Journal of Thrombosis and Circulation: Open Access

Comparative Evaluation of In Vitro Thrombolytic Activity of Four Medicinal Plants

Naga Bharathi M^*, Devi Sirisha G, Manoj kumar M, Sai Kanaka durga L, Harshitha K, Satyasree D and Srinivas K Department of Pharmacology, Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Tadepalligudem, Andhra Pradesh, India
^*Correspondence: Naga Bharathi M, Department of Pharmacology, Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Tadepalligudem, Andhra Pradesh, India, Email:

Received: 14-Jun-2022, Manuscript No. JTCOA-22-001-PreQC-22; Editor assigned: 17-Jun-2022, Pre QC No. JTCOA-22-001-PreQC-22(PQ); Reviewed: 01-Jul-2022, QC No. JTCOA-22-001-PreQC-22; Revised: 08-Jul-2022, Manuscript No. JTCOA-22-001-PreQC-22(R); Published: 18-Jul-2022, DOI: 10.35248/2572-9462.22.8.196

Introduction

Thrombus or embolus hampers the blood flow in the blood vessels therefore tissue lack normal blood flow and oxygen. Lack of oxygen causes necrosis of particular area which may result in complication like Myocardial infarction. Thrombolytic drugs used for dissolution of clot, which include tissue Plasminogen Activator (t-PA), urokinase, streptokinase etc. act by activating plasminogen which can cause lysis fibrin. All available thrombolytic agents have significant limitations like fewer outcomes, lack of specificity and efficiency at low doses and on other hand if given in large doses their safety is questionable and bleeding tendency at times leading to death [1]. Drugs based on herbs have become a common form of therapy due to less adverse effects. Today, they are one of the promising agents in the field of herbal therapeutics [2] Phyllanthus niruri is a widespread tropical plant commonly found in coastal areas, known by the common names gale of the wind, stonebreaker. It belongs to the Phyllanthus genus of Family Phyllanthace,it is an important plant of Indian Ayurvedic system of medicine in which it is used for problems of the stomach, genitourinary system, liver, kidney and spleen [3,4] . Syzigium cumini is also known as java pulp, jamun, jambal, Indian black pulp. It belongs to family Myrtaceae; traditionally the jambul fruits, leaves, seeds, and bark are all used in ayurvedic medicine. The bark contains tannins and carbohydrates, accounting for its long-term use as an astringent to combat ailments like dysentery and seeds used for diabetes, inflammation [5].

Nyctanthes arbor-tristis is known as Night- flowering Jasmine. The leaves have been used in Ayurvedic medicine and Homoeopathy for sciatica, arthritis, fevers, and as a laxative. Its leaves are also blend into a paste and used in skin related troubles, especially ringworm. The fresh leaves are boiled in mustard oil and used externally for treating ringworm. The leaf juice is mixed with common salt to treat intestinal worms. Coral jasmine is used in the treatment of fungal skin infection, dry cough. [6-8]. Cleome gynandra is used as a medicinal plant and can be found in all over world. It belongs to the family of Capparaceae, it also known as wild spider flower, African spider flower, cat whiskers, and Bastard mustard. Various parts of the plant are used as a folk therapy for various activities like free radical scavangic activity, Anti-cancer, Immuno modulatory effect, Anti-microbial, Anti-diabetic, Anti-diarrhoeal, Anthelmintic, Anti-urolithiatic effect [9].

Materials and Methods

Collection of plant materials

Leaves of Phyllanthusniruri, Nyctant hesarbor-tristis, Syzygium cumini and Clome gynandra were collected from Eluru and Tadepalligudem, during the month of February 2016. The above plants were authentificated by botany department of Dr. Rajeshekar Reddy horticulture university Leaves were collected from the plants by hand pricking method and were washed with clean water to discard dirt materials. They were dried under shade for 4-5 days. After the leaves are dried they were ground into coarse powder and preserved in a closed container at 25°C for further use.

Preparation of extracts of plant material

The extraction was performed using cold maceration technique. 25 gms powder of each plant was weighed and soaked in 250 ml of methanol and 250 ml of water separately for about 3 days at room temperature with occasional stirring. After 3 days the solution was filtered through filter cloth followed by Whatman’s filter paper and the filtrate thus obtained was collected into a beaker. This filtrate was concentrated by heating at a temperature of 60ᵒC-70ᵒC. The filtrate was completely dried; flakes of the extract were obtained. These were collected into amber colored container and preserved.

Preparation of standard (streptokinase) solution

To the commercially available lyophilized SK vial (Thromboflux) 15,00,000 I.U. used as a standard which was collected from Bharat serum and vaccines Ltd, Ambernath (Figure 1). 5 ml sterile distilled water was added to streptokinase vial and mixed properly. From this suspension 100 μl (30,000 I.U) was used for in vitro thrombolytic activity [10].

Figure 1: Microcentrifuge tubes.

Preparation of extract for thrombolytic test

The thrombolytic activities of all methanolic and aqueous leaf extracts were evaluated by a method using Streptokinase (SK) as a reference standard. 100 mg of methanolic extract and 100 mg of aqueous extract of all plants were dissolved separately in 10 ml of methanol and distilled water respectively (Figure 2). They were kept overnight. Then the solution were filtered and further used for study [11].

Figure 2: Micro centrifuge tube with blood clot.

Specimen

4 ml venous blood was collected from healthy human volunteers (n=4) by maintaining aseptic condition without a history of oral contraceptive or anticoagulant therapy. 0.5 ml of blood was blood was transferred to each tube according to protocol accepted by institutional committee of Sri Vasavi institute of pharmaceutical sciences.

Thrombolytic activity procedure

Micro centrifuge tubes were taken and empty weight of each tube was noted. Note it as W1. (W1=empty weight of micro centrifuge tube). Then 0.5 ml of blood was transferred to each tube and it was incubated at 37°C for 45 minutes for clot formation. After clot formation, fluids were completely released from each micro centrifuge tubes and weigh the tubes along with the clot formed. Note it as W2. (W2= weight of micro centrifuge tube with clot – empty weight). As a positive control, 100 μl of streptokinase (SK) was used and as a negative control 100 μl of distilled water was used. 100 μl of each samples (methanolic and aqueous) were separately added to the micro centrifuge tubes containing clot (Figure 3). All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, the released fluid was discarded and tubes were again weighed to observe the difference in weight after clot disruption, Note it as W3. Finally percentage of clot lysis was determined as followings: Wt of released clot = (W2-W3), Clot weight = (W2-W1). Therefore (W3-W2) / (W2-W2) × 100 gives us % clot lysis. % of clot lysis = (released clot /clot wt) × 100 [11].

Figure 3: Comparative thrombolytic activity.

Statistical analysis

All values are expressed as average % clot lysis for three replicates.

Results and Discussion

A number of studies conducted earlier by different researchers reveals that plants also possess thrombolytic active principles. A study was done using Boganvellia spectata leaves contain high content of phytochemical constituents like flavanoids , alkaloids , terpiniods which are responsible for thrombolytic potential. The present study was carried out to investigate thrombolytic activity of Ncyranthus arbortritis, clome gynandra , phyllanthus niruri, syzyuim cumini , where Streptokinase (SK), a known thrombolytic drug is used as a positive control [9]. Water, on the other hand, was used as a negative control. Among the four medicinal plants Nyctanthes arbor- tristis(aqueous) extract exhibited highest thrombolytic activity with 14.2% of clot lysis , followed by Cleome gynandra(aqueous) extract with 7.7% of clot lysis and Phyllanthus niruri(methanol) extract with 6.7% clot lysis when compared with Standard streptokinase showed clot lysis of 22.3% while the negative control water 2.2% lysis of clot. By comparising the results of positive control with negative control clearly confirmed that clot dissolution does not occur when water was added to the clot. All the above plants contain flavonids, alkaloids and terpinoids as phytochemical constituents hence these plants also possess antioxidant property (Table 1). This might be a reason for their thrombolytic potential.phytochemical constituents like flavonids, phenols are rich in Nyctanthes arbor- tristis, when compared with other plants, and thus it exhibited the highest thrombolytic potential.

Table 1: Effect of different plant extracts on in vitro thrombolytic activity.

Conclusion

In conclusion from our results, it can be demonstrated that our findings may help scientific community in developing novel thrombolytic compounds with less side effects from Ncyranthus arbortritis, clome gynandra, phyllanthus niruri, syzyuim cumini extracts. Further studies are necessary to isolate and characterize the compounds and to explore the possible mechanism of action for thrombolytic activity. The present study was aimed to investigate in vitro thrombolytic activity of methanolic and aquoes extract of Phyllanthus niruri, Nyctanthes arbor- tristis, Syzygium cumini and Clome gynandra, where streptokinase and water were used as positive and negative control.

Acknowledgements

The authors wish to thank Dr. K. Srinivas prinicipal of sri vasavi institute of pharmaceutical sciences for providing facilities.

Conflicts of Interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

REFERENCES

1. Collen D. Coronary thrombolysis: Streptokinase or recombinant tissue-type plasminogen activator. Ann Intern Med. 1990; 112(7):529-538.

   [Crossref], [Google Scholar], [Pubmed]

2. Marder VJ. Recombinant streptokinase: Opportunity for an improved agent. Blood Coagul Fibrinolysis. 1993; 4(6):1039-1040.

   [Google Scholar], [Pubmed]

3. Calixto JB, Santos AR, Filho VC, Yunes RA. A review of the plants of the genus Phyllanthus: Their chemistry, pharmacology, and therapeutic potential. Med Research Rev. 1998; 18(4):225-258.

   [Google Scholar], [Pubmed]

4. Thyagarajan. Phyllanthus Species: Scientific evaluation and medicinal. Applications. 1987.

5. Shweta S. A review on pharmacological activity of syzygium cumini extracts using different solvent and their effective doses. Int Res J Pharm. 2012; 3(12):25-29.

   [Google Scholar]

6. Saxena RS, Gupta B, Saxena KK, Srivastava VK, Prasad DN. Analgesic, antipyretic and ulcerogenic activity of nyctanthes arbor tristis leaf extract. J Ethnopharmacol. 1987; 19(2):193-200.

   [Crossref], [Google Scholar], [Pubmed]

7. Sodipo OA, Akanji MA, Kolawole FB, Odutuga AA. Saponin is the active antifungal principle in garcinia kola, heckle seed. Biosci Res Commun. 1991; 3:171.

   [Google Scholar]

8. Simonsen HT, Nordskjold JB, Smitt UW, Nyman U, Palpu P, Joshi P et al. In vitro screening of Indian medicinal plants for antiplasmodial activity. J Ethnopharmacol. 2001; 74(2):195-204.

   [Crossref], [Google Scholar], [Pubmed]

9. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb J. 2006; 4(1):1-4.

   [Crossref], [Google Scholar], [Pubmed]

10. Ramjan A, Hossain M, Runa JF, Md H, Mahmodul I. Evaluation of thrombolytic potential of three medicinal plants available in Bangladesh, as a potent source of thrombolytic compounds. Avicenna J Phytomed. 2014; 4(6):430.

    [Google Scholar], [Pubmed]

11. Wazir A, Sherwani SK, Ahmad M. Antibacterial, antifungal and antioxidant activities of some medicinal plants. Pak J Pharm Sci. 2014; 27(6).

    [Crossref], [Google Scholar], [Pubmed]