The whole plant of Asparagus racemosus was collected from Pabna and identified by taxonomist of National Herbarium, Bangladesh situated in Mirpur in Dhaka. The sample is preserved in the Phytochemical Laboratory of the World University of Bangladesh for further reference, Fig. (1).

Streptokinase vial was commercially purchased from (Trade name-S-Kinase from Popular pharmaceutical Ltd.) and Ciprofloxacin disk from Biotech Ltd. (India). Phosphate buffer, sodium hydroxide, hydrochloric acid, sodium carbonate, solvents (Ethanol, Pet-ether, Carbon tetrachloride, Chloroform) and also other necessary chemicals and reagents were of analytical grade.

The collected plants were shade dried for several days and dehydrated at 37°C overnight to aid crushing. The powders (200 gm) were soaked in 2000 ml of 90% ethyl alcohol. The filtrate was condensed by a vacuum rotary evaporator at 40°C. Concentrated aqueous ethanol extractive was fractionated by Kupchan protocol et al. protocol [4Kupchan SM, Britton RW, Ziegler MF, Sigel CW. Bruceantin, a new potent antileukemic simaroubolide from Brucea antidysenterica. J Org Chem 1973; 38(1): 178-9.
[http://dx.doi.org/10.1021/jo00941a049] [PMID: 4682660] ] and modified by Vanwagnen et al. method [5VanWagenen BC, Larsen R, Cardellina JH, Randazzo D, Lidert ZC, Swithenbank C. Ulosantoin, a potent insecticide from the sponge Ulosa ruetzleri. J Org 1993; 58(2): 335-7.
[http://dx.doi.org/10.1021/jo00054a013] ] to yield petroleum ether (PSF), carbon tetrachloride (CTSF), chloroform (CSF), and aqueous (AQSF) soluble fractions.

The freshly prepared crude extract was qualitatively tested for the presence of various phytochemicals. These were identified by characteristic color changes using standard procedures, and the previously described standard method [6Sofowora A. Medicinal Plants and Traditional Medicinal in Africa. 2nd Ed. Sunshine House, Ibadan, Nigeria: Spectrum Books Ltd. Screening Plants for Bioactive Agents, 1993; 134-56.].

Commercially available lyophilized Alteplase (Streptokinase) vial of 15, 00,000 I.U, was collected and 5 ml 0.9% NaCl was added and mixed properly. This suspension was used as a stock from which 100 µl (30,000 I.U) was used for in vitro thrombolysis.

The thrombolytic activity of the prepared extracts was evaluated by the method of Daginawala, where streptokinase (SK) was used as a standard drug [7Laboni FR, Karim S, Uddin J, Labu ZK. Bioactivities and chemical profiling of sesbania grandiflora (L.) poir. leaves growing in Bangladesh. Dhaka University J Pharm Sci 2017; 15(2): 173-6.
[http://dx.doi.org/10.3329/dujps.v15i2.30932] , 8Prasad 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): 14.
[http://dx.doi.org/10.1186/1477-9560-4-14] [PMID: 16968529] ]. Venous blood (5 ml) was drawn from healthy volunteers, and transferred in different pre-weighed sterile tubes (1 ml/tube) to form clots. Then it was incubated at 37°C for 45 minutes. After clot formation, the serum was completely removed without disturbing the clot formed. As a positive control, 100 μl of SK and 100 μl of isotonic (a negative non thrombolytic control) solution were separately added to two different clot containing tubes. All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, the released fluid was fully removed and vials were again weighed carefully to observe the difference in weight after clot disruption. Differences obtained in weight noted before and after clot lysis were expressed as percentage of clot lysis. The significance of the percentages of clot lysis of crude extract of weight difference was tested by the paired t-test analysis. Then statistical analysis was performed with the results of percentage of clot lysis by different fractionates, streptokinase and sterile water, where p value < 0.01 was considered statistically significant.

The membrane stabilizing activity of the extract was evaluated by their ability to inhibit the lysis of human red blood cells, provoked osmotically by hypotonic solution or heat by the method developed by Omale [9Omale J, Okafor PN. Comparative antioxidant capacity, membrane stabilization, polyphenol composition and cytotoxicity of the leaf and stem of Cissus multistriata. Afr J Biotechnol 2008; 7(17)].

Stock erythrocyte (RBC) suspension (0.5 ml) was prepared by mixing with 5 ml hypotonic solution (50 mM NaCl in 10 mM sodium phosphate buffered saline, pH 7.4), crude ethanolic extract (CEE) and their fractions (2 mg/ml each) and acetyl salicylic acid (0.1 mg/ml) was taken in different centrifuge tubes. The reference standard was used as the acetyl salicylic acid. In the Centrifuge Machine, the mixtures were centrifuged for 10 min at 3000 rpm, and incubated for 10 min at 25°C. The absorbance of supernatant part of the mixture was measured at 540 nm using UV/Visible spectrophotometer.

Two duplicate sets of centrifuge tubes were taken containing 2 mg/ml of crude ethanolic extract (CEE) of A. racemosus and its fractions (PSF, CSF, CTSF, and AQSF) solution and 5 ml of buffered isotonic NaCl solution. ASA at a concentration of 0.1 mg/ml for positive control and negative control containing only 5 ml of the isotonic buffered solution was maintained. By inversion, erythrocyte suspension (30 µl) was added to all the tubes and mixed gently. First, one set of tubes was incubated in water bath at 54°C for 20 min and followed by the other set of tubes kept in an ice bath (0-5°C). The samples were centrifuged for 10 min at 3000 rpm at the end of the incubation period and the absorbance at 540 nm of the supernatant was measured [10Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf MN. Membrane stabilizing activity-a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoterapia 1999; 70(3): 251-7.
[http://dx.doi.org/10.1016/S0367-326X(99)00030-1] , 11Uddin J, Julie AS, Ali MH, Islam MN, Khan SA, Labu ZK. Antimicrobial, thrombolytic, membrane stabilizing activities and total flavonoid content of various partitionates of aerial parts of Eclipta alba (L.) Hassk. Dhaka University J Pharm Sci 2016; 14(2): 207-13.
[http://dx.doi.org/10.3329/dujps.v14i2.28512] ].

The crude ethanolic extract and their fractions were screened for antimicrobial activity in comparison with the standard antibiotic Ciprofloxacin (5 µg/disc) in vitro by disc diffusion method using known bacterial strains [12Rahman T, Karim S, Labu ZK. Quantification of total tannin, flavonoid contents and pharmacognostic study of the seeds of Swietenia mahagoni (Linn.). J Coast Life Med 2016; 4(12): 961-4.
[http://dx.doi.org/10.12980/jclm.4.2016J6-221] ]. About 10 µl of crude extract and its fractionates were added in the paper discs, then the dried paper disc with concentration of 400 µg/mL plant extract was placed aseptically on the organism containing agar surface. It was then kept at 4°C overnight followed by incubation at 37°C for 16 h to promote bacterial growth.

The total phenol contents were determined by using Folin-Ciocalteu reagent followed by the standard method [13Dewanto V, Wu X, Adom KK, Liu RH. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem 2002; 50(10): 3010-4.
[http://dx.doi.org/10.1021/jf0115589] [PMID: 11982434] , 14Škerget M, Kotnik P, Hadolin M, Hraš AR, Simonič M, Knez Ž. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem 2005; 89(2): 191-8.
[http://dx.doi.org/10.1016/j.foodchem.2004.02.025] ]. In a concise manner, 0.3 ml of each extractive was taken in individual volumetric flask (10 ml) containing 2.7 ml of Folin-Coicalteu (1: 10) phenol mixture. After 5 minutes, 2 ml of 7.5% sodium carbonate solution was effused to each test tube and shaken and then kept for 30 min in the dark place after warming at 45°C. A standard calibration curve was prepared by a series of solutions of gallic acid. Absorbance for samples and standard solutions were determined at 725 nm with an UV/Visible spectrophotometer against the blank. The total phenolic contents of extractives were determined from extrapolation of gallic acid standardized curve.

To conduct free radical scavenging activity for antioxidant assay, free radical 2, 2-diphenyl-1- picryl-hydrazyl-hydrate (DPPH) was used. In brief, the reaction mixture consisted of 2 mL of CEE and its extractives at a concentration of 5–500 μg/mL were added to 3 mL of 0.02 mg/mL (20 µg/mL) methanolic solution of DPPH followed by thorough vortexing of mixture which was incubated for 30 min in the dark place at room temperature. Some changes were observed in the color, suggesting that the higher the decolourization, the higher the reducing ability (from deep violet to light yellow). Following this, absorbance was measured against a blank (without sample) at 517 nm using UV/Visible spectrophotometer. Low absorbance of the reaction mixture indicated high free radical scavenging activity [15Karim S, Uddin J, Laboni FR, Labu ZK. Qualitative and quantitative biological analysis of leaves of Sesbania grandiflora. J Coast Life Med 2017; 5(3): 104-8.
[http://dx.doi.org/10.12980/jclm.5.2017J6-245] ]. The results were compared with the percentage of inhibition at various concentration of positive ascorbic acid.

Three replicates of each sample were used for each test to facilitate the statistical analysis and the data are presented as mean ± standard deviation (SD).

The qualitative phytochemical screenings revealed the presence of alkaloids, glycosides, steroids, flavonoids, tannins, proteins, resins, quinones, phenols and saponins.

The extractives of plant A. racemosus were evaluated for thrombolytic activity, using a positive control Streptokinase (SK) that showed 65.16 ± 0.16% lysis of clot wherever a negative control sterile distilled water and exhibited a negligible percent of lysis of clot (8.20 ± 0.16%). The percentages of clot lysis of different extracts solution were observed in the following order, CTSF (41.48±0.43%), CSF (38.25±0.20%), CEE (36.24±0.10%), PSF (25.45±0.48%), and AQSF (17.74±0.25%). So, it was observed that significant (p value < 0.001) percentage of thrombolytic activity was exhibited by all extractives of A. racemosus Table (1). Thrombolytic agent like SK is used as a drug as it breaks the already formed thrombus (blood clot) in clinical settings where ischemia may be fatal [16Okoli CO, Akah PA, Onuoha NJ, Okoye TC, Nwoye AC, Nworu CS. Acanthus montanus: an experimental evaluation of the antimicrobial, anti-inflammatory and immunological properties of a traditional remedy for furuncles. BMC Complement Altern Med 2008; 8(1): 27.
[http://dx.doi.org/10.1186/1472-6882-8-27] [PMID: 18538006] ]. This study showed that the thrombolytic activities of our test samples (CTSF, CSF, CEE, PSF, and AQSF) revealed positive results in comparison to the positive and negative controls. Thus all the results obtained in the study clearly indicated that A. racemosus extract is a remedy of possible clot lysis agent.

Table 1
Membrane stabilizing and thrombolytic activity of crude extracts and different fractions of plant A. racemosus.

The extractives of A. racemosus significantly protected the hemolysis of human erythrocytes membrane induced by hypotonic-solution and heat as compared to the standard ASA. The membrane stabilizing activity against hypotonic solution induced stabilization obtained in the following order ASA (80.99±0.39%), CSF (80.86±0.36%), CEE (79.08±0.47%), AQSF (75.78±0.40%), PSF (74.56±0.18%) and CTSF (73.90±0.70%). In heat-induced method, the values of ASA, AQSF, PSF, CEE and CSF were (85.68±0.28%), (84.68±0.48%), (78.28±0.32%), (78.28±0.11%) and (72.05±0.39%), respectively. The presence of significant amount of phenolic compounds in the extract of A. racemosus revealed that the plant have potential to inhibit hemolysis of RBCs induced by hypotonic solution and heat induced (Table 1).

CEE and different fractions of A. racemosus were screened against the total 9 microorganisms in which 5 were gram positive and 4 were gram negative bacteria. In this study, crude extract and the fractions gave the zone of inhibition against these bacteria in the range of 6.5-14 mm (diameter) and the standard drug ciprofloxacin gave in the range of 14-22 mm (diameter) zone of inhibition. The crude extract of A. racemosus showed the maximum 14 mm zone of inhibition and the standard drug showed 19 mm of diameter zone of inhibition against Vibrio parahimolyticus in comparison to the examined Gram-negative bacteria and the results are given in Table (2). Extractives showed variable growth of inhibition against some examined bacteria and the growth inhibition was compared with the predictable standard drug ciprofloxacin. Ciprofloxacin is a broad-spectrum antibiotic; it shows its activity against both gram-positive and gram-negative bacteria. Considering the inhibited DNA gyrase, and a type II topoisomerase, topoisomerase IV, it is necessary to separate the bacterial DNA, thereby inhibiting mostly gram-negative pathogens fruitfully. According to the previous studies and literature review, a number of medicinal plants have been shown to possess anti-infective properties [17Goun E, Cunningham G, Chu D, Nguyen C, Miles D. Antibacterial and antifungal activity of Indonesian ethnomedical plants. Fitoterapia 2003; 74(6): 592-6.
[http://dx.doi.org/10.1016/S0367-326X(03)00117-5] [PMID: 12946723] , 18Arfan NB, Islam T, Mohiuddin AK, Alam Khan S, Labu K. Thrombolytic, membrane stabilizing, antidiarrhoeal, and antimicrobial properties of bioactive compounds isolated from leaves of Sesbania grandiflora naturally growing in Bangladesh. Indian J Pharm Sci 2016; 12(3): 31-46.]. The plant of A. racemosus may possess the similar properties, already found in our study. So we can expect the existence of antibacterial property in the plant of A. racemosus, which moderately inhibits the growth of both Gram (+) and Gram (–) bacteria.

Table 2
Antimicrobial activity of crude ethanol extract and various fractionates of A. racemosus.

The antioxidant potential was determined by total phenolic contents of CEE and different extractives of A. racemosus ranging from 221.16 mg of GAE/gm to 550.14mg of GAE/g of extractives (Table 3).

Table 3
Total phenol compounds and DPPH free radical scavenging activity of crude ethanol extract and its different partitionates of plant of A. racemosus.

The antioxidant potential was also determined in terms of DPPH free radical scavenging assay of A. racemosus, showing the EC₅₀ value within the range of 2.62 μg/ml to 6.20μg/ml. Among the crude ethanolic extract and fractionates, the highest free radical scavenging activity showed by AQSF which was 2.62 μg/ml followed by PSF being 3.79 μg/ml, CSF being 3.88 μg/ml, CEE being 5.70μg/ml and CTSF being 6.20 μg/ml in comparison with ascorbic acid (2.48 μg/ml) as a standard Table (3). Outcome of our study revealed that changes in color (implying that the higher the decolourisation, the higher is the reducing ability, from deep violet to light yellow) followed by low absorbance of the reaction mixture indicated a high free radical scavenging activity. AQSF contains maximum total phenolic compound of 550.12 mg of GAE/g of extractives and EC₅₀ value of AQSF was 2.62 μg/ml, which was the lowest among the extractives. So we can conclude that there exists a significant correlation between the total phenolic contents and DPPH free radical scavenging activity.