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ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 4  |  Page : 420-424  

Modulation of platelet functions by Careya sphaerica Roxb. leave extracts


1 Department of Preclinic, Faculty of Medicine, Siam University, Phasi Charoen, Bangkok, Thailand
2 Department of Thai Traditional Medicine, Thai Traditional Medicine College, Rajamangala University of Technology Thanyaburi, Pathum Thani, Thailand
3 Department of Clinical Microbiology, Faculty of Medical Technology, Nation University, Lampang, Thailand
4 Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, Thailand

Date of Submission13-Apr-2021
Date of Decision29-Jun-2021
Date of Acceptance19-Jul-2021
Date of Web Publication20-Oct-2021

Correspondence Address:
Dr. Suriyan Sukati
Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, 80160
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.japtr_95_21

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  Abstract 


Platelets form a plug to prevent blood loss and contribute to wound healing. Kradonbok, Careya sphaerica Roxb., is a Thai plant with medicinal properties. Conventionally, leaves of C. sphaerica are being used to help wound healing in Thailand. The present study was aimed to investigate the effect of C. sphaerica on the function of platelet. Four different extracts of leaves of C. sphaerica (distilled water, methanol, ethanol, and chloroform extracts) were prepared. The extracts at 5.0 mg/ml per dose were tested for the effect of C. sphaerica on platelet adhesion and aggregation properties, by employing a microtiter plate approach. The phytochemical identification was done by using gas chromatography–mass spectrometry (GC-MS). Our data revealed that chloroform extract significantly activated thrombin-induced platelet adhesion (105.27 ± 0.11%, P < 0.05). None of the extracts exhibited an improvement in platelet aggregation. Further GC-MS analysis of the chloroform extract revealed five key phytochemical constituents with potential platelet activation properties. In conclusion, our study evaluated platelet activation and potentially wound healing property of C. sphaerica. GC-MS analysis identified potential bioactive phytochemical compounds in C. sphaerica which warrant further investigation to characterize these compounds.

Keywords: Careya sphaerica roxb, platelet adhesion, platelet aggregation, primary hemostasis, wound healing


How to cite this article:
Khobjai W, Ninlaor W, Watcharasamphankul W, Thongom T, Sukati S. Modulation of platelet functions by Careya sphaerica Roxb. leave extracts. J Adv Pharm Technol Res 2021;12:420-4

How to cite this URL:
Khobjai W, Ninlaor W, Watcharasamphankul W, Thongom T, Sukati S. Modulation of platelet functions by Careya sphaerica Roxb. leave extracts. J Adv Pharm Technol Res [serial online] 2021 [cited 2021 Nov 29];12:420-4. Available from: https://www.japtr.org/text.asp?2021/12/4/420/328638




  Introduction Top


Hemostasis is an essential physiological process to stop bleeding and help wound healing. When an injury occurs, platelets play an essential role in primary hemostasis and the secondary hemostatic process of coagulation-comprising intrinsic and extrinsic pathways.[1] In addition, platelets are involved in thrombin generation.[2] Hence, platelets promote both processes of primary and secondary hemostasis. The mechanical properties of a blood clot are essential for proper hemostasis and wound healing.[3] Blood clot formation depends on several factors, most notably the structure of the fibrin polymer as well as how permeable it is, which, in turn, affects accessibility to incoming repair cells.[3]

A primary hemostatic plug is formed by aggregation of platelet whereby fibrinogen binding to platelets constitutes an important part of wound healing.[3] One of the important mechanisms of platelet function is adhesion to the damaged vessel wall, which is considered an essential process of platelet aggregation.[4] When platelets adhere to collagen at the subendothelium of vessel, they begin platelet aggregation processes. Therefore, analysis of the adhesion and aggregation functions of platelets is of great importance in the differential diagnosis and follow-up of bleeding and thrombotic syndromes.

In Thailand, many native plants are used for food and traditional medicine. Careya sphaerica Roxb, called Kradonbok in the Thai language, is an indigenous vegetable. It is commonly found in the Southern part of Thailand. Local people favorably consume the fresh young leaves and flowers. There is evidence that C. sphaerica leaves have health benefits such as accelerated wound healing.[5] Thus, this study aimed to determine the effects of C. sphaerica leaves on platelet activation.


  Materials and Methods Top


Plant extraction and preparation

C. sphaerica leaves were collected from three different trees in Na Yong District region, Trang Province, Southern Thailand, in May 2017. The collected plants were dried in a storage space at room temperature (RT). The plant specimens were identified by Walailak University (WU) Herbarium and the voucher specimens were deposited at WU Herbarium (voucher specimen number: WU1147-9), Walailak Botanic Garden, WU, Nakhon Si Thammarat, Thailand. The distilled water extraction was performed by decoction technique, using boiling with distilled water. The powder portion of C. sphaerica leaves (10% w/v) was soaked in boiling distilled water for 30 min at RT with occasional stirring. The solution was filtrated through Whatman's filter paper No. 1 and was then concentrated by lyophilization. Extraction by maceration in methanol, ethanol, and chloroform was performed at a ratio of 1:10% w/v, incubated at RT for 7 days. The precipitates were centrifuged at 2000 rpm and then evaporated by a rotary evaporator. To measure platelet activity, the platelet adhesion and platelet aggregation tests were employed.

Blood samples

Peripheral blood samples were collected from 30 healthy human volunteers. Volunteers had no history of oral contraceptive or anticoagulant therapy. The blood was placed separately in containers containing 3.2% sodium citrate. Centrifugation was carried out at 100×g for 10 min at 22°C, to separate the blood cells from plasma to harvest platelet-rich plasma (PRP). The PRP was employed for platelet adhesion and platelet aggregation tests. Study design and informed consent form for the volunteers were permitted by the Committee on Human Rights Related to Human Experimentation of Walailak University (reference number WUEC-18-024-01).

Platelet adhesion assay

The adhesion function of platelet was analyzed by a microtiter plate method using 96-well microtiter plates coated with 50 μg/ml collagen overnight.[6] The collagen solution was removed, the well washed three times with phosphate-buffered saline (PBS). Then, 25 μl of both PRP (2 × 108 cells/ml) and the sample were added to the well. After incubating for 10 min at RT, the mixtures were activated with 0.25 units/ml of thrombin. The reaction was then incubated for 30 min at RT. Absorbance at 600 nm was measured on Glomax Multi microplate reader, USA. Aspirin was used as the positive control and PBS as the negative control. The percentage of platelet adhesion was calculated by the formula following:

% platelet adhesion = As/Ac × 100

Where As is the absorbance of the sample and PRP solution, and Ac is the absorbance of the PBS and PRP without sample extract.

Platelet aggregation assay

The aggregation of platelets was determined by a microplate method.[7] Common methods for evaluating aggregation are based on the decrease of turbidity of a platelet suspension. A modification of this method, consisting of the measurement of absorptivity at 600 nm in microtiter plates, allowed detection of platelet aggregation under conditions similar to those used for the adhesion assay. The 96-well microtiter plates were added with 25 μl of 2 × 108 cells/ml PRP and 25 μl of the sample. The reaction was incubated at RT for 10 min. Then, 25 μl of 50 μg/ml collagen was added into each well of the 96-well microtiter plate. Absorbance at 600 nm was recorded at 0 and 20 min incubation time. Aspirin was used as the positive control and PBS as the negative control. Platelet aggregation percentage was calculated by the formula following:

% platelet aggregation = As/Ac × 100

Where As is platelet aggregation absorbance of sample and Ac is platelet aggregation absorbance of the PBS control.

Gas chromatography–mass spectrometry analysis

Gas chromatography–mass spectrometry (GC-MS) analysis was performed on an Agilent 7890, GC column HP-5MS, and interfaced to a 5975C inert mass selective detector with triple-axis detector. For the experiment, 2 μl of sample was injected using a 10:1 split ratio. The injection, outlet, and MS transfer line temperatures were set to 250°C, 280°C and, 250°C, respectively. The mass spectra were recorded at 70 eV and fragments from 40 to 700 Da were analyzed.[8] Identification of the constituents was conducted by comparing their mass spectra with fragment data from the National Institute of Standards and Technology (NIST).

Statistical analysis

All values are expressed as mean and standard deviation. Descriptive statistics and paired t-test were analyzed using GraphPad Prism 6 (version 6.01, GraphPad Software, CA, USA). A value of P < 0.05 was indicated statistically significant.


  Results and Discussion Top


Platelet adhesion activity

Platelet adhesion activity was determined using the collagen-coated microtiter plate. The effect of C. sphaerica extracts with different solvents at 5.0 mg/ml on human platelets adhesion activity was determined [Figure 1]. The percentage of thrombin-stimulated platelet adhesion following the addition of distilled water, methanolic, ethanolic, and chloroform extracts was 61.98 ± 1.44, 65.23 ± 0.52, 67.4 ± 2.79, and 105.27 ± 0.11, respectively. When compared to the negative control, phosphate-buffered saline (PBS), the chloroform extract could significantly increase platelet adhesion (P < 0.05). However, the distilled water, methanolic, and ethanolic extracts significantly inhibit platelet adhesion (P < 0.001).
Figure 1: Platelet adhesion activities of different extracts of Careya sphaerica. *P < 0.05 and***P < 0.001, compared with phosphate-buffered saline

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Platelet aggregation activity

The effect of the extracts with different solvents at 5.0 mg/ml on human platelet aggregation is presented in [Figure 2]. The percentage of collagen-stimulated platelet aggregation following the addition of distilled water, methanolic, ethanolic, and chloroform extracts was 46.84 ± 6.87, 52.95 ± 0.04, 96.40 ± 2.41, and 91.60 ± 3.92, respectively. No extracts could induce the aggregation of platelets when compared to control, but the distilled water and methanolic extracts surprisingly showed significant inhibition in the collagen-induced aggregation (P < 0.001).
Figure 2: Platelet aggregation activities of different extracts of Careya sphaerica. ***P < 0.001, compared with phosphate-buffered saline

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Gas chromatography–mass spectrometry analysis

Phytochemical compounds of chloroform extract were characterized and identified using GC-MS. Retention time, name, molecular formula, molecular weight, and amount (% area) of compounds are shown in [Table 1]. The chromatogram of the C. sphaerica extract was compared with the mass spectra of the compounds of NIST version 11 library. Results of the GC-MS chromatogram analysis of C. sphaerica chloroform extract showed five main constituents, including clomesone, pyrido [2,3-d] pyrimidine, 4-phenyl-, 6-nitro-1H-quinazoline-2,4-dione, 2-chloroethyl thiocyanate, and 1H-indole, 1-methyl-2-phenyl.
Table 1: Phytocomponents identified in chloroform extract of Careya sphaerica leaves by gas chromatography–mass spectrometry

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Clomesone, the 2-chloroethyl ester of methanesulfonic acid, induces the formation of DNA interstrand crosslinks and exhibits antitumor activity.[9] 4-phenyl-pyrido [2,3-d] pyrimidine has a variety of pharmacological activities such as analgesic, antimicrobial, anti-allergic, antihypertensive, antitumor, anti-leishmanial, diuretic, anti-inflammatory, anti-tuberculostatic, anticonvulsant, potassium-sparing, and anti-aggressive properties.[10] 6-nitro-1H-quinazoline-2,4-dione is quinazoline derivatives which possess anti-inflammatory and antiviral properties.[11],[12] 2-chloroethyl thiocyanate can be used for anti-inflammatory and antimicrobial action to boost the host's defense system while reducing tissue inflammation.[13] 1-methyl-2-phenyl-1H-indole is a novel compound used for dementia disorders treatment.[14] Indole and its derivatives show pharmacological activities such as virus inhibitors and peroxisome proliferator-activated receptor gamma activators.[15],[16] We assume that bioactivities exhibited by C. sphaerica in this study are correlated to the existence of one or many of these phytochemical compounds.

Wound healing, a physiological reaction to tissue injury, comprises four main processes, including hemostasis, inflammatory phagocytic, proliferative fibroblastic, and maturation remodeling.[17] Platelets play a crucial role in hemostasis which is the process of the wound being closed by clotting to stop the loss of blood. Once platelets adhere to collagen at the subendothelium of the damaged vessel, platelets are induced to release adenosine diphosphate (ADP) and thromboxane A2 (TxA2). Releasing of ADP from platelets results in platelet aggregation and initiates the process of clot formation.[18]

C. sphaerica is a Thai traditional medicine plant locally known as Kradonbok. Its leaf is used for wound healing. Therefore, it may be useful as a therapeutic agent targeted at the hemostatic process, especially platelet function. This study shows that only the chloroform extract of C. sphaerica extract could possibly activate platelet function through induction of platelet adhesion but not platelet aggregation. This observation indicates that nonpolar phytoconstituent(s) present in the chloroform extract may get involved in platelet adhesion induction. Although the chloroform extract could not activate platelet aggregation per se, induction of platelet adhesion to collagen could trigger the secretion of platelet granule substances leading to platelet aggregation. The activated platelets could play a crucial role in wound healing because they express many mediators, including multiple cytokines and vascular endothelial growth factors, that help to promote cell recruitment, tissue regeneration, and matrix remodeling.[19] In normal wound healing, the fibrinolytic pathway also plays a crucial role by removal of fibrin removal. Fibrin deposition is a feature of nonhealing wounds and may be an important pathogenic component that prolongs hemostasis.[20] The previous study has demonstrated that the methanolic extract from C. sphaerica acted as fibrinolytic enzymes.[21] Thus, its fibrinolytic property could contribute to wound healing as well. In contrast to the chloroform extract, the distilled water, methanolic, and ethanolic extracts have shown antiplatelet activity, whereas the highest inhibitory effect was observed with the extract in the distilled water. There are many phytoconstituents present in plants such as phenolics, carotenoids, and vitamins which inhibit platelet adhesion and aggregation.[22],[23],[24]


  Conclusion Top


This study demonstrated that the chloroform extract of C. sphearica leaves could possibly stimulate platelet adhesion. Bio-active phytoconstituents from nonpolar extract of C. sphearica could potentially be used for wound healing. Isolation and characterization of the specific compound (s) in the extract demonstrating platelet adhesion activity should be further investigated. Moreover, the role of C. sphearica leaves in other wound healing processes, including inflammatory, proliferative, maturation, and remodeling phases, should be studied.

Acknowledgment

This study was supported by grant IRD60001 from Rajamangala University of Technology Thanyaburi, and grant WU60307 from Walailak University. We would like to thank Dr. Gerd Katzenmeier, School of Allied Health Sciences, Walailak University, for critical reading of the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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