In recent study, the Herbal Anti-atherosclerosis Formulation comprising of Lemon, Garlic, Ginger and Apple fruit extract vinegar have been generally recognized as agents for prevention and treatment of cardiovascular and other metabolic ailments, atherosclerosis, hyperlipidemia, thrombosis, hypertension and diabetes and cholesterol bringing down impact. Accordingly, the present study was intended to investigate thrombolytic properties of Herbal Anti-atherosclerosis Formulation. Natural Antiatherosclerosis Formulation of demonstrated exceptionally huge (P &lt;0.001) clot lytic properties in different blood samples. The percent clot lytic activity was compared with negative control (water) and standard enzyme positive control (streptokinase). The mean % of clot lysis for water and streptokinase was discovered 4.71% and 86.21% individually. Then again the mean percent clot lytic activity of Herbal Anti-atherosclerosis Formulation was discovered 29.51%, which is huge contrast and the positive and negative control. The present research recommends that Herbal Anti-atherosclerosis Formulation has significant thrombolytic action. Consequently the detailing may be a wellspring of powerful natural medication

Al Mahmood, Asif

Barman, Tama

Dhar, Rana

Hossain, Mohammad Shahadat

Karim, Naymul

Sakib, Md Hossan

Ullah, Ahsan

English

European Scientific Journal, ESJ

European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 126 IN-VITRO THROMBOLYTIC ACTIVITY OF HERBAL ANTI-ARTHEROSCLEROSIS FORMULATION    Md Hossan Sakib Department of Pharmacy International Islamic University Chittagong, Bangladesh Tama Barman Department of Pharmacy BGC Trust University, Bangladesh Naymul Karim Rana Dhar Mohammad Shahadat Hossain Ahsan Ullah Department of Pharmacy International Islamic University Chittagong, Bangladesh Asif Al Mahmood Department of Pharmacy University of Science and Technology Chittagong, Bangladesh   Abstract  In recent study, the Herbal Anti-atherosclerosis Formulation comprising of Lemon, Garlic, Ginger and Apple fruit extract vinegar have been generally recognized as agents for prevention and treatment of cardiovascular and other metabolic ailments, atherosclerosis, hyperlipidemia, thrombosis, hypertension and diabetes and cholesterol bringing down impact. Accordingly, the present study was intended to investigate thrombolytic properties of Herbal Anti-atherosclerosis Formulation. Natural Anti-atherosclerosis Formulation of demonstrated exceptionally huge (P <0.001) clot lytic properties in different blood samples. The percent clot lytic activity was compared with negative control (water) and standard enzyme positive control (streptokinase). The mean % of clot lysis for water and streptokinase was discovered 4.71% and 86.21% individually. Then again the mean percent clot lytic activity of Herbal Anti-atherosclerosis Formulation was discovered 29.51%, which is huge contrast and the positive and negative control. The present research recommends that Herbal Anti-atherosclerosis European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  127 Formulation has significant thrombolytic action. Consequently the detailing may be a wellspring of powerful natural medication.  Keywords: Thrombolytic activity, Streptokinase, Herbal Anti-atherosclerosis Formulation   Introduction  Herbal medicine is utilized to treat numerous conditions, for example, asthma, dermatitis, premenstrual disorder, rheumatoid joint pain, headache, menopausal manifestations, unending weariness, fractious inside disorder, and growth, among others [1]. In many situations, researchers aren't sure what particular ingredient in a specific herb attempts to treat a condition or ailment. Entire herbs contain numerous ingredients, and they may cooperate to deliver a valuable effect. Frequently, herbs may be utilized together on the grounds that the mixture is more effective and may have less effects [2] . The point of the present research is to find out the thrombolytic effect of Herbal Anti-atherosclerosis Formulation. Therefore the formulation may be a source of powerful natural medication.  A blood clot (thrombus) produce in the blood circulatory system due to the failure of homeostasis reason vascular blockage and while recovering leads to serious consequences in atherothrombotic diseases such as acute myocardial or cerebral infarction, at times prompting to death. Usually utilized thrombolytic agents are alteplase, anistreplase, streptokinase, urokinase and tissue plasminogen activator (tPA) to dissolve clots [3]. All available thrombolytic agents still have significant shortcomings, including the need for large doses to be vast effective, limited fibrin specificity and bleeding tendency. In view of the inadequacies comings of the available thrombolytic drugs, attempts are in progress to create enhanced recombinant variations of these medications [8]. Heparin and Aspirin are just decently efficient for acceleration of lysis and prevention of reocclusion, however are safe. More particular thrombin inhibitors and antiplatelet agents are more powerful, yet their safety stays to be affirmed [4]. Proceeded investigation in this area will provide new insights and advance advancement toward the improvement of the perfect thrombolytic treatment, characterized by maximized stable coronary arterial thrombolysis with minimal bleeding [5].   Materials and Method Reagents and chemicals  Streptokinase(SK) vials of 15, 00, 000 I.U.10 blood (5ml) sample drawn from healthy human volunteers, Herbal antiatherosclerosis formulation, Distilled Water.  European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 128 Apparatus  Micro centrifuge tube (0.5ml/tube), Micropipette, Vortex mixer, 0.22-micron syringe filter, Beaker, Electric Balance, Incubator.  Experimental procedure Streptokinase (SK)  To the commercially available lyophilized SK vial (Durakinase, Dongkook Pharma. Co. Ltd. South Korea) of 15, 00,000 I.U., 5 ml sterile distilled water 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.  Specimen  Whole blood (4 ml) was drawn from healthy human volunteers (n = 20) without a history of oral contraceptive or anticoagulant therapy (using a protocol approved by the Institutional Ethics Committee of Central India Institute of Medical Sciences, Nagpur). 500 μl (0.5 ml) of blood was transferred to each of the three previously weighed microcentrifuge tubes to form clots.  Herbal preparation  100 mg Herbal Antiatherosclerosis formulation was suspended in 10 ml distilled water and the suspension was shaken vigorously on a vortex mixer. The suspension was kept overnight and decanted to remove the soluble supernatant, which was filtered through a 0.22-micron syringe filter. 100µl of this aqueous preparation of herb was added to the microcentrifuge tubes containing the clots to check thrombolytic activity [7].  Clot lysis  4 ml venous blood drawn from healthy volunteers was distributed in three different pre weighed sterile microcentrifuge tube (0.5 ml/tube) and incubated at 37°C for 45 minutes. After clot formation, serum was completely removed without disturbing the clot and each tube having clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube – weight of tube alone). To one microcentrifuge tube containing pre-weighed clot, 100 μl of aqueous extract of Herbal Anti-atherosclerosis Formulation was added. As a positive control, 100 μl of SK and as a negative non thrombolytic control, 100 μl of distilled water were separately added to the control tubes numbered. All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, fluid released was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  129 lysis. The experiment was repeated 20 times with the blood samples of 20 volunteers [10, 11, 12, 13, 14, and 15]. % Clot lysis == 2nd wt.−3rd wt.×100   2nd wt.− 1st wt.[15]  Figure 1: Micro centrifuge tubes used in the experiment. Result and Observation  Effect of Herbal Anti-atherosclerosis Formulation on in vitro thrombolysis: Blood sample   % of Clot lysis Control (water) Streptokinase Formulation 1 4.20 87.20 37.22 2 5.73 85.00 35.22 3 4.58 86.80 30.24 4 4.89 87.36 24.31 5 5.26 84.60 33.41 6 4.13 85.10 25.26 7 4.27 84.18 26.31 8 4.00 86.16 27.34 9 5.65 85.12 28.66 10 5.28 86.2 27.22 Mean 4.706 86.2075 29.519 Table 1: Comparing the data of % of Clot lysis using SPSS 11.5      European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 130 Group Statistics (Control vs. Streptokinase)   Control, Streptokinase N Mean Std. Deviation Std. Error Mean % of Clot lysis Control 10 4.7990 .65113 .20590 Streptokinase 10 85.7720 1.12211 .35484  Group Statistics (Control vs. Formulation)  Control, Streptokinase N Mean Std. Deviation Std. Error Mean % of Clot lysis Control 10 4.7990 .65113 .20590 Formulation 10 29.5190 4.39455 1.38968  Group Statistics (Formulation vs. Streptokinase)  Control, Streptokinase N Mean Std. Deviation Std. Error Mean % of Clot lysis Streptokinase 10 85.7720 1.12211 .35484 Formulation 10 29.5190 4.39455 1.38968    Here, all values are expressed as MEAN ± SEM (n=20). ***P<0.001 significant compared to negative control. Effect of Clot lysis Sample Result Streptokinase (STD) 86.21.56% Distilled water (control) 4.71 % Sample (Herbal anti-atherosclerosis formulation) 29.51 %  % Clot lysis activity of herbal formulation:  Figure 2: Comparative % in vitro thrombolytic effect of formulation, streptokinase and water (negative control). Control (Water), 4.71%  Streptokinase, 86.21%  Formulation, 29.51% % Clot Lysis Treatment % Clot Lysis Activity of Herbal Formulation European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  131 Discussion  Addition of 100µl Streptokinase (Durakinase, Dongkook Phama. Co. Ltd, South Korea) , a positive control (30,000I.U.) to the clots along with 90 minutes incubation at 37ºC, showed 86.21% clot lysis. On the other hand, clots when treated with 100µl sterile distilled water (negative control) showed only negligible clot lysis which was only 4.71%.The mean difference in clot lysis percentage between positive and negative control was very significant (***p value<0.001).But when 100µl Herbal Anti-atherosclerosis Formulation was added to 20 different clots, 29.51% clot lysis were obtained and when compared  with the negative control(water) the mean clot lysis percentage differences was significant (***P value<0.001).Percent clot lysis obtained after treating with water, streptokinase and Herbal Anti-atherosclerosis Formulation shown in Fig.4.35 .Statistical representation(Student’s t-test) of the effective clot lysis percentage by Herbal anti-atherosclerosis formulation, positive thrombolytic control (Streptokinase) and negative control (sterile distilled water) is tabulated in Table 4.17. Percentage of clot lysis of 20 different blood samples after treated with water, streptokinase and Herbal Anti-atherosclerosis Formulation is shown in Table.  Here, Clot weight = weight of clot containing tube – weight of tube alone.  All the tubes were incubated at 37ºC for 90 minutes and observe for clot lysis. After Incubation fluids released was removed and tubes were again weighted to observe the difference in weight after clot disruption. Differences obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis.     Conclusion  Atherosclerosis-induced heart attacks and strokes are leading reasons of morbidity and mortality. Current essential and auxiliary prevention strategies emphasize control of different atherosclerotic danger components, including smoking, hypertension, hypercholesterolemia, diabetes mellitus, weight, irritation, and homocysteine [16]. Current pharmacological studies recommend remedial estimations of these natural preparations, including lowering of blood pressure and lipids, antioxidation, thrombolytic activity and the promotion of microcirculation. There is a requirement for more goal and scientific approaches to authenticate individual herbs to identify chemical constituents, detect adulteration or contamination of herbs, and screen the quality of herbs and herbal medicines [17]. There is also a need to check the consistency of different batches of herbs utilized as a part of this study and to distinguish bioactive parts in herbs reported to have physiological effects. European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 132  Under this study, Herbal Anti-atherosclerosis Formulation of demonstrated very significant (P <0.001) clot lytic properties in different blood samples. The percent clot lytic activity was compared with water (positive control) and standard enzyme streptokinase (negative control). The mean % of clot lysis for water and streptokinase was found 4.71% and 86.21% separately. Then again the mean percent clot lytic activity of Herbal Anti-atherosclerosis Formulation was found 29.51%, which is significant compare with the positive and negative control. So, the present research proposes that, the Herbal Anti-atherosclerosis Formulation has significant thrombolytic activity. Thus the formulation may be a source of effective herbal drug.  References: Andrew Vickers, Memorial-Sloan Kettering Cancer Center, New York, NY, Catherine Zollman, Clinical lecturer in general practice, Bristol University, Bristol, UK,Roberta Lee,Beth Israel Health and Healing Center, Boston, MA: Herbal medicine, (ISBN 0 727912372) London: BMJ Books; 2000, edited by Dr Zollman and Dr Vickers. Sameer Al-Asheh*, Mamdouh Allawzi, Awni Al-Otoom, Hussein Allaboun, Amani Al-Zoubi Supercritical fluid extraction of useful compounds from sage.Natural Science Vol.4 No. 8(2012), Article ID: 21991, 8 pages. Joysree Das and, Md. Muradur Rahman antioxidant and thrombolytic activity of chloroform extract of Bacopamonniera (L) Bulletin of Pharmaceutical Research 2014;4(3):133-9. Verstraete M. Third Generation thrombolytic drugs. Am J Med. 109(1): 2000; 52-58. Gesler WM. Therapeutic landscapes: medical issues in light of the new cultural geography. Soc Sci Med. 34: 1992; 735–746. Tapsell LC, Hemphill I, Cobiac L, Patch CS, Sullivan DR, Fenech M, Roodenrys S, Keogh JB, Clifton PM,Williams PG, Fazio VA, Inge KE. Health benefits of herbs and spices: The past, the present, the future. Med. J. Aust. 2006; 185(4):S4-24. Cytotoxic and thrombolytic activity of ethanolic extract of Zanthoxylumbudrunga (Fam: Rutaceae) leaves, Academia 2014 Thrombolytic &cyto-toxic property of Ethanol, Acetone & DM water extract of Tamarindusindica Linn. Fruits pulp:  Phytochemical Screening & in vitro Antioxidant and Thrombolytic Activities of Argemone mexicana Extracts”, EWU Institutional Repository, 2012-07-10 Fujita M, Hong K, Ito Y, Fujii R, Kariya K, Nishimuro S. Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. Biol Pharm Bull. 1995 Oct;18(10):1387-91. European Scientific Journal June 2015 edition vol.11, No.18  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  133 Rolf Bergkvist and Per Olov Svärd, Studies on the Thrombolytic Activity of a Protease from Aspergillus Oryzae, Article first published online: 8 DEC 2008 DOI: 10.1111/j.1748- 1716.1964.tb02899.x Md. Al Amin Sikder, Abu Bakar Siddique, A.K.M. Nawshad Hossia, Md. Kowser Miah,Mohammad A. Kaisar and Mohammad A. Rashid, Evaluation of Thrombolytic Activity Cytotoxic and thrombolytic effect of Prottium serratum 67 Of Four Bangladeshi Medicinal Plants, as a Possible Renewable Source for Thrombolytic Compounds, Journal of Pharmacy and Nutrition Sciences, 2011, 1, 4-8 Sweta Prasad, Rajpal Singh Kashyap, Jayant Y Deopujari, Hemant J Purohit, Girdhar M Taori and Hatim F Daginawala, Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis, BMC Complementary and Alternative Medicine 2007, doi:10.1186/1472-6882-7-36 P.J. Gaffney and A.N. Whitaker, Fibrin crosslinks and lysis rates, thrombosis research, volume 14 Issue 1 pages 85-94 January 1979. B N Meyer, N R Ferrigni, J E Puthnam, L B Jacobsen, D E Nichols and J L Mclaughlin, Brine shrimp: a convenient general bioassay for active plant constituents. Journal of medicinal plant research, vol 45,PP 31-34,1982, Brown MS, Goldstein JL.Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. PMID, Cell. 1975 Nov;6(3):307-16. Jukka H. Meurman Mariano Sanz , Sok-Ja Janket Oral health , atherosclearosis , And cardiovascular disease. William H. Frishman, MD, Poojitha Beravol, MS, Christine Carosella, MD Alternative and Complementary Medicine for Preventing and Treating Cardiovascular Disease.   

IN-VITRO THROMBOLYTIC ACTIVITY OF HERBAL ANTI-ARTHEROSCLEROSIS FORMULATION

In recent study, the Herbal Anti-atherosclerosis Formulation comprising of Lemon, Garlic, Ginger and Apple fruit extract vinegar have been generally recognized as agents for prevention and treatment of cardiovascular and other metabolic ailments, atherosclerosis, hyperlipidemia, thrombosis, hypertension and diabetes and cholesterol bringing down impact. Accordingly, the present study was intended to investigate thrombolytic properties of Herbal Anti-atherosclerosis Formulation. Natural Anti-atherosclerosis Formulation of demonstrated exceptionally huge (P &lt;0.001) clot lytic properties in different blood samples. The percent clot lytic activity was compared with negative control (water) and standard enzyme positive control (streptokinase). The mean % of clot lysis for water and streptokinase was discovered 4.71 % and 86.21 % individually. Then again the mean percent clot lytic activity of Herbal Anti-atherosclerosis Formulation was discovered 29.51%, which is huge contrast and the positive and negative control. The present research recommends that Herbal Anti-atherosclerosi

IN-VITRO THROMBOLYTIC ACTIVITY OF HERBAL ANTI-ARTHEROSCLEROSIS FORMULATION

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