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Antiplatelet Effects of Garlic and Chitosan: a Comparative Study between Fermented and Non-Fermented Preparations
Biomed Sci Letters 2018;24:280-284
Published online September 30, 2018;  https://doi.org/10.15616/BSL.2018.24.3.280
© 2018 The Korean Society For Biomedical Laboratory Sciences.

Muhammad Irfan1,*, Minki Kim1,*, Hyuk-Woo Kwon2,**, Man Hee Rhee1,†,**, and Hyun-Kyoung Kim3,†,**

1Laboratory of Physiology and Cell Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea,
2Department of Biomedical Laboratory Science, Far East University, Eumseong 27601, Korea,
3Department of Food Science and Engineering, Seowon University, Cheongju 28674, Korea
Correspondence to: Man Hee Rhee. Laboratory of Physiology and Cell Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea. Tel: +82-53-950-5967, Fax: +82-53-950-5955, e-mail: rheemh@knu.ac.kr
Hyun-Kyoung Kim. Department of Food Science and Engineering, Seowon University, Chungbuk 28674, Korea. Tel: +82-43-299-8474, Fax: +82-43-299-8470, e-mail: kimhk4@seowon.ac.kr
Received August 17, 2018; Accepted September 4, 2018.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract

The incidence of cardiovascular diseases (CVDs) is increasing rapidly in developed countries, with CVDs now representing the leading cause of morbidity and mortality. Natural products and ethnomedicines have been shown to reduce the risk of CVDs. Garlic is a medicinal plant used throughout the world for its anti-inflammatory, antioxidant, and antiplatelet activities. Chitosan is a natural polysaccharide obtained from chitin, and derivatives of chitosan have been shown to inhibit platelet aggregation and adhesion. We hypothesized that fermented preparations of these products may possess stronger antiplatelet effects than the non-fermented forms owing to the increased bioavailability of the bioactive compounds produced during fermentation. Therefore, we compared these compounds via in vitro and ex vivo platelet aggregation assays by using standard light transmission aggregometry and ex vivo granule secretions from rat platelets. We found that fermented preparations exerted more potent and significant inhibition of platelet aggregation both in vitro and ex vivo. Likewise, ATP release from dense granules of platelets was also significantly inhibited in fermented preparation-treated rat platelets compared to that in non-fermented preparation-treated ones. We concluded that fermented preparations exerted more potent effects on platelet function both in vitro and ex vivo, possibly as a result of the increased bioavailability of active compounds produced during fermentation. We therefore suggest that fermented products may be potent therapeutics against platelet-related CVDs and can be used as antiplatelet and antithrombotic agents.

Keywords : Platelets, Fermented garlic, Fermented chitosan, Anti-platelet
Body

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality in developed countries (Maione et al., 2014). Multiple risk factors are involved in the pathophysiology, but platelets are considered the main etiological factor of CVDs. Platelet aggregation is a key step in the development and progression of atherosclerotic plaques responsible for narrowed blood vessels that may ultimately lead to stroke and heart attack (Michel et al., 2014). Platelet function can be suppressed pharmacologically, and this has been highly successful in decreasing thrombotic events; many clinically approved antiplatelet drugs are now available for the treatment of cardiovascular ailments. Unfortunately, as these drugs can lead to serious complications, such as gastric bleeding, and are ineffective in some patients (Barrett et al., 2008), there is a need for the development of more effective and safer approaches for the treatment and prevention of CVDs. One such approach includes the use of natural products with antithrombotic and anticoagulant activities (Rastogi et al., 2016). Ethnomedicine and natural products have been the focus of many recent studies as potential therapies for CVDs (Kerver et al., 2003), and several dietary and herbal compounds have been shown to contribute to a reduced risk of CVDs (Dutta-Roy, 2002).

Garlic has been shown to reduce cardiovascular diseases and its consumption confers significant cardioprotective effects to both animals and humans (Rahman and Billington, 2000; Banerjee and Maulik, 2002). Chitosan is a natural biodegradable and non-toxic cationic polysaccharide obtained from chitin. Its derivatives are known to inhibit platelet aggregation and adhesion (Jiang et al., 2012). For a long time, fermentation of foods has been appreciated owing to its significant commercial benefits, such as improved nutrient profile and enriched flavor. There are several health benefits of consuming fermented foods beyond the traditionally recognized effects on the digestive system (Kim et al., 2016). It was previously reported that fermentation enhances the quality and efficacy of food or herbal products through an improved nutrient profile (Lee et al., 2012). Therefore, we aimed to compare the effects of fermented garlic and chitosan with those of non-fermented compounds on platelet function.

For this purpose, collagen (native collagen fibrils (type I) from equine tendons), ADP, and thrombin were purchased from Chrono-log (Havertown, PA, USA). The ATP assay kit was obtained from the Biomedical Research Service Centre (Buffalo, NY, USA) and DMSO was acquired from Sigma-Aldrich (St. Louis, MO, USA). Water was obtained from J.T. Baker (Phillipsburg, NJ, USA). All chemicals were of reagent grade.

To prepare fermented chitosan, chitosan was first dissolved in hydrochloric acid and chitosanase was added to decompose the chitosan and produce chitosan oligosaccharide. To prepare fermented garlic, unpeeled garlic was placed in an autoclave and steamed to produce black garlic. Lactic acid bacteria were added for the second round of fermentation.

Male Sprague-Dawley (SD) rats (240~260 g) were purchased from Orient Co. (Seoul, Korea) and acclimatized for 1 week prior to the experiment in an animal room with controlled environmental conditions (12/12 h light/dark cycle, 23°C±2°C temperature, and 50%±10% humidity). The experiments were conducted in accordance with the IACUC guidelines and the protocols were approved by the Ethics Committee of College of Veterinary Medicine, Kyungpook National University, Daegu, Korea. The rats were randomly divided into six groups (n = 4 in each group) for the ex vivo study and received a daily oral administration of vehicle, 300 mg/kg garlic (fermented and non-fermented), and 300 mg/kg chitosan (fermented and non-fermented) for 7 days. Two hours after the final administration, blood samples were collected from rats for testing.

To prepare washed platelets, whole blood was collected from SD rats via heart puncture and transferred to a tube containing anticoagulant acid citrate dextrose solution. The blood was centrifuged at 170 ×g for 7 min to obtain plateletrich plasma (PRP), which was then centrifuged at 350 ×g for 7 min to isolate the washed platelets. The platelet concentration was adjusted to 3 × 108 cells/mL by using Tyrode’s buffer (137 mM NaCl, 12 mM NaHCO3, 5.5 mM glucose, 2 mM KCl, 1 mM MgCl2, and 1 mM NaHPO4, pH 7.4) for use in the platelet aggregation assays. All preparation procedures were performed at room temperature (23°C±2°C).

Light-transmission aggregometry (Chrono-log, Havertown, PA, USA) was performed in accordance with a previously described method to assess platelet aggregation (Irfan et al., 2018). Briefly, the washed platelets were pre-incubated with various concentrations of either fermented garlic (F. garlic), non-fermented garlic (NF. garlic), fermented chitosan (F. chitosan), non-fermented chitosan (NF. chitosan), or vehicle for 2 min at 37°C in the presence of 1 mM calcium chloride (CaCl2), and then stimulated by the agonist collagen, ADP, or thrombin. The mixture was further incubated for 5 min with continuous stirring. To achieve ex vivo platelet aggregation, washed platelets obtained from vehicle-, F. garlic-, NF. garlic-, F. chitosan-, and NF. chitosan-treated rats were incubated at 37°C with continuous stirring and stimulated with collagen or ADP for 5 min.

To determine the ex vivo effects of F. garlic, NF. garlic, F. chitosan, and NF. chitosan on dense granule secretion, an ATP assay was performed as previously described (Jeong et al., 2016). Briefly, washed platelets obtained from vehicle-, F. garlic-, NF. Garlic-, F. chitosan-, NF. Chitosan-treated groups were incubated at 37°C with continuous stirring and stimulated with collagen or ADP for 5 min. The aggregation reaction was terminated and the platelet mixture was centrifuged. The ATP secretion of the obtained supernatant was determined by using an ATP assay kit (Biomedical Research Service Center) measured using a luminometer (GloMax 20/20, Promega, Madison, WI, USA).

The data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test to measure the statistical significance of the differences observed (SAS Institute Inc., Cary, NC, USA). All data are presented as the mean ± standard error of the mean (SEM), and P-values < 0.05 indicated statistical significance.

The in vitro effects of F. garlic, NF. garlic, F. chitosan, and NF. chitosan on platelet aggregation were assessed by using light transmission aggregometry. The effects of the compounds were tested against different ligands (collagen, ADP, and thrombin), and F. chitosan was found to be most effective against ADP-induced platelet aggregation, whereas F. garlic and NF. garlic were more potent and inhibited platelet aggregation against collagen, ADP, and thrombin (Fig. 1A-B). F. garlic and N.F. garlic markedly inhibited agonist-induced platelet aggregation in a dose-dependent manner.

Fig. 1.

Garlic and chitosan inhibit in vitro and ex vivo agoniststimulated platelet aggregation. (A-B) Washed platelets were pretreated with various concentrations of F. garlic, NF. garlic, F. chitosan, NF. Chitosan, or vehicle for 2 min in presence of 1 mM CaCl2, and then stimulated with collagen, ADP, or thrombin for 5 min. (C) Washed platelets obtained from vehicle-, F. garlic-, NF. garlic-, F. chitosan-, or NF. chitosan-treated rats (all treatments at 300 mg/kg) were incubated at 37°C with continuous stirring and then stimulated with collagen or ADP for 5 min. The graph presents the mean ± SEM of experiments performed on four independent days (n = 1 each day). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the vehicle control.


To evaluate the ex-vivo effects, the rats were treated with F. garlic, NF. garlic, F. chitosan, and NF. chitosan and the platelet aggregation assay was performed. Platelet aggregation was inhibited in all treatment groups unlike in the vehicletreated control group. Moreover, F. garlic and NF. garlic showed greater inhibition of collagen-induced platelet aggregation than of ADP-induced platelet aggregation (Fig. 1C).

Activated platelets release the contents of granules such as alpha granules and dense granules; this secretion of granule content enhances platelet activation, including intracellular signaling pathways.

The early phases of platelet activation are characterized by the rapid release of ATP (Jeong et al., 2017); therefore, we assessed collagen- and ADP-induced ATP secretion. As shown in Fig. 2, F. garlic, NF. garlic, F. chitosan, and NF. chitosan significantly inhibited ATP release from dense granules in collagen- and ADP-stimulated platelets. Although both were strong inhibitors of ATP release, garlic appeared to be slightly more potent than chitosan (Fig. 2). These data suggest that the compounds exerted antiplatelet effects through the suppression of platelet aggregation and granule secretion.

Fig. 2.

Garlic and chitosan inhibit ex vivo ATP secretion. Washed platelets obtained from vehicle-, F. garlic-, NF. garlic-, F. chitosan-, or NF. chitosan-treated rats (all treatments at 300 mg/kg) were incubated at 37°C with continuous stirring and stimulated with collagen or ADP for 5 min. Following the termination of the platelet aggregation reaction, the concentration of ATP was assessed by using a luminometer. The results are presented as the mean ± SEM of experiments performed on three independent days (n = 1 each day). **P < 0.01 and ***P < 0.001 compared with the vehicle control.


In the present study, we evaluated the inhibitory effects of fermented and non-fermented preparations of garlic and chitosan on platelet aggregation and granule secretion. We found that fermented preparations significantly inhibited platelet aggregation in vitro and ex vivo and also inhibited ex-vivo ATP release from dense granules. Many studies have shown that platelets are continuously exposed to several factors that cause their activation and aggregation, such as collagen, ADP, thrombin, fibrinogen, von Willebrand factor (vWF), and thromboxane; however, inhibitory factors are also present, such as prostacyclin (PGI2) and ADPase (Ruggeri and Mendolicchio, 2007). Any imbalance in these opposing factors may impair hemostasis; thus, a strong equilibrium is necessary for normal platelet function. Our results indicate that the pretreatment of platelets with garlic or chitosan preparations, especially fermented preparations, may help maintain this balance and hemostasis. As fermentation enhances bioavailability and the absorption of food products (Lee et al., 2012; Rein et al., 2013; Kim et al., 2016), we concluded that the observed antiplatelet effects were a result of the increased bioavailability of bioactive compounds from fermented preparations.

ACKNOWLEDGEMENT

This research was supported by the Korean technology and information promotion agency (Industry-academyresearch cooperation technology development business of Assignment number C0563925).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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