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Comparative Evaluation of Platelet Augmentation Activity of Carica Papaya Leaf Juice and Ydrocortisone in Thrombocytopenic Rats

TANSENA AKHTER
Assistant Professor, Department of Pharmacology, Tairunnessa Memorial Medical College, Gazipur, Bangladesh

MD. ISMAIL KHAN
Professor and Head, Department of Pharmacology, Principal, Dhaka Medical College, Dhaka, Bangladesh.

ELIZA OMAR EVA
Department of Pharmacology, Dhaka Medical College, Dhaka, Bangladesh.

Abstract/ Executive Summary:

Aim The study aims at determining the possible effects of Carica papaya leaf juice and hydrocortisone in increasing the platelet count in thrombocytopenia similar to dengue fever. Methodology The study was carried out on 24 Long Evans Norwegian strain healthy rats to investigate the comparative platelet increasing effect of Carica papaya leaf juice and hydrocortisone in experimentally induced thrombocytopenic rats. Thrombocytopenia was induced by 3 doses of cyclophosphamide (100mg/kg body weight) given subcutaneously. The rats were divided into 4 groups comprising of six animals in each group. These groups included a normal control and a cyclophosphamide induced thrombocytopenic control and the remaining two cyclophosphamide induced thrombocytopenic groups were administered Carica papaya leaf juice and hydrocortisone respectively. The duration of study was 15 days. The clotting time was determined on the 15th day although blood samples were obtained from the rat tails at various time intervals to determine the platelet count. Collected data was tabulated and Statistical analysis was performed by using appropriate significant test. A probability value P < 0.05 was considered to be statistically significant. Result Average platelet counts of the test and control groups were (6.89)×10 5 /µL and (6.75)×10 5 /µL respectively before the experiment. In the experiment there was no significant difference in the platelet counts during the first 3 days in either group. However, platelet counts within the test group started to fall after Day 3 and remarkable thrombocytopenia developed after 7days and the average value was (2.68)×10 5 /µL. From day 11 Carica papaya leaf juice and Hydrocortisone was given in two test groups following cyclophosphamide and platelet counts observed to reach a peak level at Day 14 (7.83)×10 5 /µL in case of following Carica papaya leaf juice, and (4.05)×10 5 /µL in case of following Hydrocortisone. The clotting time was determined on day 15 and it was found to be considerably lower (95.5 ± 2.762 sec) in case of Carica papaya leaf juice than (129±3.124 sec) in Hydrocortisone. Conclusion Carica papaya leaf juice has more platelet increasing effect than hydrocortisone and this study also reflects that Carica papaya leaf juice may be a potential candidate for further research leading to the development of a herbal therapeutic agent for thrombocytopenia manifested in diseases such as dengue.

Keywords: Thrombocytopenia, Platelet, Carica papaya leaf juice, Hydrocortisone, Cyclophosphamide, Dengue fever.

Location: Department of Pharmacology of Dhaka Medical College, Dhaka

Start Date: 00-01-2013

End Date: 00-12-2013

Program Area: Resource Development and Management

Commodity: Papaya

Objectives:

In the current study, the competitive ability of Carica papaya leaf juice and hydrocortisone in increasing the platelet count in cyclophosphamide induced thrombocytopenic rats were evaluated. The clinical usage of Carica papaya leaves in dengue fever patient should be evaluated in large comparative trial.

Methodology:

Experimental Animals From January 2013 to December 2013 an experimental study in animals (rats) carried out in the Department of Pharmacology of Dhaka Medical College, Dhaka. The experiment was carried out on a total number of 24 healthy rats of Long Evans Norwegian strain weighing between 150-200 gm and age between 8-10 weeks. These rats were collected from Bangladesh Council of Scientific and Industrial Research (BCSIR) after proper authentication. They were kept in metallic cages (6 rats in each cage) in the animal house of department of pharmacology at Dhaka Medical College. The animals were allowed to live at room temperature, given standard rat foods and allowed to drink water. Proper cleaning measures were taken regularly. Preparation and Dosage of Carica papaya Leaf Juice Fresh, middle stage age, Carica papaya leaves were picked daily for 3 days. For the purpose of the study, a private plantation was identified to provide the leaves for the entire duration of the study.The plant was authenticated as Carica papaya by National Herbarium, Mirpur Dhaka. DACB. Accession number is 39501. The trees in this plantation were kept free of herbicides, pesticides, and insecticides. Juice was prepared fresh from the leaves that were washed thoroughly. The veins and the petiole of the Carica papaya leaves selected for the tests was removed and Juice was extracted from fresh leaves using a juice extractor without any addition of water, under sterile conditions and the mucilaginous (with mucus) juice concentrate manually was separated (like squeezing coconut milk) from the leaf-residue.17 Then the mixture was filtered to obtain a pure extract of Carica papaya leaves. Finally, the volume of the extract was measured and the extracts were stored at 4°C until use and administered orally 2ml/day per rat.18 Preparation and Dosage of Hydrocortisone 100mg Hydrocortisone vial was dissolved in 20ml distilled water and 0.1ml of that solution (containing 0.5mg Hydrocortisone) was given subcutaneously daily to each rat.19 Establishment of Thrombocytopenia in the Rat Model Induction of thrombocytopenia was performed by the subcutaneous administration of freshly prepared Cyclophosphamide. Each rat received 100mg/kg body weight dose of Cyclophosphamide. It was dissolved with normal saline and administered by subcutaneous injection once a day for consecutive 3 days. Then it successfully initiated thrombocytopenia as the platelets number dropped at the 7th day.16 Experimental Procedure Experiment was comprised of 24 rats which were divided into 4 groups each having 6 rats. Groups were labeled as Group-A, Group–B, Group-C, and Group-D. It was carried out to demonstrate the effect of Carica papaya leaf juice in group C and Hydrocortisone in group D in increasing platelet count following cyclophosphamide induced thrombocytopenia and compare the platelet increasing effect between group C and group D. Group A (Control group): Rats (n=6) received normal saline (10ml/kg p.o) for a period of 15days.20 Group-B (Only Cyclophosphamide group): Rats (n=6) received Cyclophosphamide 100g/kg s/c for 1st 3days16 and after that they received normal saline up to 15 days.20 Group-C (Group for Papaya leaf juice): Rats(n=6) received Cyclophosphamide 100g/kg s/c for 1st 3days and since day 4 they received normal saline (10 ml/kg p.o) up to day 10 and observed for development of thrombocytopenia. From day 11 they received Papaya leaf juice 2ml/day orally through ryles tube for next 3days.16,18 Group-D (Group for Hydrocortisone): Rats (n=6) received Cyclophosphamide 100mg/kg s/c for 1st 3days and since day 4 they received normal saline (10 ml/kg p.o) up to day 10 and observed for development of thrombocytopenia and from day 11 they received inj. hydrocortisone 0.5mg per day s/c for 3days.16,19 Blood collection Blood was collected from each of the rats by aseptically cutting the tail at the tip with a sharp sterile blade on the 1st, 4th, 11th and 14th day of study. On the 15th day, the clotting time of blood was determined by capillary method.20 Platelet Examination Induction of thrombocytopenia was established by determining platelet counts with the use of Neubauer’s improved Haemocytometer according to standard protocols. Briefly, blood obtained from the tail bleed of rats was added to EDTA containing tubes. Twenty microliters of blood was mixed thoroughly with 0.38 ml of diluting fluid (1% ammonium oxalate freshly prepared) for at least 10 minutes. The haemocytometer filled with this mixture was incubated in a moist chamber for 10-20 minutes to allow the platelets to settle down. Round to oval shaped platelets were counted in the triple laminated middle 25 squares of the haemocytometer, using oil immersion light microscopy.

Source of Information: Bangladesh J Physiol Pharmacol 2014;30(2):32-40

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Achievement/Findings:

Carica papaya leaf juice has more platelet increasing effect than hydrocortisone and this study also reflects that Carica papaya leaf juice may be a potential candidate for further research leading to the development of a herbal therapeutic agent for thrombocytopenia manifested in diseases such as dengue. However, this is a preliminary study and more work is needed to isolate and identify the biologically active ingredients of Carica papaya leaves to create a suitable formula responsible for the release and/or production of the thrombocytes (platelets).

Knowledge Management: Journal