (10)	≥300	110	≥300	≥300
Icariside DC (11)	≥300	126	≥300	≥300
Dihydrocinnacasside (12)	72.8	95.3	≥300	210
Lyoniresinol 3α-O-β-d-glucopyranoside (13)	≥300	≥300	≥300	≥300

Platelet aggregation was monitored with the use of a platelet aggregometor. The platelet aggregation was induced in the adjusted PRP with the addition of an aggregation agent, collagen (2.5 μg/mL). AA (50 μM), U46619 (3 μM), or epinephrine (3 μM) was added in the presence of the threshold concentration (1.2 μg/mL) of collagen.

IC₅₀, 50% inhibitory concentration.

TXA₂ formation

The washed rat platelets were treated with AA, and the formation of TXA₂ was determined by measuring TXB₂, the stable metabolite of TXA₂. Compounds 5, 6, 7, 8, 9, and 10, at concentrations of 80, 120, or 240 μM, did not decrease TXA₂ formation (Fig. 2).

FIG. 2.

Effects of the compounds from C. cassia on thromboxane B₂ (TXB₂) formation. The formation of thromboxane A₂ was determined by measuring the concentration of TXB₂, the stable metabolite of thromboxane A₂. Adjusted washed platelets were preincubated with vehicle (dimethyl sulfoxide) or compounds 5–10 and stimulated with 100 μM AA. The concentration of TXB₂ was analyzed by an enzyme immunoassay kit according to the manufacturer's (GE Healthcare's) instructions. C, control.

Anticoagulant activity

The 70% EtOH extract showed strong anticoagulation effects, at a concentration of 2 mg/mL, with a 32–53% increase in coagulation time in aPTT (47.3 ± 2.3 vs. 32.3 ± 1.2 seconds), PT (17.0 ± 0.8 vs. 12.9 ± 0.1 seconds), and TT (28.6 ± 2.2 vs. 18.8 ± 0.4 seconds) times. However, the 13 compounds were only very mildly effective on blood coagulation, if not at all, as shown in Table 3. Compound 3 elongated the aPPT time by approximately 10% (35.6 ± 0.7 vs. 32.3 ± 1.2 seconds) from the control. The PT time was 12–15% elongated by compounds 1, 9, and 11 (14.4 ± 1.0, 14.5 ± 0.9, and 14.8 ± 0.6, respectively, vs. 12.9 ± 0.1 seconds) from the control. None of the 13 compounds elongated TT time.

Table 3.

Effects of the Compounds from C. cassia on Blood Coagulation

	Clotting time (seconds)
Compound ^a	aPTT	PT	TT
Control	32.3 ± 1.2	12.9 ± 0.1	18.8 ± 0.4
Positive control^b	76.3 ± 2.0	37.7 ± 1.5	46.4 ± 2.2
70% EtOH extract^c	47.3 ± 2.3	17.0 ± 0.8	28.6 ± 2.2
4-Hydroxybenzoic acid (1)	33.0 ± 0.2	14.4 ± 1.0^*	15.9 ± 1.0
Eugenol (2)	35.0 ± 0.4	12.7 ± 0.2	19.7 ± 0.4
Coumarin (3)	35.6 ± 0.7^#	12.7 ± 0.2	19.0 ± 0.4
Amygdalactone (4)	31.7 ± 0.0	14.0 ± 0.1	19.5 ± 2.1
Cinnamic alcohol (5)	31.6 ± 0.6	13.5 ± 06	19.1 ± 0.8
Cinnamaldehyde (6)	32.6 ± 0.6	12.3 ± 0.2	19.1 ± 0.2
2-Hydroxycinnamaldehyde (7)	34.3 ± 1.0	13.1 ± 0.2	18.7 ± 0.2
2-Methoxycinnamaldehyde (8)	35.2 ± 0.3	12.9 ± 0.2	19.3 ± 0.1
Coniferaldehyde (9)	31.8 ± 0.3	14.5 ± 0.9^*	18.5 ± 0.7
Cinnamamic acid (10)	35.3 ± 0.9	12.5 ± 0.3	19.2 ± 0.2
Icariside DC (11)	31.7 ± 0.3	14.8 ± 0.6^*	19.3 ± 1.8
Dihydrocinnacasside (12)	31.5 ± 0.3	14.1 ± 0.0	19.5 ± 1.2
Lyoniresinol 3α-O-β-d-glucopyranoside (13)	31.5 ± 0.4	13.4 ± 0.4	19.4 ± 04

Coagulation was measured with a fibrometer. It was initiated by addition of prewarmed Neoplastin Cl Plus (prothrombin time [PT] reagent) or STA-Thrombin (thrombin time [TT] reagent) to the preincubated plasma. Plasma was incubated with STA-PTT Automate 5 (activated partial thromboplastin time [aPTT] reagent) and initiated by addition of CaCl₂ (25 mM) to the plasma. All data were standardized to the clotting time of the control plasma. Data are expressed as mean ± SD values (n = 3).

Sample concentration, 300 μM.

Concentration of heparin: PT, 2 U/mL; TT, 0.1 U/mL; aPTT, 0.05 U/mL.

Concentration of 70% EtOH extract, 2 mg/mL.

P < .05 significantly different from aPTT time with dimethyl sulfoxide control.

P < .05 significantly different from PT time with dimethyl sulfoxide control.

Discussion

The antiplatelet, anticoagulant, and antithrombic effects of the extract of Cinnamomi cortex (cortex of C. cassia) have been reported.^19,20 Moreover, the extract of this plant showed both platelet anti-aggregation and blood anticoagulation effects in our preliminary testing. Thus, the present work was undertaken to identify the active constituents in the C. cassia extract. Although the isolation of a large number of compounds from C. cassia has been reported, 10 phenolic compounds—4-hydroxybenzoic acid (1), eugenol (2), cinnamic alcohol (5), cinnamaldehyde (6), 2-hydroxycinnamaldehyde (7), 2-methoxycinnamaldehyde (8), coniferaldehyde (9), cinnamic acid (10), dihydrocinnacasside (12), and lyoniresinol 3α-O-β-D-glucopyranoside (13)—along with coumarin (3), amygdalactone (4), and icariside DC (11) were secured in sufficient quantities to proceed with our evaluation.

All of the compounds tested, with the exception of compounds 1 and 13, inhibited platelet aggregation induced by at least one of the agonists. In addition, compounds 2, 4, 8, and 9 exhibited strong inhibitory effects on AA-, U46619-, and epinephrine-induced aggregation. In particular, compounds 2 (eugenol) and 9 (coniferaldehyde) with two adjacent functional groups, i.e., hydroxyl and methoxy groups on the phenyl ring, were more potent than the other compounds. Compounds 7 and 8 with one functional group, either a hydroxyl or methoxy group on the phenyl ring, were less effective than compound 9. Eugenol (2) and compounds 5–10, with cinnamic moieties, exhibited either approximately equal or stronger inhibitory potencies against U46619-induced aggregation than against AA-induced aggregation. Eugenol was previously reported to suppress TXA₂ formation from AA in platelets^30,31 and thus was presumed to decrease platelet aggregation indirectly by inhibiting the synthesis of one of the strongest agonists and directly by blocking the TXA₂ receptor. However, the TXA₂ production from AA (100 μM) in platelets was not decreased even at comparatively high concentrations (80–240 μM) of compounds 5–10 (Fig. 2), suggesting that these compounds exhibit antiplatelet effects by directly interfering with TXA₂ receptors rather than inhibiting the synthesis of TXA₂. Amygdalactone (4) was another constituent of this plant with strong inhibitory potency against AA-, U46619-, and epinephrine-induced aggregation. None of the compounds showed a strong anticoagulant effect (Table 3), although the 70% EtOH extract markedly prolonged the clotting times. Compounds 1, 3, 9, and 11 extended either PT or aPTT times by approximately 10%. It was assumed that the strong anticoagulant effect of the extract may be the result of additive effects of all or several of the compounds. However, the presence of other constituents in the extract with stronger anticoagulant activity could not be excluded.

In summary, 11 compounds showed antiplatelet effects in at least one of the agonist-induced aggregation determinations. Compounds 2, 4, 8, and 9 showed stronger inhibitory potencies than the other compounds against AA-, U46619-, and epinephrine-induced aggregation. The results suggested that eugenol (2) and coniferaldehyde (9) are the most active antiplatelet constituents of C. cassia.

Footnotes

Acknowledgments

This work was supported by research funding from the Korean Food and Drug Administration (2007 and 2008) and the BK21 project of the Ministry of Education, Science and Technology, Republic of Korea. The authors are grateful for their financial support.

Author Disclosure Statement

No competing financial interests exist.

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Platelet Anti-Aggregation Activities of Compounds from Cinnamomum cassia

Abstract

Introduction

Materials and Methods

Instruments and reagents

Animals

Preparation of rat platelet-rich plasma

Smear screening test

Platelet aggregation (turbidimetric method)

Measurement of TXB2

Measurement of blood coagulation time

Statistical analysis

Results

Platelet anti-aggregatory activity

TXA2 formation

Anticoagulant activity

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Measurement of TXB₂

TXA₂ formation