Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 2  |  Issue : 2  |  Page : 86-89

Protective effect of Euphorbia neriifolia extract on experimentally induced thrombosis in murine model


1 Drug Discovery Laboratory, Shri Ram Institute of Technology-Pharmacy, Jabalpur, Madhya Pradesh, India
2 Pharmaceutics Research Laboratory, Shri Ram Institute of Technology-Pharmacy, Jabalpur, Madhya Pradesh, India

Date of Web Publication17-Nov-2014

Correspondence Address:
Aditya Ganeshpurkar
Shri Ram Institute of Technology-Pharmacy, Jabalpur - 482 002, Madhya Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-0149.144842

Rights and Permissions
  Abstract 

Background: Thrombo-embolic disorders are one of the major causes of 'morbidities and mortality' worldwide. Antithrombotic therapy which comprise use of anti-platelet agents, fibrinolytic agents and anti-coagulants seem to be ineffective in the treatment of thrombi due to side effects and delayed onset of effects, along with chances of hemorrhage. Euphorbia neriifolia Linn is a large succulent shrub, with stipular thorns and is found throughout the Deccan peninsula of India. The plant is known for its medicinal value. A number of evidence about resins and flavonoids has demonstrated potent antithrombotic effects in vitro and in vivo due to prevention of the actions of 'cyclooxygenase and lipoxygenase pathways'. Aim: The present work was designed study antithrombotic potential of this E. neriifolia Linn. Materials and Methods: The whole plant of E. neriifolia Linn were shade dried at 37 o C to 40 o C and coarsely powdered through mesh 20. Powder was defatted with petroleum ether and extracted with ethanol by soxhlet apparatus and subjected to phytochemical analysis. Acute toxicity of extract was determined by Organization for Economic Cooperation and Development guideline No 423. Carrageenan-induced rat tail thrombosis model was used to study the anti-thrombotic effect by measuring thrombosis of tail and bleeding time as well as clotting time. Result: The extract was found to be rich in flavonoids, polyphenols, alkaloids, tannins and resins. Swelling and redness were observed within 2-3 h after intravenous injection of carrageenan, and the tail appeared auburn after 6 h, indicating that thrombosis had formed in the tail. Extract significantly (*P < 0.05, **P < 0.01) increased the bleeding and clotting time of the animals subjected to the test. Discussion and Conclusion: In the present work, phyto-constituents like flavonoids and polyphenols could be responsible to demonstrate protective effect on experimentally induced thrombosis in murine model.

Keywords: Anti-thrombotic, bleeding time, clotting time, Europhobia neriifolia, rat tail


How to cite this article:
Hasan M, Ganeshpurkar A, Bansal D, Dubey N. Protective effect of Euphorbia neriifolia extract on experimentally induced thrombosis in murine model . Niger J Exp Clin Biosci 2014;2:86-9

How to cite this URL:
Hasan M, Ganeshpurkar A, Bansal D, Dubey N. Protective effect of Euphorbia neriifolia extract on experimentally induced thrombosis in murine model . Niger J Exp Clin Biosci [serial online] 2014 [cited 2017 Apr 28];2:86-9. Available from: http://www.njecbonline.org/text.asp?2014/2/2/86/144842


  Introduction Top


Thrombo-embolic disorders are one of the major causes of 'morbidity and mortality' worldwide. Thrombosis is the result of solid mass formation within the circulation due to accumulation of flowing blood constituents. The mass thus formed is termed 'thrombus'. Hemostatic plugs and clots of blood produced in healthy person during bleeding aid in preventing loss of blood and plasma. However, thrombi, formed in 'unruptured' blood vessel could be fatal. [1]

Changes in 'vascular endothelium', triggering of 'coagulation system and/ or reduction in blood flow' are the three prime episodes that may lead to thrombus formation. [2] Thrombus is composed of fibrin, platelets and red blood corpuscles. Arterial thrombi are evident after 'endothelial injury' owing to hastily flowing blood. Venous thrombus is the result of blood blockade in veins. Antithrombotic therapy comprise of use of anti-platelet agents, fibrinolytic agents and anti-coagulants. [3],[4] These drugs are effective in treating thrombi; however, side effects, delayed onset of effects and chances of hemorrhage are few of the untoward effects required to be considered. [5]

Euphorbia neriifolia Linn (Hindi: Sehund; Family: Euphorbiaceae) is a large succulent shrub, with stipular thorns and is found throughout the Deccan peninsula of India. The plant is known for its medicinal value, such as antibacterial, antifungal, antiviral, antiparasitic, antiarthritic, antidiabetic, anticonvulsant, antioxidant, wound healing and immuno-modulatory, radioprotective, spasmodic, aphrodisiac, anticancer and purgative properties. [6] Such properties are attributed due to the presence of phytoconstituents like lectin, quercetin, saponin, flavonoids, triterpenes, diterpenes, anthocyanins, etc. [7]

Resins and flavonoids are two of the phytoconstituents which are known to exert antithrombotic effect due to scavenging of free radicals which aids in regulating the necessary levels of 'endothelial prostacyclin and nitric oxide'. [8] Scientific evidence about resins and flavonoids have demonstrated their potent antithrombotic effects in vitro and in vivo which is due to prevention of the actions of 'cyclooxygenase and lipoxygenase pathways'. [9],[10],[11] Key antiaggregatory effect of resins and flavonoids is due to inhibition of thromboxane A formation. [12],[13],[14],[15] In view of these facts, the present work was designed to investigate the antithrombotic potential of E. neriifolia Linn.

Experimental

Plant collection and authentication


The whole plant of E. neriifolia was collected in the month of November from hilly grounds near Tilwaraghat, Narmada River Bank, Jabalpur (M.P.). The whole plant was identified and authenticated by Dr AB Tiwari Sr. Scientist, of Department of Crop and Herbal Physiology, Jawaharlal Nehru Krishi Vishwavidyalaya Jabalpur (M.P.).

Chemicals

Bulk solvents like petroleum ether (60-80 o C), Ethyl alcohol, and routine chemicals were obtained from Central Drug House, India. All chemicals used were of analytical grade.

Extraction procedure

Roots and leaves of E. neriifolia Linn were shade dried at 37 o C to 40 o C and coarsely powdered through mesh 20. Powder was defatted with petroleum ether and extracted with ethanol by soxhlet apparatus. The extract was dried at 40°C, and stored properly.

Phytochemical analysis

Phytochemical analysis of extract was done by standard method. [16]

Animals

Wistar rats weighing 200-250 g were used in the study. Animals were maintained in rooms with a 12-h light/dark cycle at 22 ± 3 o C. All animals were given tap water to drink and were maintained on a normal pellet diet for at least 2 weeks before the experiments. All the experiments were approved by Institutional Animal Ethics Committee.

Acute toxicity studies

Extracts of EN (E. neriifolia) were studied for acute oral toxicity as per revised OECD (Organization for Economic Cooperation and Development) guidelines No. 423. [17] Extracts were devoid of any toxicity in rats when given in doses up to 2000 mg/kg by oral route. Hence a dose of 200 mg/kg and 400 mg/kg were used for this study.

Carrageenan-induced rat tail thrombosis

The antithrombotic activity of Euphorbia neriifolia extract in carrageenan-induced rat tail thrombosis model was carried out. Animals were divided into five groups of four animals each whose tails were of above 13-cm length. [18] Carrageenan was dissolved in saline and injected into the dorsal tail vein in all animals at a dose of 2 mg/kg. The tails were then ligated in a region of about 13 cm from tail tip.

Animal grouping

Group-A: Saline (2 ml/kg)

Group-B: Carrageenan (2 mg/kg)

Group-C: Carrageenan (2 mg/kg) + Heparin (100 IU)

Group-D:

Carrageenan (2 mg/kg) + Euphorbia neriifolia extract (200 mg/kg)

Group-E:

Carrageenan (2 mg/kg) + Euphorbia neriifolia extract (400 mg/kg)

After 10 minutes, heparin was administered in Group C at a dose of 300 IU/kg, E. neriifolia extract was administered at a dose of 200 and 400 mg/kg of body weight in Group D and Group E, respectively. The frequency of infarction and the length of the infarcted region of the tail tip were recorded at 6, 24 and 48 h after the carrageenan injection. The control group was administered only saline.

Evaluation of thrombosis and antithrombotic activity

Thrombosis of tail

Rat's tails were observed for a time period of 6 h, 12 h, 24 h, 48 h and 72 h for redness and swelling. [19]

Bleeding and clotting time

Bleeding time and clotting time [19] were determined by reported method as follows.

Bleeding time

Animal was kept in a restrainer. Tail was passed through one end of the opening. Tail of the animal was massaged at the part where the tail joins the pelvis. The tail was disinfected with spirit, dried and pricked with a sterile needle about 4-mm deep. The oozing blood was wiped with a filter paper at 15-sec intervals. The exercise was repeated every 15 seconds on fresh spots of the filter paper until the bleeding stopped. Time was recorded by stop-watch.

Clotting time

Animal was kept in a restrainer. Tail was passed through one end of the opening. Tail of the animal was massaged at the part where the tail joins the pelvis. The tail was disinfected with spirit, dried and pricked with a sterile needle about 4-mm deep. A blood drop was kept in the middle of the slide. Blood drop was gradually lifted up at 15 second intervals by use of a pin. This was continued until coagulation was noticed. The time for the coagulation (in seconds) was noted.

Statistical analysis

The results of study are expressed as mean ± standard error of mean (n = 5). Statistical comparison was performed using analysis of variance (ANOVA) followed by Dunnett test. Significance was set at P < 0.05 and P < 0.01.


  Results Top


Phytochemical Studies

The yield of the extract was 5% w/w of the powdered plant material. The extract was found to be rich in flavonoids, polyphenols, alkaloids, tannins and resins.

Thrombosis of Tail

Swelling and redness were observed within 2-3 h after intravenous injection of carrageenan, and the tail appeared auburn after 6 h, indicating that thrombosis had formed in the tail. The mean length of the infarcted region in the rat tail of the responded rats was 10.1 ± 0.6 cm in 200 mg/kg extract treated rats, 9.2 ± 0.3 cm in 400 mg/kg extract treated rats and 6.5 ± 0.7 cm in heparin-treated rats. There was an infarction of about 11.1 ± 0.3 cm in saline-treated group. No infarction was observed in the control group [Figure 1] and [Figure 2].
Figure 1: Evidence of protective effect of E. neriifolia extract on progression of rat tail thrombosis

Click here to view
Figure 2: Effect of E. neriifolia extract on progression of rat tail thrombosis. Results are given as mean ± SEM of four animals in each group. Standard treated group compared with extract treated groups. Significance at *P < 0.05; ** P < 0.01

Click here to view


Bleeding Time and Clotting Time

It was observed by Ivy's bleeding time method and plain slide technique for clotting time that the EN extract significantly (*P < 0.05, ** P < 0.01) increased the bleeding and clotting time of the animals subjected to the test [Figure 3].
Figure 3: Effect of E. neriifolia extract on bleeding time and clotting time. Results are given as mean ± SEM of four animals in each group. Standard treated group compared with extract treated groups. Significance at * P < 0.05; ** P < 0.01

Click here to view



  Discussion Top


Cardiovascular diseases are 'chronic diseases' widespread throughout the globe. A number of antithrombotic drugs are available to treat thrombotic diseases; however, the major drawback being the cost and various side effects. [20] Thus it becomes an urgent issue to explore safe and sound, reasonably priced and naturally occurring molecules, preferably from plant source.

In the present work, assessment of rat tail thrombosis was measured by measuring the redness and swelling caused by carrageenan at a dose of 2 mg/kg that tends to damage walls of the caudal vein of rat tail. It was observed that the extract of E. neriifolia at a dose of 400 mg/kg significantly (**P < 0.01) reduced rat tail thrombosis.

'Swelling' and 'redness' are two of the considerations that are observed during the progression of thrombosis all along the rat tail length. Group B animals in which only carrageenan was administered, showed a high degree of thrombosis as there was only carrageenan to cause thrombosis for long duration and for longer extent. In Group C where heparin was administered, There was profound antithrombotic effect than extract treated groups, while the animals which were administered with the ethanol extract also showed a reduced length of redness.

Based on the result obtained, as well as from further statistical evaluation, the ethanolic plant extract of E. neriifolia demonstrated its ability to influence the hematological indices of bleeding time as well as clotting time, when compared with values obtained from rats treated with the control group normal saline and heparin.

In bleeding time studies, doses of 200 mg/kg and 400 mg/kg of the extract of E. neriifolia caused significant (**P < 0.05) increase in the time required to stop bleeding when compared with the values obtained from treatment with the negative control-normal saline. The prolongation in the bleeding time proves to be dose dependent. Similar results were observed with clotting time.

Thus, when compared statistically, there was a significant difference.

Polyphenols and flavonoids are naturally occurring compounds from plants. A number of flavonoids are known to contribute to antithrombotic activity. [18],[19],[20],[21] In the present work, these constituents might be responsible for such effects. In nut shell, it can be concluded that E. neriifolia which is rich source of phytoconstituents like resins, flavonoids and polyphenols demonstrated protective effect on experimentally induced thrombosis in murine model. However, more systematic studies on each phytoconstituents seem to be necessary.

 
  References Top

1.
Sikka P, Bindra VK. Newer antithrombotic drugs. Indian J Crit Care Med 2010;14:188-95.  Back to cited text no. 1
[PUBMED]  Medknow Journal  
2.
del Zoppo GJ. Virchow's triad: The vascular basis of cerebral injury. Rev Neurol Dis 2008;5 Suppl 1:S12-21.  Back to cited text no. 2
[PUBMED]    
3.
Becker RC, Meade TW, Berger PB, Ezekowitz M, O'Connor CM, Vorchheimer DA, et al. The primary and secondary prevention of coronary artery disease: American College of Chest Physicians Evidence Based Clinical Practice Guidelines (8th Edition). Chest 2008;133:776-814S.  Back to cited text no. 3
    
4.
Kearon C, Kahn SR, Agnelli G, Goldhaber S, Raskob GE, Comerota AJ, et al. Antithrombotic therapy for venous thromboembolic disease: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008;133:454-545S.  Back to cited text no. 4
    
5.
Sikka P, Bindra VK. Emerging antithrombotic drugs: A review. Ann Trop Med Public Health 2011;4:138-42.  Back to cited text no. 5
  Medknow Journal  
6.
Pracheta P, Sharma V, Singh L, Paliwal R, Sharma S, Yadav S, et al. Chemopreventive effect of hydroethanolic extract of Euphorbia neriifolia leaves against DENA-induced renal carcinogenesis in mice. Asian Pac J Cancer Prev 2011;12:677-83.  Back to cited text no. 6
    
7.
Norskhydro AS, Kiellant S. W.O. 9321925 (ICI A61K 31/56) 1993;l:921666.  Back to cited text no. 7
    
8.
Osman HE, Maalej N, Shanmuganayagam D, Folts JD. Grape juice but not orange or grapefruit juice inhibits platelet activity in dogs and monkeys. J Nutr 1998;128:2307-12.  Back to cited text no. 8
    
9.
Gryglewski RJ, Korbut R, Robak J, Swies J. On the mechanism of antithrombotic action of flavonoids. Biochem Pharmacol 1987;36:317-22.  Back to cited text no. 9
[PUBMED]    
10.
Alcaraz MJ, Ferrándiz ML. Modification of arachidonic metabolism by flavonoids. J Ethnopharmacol 1987;21:209-29.  Back to cited text no. 10
    
11.
Carrieri C, Milella RA, Incampo F, Crupi P, Antonacci D, Semeraro N, et al. Antithrombotic activity of 12 table grape varieties. Relationship with polyphenolic profile. Food Chem 2013;140:647-53.  Back to cited text no. 11
    
12.
Mower RL, Landolfi R, Steiner M. Inhibition in vitro of platelet aggregation and arachidonic acid metabolism by flavone. Biochem Pharmacol 1984;33:357-63.  Back to cited text no. 12
[PUBMED]    
13.
Harborne JB. Phytochemical method: A guide to modern techniques of plants analysis. New York: Chapman and Hall, 1983; p. 16-30.  Back to cited text no. 13
    
14.
Organization for Economic Cooperation and development (OECD). Guideline 423 for testing chemicals: Paris; 2001. p. 1-14.  Back to cited text no. 14
    
15.
Bekemeier H, Hirschelmann R, Giessler AJ. Carrageenan-induced thrombosis in rats and mice: A model for testing antithrombotic substances? Agents Actions 1985;16:446-51.  Back to cited text no. 15
[PUBMED]    
16.
Ivy AC, Welson D, Bucher G. The standardisation of certain factors in the cutaneous. Venostasis bleeding time technique. J Lab Clin Med 1989;26:1812.  Back to cited text no. 16
    
17.
Dub AM, Dugani AM. Antithrombotic effect of repeated doses of the ethanolic extract of local olive (Olea europaea L.) leaves in rabbits. Libyan J Med 2013;8:20947.  Back to cited text no. 17
    
18.
Kang WS, Lim IH, Yuk DY, Chung KH, Park JB, Yoo HS, et al. Antithrombotic activities of green tea catechins and (-)-epigallocatechin gallate. Thromb Res 1999;96:229-37.  Back to cited text no. 18
    
19.
Singh I, Mok M, Christensen AM, Turner AH, Hawley JA. The effects of polyphenols in olive leaves on platelet function. Nutr Metab Cardiovasc Dis 2008;18:127-32.  Back to cited text no. 19
    
20.
Léger CL, Carbonneau MA, Michel F, Mas E, Monnier L, Cristol JP, et al. A thromboxane effect of a hydroxytyrosol-rich olive oil wastewater extract in patients with uncomplicated type I diabetes. Eur J Clin Nutr 2005;59:727-30.  Back to cited text no. 20
    
21.
Yang SA, Im NK, Ji YJ, Yoo DC, Jhee KH, Lee IS. Radical scavenging and inhibition of platelet function by a polyphenol-rich fraction from Salvia miltiorrhiza Bunge. Open Nat Prod J 2008;1:7-13.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
   Abstract
  Introduction
  Results
  Discussion
   References
   Article Figures

 Article Access Statistics
    Viewed1130    
    Printed67    
    Emailed0    
    PDF Downloaded175    
    Comments [Add]    

Recommend this journal