Procoagulant Substance and Mechanism of Myristica fragrans

Abstract

The effect and mechanisms of Myristica fragrans on blood clotting were evaluated by evaluating blood coagulation time and the fibrinolytic system. The compounds 2 and 5 were isolated from the herbal extract and their activities were assessed for the first time. None of the tested compounds had fibrinolytic activity, but could inhibit the fibrinolytic activity of urokinase. Compound 2 showed the highest inhibitory activity (IC₅₀ = 1.747 mg·mL⁻¹) followed by compounds 4 (IC₅₀ = 1.818 mg·mL⁻¹) and 1 (IC₅₀ = 2.407 mg·mL⁻¹), which were higher than that of the compound in Danshen drug tablets (IC₅₀ = 6.577 mg·mL⁻¹) used in China. Moreover, compounds 1 and 2 showed strong α-glucosidase inhibitory activity in a dose-dependent manner with IC₅₀ values 21.76 ± 0.59 and 21.31 ± 0.00 μg·mL⁻¹, respectively. These results demonstrated that the compounds are promising candidates as procoagulant and antidiabetic agents.

Introduction

The genus Myristica, belonging to the Myristicaceae family, consists of about 120 species and is mostly distributed in South Asia, from west Polynesia, Oceania, and eastern India to the Philippines. In China, 4 species are found in Taiwan and Yunnan province.¹ Myristica fragrans Houtt., an aromatic green tree, is known as a source of nutmeg and mace, which are source popular tropical spices and medicines.² In folk medicine, oral nutmeg and mace are for the treatment of stomach cramps, flatulence, inflammation of gastric mucosa, and can be used as an external analgesic, especially for rheumatism.³ Pharmacological investigations indicate that nutmeg and mace possess antioxidant,⁴ antimicrobial,⁵ anti-inflammatory,⁶ antitumor,⁷ hepatoprotective,⁸ hyperglycemic,⁹ antiplatelet aggregation,¹⁰ and acetylcholinesterase inhibitory activities.¹¹ Lignans are characteristic constituents in M. fragrans, but there are also other bioactive compounds, such as flavonoids,¹² phenols,¹³ and terpenes.¹⁴

Coagulation of blood after vascular spasm and platelet aggregation involves formation of prothrombinase and the conversion of prothrombin into thrombin, which helps the conversion of fibrinogen into fibrin (clot).¹⁵ Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) tests are the most commonly clinically used coagulation tests.

PT is used in the screening test for the extrinsic coagulation system.¹⁶ APTT is a commonly used screening test for the endogenous coagulation system.¹⁷ TT reflects the time required for fibrinogen to be converted to fibrin in the common pathway of the coagulation process.¹⁸ FIB mainly reflects the content of fibrinogen, which is an acute-phase protein produced during the process of coagulation.¹⁹

Plasminogen and fibrinolytic enzyme are the main components of the fibrinolytic system, and fibrinolytic enzyme is an important protease involved in many physiological and pathological processes.²⁰ Urokinase-type plasminogen activator is a multifunctional multidomain protein that is not only a regulator of fibrinolysis but also associated with several acute and chronic pathological conditions.²¹

Abnormal changes in the blood coagulation and fibrinolysis system are closely related to the damage from diabetes mellitus.²² AMPK (protein kinase),²³ PPARγ receptor,⁹ and protein tyrosine phosphatase PTP1B²⁴ might be potential mechanistic targets for antidiabetic agents for the treatment of type 2 diabetes. By slowing down the digestion and absorption of dietary carbohydrates either by means of dietary manipulations or intestinal,²⁵ α-glucosidase inhibitory drugs such as acarbose, miglitol, and voglibose have shown the ability to reduce blood glucose levels in patients with type 2 diabetes.²⁶ So, α-glucosidase inhibitors have been used as a new class of antidiabetic drugs, and many are found in medicinal plants.²⁷

Our study was undertaken to investigate the blood clotting mechanism of compounds from M. fragrans by observing the effects on blood coagulation time and the fibrinolytic system. In addition, the α-glucosidase inhibitory activities of M. fragrans were screened.

Materials and Methods

Collection and identification of plant samples

The pericarp parts of M. fragrans were collected in December 2012 from the Wanning Region, Hainan Province, China, and a voucher specimen was identified by Prof. Chang-qin Li of Henan University and deposited in the Herbarium of the Institute of Natural Products, Henan University.

General

Column chromatography was performed with different types of D-101 macroporous resin (Tianjin Haiguang Chemical, Inc., China), Sephadex LH-20 (Pharmacia, Sweden), 200–300, 40–80 mesh silica gel (Ding Kang Silica Gel Co., Ltd., Qingdao, China), silica gel H (Qingdao Marine Chemical Co., Qingdao, China), and 40–63 μm silica gel (Merck, Germany).

Deuterated chloroform (Beijing SeaSky Bio Technology Co., Ltd.) and analytical grade solvents were purchased from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). α-Glucosidase (22003401), 4-N-trophenyl-α-D-glucopyranoside (PNPG, D00129651), and acarbose (100434) were from Sigma (USA). Saline (Qi Pharmaceutical Co., Ltd., Shandong, 2012053107); vitamin K₁ injection (Tianjin Pharmaceutical Group Co., Ltd., Xinzheng, 1109051); injection breviscapine (Hang Sheng Pharmaceutical Co., Ltd., Hunan, 20110202); PT (Lot: 105227), APTT (Lot: 112163), TT (Lot: 121116), FIB (Lot: 132058) assay kits (Shanghai Sun Biotech Co., Ltd., China); bovine fibrinogen (Sigma, F8630); thrombin (Sigma, T4648); urokinase (Biotechnology Co., Ltd., Hangzhou, Macao and Asia, Lot: 1201166); agarose (GENE Company, 122015); and complex tablet (Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co., Ltd., China, 20130326).

Animals

The animal experimental protocol was approved by the Ethics Committee on Animal Experiments in Henan University. The rabbit license of the laboratory (No. 15-2-6) was issued by the Experimental Animal Center of Henan Province (Zhengzhou, China). All the procedures were carried out according to the guidelines of the National Institutes of Health for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of Henan University.

There was one male Rex rabbit (Institute of Henan University of Traditional Chinese Medicine, Henan University) weighing from 2.0 to 2.5 kg. The rabbit, given free access to food and water, was raised in a controlled environment at 25°C, 45–65% humidity, and 12 h of light/dark.

Extraction and isolation

The extraction was executed according to published methods with some modifications. The dried pericarp parts of M. fragrans (1.2 kg) were extracted twice (1 h each time) with ethanol (70%) at 60°C.²⁸ After evaporation of ethanol, the residue was dispersed in water and adsorbed in D-101 (Tianjin Haiguang Chemical) macroporous resin column chromatography with water, and then eluted using a stepwise gradient of 20%, 40%, and 60% methanol. The solution was concentrated under reduced pressure to yield the methanol fraction (10.7 g). The methanol fraction was subjected to medium pressure liquid chromatography (Beijing ChuangXinTongHeng Science and Technology, China) over silica gel H and developed with petroleum ether/acetone to yield five fractions.

Fraction 1 was chromatographed on Sephadex-LH-20 (petroleum ether-CHCl₃-MeOH 9:9:2, v/v/v) and further separated on silica gel H with petroleum-EtOAc (8:2, v/v) to yield compound 1 (19.6 mg). Fraction 2 was chromatographed on Sephadex-LH-20 (petroleum ether-CHCl₃-MeOH 9:9:2, v/v/v) and further separated on silica gel H with petroleum/acetone (8:2–6:2, v/v) to yield compounds 2 (42.9 mg) and 3 (5.3 mg). Fraction 3 was chromatographed on Sephadex-LH-20 (petroleum ether-CHCl₃-MeOH 9:9:2, v/v/v) and further separated on silica gel H with petroleum ether/EtOAc/acetone (8:1:1, v/v/v), then chromatographed on Sephadex-LH-20 (acetone) and separated on silica gel H with petroleum/acetone (8:2, v/v) to yield compound 4 (15.6 mg).

Fraction 4 was separated on silica gel H with petroleum ether/CHCl₃/acetone (8:1:1, v/v/v) and further chromatographed on Sephadex-LH-20 (petroleum ether-CHCl₃-MeOH 9:9:2, v/v/v) and separated on silica gel H with petroleum ether/acetone (8:2, v/v) to yield compound 5 (0.9 mg). Fraction 5 was separated on silica gel H with petroleum ether/EtOAc (20:1–10:1, v/v) and further chromatographed on Sephadex-LH-20 (acetone) to yield compound 6 (15.0 mg).

Biological study

Coagulation time test

All tested compounds and the positive control were dissolved by a mixed solution (propanediol:ethanol:saline = 1:1:3). Blood samples were taken from the rabbit's auricular vein, and we disinfected the site of collection with physiological saline and then covered with multilayer sterile gauze bandage tightly wound. The blood was then decalcified by sodium citrate (38 g·L⁻¹) to prevent blood clotting and serum was separated from the plasma by centrifugation (894.4 g) in TGL-16 high-speed centrifuge (Zhongda Instrument Factory, Jintan, China) for 15 min at 25°C.²⁹ APTT and PT were determined according to the method as described previously.³⁰ In brief, serum (50 μL) was mixed with 25 μL of samples, after APTT assay reagent (50 μL) was added, and incubated in LRH-150 incubator (Shanghai Yiheng Technology Co., Ltd., China) for 5 min at 37°C, and then 25 mM CaCl₂ (2.775 g·L⁻¹, 100 μL) was added. Clotting times were recorded. In PT assays, serum (50 μL) was mixed with 25 μL of samples and incubated for 3 min at 37°C. PT assay reagent (50 μL), which has been preincubated for 10 min at 37°C, then was added and clotting time was recorded.

TT and FIB were determined according to the manufacturer's recommendations (Shanghai Sun Biotech). In the above tests, blank solvent was used as the blank control group; breviscapine and vitamin K₁ were used as positive controls. PT, APTT, TT, and FIB tests were conducted using a HF6000 Semi-Automated Coagulation Analyzer (Chinese Prescription Medical Instrument Co., Ltd., Jinan, China).

Data are expressed as mean ± standard deviation of three independent experiments and subjected to ANOVA to assess significant differences using SPSS (Version 19.0; SPSS, Inc., Chicago, IL, USA).

Effect on fibrinolytic and urokinase fibrinolytic activity

Activity was measured by the fibrin plate method³¹ as described previously.³² It was performed as follows: 30 BP/mL thrombin (500 μL) was used to cover the entire surface of a sterile Petri dish (9 cm in diameter). Fibrinogen solution (8 mL) and agarose solution (7 mL), which were preincubated at 37°C, were added. After the sample was solidified into the fibrin plate by rapid mixing, it was allowed to sit for 30 min, incubated for 1 h at 37°C, and holes were punched in the surface.

Fibrinolytic activity

A twenty-microliter sample was added to each hole in the fibrin plates at different positions, and then, 20 μL of blank solvent and 20 μL of urokinase solution were added as blank group and positive group, respectively. After being cultured for 16 h at 37°C, a Vernier caliper was used to measure the vertical circle diameter for the average.

Urokinase fibrinolysis activity

A ten-microliter sample and 20 μL of urokinase solution were added to each hole in the fibrin plates' different positions, and then, 10 μL of blank solvent and 20 μL of urokinase solution were added again in the blank group. After being cultured for 16 h at 37°C, a Vernier caliper was used to measure the vertical circle diameter for the average.

According to the above method, the standard urokinase was diluted into five concentrations with gradient dilution, then diameter square as the abscissa and circle soluble vitality as the ordinate were to make a standard curve and calculate the regression equation. Taking the sample's circle soluble diameter into the equation to draw the circle soluble vitality (A) value, the inhibitory rates were calculated according to the formula: inhibition rate = 1-A (sample)/A (blank).

α-Glucosidase inhibitory screening assay

The bioassay was executed according to a method described previously.³³ Briefly, α-glucosidase inhibitory activity was measured spectrophotometrically in a 96-well plate based on PNPG as substrate. The assay mixture (160 μL) containing 8 μL of a sample in dimethyl sulfoxide (DMSO) (or DMSO itself as blank control), 112 μL phosphate buffer (pH 6.8), and 20 μL enzyme solution (0.2 U·mL⁻¹ α-glucosidase in phosphate buffer) was mixed and incubated at 37°C for 15 min, and then, 20 μL substrate solution (2.5 mmol·L⁻¹ PNPG prepared in the same buffer) was added. The reaction was processed at 37°C for 15 min and stopped by adding 80 μL of 0.2 mol·L⁻¹ Na₂CO₃ solution. The amount of p-nitrophenol released from PNP-glycoside was quantified on a 96 microplate reader at 405 nm by 1510-Multiskan Spectrum (Thermo Fisher Scientific, Inc., USA).

The inhibitory rates (%) were calculated according to the formula: I% = [1-(OD_test–OD_{test blank})/(OD_control–OD_{control blank})] × 100%. Acarbose was used as positive control. All the field experiments were carried out with three replications and then the data were expressed as mean ± standard deviation.

Results

Isolation

Six compounds were isolated from episperms of M. fragrans. Compounds 2 and 5 were isolated from the genus for the first time (Fig. 1). The chemical structures of the compounds were identified as (benzo[3′,4′]dioxol-1′-yl)-7-hydroxypropyl)benzene-2,4-diol (1),¹³ odoratisol A (2),³⁴ dehydrodiisoeugenol (3),³⁵ erythro-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(4-hydroxy-3-rnethoxyphenyl)propan-1-ol (4),³⁶ bis(2-ethylhexyl)phthalate (5),³⁷ and β-sitosterol (6).³⁸ Melting points of these compounds were determined on an XRC-1 apparatus. 1D NMR spectra was recorded on a Bruker-AM-400 spectrometer (Bruker, Billerica, MA, USA) in deuterated chloroform (CDCl₃) using tetramethylsilane (TMS) as internal standard. Electron ionization–mass spectrometry (EI-MS) spectra were obtained using a Thermo Trace DSQ II—mass spectrometer (Thermo Fisher Scientific).

FIG. 1.

Chemical structures of compounds 1 to 6.

(benzo[3′,4′]dioxol-1′-yl)-7-hydroxypropyl)benzene-2,4-diol (1)

C₁₆H₁₆O₅; Colorless oil; EI-MS m/z: 288[M]⁺; ¹H-NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 6.38 (1H, d, J = 6.8 Hz, H-5), 6.80 (1H, d, J = 8.0 Hz, H-5′), 6.86 (1H, d, J = 8.0 Hz, H-6), 6.92 (1H, d, J = 8.0 Hz, H-6′), 4.93 (1H, d, J = 9.6 Hz, H-7), 2.12 (1H, m, H-8), 1.98 (1H, m, H-8), 2.85 (1H, m, H-8′), 2.70 (1H, d, J = 1.6 Hz, H-8′), 5.96 (2H, s, O-CH₂-O). ¹³C-NMR (100 MHz, CDCl₃, δ, ppm): 135.7 (C-1), 156.0 (C-2), 103.7 (C-3), 156.0 (C-4), 108.3 (C-5), 130.3 (C-6), 77.9 (C-7), 30.2 (C-8), 114.3 (C-1′), 106.9 (C-2′), 148.0 (C-3′), 147.4 (C-4′), 108.3 (C-5′), 119.7 (C-6′), 25.4 (C-8′), OCH₂O (101.2).

Odoratisol A (2)

C₂₁H₂₄O₅; Yellow oil; EI-MS m/z: 356 [M]⁺; ¹H-NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 6.67 (2H, s, H-2,6), 5.06 (1H, d, J = 9.6 Hz, H-7), 6.34 (1H, d, J = 16.0 Hz, H-7′), 3.45 (1H, m, H-8), 6.08 (1H, m, H-8′), 1.38 (3H, d, J = 6.8 Hz, H-9), 1.86 (3H, d, J = 6.0 Hz, H-9′), 3.89 (6H, s, MeO-3, and MeO-5), 3.88 (3H, s, MeO-3′). ¹³C-NMR (100 MHz, CDCl₃, δ, ppm): 133.2 (C-1), 103.5 (C-2), 147.1 (C-3), 134.9 (C-4), 147.1 (C-5), 103.5 (C-6), 93.9 (C-7), 45.6 (C-8), 17.5 (C-9), 132.2 (C-1′), 109.4 (C-2′), 144.1 (C-3′), 146.5 (C-4′), 134.9 (C-5′), 113.3 (C-6′), 131.2 (C-7′), 123.4 (C-8′), 18.3 (C-9′), 56.3 (MeO-3), 56.3 (MeO-5), 53.4 (MeO-3′).

Dehydrodiisoeugenol (3)

C₂₀H₂₂O₄; Yellow crystalline; EI-MS m/z: 326 [M]⁺; ¹H-NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 1.39 (3H, d, J = 6.4 Hz, 3-Me), 1.87 (3H, d, J = 6.0 Hz, H₃-10), 3.45 (1H, t, H-3), 3.88 (3H, s, 3′-OMe), 3.89 (3H, s, 7-OMe), 5.08 (1H, d, J = 9.2 Hz, H-2), 5.58 (1H, s, 4′-OH), 6.11 (1H, m, H-9), 6.37 (1H, d, J = 15.6 Hz, H-8), 6.76 (1H, s, H-6), 6.78 (1H, s, H-4). ¹³C-NMR (100 MHz, CDCl₃, δ, ppm): 94.1 (C-2), 45.2 (C-3), 131.4 (C-3a), 112.1 (C-4), 130.4 (C-5), 109.4 (C-6), 144.3 (C-7), 146.7 (C-7a), 130.9 (C-8), 123.6 (C-9), 17.8 (C-10), 132.4 (C-1′), 103.5 (C-2′), 147.2 (C-3′), 130.4 (C-4′), 146.7 (C-5′), 103.5 (C-6′), 17.7 (C₃-Me), 56.1 (3′-OMe), 56.3 (7-OMe).

Erythro-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(4-hydroxy-3-rnethoxyphenyl)propan-1-ol (4)

C₂₁H₂₆O₆; Yellow oil; EI-MS m/z: 374 [M]⁺; ¹H-NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 1.12 (3H, d, J = 6.4 Hz, γ-H × 3), 3.36 (2H, d, J = 6.8 Hz, α′-H × 2), 3.87 (6H, s, MeO × 2), 3.89 (3H, s, MeO), 4.33 (1H, q, β-H), 4.80 (1H, brs, OH), 5.10–5.15 (2H, t, γ′-Hb, and γ′-Ha), 5.84–6.03 (1H, m, β′-H), 6.46 (2H, s, 3′-H, and 5′-H), 6.67–6.97 (3H, ABX, 2-H, 5-H, and 6-H). ¹³C-NMR (100 MHz, CDCl₃, δ, ppm): 133.2 (C-1), 108.8 (C-2), 146.6 (C-3), 146.6 (C-4), 114.0 (C-5), 119.0 (C-6), 82.5 (C-α), 73.0 (C-β), 12.9 (C-γ), 136.3 (C-1′), 153.6 (C-2′), 105.7 (C-3′), 132.2 (C-4′), 105.7 (C-5′), 153.6 (C-6′), 40.7 (C-α′), 137.2 (C-β′), 116.3 (C-γ′), 56.1 (MeO × 3).

Bis(2-ethylhexyl)phthalate (5)

C₂₄H₃₈O₄; Yellow oil; EI-MS m/z: 390 [M]⁺; ¹H-NMR (400 MHz, CDCl₃, δ, ppm): 7.69 (2H, q, H-3,6), 7.54 (2H, q, H-4,5), 4.23 (4H, m, H-1′), 1.70 (2H, m, H-2′), 1.44 (4H, m, H-1″), 1.35 (4H, m, H-3′), 1.32 (4H, m, H-4′), 0.94 (6H, m, H-2″), 0.88 (6H, m, H-6′). ¹³C-NMR (100 MHz, CDCl₃, δ, ppm): 167.9 (COO-), 132.7 (C-1,2), 131.0 (C-4,5), 129.0 (C-3,6), 68.3 (C-1′), 38.9 (C-2′), 30.5 (C-3′), 29.1 (C-4′), 23.9 (C-1″), 23.1 (C-5′), 14.2 (C-6′), 11.1 (C-2″).

β-Sitosterol (6)

C₂₉H₅₀O; White needle crystals; mp 138–141°C; EI-MS m/z: 414.7 [M]⁺; ¹H-NMR (CDCl₃, 400 MHz) δ:5.36 (1H, brs, H-6), 3.52 (1H, m, H-3α), 0.68 (3H, s, CH₃-18), 0.80 (3H, s, CH₃-27), 0.82 (3H, d, J = 8 Hz, CH₃-26), 0.86 (3H, s, CH₃-29), 0.93 (3H, d, J = 8 Hz, CH₃-21), 1.01 (3H, s, CH₃-19). ¹³C-NMR (CDCl₃, 100 MHz) δ:37.4 (C-1), 32.1 (C-2), 72.0 (C-3), 42.5 (C-4), 140.9 (C-5), 121.9 (C-6), 31.8 (C-7), 32.1 (C-8), 50.3 (C-9), 36.7 (C-10), 21.2 (C-11), 39.9 (C-12), 42.5 (C-13), 56.9 (C-14), 24.5 (C-15), 28.4 (C-16), 56.2 (C-17), 12.0 (C-18), 20.0 (C-19), 36.3 (C-20), 19.5 (C-21), 34.1 (C-22), 26.3 (C-23), 46.0 (C-24), 29.3 (C-25), 18.9 (C-26), 19.2 (C-27), 23.2 (C-28), 12.1 (C-29).

The effects on blood coagulation time

Compared with the blank group, compounds 1 and 4 significantly shortened the PT, indicating that they had certain effects on promoting exogenous coagulation, but the effects were not very large compared with vitamin K₁ as a positive control. Compared with breviscapine, the anti-exogenous coagulation effect of compound 2 was modest. For APTT, compared with the blank group, the endogenous coagulation effects of compounds 1, 2, and 4 were significantly different (P < .001), but less than that of vitamin K₁.

The value of TT showed that compounds 1, 2, and 4 could promote transfer of fibrinogen to fibrin, but the effects were less than that of vitamin K₁ (P < .001). The contents of FIB from three compounds were not different from the blank group (P ≥ .05), but were significantly different from breviscapine (P < .001). Results indicated that compounds 1, 2, and 4 had no impact on plasma fibrinolysis enzyme activity (Table 1) and suggested that compounds 1 and 4 promoted blood coagulation by both endogenous and exogenous pathways, whereas compound 2 was mainly through the intrinsic coagulation pathway.

Table 1.

The Effect of Compounds on Blood Coagulation

Group	Prothrombin time (s)	Activated partial thromboplastin time (s)	Thrombin time (s)	Fibrinogen (g/L)
Blank	10.27 ± 0.09	25.80 ± 0.22	32.78 ± 0.10	3.38 ± 0.02
Breviscapine	11.30 ± 0.08	34.70 ± 0.48	39.55 ± 0.13	5.29 ± 0.17
vK₁	7.42 ± 0.09	12.60 ± 0.18	19.67 ± 0.17	—
1	9.17 ± 0.17^c,i	22.90 ± 0.45^c,i	27.25 ± 0.96^{b, i}	3.33 ± 0.24^f
2	10.65 ± 0.13^c,f	17.45 ± 0.34^c,i	27.48 ± 0.46^c,i	3.86 ± 0.41^f
4	8.10 ± 0.18^c,i	24.32 ± 0.19^c,i	28.23 ± 0.87^{b, i}	3.47 ± 0.18^f

Mean value ± standard error of mean. Compared with Blank (0.01 < ^a P < .05, 0.001 < ^b P < .01, ^c P < .001); Compared with Breviscapine (0.01 < ^d P < .05, 0.001 < ^e P < .01, ^f P < .001); Compared with vitamin K₁ (0.01 < ^g P < .05, 0.001 < ^h P < .01, ⁱ P < .001).

a,d,g

: significant difference.

b,e,h

: very significant difference.

c,f,i

: highly significant difference.

Urokinase fibrinolytic activity assay

The fibrin plate assay was used to assay the urokinase fibrinolytic activity (Fig. 2). There was a good linear relationship between the urokinase activity and dissolved ring indicating the mass concentration (3.125–50 IU·mL⁻¹). The thrombolysis properties are also revealed in Figure 2.

FIG. 2.

The urokinase activity standard curve and the fibrin plate images a1, a2: 50 IU•mL-1; b1, b2: 25 IU•mL-1; c1, c2: 12.5 IU•mL-1; d1, d2: 6.25 IU•mL-1; e1, e2: 3.125 IU•mL-1.

The series of Compound Danshen tablet concentrations and its fibrinolytic inhibitions are presented in Table 2. Origin 8.0 software was used to make a scatter diagram and mark the half-maximal inhibitory concentration (IC₅₀) value of 6.577 mg/mL. The fibrinolytic inhibition activity was described in the fibrin plate (Fig. 3).

FIG. 3.

Fibrinolytic activity inhibition by complex tablets in the fibrin plate a1, a2: 1.875 mg/mL; b1, b2: 3.75 mg/mL; c1, c2: 7.5 mg/mL; d1, d2-15 mg/mL; e1, e2: blank control.

Table 2.

Fibrinolytic Inhibitive Effect by Complex Tables

No.	Concentration (mg/mL)	Square diameter (cm²)	Enzyme activity (IU)	Inhibition rate (%)
1	6.67	1.47 ± 0.01	78.99	52.07
2	5.0	1.82 ± 0.01	100.6	38.95
3	2.5	2.00 ± 0.02	111.7	32.22
4	1.25	2.26 ± 0.01	127.8	22.45
5	0.625	2.60 ± 0.02	148.7	9.769
6	Blank	2.86 ± 0.01	164.8	NT

NT, unable to provide data or calculation.

The fibrinolytic activity from M. fragrans

Three compounds did not show any fibrinolytic activity (Fig. 4).

FIG. 4.

Fibrinolytic activity from Myristica fragrans in the fibrin plate a1, a2: urokinase; b1, b2: blank control; c1, c2: compound 1; d1, d2: compound 2; and e1, e2: compound 4.

The fibrinolytic inhibition activity from M. fragrans

According to the method, prescreening fibrinolytic inhibition rates of the three compounds exceeded 50% and entered into secondary screening (Fig. 5; Table 3).

FIG. 5.

Prescreening fibrinolytic inhibition activity from Myristica fragrans in the fibrin plate a1, a2: blank control; b1, b2: compound 1; c1, c2: compound 2; d1, d2: compound 4; and e1, e2: complex tablets.

Table 3.

Fibrinolytic Inhibitive Effect by Compounds of Myristica fragrans

No.	Sample	Initial concentration (mg/mL)	Square diameter (cm²)	Enzyme activity (IU)	Inhibition rate (%)	IC₅₀ (mg/mL)
1	1	5	0.3164 ± 0.036	7.784	75.33	2.407
2	2	5	0.3393 ± 0.021	9.198	70.85	1.747
3	4	5	0.3192 ± 0.012	7.958	77.78	1.818
4	Complex tablet	20	0.1913 ± 0.010	0.0631	99.98	6.577

All three compounds had high inhibitory effects on the fibrinolytic pathway activated by urokinase. Compound 2 exhibited the highest inhibitory activity (IC₅₀ = 1.747 mg·mL⁻¹) followed by compounds 4 (IC₅₀ = 1.818 mg·mL⁻¹) and 1 (IC₅₀ = 2.407 mg·mL⁻¹), which were higher than that of Compound Danshen Tablets (IC₅₀ = 6.577 mg·mL⁻¹) (Table 3; Fig. 6).

FIG. 6.

Secondary screening of fibrinolytic inhibition activity from Myristica fragrans in the fibrin plate a1, a2: 1/2 initial concentration; b1, b2: 1/4 initial concentration; c1, c2: 1/8 initial concentration; d1, d2: 1/16 initial concentration; and e1, e2: blank control.

α-Glucosidase inhibitory activity

Compounds 1, 2, and 4 were tested on α-glucosidase inhibitory activity for the first time. The α-glucosidase inhibitory activity of compounds 1 and 2 was higher than that of acarbose as the positive control (Table 4), and the activity of 1 and 2 showed a concentration dependence (Fig. 7). Within a certain range of mass concentration, the α-glucosidase inhibitory rates showed a linear relationship with the concentration, but upon increasing the concentration further, the inhibitory rates changed a little.

FIG. 7.

Effect at different compound concentrations of Myristica fragrans on α-glucosidase inhibitory activity.

Table 4.

Inhibitory Effect of Compounds 1, 2, and 4 from Myristica fragrans Against α -Glucosidase

Compound^a	Prescreening concentration (μg·mL⁻¹)	Inhibition rate (I%)	IC₅₀ (μg·mL⁻¹)
1	100	93.18	21.76 ± 0.59
2	100	82.70	21.31 ± 0.00
4	100	11.54	NT
Acarbose^a	1500	61.23	1136.41 ± 150.26

Prescreening concentration 100 μg·mL⁻¹.

NT, not available because of low activity (I% < 50%).

Discussion

The body's blood clotting response is normally precisely controlled by three systems: (1) serine protease inhibitors, such as the plasminogen activator inhibitor; (2) the protein C/S anticoagulant system; and (3) surface binding and coagulation inhibitors.³⁹ Urokinase as one plasminogen activator launches the fibrinolytic function by activating plasminogen into plasmin.³² Figure 8 shows the schematic diagram of blood coagulation.

FIG. 8.

Schematic diagram of blood coagulation.

The three active compounds identified in the study belong to phenol and lignan, however, no studies have been conducted to investigate their procoagulant effects. On the contrary, several studies have reported on the anticoagulant activity of lignans. It has been reported that the inhibitory mechanism of erythro-(7S,8R)-7-acetoxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan (EATN) on platelet aggregation might increase cyclic adenosine monophosphate levels and subsequently inhibit intracellular Ca²⁺ mobilization by interfering with a common signaling pathway rather than by directly inhibiting the binding of thrombin or platelet activating factor (PAF) to their receptors.⁴⁰ Specifically, bicycle (3,2,1) octane neolignans, 8,3′-neolignans, and 8-O-4′-neolignans were demonstrated to possess potent PAF antagonistic properties. However, it also revealed that 8-O-4′-neolignan activities were not obvious, especially when C-7 of the compounds was replaced by a hydroxyl group.⁴¹

The reason for the coagulation activity difference may be that they belong to a different type of lignans. In a previous study, three phenolic acids, vanillic acid, p-coumaric acid, and protocatechuic acid, possessed procoagulant activity.⁴² Lignans are the most common constituents of M. fragrans. Our research objectives of the procoagulant ingredients in pericarps of M. fragrans and the structure–activity relationship are further discussed.

Clinically used procoagulant medicines can be divided into four categories according to their mechanisms, including promoting clotting factor activity, inhibition of fibrinolytic system, effect on blood vessels, platelets, and topical hemostatic or others, such as vitamin K, aminomethylbenzoic acid, adenosine, and thrombin, respectively.⁴³

Currently, various types of hemostatic agents have been explored; unfortunately, each of them has its own shortcomings. For example, the fibrinogen or thrombin is mainly obtained from animal or human blood; they are expensive and include the potential risk of viral infection. The adhesive properties of collagen and gelatin sponges to tissues are poor. Water-soluble collagen is a toxicoid, and the use of this material in human body may cause inflammatory responses.^44,45

Therefore, it is particularly important to develop safe and new hemostatic agents for clinical use with better hemostatic properties and reduced tissue response.

This is the first study to report the procoagulant mechanism of (benzo[3′,4′]dioxol-1′-yl)-7-hydroxypropyl)benzene-2,4-diol (1), odoratisol A (2), and erythro-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(4-hydroxy-3rnethoxyphenyl)propan-1-ol (4), which could mainly activate many kinds of cruor factors in the endogenous coagulation/common coagulation pathway and inhibit the fibrinolytic pathway activated by urokinase. In other words, they inhibited thrombus dissolution and promoted blood coagulation. Moreover, compounds 1 and 2 showed a strong α-glucosidase inhibitory activity in a dose-dependent manner, suggesting for the first time that M. fragrans might protect against type 2 diabetes.

Pericarps peeled off after maturity, while the isolated pericarp compounds are still active, could promote coagulation, and inhibit α-glucosidase activity, This research suggests that M. fragrans pericarp has the potential to be developed into a new hemostatic therapeutic intervention that is effective, inexpensive, and derived from easily obtainable sources.

Footnotes

Acknowledgments

This work was supported by the Henan Province University Science and Technology Innovation Team (16IRTSTHN019), Industry & University Research Project in Henan Province (162107000038 and 152107000051), Kaifeng City Science and Technology Innovation Talent (1509010), National cooperation project of Henan Province (2015GH12), Key project in Science and Technology Agency of Zhengzhou City (20150341), and Science Research Foundation of Henan University (ZZJJ20140047).

Author Disclosure Statement

No competing financial interests exist.

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