In Vivo Hemostatic Activity Screening of the Decoction of the Peel of Musa errans (Blco.) Teod. Var. Botoan Teod. on Sprague-Dawley Rats

Transcription

1 In Vivo Hemostatic Activity Screening of the Decoction of the Peel of Musa errans (Blco.) Teod. Var. Botoan Teod. on Sprague-Dawley Rats Florano, Solmuell M. Sigua, Patricia Marie D. Tabi, Maria Jo Fatima J. Trinidad, Aleanor Josyl C. Vergara, Ina Marie Angela M.

2 Circulatory System Organ system that permits blood and lymph movement to the different parts of the body.

3 Circulatory System Transports gases, nutrients, waste materials Regulates internal temperature and several physiologic functions Protects against microbes and/or toxins.

4 Bleeding Tendencies In general, bleeding tendencies occur when there are pathologic conditions that affect: platelet adhesion to damaged vascular walls and aggregation of platelets to one another, the coagulation factor cascade and its fibrinolytic counterpart pathways fragility of small vessel walls

5 Disorders/Events Which May Cause Bleeding Dengue Hemorrhagic Shock Hemorrhage Surgery Trauma to blood vessels

6 Hemostasis Hemostasis is the biological process by which the body compensates to an injury to attain homeostasis.

7 Hemostasis Vasoconstriction Platelet Plug Formation Blood Coagulation

8 Vasoconstriction Reduction of blood flow

9 Platelet Plug Formation

10 Blood Coagulation Fibrin Clot Stabilize the clot

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12 Uncontrolled hemorrhage accounts for over 35% of pre-hospital deaths. Excessive bleeding leads to impaired resuscitation, shock and coagulopathy. Low blood pressure is an effect of severe bleeding which may cause immediate multiple organ failure and lifethreatening complications.

13 According to the World Health Organization, 80% of the total population depends on traditional medicine. 85% of medicines used nowadays are derived from plants.

14 MUSACEAE 40 species Musa Ensente An article by Benett (2007) listed Musaceae as one of the most economically important plant families.

15 Musaceae Musa sapientum Musa paradisiaca Hemostatic activity (Dougnon et. al., 2012) Antibacterial and antioxidant (Sahaa et al., 2013) Analgesic activity (Gangwaret al., 2012) Hepatoprotective activity (Dikshitet al., 2001) Anti-ulcer activity (Prabhaet al., 2011) Hemostatic activity (Weremfo et. al., 2011) Promotes wound healing and skeletal muscle contraction (Kumar et al., 2012) Hepatoprotective and anti-ulcer activity. (Kumar et al., 2012).

16 Plants under the same family tend to share the same characteristics and properties Rollins, as cited by Weber (2001)

17 Musa errans Endemic in the Philippines

18 Musa errans Traditional Uses: Leaves for chest pains (topical) Juice of corms as anti-tubercular agents Sap used for wound healing and to treat gonorrhea Quisumbing, 1978

19 Statement of the Problem This study aims to determine the hemostatic potential of the peel of Musa errans.

20 RESEARCH METHODS The researchers used Experimental Study as their research design.

21 Plant Material Procedure Plant Material Collection The plant material (unripe fruit) was collected from Malolos, Bulacan. Plant Authentication The fruit was authenticated by the National Museum. Plant Preparation The unripe peels were washed with distilled water, decocted and lyophilized.

22 Plant Preparation Preparation Decoction Lyophilization The unripe peels were washed with distilled water and cut into smaller pieces. The peels were decocted for two hours at boiling temperature with a peel-water ratio of 1kg : 7.2 L of distilled water. The decoction was lyophilized at De La Salle Chemistry Laboratory, Taft, Manila.

23 Phytochemical Test The extract was tested for the presence of tannins using the Gold-Beater s Test.

24 Acute Toxicity Test Limit Test Administer 2000mg/kg BW to 1 rat Death of Animal Survival of Animal Main Test Administer dose to 4 test animals Death (3 or more rat) Main Test Survival (3 or more rats) LD50>2000 mg/kg

25 Acute Toxicity Test Main Test: Administer 175mg/kg of extract to 1 rat Died Survived Decrease dose by a progression of 3.2 Increase dose by a progression of 3.2

26 Pharmacological Test Baseline Determination Administration of Extract Post Treatment Data Collection

27 Baseline Determination Blood Collection Bleeding Time Clotting Time Prothrombin Time Activated Partial Thromboplastin Time

28 Determination of Bleeding Time The Modified Duke s Method was done to determine the rat s bleeding time.

29 Determination of Clotting Time The Slide Method was done to determine the rat s clotting time

30 Determination of Prothrombin Time Sufficient amount of blood was collected using the Tail Tip method in order to determine the Prothrombin Time.

31 Determination of Activated Partial Thromboplastin Time Sufficient amount of blood was collected using the Tail Tip method in order to determine the Activated Partial Thromboplastin Time.

32 Administration of Extract 5 rats/group Negative Control Distilled Water Musa Musa Musa errans errans errans Decoction Decoction (MED 2) Decoction mg/kg mg/kg (MED 1) 100 mg/kg (MED 3)

33 Administration of Extract Route of Administration: Oral (via oral gavage) Duration of Administration: Ten (10) days Dose Administered: determined through the Log Dose Interval Method

34 Administration of Extract

35 Post Treatment Data Collection Blood Collection Bleeding Time Clotting Time Prothrombin Time Activated Partial Thromboplastin Time

36 Statistical Analysis The mean and standard deviation of the different groups were calculated. The data of each group for the pre and post administration were compared using the Paired T-Test. Tukey s Post hoc test was used to see if there was a statistical difference among the hemostatic effects of the different dose levels.

37 Results and Discussion

38 Test for Tannins Goldbeater s Skin Test Pig intestinal membrane before (left) and after (right) the Gold eater s skin test

39 Goldbeater s Skin Test The Gold eater s skin test done using the decocted extract of Musa errans revealed a rownish stain in the pig s skin indi ating the presence of tannins. Tannins contain hemostatic properties. (Rangari, 2007)

40 Acute Toxicity Test The Median Lethal Dose (LD50) of the plant material is greater than 2000 mg/kg since no subject animal died during the treatment period.

41 Comparison between Pre and Post Administration Data of Extract Bleeding Time Mean 350 Bleeding 300 Time in 250 seconds % reduction % reduction 78% reduction Negative control MED 1 (100mg/kg) pre administration MED 2 (199.53mg/kg) negative control MED 3 post administration

42 Comparison between Pre and Post Administration Data of Extract Clotting Time % reduction 80 Mean Clotting Time in seconds 7% reduction 14% reduction Negative control pre administration MED 1 (100mg/kg) MED 2 (199.53mg/kg) negative control MED 3 (398.11mg/kg) post administration

43 Comparison between Pre and Post Administration Data of Extract Prothrombin Time Mean PT in seconds % reduction 20 54% reduction 54% reduction Negative control MED 1 (100mg/kg) pre administration MED 2 (199.53mg/kg) negative control MED 3 (398.11mg/kg) post administration

44 Comparison between Pre and Post Administration Data of Extract Activated Partial Thromboplastin Time Mean aptt in second s % reduction 66% reduction 15 66% reduction Negative control MED 1 (100mg/kg) pre administration MED 2 (199.53mg/kg) negative control MED 3 (398.11mg/kg) post administration

45 Statistical Analysis Result Pre administration data of extract were significantly different from the Post administration data extract after the ten-day administration Therefore, it has been proven that the Musa errans contain hemostatic activity, not only affecting the platelets, but also affecting the clotting factors.

46 Statistical Analysis Result No significant difference observed in the three treatment groups given with different doses of the decoction Therefore, hemostatic activity of the Musa errans is dose-independent showing that even at 100 mg/kg concentration the hemostatic effect is still observed and is the same as with that of mg/kg concentration.

47 Conclusion The aforementioned results of the investigation proved that the decoction of the peel of Musa errans exhibits dose-independent hemostatic activity. The hemostatic potential it conferred was reflected by the significant decrease in: Bleeding time Clotting time Prothrombin Time; and Activated Partial Thromboplastin Time

48 Conclusion The mechanism of the hemostatic action of the unripe peel of Musa errans is due to its ability to affect platelet action, and both the extrinsic and intrinsic pathways of coagulation. The presence of tannins may also play a part in its hemostatic activity since tannins can precipitate protein and a study conducted by Dougnon et al (2012), stated the role of tannins on platelet aggregation.

49 Recommendation With the observations of the hemostatic activity of the decoction of Musa errans, the group recommends the following for future researchers: 1. Conduct more hematological and biochemical parameters of hemostasis such as Platelet count and Milk Precipitation of Milk test (for astringent property); 2. Use human blood sample to determine the effects of the plant material on human subjects;

50 Recommendation 3. Employ animals and methods specific to a disease model in which the control of bleeding is of primary importance; and 4. Compare the hemostatic activity other Musa species to Musa errans to determine the best source of extract for the development of a hemostatic agent.

51 References Atzingen, Gragnani, Veiga et al. (2011). Unripe Musa sapientum peel in the healing of surgical wounds in rats. Retrieved from: accessed on October 2, 2013 Weremfo, M.B. Adinortey and A.N.M. Pappoe(2011) Haemostatic Effect of the Stem Juice of Musa paradisiaca L. (Musaceae) in Guinea Pigs Retrieved from: accessed on October 2, 2013 Bamidele, O., et. al. (2010). Hemostatic effect of the methanolic leaf extract of Ageratum conyzoides in albino rats. Journal of Medicinal Plants Research Vol. 4(20), pp , 18 October, Retrieved from pdf accessed on October 10, B.C. Bennett(2007).Twenty five economically important plant Families. Retrieved from: accessed on October 10, 2013

52 References Bleeding time (2006) In Gale Encyclopedia of Medicine. Retrieved from accessed on October 9, 2013 Chua, J.E. et al (2007). A triterpene from Musa errans. Retrieved from: ans.pdf accessed on October 9, 2013 Daswani, P.G.(2007). Preparation of Decoction of Medicinal Plants: A Self-Help Measure?. Retreved from: accessed on: October 11, 2013 Eversole, L. Bleeding Disorders retrieved from accessed on October 9, 2013

53 References Atzingen, Gragnani, Veiga et al. (2011). Unripe Musa sapientum peel in the healing of surgical wounds in rats. Retrieved from: accessed on October 2, 2013 Weremfo, M.B. Adinortey and A.N.M. Pappoe(2011) Haemostatic Effect of the Stem Juice of Musa paradisiaca L. (Musaceae) in Guinea Pigs Retrieved from: accessed on October 2, 2013 Bamidele, O., et. al. (2010). Hemostatic effect of the methanolic leaf extract of Ageratum conyzoides in albino rats. Journal of Medicinal Plants Research Vol. 4(20), pp , 18 October, Retrieved from pdf accessed on October 10, B.C. Bennett(2007).Twenty five economically important plant Families. Retrieved from: accessed on October 10, 2013

54 References General Principles of Freeze Drying. Retrieved from: Accessed on: October 13, 2013 Hawiger, J (1987). Formation and regulation of platelet and fibrin hemostatic plug Retrieved from: accessed on October 10, 2013 Hemorrhage.(nd).In National Trauma Institute. Retreieved from: accessed on October 5, 2013 Henry, J. B. (1996) Clinical Diagnosis and Management by Laboratory Methods. Philadelphia: W. B. Saunders Co., Klotoé JR et al (2012) Hemostatic potential of the sap of Musa sapientum L. (Musaceae) Retrieved from: accessed on October 9, 2013

55 References Lino, P., Correa, C., Archondo, M., Dellova, D. (2011). Evaluation of post-surgical healing in rats using a topical preparation based on extract of Musa sapientum L., Musaceae, epicarp. Revista Brasileira de Farmacognosia, 21(3), Epub April 01, Retrieved from /S X accessed on October 10, Nieuwenhuis, H. et al (2011). Hemostasis and Thrombosis: Basic Principles and Clinical Practice Retrieved from: q=nieuwenhuis+2011+bleeding+time&source=bl&ots=jcpqldc68&sig=g5ty5wfbrqybtuol1gvlm6cp720&hl=en&sa=x&ei=p-xauv66kcmziaehyhydg&ved=0ceuq6aewba#v=onepage&q=nieuwenhuis%202011%20bleeding%2 0time&f=false accessed on October 8, 2013

56 References Pal, G. & Pal, P. (2001) Bleeding Disorders. Retrieved from: accessed on October 9, 2013 Patel, S.M., & PIkal, M.J. (2013) Lyophilization Process Design Space. Retrieved from: accessed on October 10, 2013 Okon, J. E.; Esenowo, G. J.Afaha, I. P. and Umoh, N. S. (2012).Haematopoietic Properties of Ethanolic Fruit Extract of Musa Acuminata on Albino Rats. Retrieved from: Quisumbing (1919). Studies of Philippine bananas. Philippine Agricultural Review 12: Rodeghiro, F. (1992) The bleeding time in normal subjects is mainly determined by platelet von Willebrand factor and is independent from blood group. Retrieved from: accessd on October 9, 2013

57 References Seeley, R., Stephens, T., and Tate, P. (2007) Anatomy and Physiology. McGraw-Hill Higher Education Tan (1980) Philippine Medicinal Plants in Common Use Retrieved from: accessed on october 10, 2013 Traditional Medicine (2008). Retrived from: Teodoro (1915). A preliminary study of Philippine bananas. Philippine Journal of Science 10, section c (Botany): pls.