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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access   Full Text Article                                                                                                                                         Research Article 

Evaluation on In Vitro Blood Clot Dissolving Potential of Aqueous Extract of Sida acuta Burm. F. Leaves

Anita Mishra*¹, Shadma Siddiqui² and Shreya Tiwari^3,4

¹ Department of Biotechnology, Vidyadayini Institute of Science, Management and Technology, Bhopal M.P. India

² Department of Paramedical Sciences, SAM Global University, Raisen, M.P. India

³ Department of Biotechnology, Shoolini University, Solan, H.P. India

⁴ Biomedical & Biopharmaceutical Division, Lenience Biotech Lab, Bhopal, M.P. India

INTRODUCTION

Blood clotting is one of the vital processestakes place in humans, animals and birds¹. Clotting of blood is a perplexing interaction of various mechanisms, where enactment of the coagulation, fibrinolytic frameworks, disruption of the vascular endothelium, and the generalized triggering of cellular mechanisms prompts to clotting on the surface of monocytes and platelets available in flow². Sometimes blood clots are considered as a concern when formed in the circulatory system due to disturbances in hemostasis resultingin vascular obstruction and causingstern consequences in thrombolytic diseases like acute myocardial or cerebral infarction which might cause demise³.Hereditary factors, primary or acquired, play a role in the development of thrombosis². In pharmacological terms the dissolution ofblood clots is regarded as thrombolysisachieved by secondary fibrinolysis by plasmin through application of tissue plasminogen activator equivalents which are plasmin activator proteins. Alteplase, anistreplase, streptokinase, urokinase and tissue plasminogen activator are available in the market as clot dissolving agents⁴. Due to certain negative and life-threatening side effectsanticoagulants currently in use scientists and are interested in finding new alternatives to these anticoagulants of natural and organic origin⁵.

Medicinal plants are the preeminentre source of drugs and pharmacological components as phytomedicine, derived from different parts of the plants⁶.

Sida acuta Burm. f (Malvaceae) is one of those plants as of now utilized by native individualsto manage health issues. This plantis an erect, small perennial herb or small shrub of about 1.5 m height with branches⁷. It grows abundantly on squander regions, cultivated fields and roadsides.

As a therapeutic all parts of the S. acuta are used, however the leaves are the most often demand.Leaves are considered to havediuretic, anthelmintic, calmative and wound healing properties, and are utilized in treating rheumatism^{8, 9}.Stomach torment, hemorrhoids, azoospermia and oligospermia are being used to treat with leaf decoctions of this plant¹⁰. While, vomiting and gastric disorders were controlled using its leaf juice¹¹.Roots are mostly used as a stomachic, diaphoretic and antipyretic in Indian traditional medicine. S. acuta also used for disorders like blood, bile, liver,nervous and urinarydiseases,while the hot water extract of whole dried plant is orally used as febrifuge, and abortifacient^{12, 13}. Sida acuta is a constituent in Siddha formulation recommended in pulmonary tuberculosis, rheumatism, facial paralysis, sciatica, haemorrhage, spermatorrhoea, leucorrhoea and gonorrhea¹⁴. Folks say this plant also have thrombolytic activity to some extent. So, the present work was aimed at phytochemical investigation andblood clot dissolving activity of aqueous extract of leaves of Sida acuta under in vitro conditions.

MATERIALS AND METHODS

Sample Collection and Processing 

The plant material as leaves of Sida acuta (Burm.) were collected from road side in Bhopal City and were authenticated bybotanical literatures and web resources. The collected leave were thoroughly washed in tap water, drained and allowed to dry at room temperature. The dried leaves were then grounded into fine powder followed by defatting with petroleum ether overnight atambient temperature.

Phytochemical Extraction and Analysis 

The defatted leaf powder of S. acuta plant was then subjected to phytochemical extraction with pure distilled water by soxhletion method in order to prepare their aqueous extract. The obtained aqueous extract was then concentrated by evaporating the solvent in a boiling water bath. The preliminary phytochemical analysis was done according to the methods described by Harborne¹⁵, Khandelwal¹⁶, and Tenguria et al.,⁶ which are described as follows;

1. Test for alkaloids: Few drops of Dragendorff’s reagent was mixed in diluted stock of extract if yields orange red precipitate demonstrates the presence of alkaloids.
2. Test for tannins: Small amount of diluted extract stockis whenwarmed and followed by addition of 2-3 drops ferric chloride solution gives a dark green colour in solution demonstrates the existence of tannins in extract.
3. Test for terpenoids: About 100 µl of stock was diluted with distilled water in a test tubeand carefully 2 ml chloroform (CHCl₃) was added to it by the side wall of test tube followed by addition of concentrated H₂SO₄ (3ml) in the same way to form a layer. The formation of a reddish brown coloration or a ring at the interface is the indicator for the presence of terpenoids in extract.
4. Test for saponins: The diluted stock of extractwhen warmed a little then had shaken vigorously. The formation of froth or bubbles that stays for 5 minutes at least indicates the presence of saponins.
5. Test for flavonoids: To the diluted stock of extract in a test tube 2-3 drops of 10% lead acetate was added. The appearance of a creamy white dirty precipitate demonstrates the presence of flavonoids in extract.
6. Test for glycosides: Benedict reagent was used to check the presence of glycosides in diluted extract. The diluted extract was first warmed they 2-3 drops of Benedicts reagent were added to the reaction tube. The appearance of yellow or orange precipitate indicates the presence of glycoside in extract.

Total Phenolic Content: The total phenolic content (TPC) of the extract was assessed through Folin-Ciocalteu method with suitable modification^{17, 18}. The suitably diluted extract was made upto 3 ml with distilled water andwas oxidized 0.5 ml of with Folin-Ciocalteu reagent, and the reaction was neutralized by addition of 2 ml of 20% sodium carbonate solution. The reaction was permitted to stand for a 60 min in the dark at room temperature, and absorbance of the resulting blue colour was measured at 650 nm. The TPC was evolved from the calibration curve of gallic acid (using concentrations 0-2 mg/ml), and the outcomes were stated as mg of gallic acid equivalent per g dry weight was expressed as mg GA equivalent/L of extract.

In vitro Clot Dissolving Activity

 In vitro experiment was thrombolytic activity was planned for present investigation according to the methods suggested by Sweta et al., (2007), Fatema et al., (2017) and Alawa, et al., (2018) with suitable modification as per present study^{19, 4, 5}.The goat blood samples were collected from local slaughter house and 0.5 ml of poured into 5 different 1.5 ml microfuge and incubated at 37°C for 3 hours to allow the clot formation followed by removal of serum carefully and weight of the clot was measured. Three serial dilutions were made from the 100 mg/ml stock solution ofaqueous extract of S. acuta was. With the help of micropipette, 100 μl of aqueous extract from each dilution wasadded in each corresponding separated microfuge touching the surface of blood clot. Streptokinase and sterile distilled water were used in each separate microfuge withblood clot was used as positive and negative control respectively. The set of experiment was incubated at 37 °C for 2 hours followed by observation in vitro thrombolytic activity. The liquid released after incubation was drained and residual clot was washed with sterile distilled water and moisture was removed by drying. Final weights of microfuge were taken to determine the percentage thrombolytic activity of extract compared to standard through following formulae;  

RESULTS AND DISCUSSION

Preliminary Phytochemical Analysis 

The results of phytochemical analysis of aqueous extract of S. acuta leaves are depicted in table 1 which indicates the aqueous extract is rich in phytoconstituents like alkaloids, flavonoids, tannins, terpenoids and glycosides. Though saponins were reported to be absent in aqueous extracts.

Table 1: Phytochemical Analysis of Sidaacuta (Burm) leaf aqueous extracts

(+) means present, & (-) means absent

In an earlier investigation Senthil kumar et al., (2018) also reported more or less similar phytochemical profile for aqueous extract of Sidaacuta leaves²⁰. Various phytoconstituents chiefly polyphenols and flavonoids are responsible for the biological and pharmacological activity of medicinal plants^{18, 6}.

Estimation Total Phenolic Content 

In present investigation 20 times diluted 100 mg/ml stock solution of aqueous extract of S. acuta leaves gave an absorbance reading of 0.830 in reaction mixture in digital micro processed spectrophotometer (Electronic India model EI-2305) when subjected to estimation of phenolic content through calibration curve of standard plot with phenol equivalent to gallic acid (table 2 and figure 1) indicates the presence of 17.48 mg/100 mg aqueous extract of S. acuta leaves or simply the total phenolic content in aqueous extract of S. acuta leaves was reported to be 17.48 %.Most of the earlier investigation reported TPC in extracts other than aqueous extracts. Muneeswari et al., (2019) reported 31±0.15 mg/g TPC in ethanolic extract of S. acuta leaves²¹.The presence of tannins and phenolics are responsible of the anti-inflammatory, antimicrobial and free radical scavenging property of any medicinal plant or their extracts^{22, 23, 24}. 

Table 2: Gallic acid as standard concentration vs absorbance at 650 nm to plot standard curve for estimation of phenolics in samples Using Folin-Coeucaltue’s Method.

Instrument Used

Single beam visible range digital micro processed spectrophotometer from Electronic India model EI-2305.

Figure 1: Standard Plot for known concentration of Gallic acid Standardat 650 nm. The Graph is obtained from Excel 2013 linear regression function

In vitro Clot Dissolving Activity 

The in vitro clot dissolving activity of aqueous extract of test plant considered in present study indicates theencouragingoutcomes in prospect of developing of new therapeutic substances. There were 3 different concentrations of extracts used whose percentage blood clot dissolving activity are illustrated in table 3 compared to the standard streptokinase.

Table 3: In vitro clot dissolving activity of aqueous extract of S. acuta leavescompared to gel containing Streptokinase

In present investigation the in vitrothrombolytic activity of aqueous leaf extract of S. acuta at a concentration of 10 mg/100µl solution is reported to be 41 % which reduces to 34 % and 12 % when 5.0 mg/100µl and 2.5 5.0 mg/100µl extract solution were used respectively. Streptokinase at a concentration of 0.1 mg/ml generally produces a thrombolytic activity in range from 80 to 95 % ^{25, 26}. In present study, Streptokinase displayed an activity of 73% as positive control at similar concentration that is optimum. The activity of varied concentration of aqueous extract of S. acuta leaves and streptokinase, where the dissolution of blood clot due to extracts is considerable (Figure 2).

In terms of blood related studies, Eze, and Nwodo, (2016) reported the significant inhibition of in-vitro haemolysis while investigating the potentiality of membrane stability; platelet aggregation and activities of phospholipase A₂and prostaglandin in ethanol extract leaves of Sida acuta²⁷. While working on methanolic extract S. acuta leaves, Bahar, et al., (2013) reported 24.786 % of thrombolytic activity using 100 µl of 1 mg/ml and 0.5 mg/ml concentration²⁸.Heavy external blood clots due to any tissue tear or rupture or large accidental wound development is also a problem sometimes when causing painful bandage.  Though in present study the thrombolytic activity is reported at higher concentrations, but it is easy and cost effective to obtain the aqueous extracts of any plant material.

Figure 2: Graphical representation of in vitro blood clot dissolving potential of S. acuta aqueous leaf extractand standard streptokinase.

CONCLUSION

The outcomes of present investigation indicates that the aqueous extract of S. acuta leaves is significantly rich in phenolic contents and variety of phytochemical constituents that could be explored for pharmacological properties in order to develop new drug for the cure of variety of pathological conditions in humans and animals. In present investigation the aqueous extract of S. acuta leaves were observed to be promising sources of thrombolytic drug under in vitro experiments. After this preliminary study, further extensive and stringent studies related to their cytotoxic & genotoxic studies, and physiological compatibility and in vivo pharmacological studies, the thrombolytic property of aqueous extract of S. acuta leaves could be exploited in development of new biopharmaceutical and therapeutic agents which could be easily accessible, cost effective and probably safe due to their natural origin. 

ACKNOWLEDGEMENT

The authors are thankful to Mr. Mayank Tenguria (Director & Scientist) from Lenience Biotech Lab for designing research work, providing necessary funding and laboratory facility to pursue our study.

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