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��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������	����bjbj+�+�	ʼnI�I��-+0��������	�	""����������8�l��T�n�"8884FNFZF�S�S�S�S�S�S�S$W�1ZV�Si�bFF"4FbFbF�S""88�*IT�J�J�JbF>"l8�8JRp�JbF�S�J�J:�N,�,
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O��Z��OdbFbF�JbFbFbFbFbF�S�S�JbFbFbF�TbFbFbFbF���������������������������������������������������������������������ZbFbFbFbFbFbFbFbFbF�	 �:	In Vitro Experimental Evaluation of Thrombolytic Potentiality of Vanda roxburghii 
Md. Rezanur Rahman1, Tania Islam1, Sharmin Akhter2, Nilufa Akhter Banu1, Md. Anwarul Haque1, Satya Prya Sharma3, Md. Abu Hena Mostofa Jamal1,3*
1. Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh
2. Department of Applied Food and Nutrition, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh
3Department of Biomedical Sciences, Chonbuk National University Medical School, Jeonju, South Korea. 

*Corresponding Author: Md. Abu Hena Mostofa Jamal
Associate professor
Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh, E-mail: jamalbtg@gmail.com


Abstract
Thrombus formation in blood vessels leads to serious medical consequences such as myocardial or cerebral infarction. Thrombolytic agents are exploited to degrading the already formed clots in the blood vessels despite having serious adverse effects of these drugs which could lead to fatal consequences. Plants are traditionally used since ancient times for treatment of different disease; and different plants possessing thrombolytic properties have been reported previously, however, the medicinal plant Vanda roxburghii R. Br., an epiphyte, is commonly known as Rasna usually distributed throughout Bangladesh has not been reported earlier. The aim of the study was to investigate whether the Vanda roxburghii possesses thrombolytic properties or not. The crude methanol extract of Vanda roxburghii exhibited highest thrombolytic activity with clot lysis value of 32.1 � 0.59%. Standard Streptokinase and water were used as the positive and negative control which demonstrated 55.11 � 0.06% and 7.917 � 0.04% lysis of clot of human blood respectively. Through our study it was found that Chloroform extract of V. roxburghii possesses significant thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and an active component(s) of V. roxburghii for the clot lysis is yet to be discovered. Once found V. roxburghii could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.
Keywords: Atherothrombotic ,Vanda roxburghii, Clot lysis.

1. Introduction
Blood clot can be harmful in HYPERLINK "https://en.wikipedia.org/wiki/Thrombosis" \o "Thrombosis"thrombosis, when clots obstruct blood flow through healthy blood vessels while recovering leads to serious consequences in atherothrombotic diseases such as myocardial or cerebral infarction, pulmonary embolism, at times leading to death ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.2165/00002018-199513020-00002", "ISSN" : "11791942", "abstract" : "Streptokinase is an antigenic thrombolytic agent used for the treatment of acute myocardial infarction. It reduces mortality as effectively as the nonantigenic alteplase in most infarct patients while having the advantage of being much less expensive. This cost implication is important since myocardial reinfarction is common, with fibrinolytic therapy indicated in many patients with reinfarction. Following streptokinase, antistreptokinase antibodies and neutralisation titres can rise to significant levels from 4 days after the initial dose. These antibodies can presist for at least 4 years in up to 50{%} of patients. It is possible that these antibodies may cause allergic reactions or neutralisation of a further dose of streptokinase, rendering it ineffective for the treatment of myocardial reinfarction. To date, 2 small studies of patients without previous streptokinase exposure suggest that higher antibody titres are associated with a lower rate of coronary reperfusion, while a further study suggests that high titres are associated with hypersensitivity reactions. At present the readministration of streptokinase cannot be recommended from 4 days after a first dose. Further larger studies are needed to assess the effect of high neutralisation titres on coronary reperfusion.", "author" : [ { "dropping-particle" : "", "family" : "Lee", "given" : "Hai Shiang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Drug Safety", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "1995" ] ] }, "page" : "76-80", "title" : "How Safe is the Readministration of Streptokinase?", "type" : "article-journal", "volume" : "13" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=5829a707-09a7-496d-bfee-e5d1bb2a1946" ] } ], "mendeley" : { "formattedCitation" : "(Lee, 1995)", "plainTextFormattedCitation" : "(Lee, 1995)", "previouslyFormattedCitation" : "(Lee, 1995)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Lee, 1995). Different types of thrombolytic agents such as tissue plasminogen activator, Urokinase (UK), Streptokinase (SK) etc. are used globally for the treatment of these diseases as first-line clinical thrombolytic agents. Due to the lower cost of UK and SK are extensively used in Bangladesh, India and other developing countries ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISSN" : "0959-8138", "PMID" : "8520368", "author" : [ { "dropping-particle" : "", "family" : "Mucklow", "given" : "J C", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMJ (Clinical research ed.)", "id" : "ITEM-1", "issue" : "7018", "issued" : { "date-parts" : [ [ "1995", "12", "2" ] ] }, "page" : "1506", "publisher" : "BMJ Publishing Group", "title" : "Thrombolytic treatment. Streptokinase is more economical than alteplase.", "type" : "article-journal", "volume" : "311" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=27b95a8c-13ca-3953-9b56-f91600a9c1a6" ] } ], "mendeley" : { "formattedCitation" : "(Mucklow, 1995)", "plainTextFormattedCitation" : "(Mucklow, 1995)", "previouslyFormattedCitation" : "(Mucklow, 1995)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Mucklow, 1995). In addition, exploitation of these drugs remains associated with complications, including intracranial haemorrhage, severe anaphylactic reaction, and lack of specificity ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1111/j.1365-2044.1982.tb00985.x", "ISSN" : "13652044", "abstract" : "The fibrinolytic effects of six different anaesthetic agents were studied in 36 patients undergoing abdominal hysterectomy. Fibrinolytic activity was measured by dilute whole blood and euglobulin clot lysis times. Concentrations of plasminogen, fibrinogen and fibrin degradation products were also determined. Significant enhancement of fibrinolytic activity, measured as a decrease in clot lysis time, was seen during anaesthesia and surgery with all agents studied. Halothane produced the least effect and the greatest increase in fibrinolytic activity was seen during trichloroethylene anaesthesia. Lumbar extradural anaesthesia also consistently produced enhancement of fibrinolysis. The reasons for these results and their clinical implications are discussed.", "author" : [ { "dropping-particle" : "", "family" : "SIMPSON", "given" : "P. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "RADFORD", "given" : "SHEILA G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "FORSTER", "given" : "S. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "COOPER", "given" : "GRISELDA M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "HUGHES", "given" : "A. O.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Anaesthesia", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "1982" ] ] }, "page" : "3-8", "title" : "The fibrinolytic effects of anaesthesia", "type" : "article-journal", "volume" : "37" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=f1771f5d-aa45-46f2-b6ba-56d76d34438a" ] } ], "mendeley" : { "formattedCitation" : "(SIMPSON <i>et al.</i>, 1982)", "plainTextFormattedCitation" : "(SIMPSON et al., 1982)", "previouslyFormattedCitation" : "(SIMPSON <i>et al.</i>, 1982)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(SIMPSON et al., 1982) [3]. Moreover, these drugs are not used in patients who had undergone surgery or those with a history of nervous lesions, gastrointestinal bleeding, or hypertension ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1111/j.1365-2044.1982.tb00985.x", "ISSN" : "13652044", "abstract" : "The fibrinolytic effects of six different anaesthetic agents were studied in 36 patients undergoing abdominal hysterectomy. Fibrinolytic activity was measured by dilute whole blood and euglobulin clot lysis times. Concentrations of plasminogen, fibrinogen and fibrin degradation products were also determined. Significant enhancement of fibrinolytic activity, measured as a decrease in clot lysis time, was seen during anaesthesia and surgery with all agents studied. Halothane produced the least effect and the greatest increase in fibrinolytic activity was seen during trichloroethylene anaesthesia. Lumbar extradural anaesthesia also consistently produced enhancement of fibrinolysis. The reasons for these results and their clinical implications are discussed.", "author" : [ { "dropping-particle" : "", "family" : "SIMPSON", "given" : "P. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "RADFORD", "given" : "SHEILA G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "FORSTER", "given" : "S. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "COOPER", "given" : "GRISELDA M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "HUGHES", "given" : "A. O.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Anaesthesia", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "1982" ] ] }, "page" : "3-8", "title" : "The fibrinolytic effects of anaesthesia", "type" : "article-journal", "volume" : "37" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=f1771f5d-aa45-46f2-b6ba-56d76d34438a" ] } ], "mendeley" : { "formattedCitation" : "(SIMPSON <i>et al.</i>, 1982)", "plainTextFormattedCitation" : "(SIMPSON et al., 1982)", "previouslyFormattedCitation" : "(SIMPSON <i>et al.</i>, 1982)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(SIMPSON et al., 1982). Therefore, the quest for novel medicinal agents may let obtaining new drugs with enhanced pharmacological properties, extensive range of therapies, efficacy, and safety.
Plants are the natural sources of medicinal agents include antimicrobial, anticancer agents, analgesics and so on. Traditionally a large number of plant-derived medicines had been used without any adverse effects. A wide array of plants represents the natural source of valuable compounds that might provide as the lead for the development of novel drugs ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/s12906-015-0833-y", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "26335564", "abstract" : "BACKGROUND: Ethnopharmacological approach has explored several leads from plant sources to identify potential new drugs for various diseases including pain. Vanda roxburghii R. Br., an epiphytic orchid is widely distributed throughout Bangladesh. The root of this plant has a folkloric reputation to treat inflammations, fever, dyspepsia, bronchitis, hiccough, piles, snake bites, and diseases of the nervous system. In this study therefore, we aimed to investigate antinociceptive and cytotoxic properties of the extracts from the root of V. roxburghii.\\n\\nMETHODS: Dried powder of aerial root of V. roxburghii was extracted with methanol (VRM) and the resultant was fractionated into petroleum ether (VRP), chloroform (VRC), ethyl acetate (VRE), and residual aqueous fraction (VRA). The antinociceptive effect of the extracts was evaluated in mice using acetic acid-induced writhing, formalin injection, and hot plate tests. The brine shrimp lethality bioassay in vitro was used to evaluate cytotoxic activity of the plant extracts.\\n\\nRESULTS: In the acetic acid-induced writhing test, mice treated with different fractions (12.5, 25, and 50\u00a0mg/kg, i.p.) exhibited reduced number of writhing. Amongst, VRE showed the highest activity at all three concentrations (43.65, 71.34, and 80.23\u00a0%, respectively) in a dose-dependent manner. Secondly, VRC (12.5, 25, and 50\u00a0mg/kg, i.p.) displayed the highest reduction of paw licking time in mice during the first phase of the formalin test (by 15.00, 37.05, and 56.44\u00a0%, respectively) as well as during the second phase of the test (by 20.55, 49.08, and 59.81\u00a0%, respectively). In hot plate test, VRE treatment at doses of 25 and 50\u00a0mg/kg both increased the highest latency time after 30\u00a0min. All fractions showed lower cytotoxicity compared with the standard drug vincristine sulfate in the brine shrimp bioassay.\\n\\nCONCLUSION: Overall studies suggest that the root of V. roxburghii is effective as a potent analgesic with lower toxicity. Our findings support previous claims of traditional uses of V. roxburghii in different inflammatory disorders.", "author" : [ { "dropping-particle" : "", "family" : "Uddin", "given" : "Md Josim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahman", "given" : "Md Masudur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abdullah-Al-Mamun", "given" : "Md", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sadik", "given" : "Golam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC complementary and alternative medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "305", "publisher" : "BMC Complementary and Alternative Medicine", "title" : "Vanda roxburghii: an experimental evaluation of antinociceptive properties of a traditional epiphytic medicinal orchid in animal models.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=c38e8eda-83ba-4c08-98b9-b044d4d3d0ed" ] } ], "mendeley" : { "formattedCitation" : "(Uddin <i>et al.</i>, 2015)", "plainTextFormattedCitation" : "(Uddin et al., 2015)", "previouslyFormattedCitation" : "(Uddin <i>et al.</i>, 2015)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Uddin et al., 2015). It is, therefore, a great attention should be concentrated to introduce new medicinal agents to develop more effective and cheaper drugs. Thus, traditional medicine has been paid great consideration because they are economical, obtainable, and have minute side effects which arguede WHO that around 80 % of the world population still rely mainly on plant-based drugs ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/s12906-015-0833-y", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "26335564", "abstract" : "BACKGROUND: Ethnopharmacological approach has explored several leads from plant sources to identify potential new drugs for various diseases including pain. Vanda roxburghii R. Br., an epiphytic orchid is widely distributed throughout Bangladesh. The root of this plant has a folkloric reputation to treat inflammations, fever, dyspepsia, bronchitis, hiccough, piles, snake bites, and diseases of the nervous system. In this study therefore, we aimed to investigate antinociceptive and cytotoxic properties of the extracts from the root of V. roxburghii.\\n\\nMETHODS: Dried powder of aerial root of V. roxburghii was extracted with methanol (VRM) and the resultant was fractionated into petroleum ether (VRP), chloroform (VRC), ethyl acetate (VRE), and residual aqueous fraction (VRA). The antinociceptive effect of the extracts was evaluated in mice using acetic acid-induced writhing, formalin injection, and hot plate tests. The brine shrimp lethality bioassay in vitro was used to evaluate cytotoxic activity of the plant extracts.\\n\\nRESULTS: In the acetic acid-induced writhing test, mice treated with different fractions (12.5, 25, and 50\u00a0mg/kg, i.p.) exhibited reduced number of writhing. Amongst, VRE showed the highest activity at all three concentrations (43.65, 71.34, and 80.23\u00a0%, respectively) in a dose-dependent manner. Secondly, VRC (12.5, 25, and 50\u00a0mg/kg, i.p.) displayed the highest reduction of paw licking time in mice during the first phase of the formalin test (by 15.00, 37.05, and 56.44\u00a0%, respectively) as well as during the second phase of the test (by 20.55, 49.08, and 59.81\u00a0%, respectively). In hot plate test, VRE treatment at doses of 25 and 50\u00a0mg/kg both increased the highest latency time after 30\u00a0min. All fractions showed lower cytotoxicity compared with the standard drug vincristine sulfate in the brine shrimp bioassay.\\n\\nCONCLUSION: Overall studies suggest that the root of V. roxburghii is effective as a potent analgesic with lower toxicity. Our findings support previous claims of traditional uses of V. roxburghii in different inflammatory disorders.", "author" : [ { "dropping-particle" : "", "family" : "Uddin", "given" : "Md Josim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahman", "given" : "Md Masudur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abdullah-Al-Mamun", "given" : "Md", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sadik", "given" : "Golam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC complementary and alternative medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "305", "publisher" : "BMC Complementary and Alternative Medicine", "title" : "Vanda roxburghii: an experimental evaluation of antinociceptive properties of a traditional epiphytic medicinal orchid in animal models.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=c38e8eda-83ba-4c08-98b9-b044d4d3d0ed" ] } ], "mendeley" : { "formattedCitation" : "(Uddin <i>et al.</i>, 2015)", "plainTextFormattedCitation" : "(Uddin et al., 2015)", "previouslyFormattedCitation" : "(Uddin <i>et al.</i>, 2015)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Uddin et al., 2015).
The herb Vanda roxburghii R. Br., an epiphyte belonging to the family Orchidaceae, is commonly known as Rasna usually distributed throughout Bangladesh. In all over Bangladesh, it is grown on Mango, Black berry, and Guava trees. The medicinal properties of V. roxburghii are well described in Ayurveda, traditional Indian medicine ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/s12906-015-0833-y", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "26335564", "abstract" : "BACKGROUND: Ethnopharmacological approach has explored several leads from plant sources to identify potential new drugs for various diseases including pain. Vanda roxburghii R. Br., an epiphytic orchid is widely distributed throughout Bangladesh. The root of this plant has a folkloric reputation to treat inflammations, fever, dyspepsia, bronchitis, hiccough, piles, snake bites, and diseases of the nervous system. In this study therefore, we aimed to investigate antinociceptive and cytotoxic properties of the extracts from the root of V. roxburghii.\\n\\nMETHODS: Dried powder of aerial root of V. roxburghii was extracted with methanol (VRM) and the resultant was fractionated into petroleum ether (VRP), chloroform (VRC), ethyl acetate (VRE), and residual aqueous fraction (VRA). The antinociceptive effect of the extracts was evaluated in mice using acetic acid-induced writhing, formalin injection, and hot plate tests. The brine shrimp lethality bioassay in vitro was used to evaluate cytotoxic activity of the plant extracts.\\n\\nRESULTS: In the acetic acid-induced writhing test, mice treated with different fractions (12.5, 25, and 50\u00a0mg/kg, i.p.) exhibited reduced number of writhing. Amongst, VRE showed the highest activity at all three concentrations (43.65, 71.34, and 80.23\u00a0%, respectively) in a dose-dependent manner. Secondly, VRC (12.5, 25, and 50\u00a0mg/kg, i.p.) displayed the highest reduction of paw licking time in mice during the first phase of the formalin test (by 15.00, 37.05, and 56.44\u00a0%, respectively) as well as during the second phase of the test (by 20.55, 49.08, and 59.81\u00a0%, respectively). In hot plate test, VRE treatment at doses of 25 and 50\u00a0mg/kg both increased the highest latency time after 30\u00a0min. All fractions showed lower cytotoxicity compared with the standard drug vincristine sulfate in the brine shrimp bioassay.\\n\\nCONCLUSION: Overall studies suggest that the root of V. roxburghii is effective as a potent analgesic with lower toxicity. Our findings support previous claims of traditional uses of V. roxburghii in different inflammatory disorders.", "author" : [ { "dropping-particle" : "", "family" : "Uddin", "given" : "Md Josim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahman", "given" : "Md Masudur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abdullah-Al-Mamun", "given" : "Md", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sadik", "given" : "Golam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC complementary and alternative medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "305", "publisher" : "BMC Complementary and Alternative Medicine", "title" : "Vanda roxburghii: an experimental evaluation of antinociceptive properties of a traditional epiphytic medicinal orchid in animal models.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=c38e8eda-83ba-4c08-98b9-b044d4d3d0ed" ] } ], "mendeley" : { "formattedCitation" : "(Uddin <i>et al.</i>, 2015)", "plainTextFormattedCitation" : "(Uddin et al., 2015)", "previouslyFormattedCitation" : "(Uddin <i>et al.</i>, 2015)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Uddin et al., 2015). The root of this plant has a folkloric reputation for alleviating inflammations. Traditionally, the root of V. roxburghii has been used to treat fever, dyspepsia, bronchitis, hiccough, piles, and snake bites ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Ghani", "given" : "A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "1998" ] ] }, "publisher" : "Asiatic Society of Bangladesh", "publisher-place" : "Dhaka, Bangladesh", "title" : "Medicinal plants of Bangladesh: chemical constituents and uses.", "type" : "book" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=ac11f44f-bc0f-45e5-85e4-3392dcc9396c" ] } ], "mendeley" : { "formattedCitation" : "(Ghani, 1998)", "plainTextFormattedCitation" : "(Ghani, 1998)", "previouslyFormattedCitation" : "(Ghani, 1998)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Ghani, 1998). In addition, externally the root is used in rheumatism and allied disorders, and diseases of the nervous system ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/s12906-015-0833-y", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "26335564", "abstract" : "BACKGROUND: Ethnopharmacological approach has explored several leads from plant sources to identify potential new drugs for various diseases including pain. Vanda roxburghii R. Br., an epiphytic orchid is widely distributed throughout Bangladesh. The root of this plant has a folkloric reputation to treat inflammations, fever, dyspepsia, bronchitis, hiccough, piles, snake bites, and diseases of the nervous system. In this study therefore, we aimed to investigate antinociceptive and cytotoxic properties of the extracts from the root of V. roxburghii.\\n\\nMETHODS: Dried powder of aerial root of V. roxburghii was extracted with methanol (VRM) and the resultant was fractionated into petroleum ether (VRP), chloroform (VRC), ethyl acetate (VRE), and residual aqueous fraction (VRA). The antinociceptive effect of the extracts was evaluated in mice using acetic acid-induced writhing, formalin injection, and hot plate tests. The brine shrimp lethality bioassay in vitro was used to evaluate cytotoxic activity of the plant extracts.\\n\\nRESULTS: In the acetic acid-induced writhing test, mice treated with different fractions (12.5, 25, and 50\u00a0mg/kg, i.p.) exhibited reduced number of writhing. Amongst, VRE showed the highest activity at all three concentrations (43.65, 71.34, and 80.23\u00a0%, respectively) in a dose-dependent manner. Secondly, VRC (12.5, 25, and 50\u00a0mg/kg, i.p.) displayed the highest reduction of paw licking time in mice during the first phase of the formalin test (by 15.00, 37.05, and 56.44\u00a0%, respectively) as well as during the second phase of the test (by 20.55, 49.08, and 59.81\u00a0%, respectively). In hot plate test, VRE treatment at doses of 25 and 50\u00a0mg/kg both increased the highest latency time after 30\u00a0min. All fractions showed lower cytotoxicity compared with the standard drug vincristine sulfate in the brine shrimp bioassay.\\n\\nCONCLUSION: Overall studies suggest that the root of V. roxburghii is effective as a potent analgesic with lower toxicity. Our findings support previous claims of traditional uses of V. roxburghii in different inflammatory disorders.", "author" : [ { "dropping-particle" : "", "family" : "Uddin", "given" : "Md Josim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahman", "given" : "Md Masudur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abdullah-Al-Mamun", "given" : "Md", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sadik", "given" : "Golam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC complementary and alternative medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "305", "publisher" : "BMC Complementary and Alternative Medicine", "title" : "Vanda roxburghii: an experimental evaluation of antinociceptive properties of a traditional epiphytic medicinal orchid in animal models.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=c38e8eda-83ba-4c08-98b9-b044d4d3d0ed" ] } ], "mendeley" : { "formattedCitation" : "(Uddin <i>et al.</i>, 2015)", "plainTextFormattedCitation" : "(Uddin et al., 2015)", "previouslyFormattedCitation" : "(Uddin <i>et al.</i>, 2015)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Uddin et al., 2015). The plant has also been reported to possess important pharmacological effects including anti-arthritic ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/s12906-015-0833-y", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "26335564", "abstract" : "BACKGROUND: Ethnopharmacological approach has explored several leads from plant sources to identify potential new drugs for various diseases including pain. Vanda roxburghii R. Br., an epiphytic orchid is widely distributed throughout Bangladesh. The root of this plant has a folkloric reputation to treat inflammations, fever, dyspepsia, bronchitis, hiccough, piles, snake bites, and diseases of the nervous system. In this study therefore, we aimed to investigate antinociceptive and cytotoxic properties of the extracts from the root of V. roxburghii.\\n\\nMETHODS: Dried powder of aerial root of V. roxburghii was extracted with methanol (VRM) and the resultant was fractionated into petroleum ether (VRP), chloroform (VRC), ethyl acetate (VRE), and residual aqueous fraction (VRA). The antinociceptive effect of the extracts was evaluated in mice using acetic acid-induced writhing, formalin injection, and hot plate tests. The brine shrimp lethality bioassay in vitro was used to evaluate cytotoxic activity of the plant extracts.\\n\\nRESULTS: In the acetic acid-induced writhing test, mice treated with different fractions (12.5, 25, and 50\u00a0mg/kg, i.p.) exhibited reduced number of writhing. Amongst, VRE showed the highest activity at all three concentrations (43.65, 71.34, and 80.23\u00a0%, respectively) in a dose-dependent manner. Secondly, VRC (12.5, 25, and 50\u00a0mg/kg, i.p.) displayed the highest reduction of paw licking time in mice during the first phase of the formalin test (by 15.00, 37.05, and 56.44\u00a0%, respectively) as well as during the second phase of the test (by 20.55, 49.08, and 59.81\u00a0%, respectively). In hot plate test, VRE treatment at doses of 25 and 50\u00a0mg/kg both increased the highest latency time after 30\u00a0min. All fractions showed lower cytotoxicity compared with the standard drug vincristine sulfate in the brine shrimp bioassay.\\n\\nCONCLUSION: Overall studies suggest that the root of V. roxburghii is effective as a potent analgesic with lower toxicity. Our findings support previous claims of traditional uses of V. roxburghii in different inflammatory disorders.", "author" : [ { "dropping-particle" : "", "family" : "Uddin", "given" : "Md Josim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahman", "given" : "Md Masudur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abdullah-Al-Mamun", "given" : "Md", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sadik", "given" : "Golam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC complementary and alternative medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "305", "publisher" : "BMC Complementary and Alternative Medicine", "title" : "Vanda roxburghii: an experimental evaluation of antinociceptive properties of a traditional epiphytic medicinal orchid in animal models.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=c38e8eda-83ba-4c08-98b9-b044d4d3d0ed" ] } ], "mendeley" : { "formattedCitation" : "(Uddin <i>et al.</i>, 2015)", "plainTextFormattedCitation" : "(Uddin et al., 2015)", "previouslyFormattedCitation" : "(Uddin <i>et al.</i>, 2015)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Uddin et al., 2015), wound healing ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1177/1534734605282994", "ISSN" : "1534-7346", "abstract" : "The extract of Vanda roxburghii was administered topically to rats at a dose of 150mgkg - 1 day - 1 for 10daysandwas studied for its effect on wound healing, using the excision wound model. A 60% reduction in wound diameter was observed in the test group rats receiving the extract compared to controls (48%). Significant increases in wet and dry granulation tissue weights (P < .001), hydroxyproline (P < .001), and hexosamine (P < .003) contents were detected. An increase in protein content was also detected in the test group (P > .05, ns). These findings are consistent with wound healing at cellular levels. The pro-healing action may be attributed either to increased collagen deposition or to better alignment and maturation or both. The test wounds (extract-treated wounds) were, on average, fully healed by the 13th day, whereas the control group healed, on average, by the 20th day. These data suggest that the extract ofVanda roxburghii administered topically has wound-healing potential in rats", "author" : [ { "dropping-particle" : "", "family" : "Nayak", "given" : "B. 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}(Anwar et al., 2013). Although the root of V. roxburghii has extensive medicinal use to treat pain and inflammation, to date, no studies have yet examined its thrombolytic capabilities.  But it�s thrombolytic effects and usability has not been studied at all. Therefore, the aim of this study was carried out to evaluate the clot lysis (thrombolytic activity) properties of different fractions of V. roxburghii root methanol extract by using in vitro models.

2. Materials and Methods
2.1 Plant Materials
The root of V. roxburghii plant was collected from Islamic University, Kushtia, Bangladesh. The collected roots of the plant were air dried and powdered. The powdered plant (950 g) was subjected to extraction with methanol at room temperature for 7 days with occasional shaking. The extractive solution was filtered and concentrated under vacuum in a rotary evaporator at 45 �C to yield 17 g (1.79% w/w) of the crude extract. 
2.2 Streptokinase (SK)
Commercially available lyophilized Streptokinase vial (Beacon pharmaceutical Ltd., Mymensingh, Bangladesh) of 15, 00,000 I.U., was collected and 5 ml sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100�l (30,000 I.U) was used for in vitro thrombolysis.
2.3 Blood sample and Thrombolytic Activity
Thrombolytic potentiality of the plant was estimated by the method described by Prasad et al ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/1472-6882-7-36", "ISBN" : "1472-6882 (Electronic)\\r1472-6882 (Linking)", "ISSN" : "1472-6882", "PMID" : "17986325", "abstract" : "Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components possessing antithrombotic activity have been reported before; however, herbs that could be used for thrombolysis has not been reported so far. This study's aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not. An in vitro thrombolytic model was used to check the clot lysis effect of six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control. Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively . Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%). Through our study it was found that Dhamasa possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component(s) of Dhamasa for clot lysis are yet to be discovered. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.", "author" : [ { "dropping-particle" : "", "family" : "Prasad", "given" : "Sweta", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kashyap", "given" : "Rajpal Singh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Deopujari", "given" : "Jayant Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Purohit", "given" : "Hemant J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taori", "given" : "Girdhar M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Daginawala", "given" : "Hatim F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC Complementary and Alternative Medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2007", "3", "1" ] ] }, "page" : "36", "publisher" : "BioMed Central", "title" : "Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis", "type" : "article-journal", "volume" : "7" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=163b5b84-709e-3338-8d60-7cd6b7407e27" ] } ], "mendeley" : { "formattedCitation" : "(Prasad <i>et al.</i>, 2007)", "plainTextFormattedCitation" : "(Prasad et al., 2007)", "previouslyFormattedCitation" : "(Prasad <i>et al.</i>, 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Prasad et al., 2007) with approved protocol for ethical issues. A 4 ml venous blood pinched from the healthy volunteers was dispersed in seven different pre weighed sterile microcentrifuge tube (0.5 ml/tube) and incubated at 37�C for 45 min. 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). Five fractions (100 �l) of the plant were added to each microcentrifuge tube separately containing pre-weighed clot. As a positive control, 100 �l of streptokinase (SK) and as a negative non-thrombolytic control, 100 �l of distilled water were separately added to the control tubes. All the tubes were then incubated at 37�C for 90 minutes and observed lysis of the clot. After incubation, fluid released was removed and tubes were again weighed to study the difference in weight after clot disruption. The difference obtained in weight taken before and after the clot lysis was expressed as the percentage of clot lysis. The experiment was repeated with the blood samples of the 10 volunteers without a history of oral contraceptive or anticoagulant therapy since two weeks.
2.4 Statistical analysis
All values of thrombolytic activity were calculated as mean�SEM of three parallel determinations and were evaluated using t-test implemented by GraphPad Prism Data Editor for Windows, version 7.0 (GraphPad Software Inc., San Diego, CA, USA). P-Values <0.05 were regarded as statistically significant.

3. Results 
Thrombolytic activity
The thrombolytic activity of different extracts of V. roxburghii was assessed and the results are presented in Table 1 and summarized in Figure 1. In this study, the Streptokinase (Positive control) exhibited highest thrombolytic activity (55.11 � 0.06) % while Chloroform fraction exhibited a (32.106�0.59) % clot lysis which is statistically highly significant (P value <0.0001). In addition, significant thrombolytic activity was demonstrated by the Petroleum Ether fraction (30.72�0.55) % and Methanol crude extract exhibited (21.56�0.77) % respectively which is also statistically significant ((P value <0.0001)). However, the clot lysis activity by the Ethyl Acetate extract was found (19.25�0.57) % which is statistically significant (P value <0.0001). The mean difference between positive and negative controls in clot lysis percentage was found statistically significant. Statistical calculations of the effective clot lysis in percentage by all plant extract preparations, positive control (Streptokinase) and negative control (sterile distilled water) done by t-test analysis. Percent of clot dissolution is represented as mean�SEM and P values of all the plant preparations were considered as significant (P<0.05). 

4. Discussion
Medicinal derivatives from plants have a long history of use for the prevention and management of human diseases ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/1472-6882-13-25", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "23363538", "abstract" : "BACKGROUND: Thrombus formed in blood vessels lead to atherothrombotic diseases such as myocardial or cerebral infarction. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs sometimes cause serious and fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases although they show little toxicity in some cases. Aqueous extracts of herbs used in thrombolysis have been reported before with cytotoxic data, however, the organic extracts of herbs have not been documented. This study aims to investigate whether organic extracts possess thrombolytic properties with minimal or no toxicity.\\n\\nMETHODS: An in vitro thrombolytic model was used to check the clot lysis effect of six Bangladeshi herbal extracts viz., Ageratum conyzoides L., Clausena suffruticosa, Leea indica (Burm.f.) Merr., Leucas aspera Willd., Senna sophera L. Roxb., and Solanum torvum Swartz. using streptokinase as a positive control and water as a negative control. Briefly, venous blood drawn from twenty healthy volunteers was allowed to form clots which were weighed and treated with the test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis. Cytotoxicity was screened by brine shrimp lethality bioassay using vincristine sulfate as positive control.\\n\\nRESULTS: Using an in vitro thrombolytic model, Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed 18.12 \u00b1 2.34%, 48.9 \u00b1 2.44%, 39.30 \u00b1 0.96%, 37.32 \u00b1 2.00%, 31.61 \u00b1 2.97% and 31.51 \u00b1 0.57% and clot lysis respectively. Among the herbs studied Clausena suffruticosa, Leea indica and Leucas aspera showed very significant (p < 0.0001) percentage (%) of clot lysis compared to reference drug streptokinase (75.00 \u00b1 3.04%). In brine shrimp cytotoxic assay, the extracts Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed LC50 values 508.86 \u00b1 6.62,41.16 \u00b1 1.26, 2.65 \u00b1 0.16, 181.67 \u00b1 1.65, 233.37 \u00b1 7.74 and 478.40 \u00b1 3.23 \u03bcg/ml, respectively, with reference to vincristine sulfate (LC50 0.76 \u00b1 0.04).\\n\\nCONCLUSION: Through our study it was found that Clausena suffruticosa, Leea indica and Leucas aspera possessed effective thrombolytic properties whereas Senna sophera and Solanum torvum showed moderate to mild thrombolytic effects while Ageratum conyzoides showed no signif\u2026", "author" : [ { "dropping-particle" : "", "family" : "Rahman", "given" : "M Atiar", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sultana", "given" : "Rabeya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Emran", "given" : "Talha", "non-dropping-particle" : "Bin", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Islam", "given" : "M Saiful", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahman", "given" : "M Ashiqur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chakma", "given" : "Joti Sankhar", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rashid", "given" : "Harun-ur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasan", "given" : "Chowdhury Mohammad Monirul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC complementary and alternative medicine", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "25", "title" : "Effects of organic extracts of six Bangladeshi plants on in vitro thrombolysis and cytotoxicity.", "type" : "article-journal", "volume" : "13" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=38121f34-8446-4962-9d6f-35e463b57467" ] } ], "mendeley" : { "formattedCitation" : "(Rahman <i>et al.</i>, 2013)", "plainTextFormattedCitation" : "(Rahman et al., 2013)", "previouslyFormattedCitation" : "(Rahman <i>et al.</i>, 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Rahman et al., 2013). Plants are the prominent source of the potentially helpful compounds for the discovery and development of novel compounds leading to drugs ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/1472-6882-13-167", "ISSN" : "1472-6882", "PMID" : "23842440", "abstract" : "In Pakistan Fagonia olivieri (Zygophyllaceae) is commonly used in the indigenous system of medicine for treatment of conditions like diabetes, cancer, fever, asthma, toothache, stomach troubles and kidney disorders. This study evaluated the crude methanol extract of F. olivieri (FOM) and its derived fractions for their antimicrobial and cytotoxic activities as well as the classes of phytochemical. Dried powder of whole plant of F. olivieri was extracted with methanol (FOM) and the resultant was fractionated to give n-hexane fraction (FOH), chloroform fraction (FOC), ethyl acetate fraction (FOE), n-butanol fraction (FOB) and residual aqueous fraction (FOA). Methanol extract and its derived fractions were subjected to phytochemical screening using standard procedures. Also the extract and fractions were assayed for antibacterial, antifungal and cytotoxic activities using agar well diffusion technique, agar tube dilution method and brine shrimps lethality test, respectively. The results obtained for phytochemical analysis indicate the presence of saponins and alkaloids in all the tested extract and fractions while anthraquinones were not detected. The results showed that all the bacterial strains tested in this study were susceptible to at least one of the fractions tested. However, FOE and FOB were the best antibacterial fractions and showed antibacterial activity against maximum number of bacterial strains. The results showed that Escherichia coli was the most sensitive bacterium while Bordetella bronchiseptica and Enterobacter aerogenes were less susceptible against various fractions. 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Globally, around 30% of the pharmaceuticals are chiefly prepared from plant sources. It is considered that the bioactive phytoconstituents are accountable for the remedial value of plants ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/1472-6882-6-35", "ISBN" : "1472-6882", "ISSN" : "1472-6882", "PMID" : "17042964", "abstract" : "Antimicrobial activity of 18 ethnomedicinal plant extracts were evaluated against nine bacterial strains (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Ervinia sp, Proteus vulgaris) and one fungal strain (Candida albicans). The collected ethnomedicinal plants were used in folk medicine in the treatment of skin diseases, venereal diseases, respiratory problems and nervous disorders.", "author" : [ { "dropping-particle" : "", "family" : "Duraipandiyan", "given" : "Veeramuthu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ayyanar", "given" : "Muniappan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ignacimuthu", "given" : "Savarimuthu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC Complementary and Alternative Medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2006" ] ] }, "page" : "35", "title" : "Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India", "type" : "article-journal", "volume" : "6" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=7fac3870-18d7-46f2-b6e0-4ba3ef948161" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.3906/biy-1112-11", "ISSN" : "13000152", "abstract" : "Alocasia macrorrhizos (L.) (Araceae), a coarse and erect plant that is widely cultivated to be eaten as a vegetable throughout Bangladesh, has been reported to possess a number of medicinal properties. Th e antihyperglycemic, antioxidant, and cytotoxic eff ects of the methanol extract of A. macrorrhizos rhizomes were investigated. Th e extract was given at a single dose (250 and 500 mg/kg) in alloxan-induced hyperglycemic mice. Th e extract at 500 mg/kg produced a signifi cant (P < 0.05) decrease in the blood glucose level (55.49%) at 8 h of treatment when compared with the control and was comparable with the reference drug, metformin. Th e extract was also subjected to antioxidant potentiality and brine shrimp lethality bioassays. Th e IC 50 value of the extract was 693.0 \u03bcg/mL and the LC 50 was 188.14 \u03bcg/mL.", "author" : [ { "dropping-particle" : "", "family" : "Rahman", "given" : "Md Masudur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hossain", "given" : "Md Aslam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Siddique", "given" : "Saiful Alam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Biplab", "given" : "Kaishar Parvej", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uddin", "given" : "Md Helal", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Turkish Journal of Biology", "id" : "ITEM-2", "issue" : "5", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "574-579", "title" : "Antihyperglycemic, antioxidant, and cytotoxic activities of Alocasia macrorrhizos (L.) rhizome extract", "type" : "article-journal", "volume" : "36" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=fee29a96-ff14-3d53-9225-1240b6bc7622" ] } ], "mendeley" : { "formattedCitation" : "(Duraipandiyan, Ayyanar and Ignacimuthu, 2006; Rahman <i>et al.</i>, 2012)", "plainTextFormattedCitation" : "(Duraipandiyan, Ayyanar and Ignacimuthu, 2006; Rahman et al., 2012)", "previouslyFormattedCitation" : "(Duraipandiyan, Ayyanar and Ignacimuthu, 2006; Rahman <i>et al.</i>, 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Duraipandiyan, Ayyanar and Ignacimuthu, 2006; Rahman et al., 2012). The bucolic population of Bangladesh still largely depends on the native system of medicine ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.17348/era.8.0.195-218", "ISSN" : "1547-3465", "abstract" : "An ethnomedicinal survey conducted in two sub-districts (upazillas) each of three districts of Bangladesh revealed that there is both inter- and intra-district variation between the choices of medicinal plants selected by folk medicinal healers ( kavirajes ) to treat any specific ailment. The variations cannot be accounted for by differences in climate or plant availability for most of the plants used by the  kavirajes  are available in all six upazillas where the survey was conducted. Most  kavirajes  claimed that they obtained information on medicinal plants either from \u2018gurus\u2019 or from elderly members of their family. However, it was also revealed by the  kavirajes  that further modes for acquiring medicinal plant knowledge were through initial experiments conducted on animals and poultry, dreams, and a perceived similarity between plants or plant parts and human body features. It is concluded that such selections are, and were, primarily made through initial experiments by traditional healers on animals and birds. Dreams and perceived similarities between plant parts and human body parts may also play an important role in such selection.", "author" : [ { "dropping-particle" : "", "family" : "Mollik", "given" : "Mohammed Ariful Haque", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hossan", "given" : "Mohammed Shahadat", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Paul", "given" : "Alok Kumar", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taufiq-Ur-Rahman", "given" : "M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jahan", "given" : "Rownak", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahmatullah", "given" : "Mohammed", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rahmatullah", "given" : "Mohammed", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Ethnobotany Research and Applications", "id" : "ITEM-1", "issue" : "0", "issued" : { "date-parts" : [ [ "2010", "7", "5" ] ] }, "page" : "195", "title" : "A Comparative Analysis of Medicinal Plants Used by Folk Medicinal Healers in Three Districts of Bangladesh and Inquiry as to Mode of Selection of Medicinal Plants", "type" : "article-journal", "volume" : "8" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=ab8408cf-0c99-3154-8da9-d057f690a986" ] } ], "mendeley" : { "formattedCitation" : "(Mollik <i>et al.</i>, 2010)", "plainTextFormattedCitation" : "(Mollik et al., 2010)", "previouslyFormattedCitation" : "(Mollik <i>et al.</i>, 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Mollik et al., 2010). In present study of thrombolysis, we investigated the different extracts of V. roxburghi in order to explore possible thrombolytic agents. The majority of the thrombolytic agents exert their useful outcome by activating the enzyme plasminogen, which solubilizes the cross-linked fibrin mesh to restore blood flow in blocked blood vessels ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1111/j.1365-2044.1982.tb00985.x", "ISSN" : "13652044", "abstract" : "The fibrinolytic effects of six different anaesthetic agents were studied in 36 patients undergoing abdominal hysterectomy. Fibrinolytic activity was measured by dilute whole blood and euglobulin clot lysis times. Concentrations of plasminogen, fibrinogen and fibrin degradation products were also determined. Significant enhancement of fibrinolytic activity, measured as a decrease in clot lysis time, was seen during anaesthesia and surgery with all agents studied. Halothane produced the least effect and the greatest increase in fibrinolytic activity was seen during trichloroethylene anaesthesia. Lumbar extradural anaesthesia also consistently produced enhancement of fibrinolysis. The reasons for these results and their clinical implications are discussed.", "author" : [ { "dropping-particle" : "", "family" : "SIMPSON", "given" : "P. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "RADFORD", "given" : "SHEILA G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "FORSTER", "given" : "S. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "COOPER", "given" : "GRISELDA M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "HUGHES", "given" : "A. O.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Anaesthesia", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "1982" ] ] }, "page" : "3-8", "title" : "The fibrinolytic effects of anaesthesia", "type" : "article-journal", "volume" : "37" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=f1771f5d-aa45-46f2-b6ba-56d76d34438a" ] } ], "mendeley" : { "formattedCitation" : "(SIMPSON <i>et al.</i>, 1982)", "plainTextFormattedCitation" : "(SIMPSON et al., 1982)", "previouslyFormattedCitation" : "(SIMPSON <i>et al.</i>, 1982)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(SIMPSON et al., 1982). Dissolution of clots, therefore, is useful for the treatment of clot-related disorders, including myocardial infarction, thromboembolic strokes, deep vein thrombosis, and pulmonary embolism, to clear a blocked artery that circumvents stable damage to the respective tissues ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/1472-6882-7-36", "ISBN" : "1472-6882 (Electronic)\\r1472-6882 (Linking)", "ISSN" : "1472-6882", "PMID" : "17986325", "abstract" : "Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components possessing antithrombotic activity have been reported before; however, herbs that could be used for thrombolysis has not been reported so far. This study's aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not. An in vitro thrombolytic model was used to check the clot lysis effect of six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control. Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively . Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%). Through our study it was found that Dhamasa possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component(s) of Dhamasa for clot lysis are yet to be discovered. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.", "author" : [ { "dropping-particle" : "", "family" : "Prasad", "given" : "Sweta", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kashyap", "given" : "Rajpal Singh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Deopujari", "given" : "Jayant Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Purohit", "given" : "Hemant J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taori", "given" : "Girdhar M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Daginawala", "given" : "Hatim F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BMC Complementary and Alternative Medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2007", "3", "1" ] ] }, "page" : "36", "publisher" : "BioMed Central", "title" : "Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis", "type" : "article-journal", "volume" : "7" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=163b5b84-709e-3338-8d60-7cd6b7407e27" ] } ], "mendeley" : { "formattedCitation" : "(Prasad <i>et al.</i>, 2007)", "plainTextFormattedCitation" : "(Prasad et al., 2007)", "previouslyFormattedCitation" : "(Prasad <i>et al.</i>, 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Prasad et al., 2007).  The comparison between positive and negative controls clearly showed that there was no clot dissolution when water (negative control) was added to the clot. SK, a known thrombolytic drug was used as a positive control. Water, on the other hand, was selected as a negative control. The comparison of positive control with negative control demonstrated that the clot dissolution does not occur when water was added to the clot. The percentage of clot lysis by both of these controls differ significantly as the p value was <0.0001 (test was extremely significant). When compared with the clot lysis percentage obtained through water, a significant thrombolytic activity was observed after treating the clots with V. roxburghi Chloroform extract (32.106�0.59%, P value <0.0001) and Petroleum Ether extract (30.72�0.55%, P value <0.0001), Methanol crude extract (21.56�0.77%, P value <0.0001) and Ethyl Acetate extract (19.25 � 0.57%, P value <0.0001) respectively. The lysis of clot by different extracts of V. roxburghi compared to the controls demonstrates its potential use in clot-related disorders.

Conclusion
It can be concluded that the extracts of all the above can be used to design different anti-thrombotic agents due to its moderate thrombolytic activity will have implication for treatment of cardiovascular health. Further work is needed to isolate the secondary metabolites and study thoroughly for more precise and accurate activity. 

References
ADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY Anwar, Mohammad; N Kumar, Santhosh/ ; Mahendran, B. (2013)  Hepatoprotective Activity of Pet-Ether Extract of Vanda , Internaitonal Ayurvedic Medical Journal, 1(3).
Basu, K. et al. (1971) �Anti-inflammatory principles of Vanda roxhburghii�, Cur Sci.
Duraipandiyan, V., Ayyanar, M. and Ignacimuthu, S. (2006) �Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India�, BMC Complementary and Alternative Medicine, 6(1), p. 35. doi: 10.1186/1472-6882-6-35.
Ghani, A. (1998) Medicinal plants of Bangladesh: chemical constituents and uses. Dhaka, Bangladesh: Asiatic Society of Bangladesh.
Lee, H. S. (1995) �How Safe is the Readministration of Streptokinase?�, Drug Safety, 13(2), pp. 76�80. doi: 10.2165/00002018-199513020-00002.
Mollik, M. A. H. et al. (2010) �A Comparative Analysis of Medicinal Plants Used by Folk Medicinal Healers in Three Districts of Bangladesh and Inquiry as to Mode of Selection of Medicinal Plants�, Ethnobotany Research and Applications, 8(0), p. 195. doi: 10.17348/era.8.0.195-218.
Mucklow, J. C. (1995) �Thrombolytic treatment. Streptokinase is more economical than alteplase.�, BMJ (Clinical research ed.). BMJ Publishing Group, 311(7018), p. 1506. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8520368 (Accessed: 22 August 2017).
Nayak, B. S. et al. (2005) �Evaluation of Wound Healing Activity of Vanda roxburghii R.Br(Orchidacea): A Preclinical Study in a Rat Model�, The International Journal of Lower Extremity Wounds, 4(4), pp. 200�204. doi: 10.1177/1534734605282994.
Prasad, S. et al. (2007) �Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis�, BMC Complementary and Alternative Medicine. BioMed Central, 7(1), p. 36. doi: 10.1186/1472-6882-7-36.
Rahman, M. A. et al. (2013) �Effects of organic extracts of six Bangladeshi plants on in vitro thrombolysis and cytotoxicity.�, BMC complementary and alternative medicine, 13, p. 25. doi: 10.1186/1472-6882-13-25.
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Rashid, U. et al. (2013) �Assessment of phytochemicals, antimicrobial and cytotoxic activities of extract and fractions from Fagonia olivieri (Zygophyllaceae)�, BMC Complementary and Alternative Medicine, 13(1), p. 167. doi: 10.1186/1472-6882-13-167.
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Competing interest
The authors declare that they have no competing interests.
Authors� contributions
M.R.R., T.I., S.A. carried out the experiment and statistical analysis. M.R.R. and T.I.  drafted the manuscript. MAH, SPS and MAHMJ participated in the design and coordination of the study and have been involved in revising the manuscript critically. All authors read and approved the final manuscript.



Table: Thrombolytic activity of different fractions of Vanda roxburghi
Sample/DrugsPercentage of clot lysis 
(Mean � SEM)P value when compared to
negative control (water)Streptokinase(Positive Control)55.11 � 0.06<0.0001Methanol Crude Extract21.57 � 0.77<0.0001Ethyl Acetate19.25 � 0.57<0.0001Chloroform32.1 � 0.59<0.0001Petroleum Ether30.72 � 0.55<0.0001Water(Negative Control)7.917 � 0.04-Note: Values are expressed as mean�SEM (n = 3). p < 0.05, significantly different from control.




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 PAGE   \* MERGEFORMAT 2



Positive 
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Extract

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