INTRODUCTION:

Cardiovascular disease like myocardial or cerebral infarction due to the blood clot (thrombus) formation in blood vessels.¹ most commonly used thrombolytic agents like: streptokinase and urokinasebut hyper risk of haemorrhage and anaphylactic reaction associated with their use.²Additionally, these agents are biotechnological products and cost of therapy is usually high.

Everyday new drugs are being discovered in the market. Medicinal plants possess variety of phytoconstituents and have significant importance in the field of biotechnology. India has been known to be well-off repository of medicinal plants since early time.³ Use of herbal drugs is common as an alternative medicine; these phytoconstituents are prominent and promising pharmacological agents with good safety profile.

Prunus avium L. also known as a “sweet cherry” is used in the Ayurvedic systems of medicine. Fruits of Prunus avium L. is used in cardiovascular, stroke, diabetes, and some cancers diseases.⁴ Discovery and development of natural products from various plant and animal sources which have large efforts and have antiplatelet,^5,6 anticoagulant,^7,8 antithrombotic,^9,10and thrombolytic activity.^10,11,12 The current research work was designed to evaluate the fruits of Prunus avium L. for their clot lysis property (thrombolytic activity) by using an in-vitro procedure.

MATERIALS AND METHODS:

Plant collection and Authentication:

Plant collection:

Fruits of Prunus avium L. (sweet cherry) were collected from local market of Rajkot

Authentication:

Authentication of fruits was done at Department of botany, School of Science R.K. University, Rajkot.

Preparation of plant extract:

Fruits were air-dried in an oven the constant weight at 45˚C.¹⁴ 1gm of sample were weight and extracted consecutively with 40ml of methanol/water (50:50, v/v) for 1hr at room temperature. Centrifuged the sample at 25,400rpm for 15 min and supernatant was received. After that 40ml acetone/water (70:30, v/v) was added into the residue at room temperature and further for 60 min samples were extracted and centrifuged then supernant was recovered. Both methanolic and acetonic supernants were mix and evaporate to dryness of solvents.¹⁵

In-vitro study of clot lysis activity of thrombolytic drugs

Procedure:

Human blood (10ml) was collected from the blood blank. In previously weighed sterile centrifuge tube 500 μl of blood was transferred.

Streptokinase (SK):

To the commercially available lyophilized streptokinase vial (15,00,000 I.U.). 5ml phosphate buffered saline solution was added and mixed accurately and this solution was used as a stock from which 100μl (30,000 I.U.) was used for in-vitro thrombolytic assay.¹⁶

Preparation of test sample (extracts):

The methanolic extracts were dissolved in DMSO respectively to prepare different concentrations (50, 25, 10 and 5μg/ml respectively)¹⁷ of the test samples. 100μl of the methanolic extracts of the plant was added to the microcentrifuge tube containing the clots to check thrombolytic activity.

Phytochemical Screening:

The extract was tested for the presence of flavonoids, saponins, alkaloids, tannins, phenols, carbohydrate, and glycoside etc.¹⁸

Study design:

All micro centrifuge tubes were sterile and weighted. 0.5 ml of human blood was transferred to different micro-centrifuge tubes then incubated all the tubes at 37°C for 45 minutes. After the clot formation, serum was totally removed without disturbing the clot formation and clot weight was determined. Clot weight = Weight of tube containing clot -weight of empty tube. Properly labeled the all the micro centrifuge tubes and 100μl of the methanolic extracts were added at various concentration (5μg/ml, 10μg/ml, 25μg/ml and 50μg/ml) respectively. 100μl of 15,00,000 I.U Streptokinase was used as standard control. 100μl of DMSO used as normal control. Micro centrifuge tubes were then incubated yet again at 37°C for 90 min. After incubation, fluid was totally removed from all the tubes and tubes were re-weighed for the determination of the difference in weight after clot disruption. % clot lysis was calculated through the difference between before and after clot weight. Triplication of assay was done. The results of thrombolytic assay are expressed as % clot lysis.

Table 1: Protocol of thrombolytic activity

Sr No	Groups	Treatment
1	Normal control	100μl DMSO
2	Standard control	100μl Streptokinase
3	MPA-1 (5µg/ml)	5μl
4	MPA-2 (10µg/ml)	10μl
5	MPA-3 (25µg/ml)	25μl
6	MPA-4 (50µg/ml)	50μl

MPA: Methanolic extract of Prunus avium L.

Statistical analysis:

All data are representing as mean ± SEM. The mean % clot lysis of streptokinase with different concentrations of MPA was compared with DMSO using one way analysis of variance (ANOVA) test by Graph Pad Prism version 8.4.2. p value ≤ 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION:

Plant extraction:

Extraction of dry powder of Prunus avium L. in methanol/water and acetone/water was performed by centrifuge method and then dryness to evaporate the solvent up to solid residue was obtained. The extract was obtained in dark brown color and semisolid nature. % yield of methanolic extract of 1 gm of dried powder of Prunus avium L. was found to be 19.6% w/w by centrifugal extraction method.

Phytochemical screening:

Following phytochemicals test were performed in methanolic extract of Prunus avium L. fruit as shown in Table 2.

Table 2: Preliminary phytochemical screening of methanolic extract of Prunus avium L. fruit

Sr No	Phytochemicals	Chemical test	Result
1	Flavanoids	Alkaline reagent test	+ +
2	Saponins	Form test	_
3	Alkaloid	Dragendroff’s test	+ +
4	Tannin	Ferric chloride test	_
6	Carbohydrate	Molisch’s test	+
7	Glycoside	Legal test	+

Methanolic extract of the Prunus avium L. Fruit shows the presence of flavoinoid, alkaloid, carbohydrate and glycoside in the sample and absence of tannin and saponin in the sample. Present study was considered on the basis of presence of phytochemicals like; Flavonoids: rutin, catechin, epicatechin, quercetin 3-glucoside, quercetin 3-rutinoside, and kaempferol 3-rutinoside.¹⁹ Rutin reported as potent natural thrombolytic agent.²⁰ Prunus avium L. Prunus avium L. fruit is rich source of Flavanoids, such as rutin. In-vitro thrombolytic activity of MPA may be because of presence of phytoconstituents like rutin.

In-vitro study of thrombolytic activity of Prunus avium L. by used streptokinase as standard:

Methanolic extract of Prunus avium L. significantly gives clot lysis activity.

Table 3: Effect of MPA various concentration on in- vitro clot lysis activity

Sr No	Groups	Mean ±SEM (% clot lysis) at 90 min
1	Normal control (DMSO)	5.4±1.7
2	Standard control (Streptokinase)	84.6±2.1
3	MPA (5μg/ml)	25.6±2.4
4	MPA (10μg/ml)	32.5±2.6
5	MPA (25μg/ml)	54.6±2.8
6	MPA (50μg/ml)	59.7±2.8

Figure 1: Effect of MPA on in vitro clot lysis activity

All values are expressed as mean± SEM (N=3) for each group.

*indicates significant difference from normal control at p<0.05

In in-vitro clot lysis study, addition of 100μl streptokinase (30,000 I.U.) This was considered as positive control. Negetive control (Clots treated with 100 μl DMSO) showed only negligible clot lysis i.e. 5.4%. The mean difference in % clot lysis between positive and negative control was very significant (p value <0.0001).

The in-vitro thrombolytic activity of Prunus avium L. different concentration (5μg/ml, 10μg/ml, 25μg/ml, and 50μg/ml) showed 25.6%, 32.5%, 54.6%, 59.7% respectively compared with negative control DMSO (5.4%). Methanolic extract of Prunus avium L. in a dose dependent manner was shown to possess thrombolytic activity in in-vitro assessment. % clot lysis was found to higher as compared to negative control sample in all concentration. MPA (25µg) and MPA (50µg) were found to have statistically significant difference (p<0.05) as compared to negative control (Table 1, Figure 1).

Statistical representation of the effective % clot lysis by herbal preparations (Prunus avium L. fruits), positive control (Streptokinase) and negative control (DMSO) is shown in Table 3. Streptokinase is reported to gives % clot lysis activity 86.2% and in present study we found that % clot lysis of streptokinase is 84.6%, which is near to reported result. The extracts of Prunus avium L. possess significant thrombolytic activity in in-vitro clot lysis activity in human blood. Rutin reported as potent natural thrombolytic agent. Prunus avium L. also reported to have rutin flavonoid in their phytoconstituents. So, we were performed; an in-vitro thrombolytic model was used to check the clot lysis Effect of methanolic extract of Prunus avium L. along with streptokinase as a positive control and DMSO as a negative control.

CONCLUSION:

Using an in-vitro thrombolytic model; methanolic extract of Prunus avium L different doses (50μg, 25μg, 10μg and 5μg) showed moderate clot lysis activity respectively (59.7%, 54.6%, 32.5%, 25.6%) with reference to DMSO (10.6%) and streptokinase (88.7%). These herbal extracts possess thrombolytic properties that could lyse blood clots in-vitro; however, in vivo clot dissolving properties and active component(s) of these extracts for clot lysis are yet to be discovered. Further Pre-clinical and Clinical studies are warranted to establise use of Prunus avium L. in thrombolytic activity.

ACKNOWLEDGEMENT:

The authors wish to thank to the B. K. Mody Govt. Pharmacy College for laboratory facility for this research work.

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Received on 21.06.2020 Modified on 25.07.2020

Asian J. Res. Pharm. Sci. 2021; 11(1):41-44.

DOI: 10.5958/2231-5659.2021.00007.2