Animal Trials of Carica Papaya Leaf Extracts for

DOI Number: 10.5958/0976-5506.2017.00431.4

Animal Trials of Carica Papaya Leaf Extracts for Increasing Platelet Count Mrinal Saraf1, Bhushan Kavimandan2 Research Scholar, 2Assistant Professor, Department of Biochemistry, Moolji Jaitha College, Jalgaon, India

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ABSTRACT Thrombocytopenia is defined as decreased platelet count. Thrombocytopenic disorders (dengue, chikungunea, malaria etc.) are spreading in urban area with the lightning speed. This is now endemic in more than 125 countries. The major mechanisms for a reduced platelet count are decreased production and increased destruction of platelets. Use of papaya leaf extract for increasing platelet count has been described by many scientists. The proposed research work has main aim to explore Carica papaya leaf extract for increase in platelet count in induced thrombocytopenic experimental animals and to study hematology and histopathology of various organs for any side-effects. So papaya leaves were extracted with n-hexane, acetone, ethanol and methanol using Soxhlet apparatus and distilled water using maceration for 8hours.and experiments on experimental animals to see increase in platelet count were carried out. Hematological and Histopathological studies on experimental animals may prove beneficial for toxicologists. Keywords: Carica papaya, extracts, organic solvents, platelet, Thrombocytopenia,

INTRODUCTION Thrombocytopenia is defined as a platelet count below the 2.5th lower percentile of the normal platelet count distribution. Results of the third US National Health and Nutrition Examination Survey (NHANES III) support the traditional value of 150 x 109/L as the lower limit of normal1. However, platelet counts between 100 and 150 x 109/L do not necessarily indicate disease if they have been stable for more than 6 months (R. Stasi et al 2006) and the adoption of a cutoff value of 100 x 109/L may be more appropriate to identify a pathologic condition2. Platelet counts above 200 x 109/L, clinical symptoms are often limited to easy bruising; however, below 100 x 109/L, the risk of spontaneous mucocutaneous bleeding increases rapidly3. Thrombocytopenia is caused by two basic reasons; one is impaired platelet production and second is excessive destruction. Impaired platelet production occurs in BM failure, megaloblastic anemia, leukemia, myeloma, myelo-fibrosis, solid tumor infiltration, aplastic anemia and paroxysmal nocturnal haemo-globinuria (PNH) 4. Excessive destruction occurs in autoimmune ITP, SLE, viral infections like EBV, dengue and HIV, DIC, TTP and in hyper-splenism4. Thrombocytopenia is the most common cause of defective primary hemostasis

that can lead to significant bleeding. Significant quantitative or qualitative platelet dysfunction causes muco-cutaneous bleeding. The most common causes of thrombocytopenia are defective production of platelets by the bone marrow, diminished platelet survival and sequestration of the platelets by the spleen, leptospirosis, malaria and dengue associated with platelet destruction and bone marrow suppression and sepsis. A combination of above facts also can lead to thrombocytopenia3. It is now known that in many cases of Thrombocytopenia, such as primary immune Thrombocytopenia (ITP) and hepatitis C virus infection, multiple mechanisms may contribute to the development of Thrombocytopenia5. Carica papaya L. belongs to the plant family Caricaceae6. The different parts of papaya leaf contains various chemical constituents that has been proved for various medicinal activities like antioxidant, antihypertensive, wound healing, hepatoprotective, antiinflammatory, antimicrobial, anti-amoebic, and antihelminthic. Recent Phytochemical examination of plants has a suitable history of use in folklore for the treatment of thrombocytopenia. Research into phytotherapy of diseases is a current trend in the management of tropical diseases and genetic disorder like sickle cell anemia, with a view to finding cheaper, alternative medicines that the wide populace can have immediate access7.

Indian Journal of Public Health Research & Development, October-December 2017, Vol.8, No. 4

The rapid recovery of platelet counts was studied in children suffering from dengue. The boys, were administered a spoonful of ground papaya leaves paste every 04 hourly. A dramatic increase in platelet counts was observed within 12 h of initiating treatment, the count increased to 100,000 in one case and in the second case, it increased within 2 days to 250,000. Patients were administered fresh juice C. papaya leaves once a day 15 min after breakfast for 3 consecutive days. The study found that there was a significant increase in the platelet counts at the end of 40 h when compared to the counts 8 h after the intervention began8. A suspension of powdered crude Carica papaya leaves in palm oil when administered to experimental animal have shown the consistent rise of the thrombocyte counts in the treated group. The effect of papaya leaf formulations was higher and significant indicating a potential in the treatment of thrombocytopenic purpura9. Various clinical and pre-clinical studies conducted demonstrate the positive beneficial trend in increasing the platelets. Significant rise was observed on the fourth day of admission. However, large scale randomized clinical trials were suggested to further establish its pivotal role in the management of dengue. The results of Zunjar et al (2016)10 experiments, suggested that Carica papaya leaves decoction has very good anti-thrombocytopenic properties. It is predicted in the study that anti-thrombocytopenic property of Carica papaya leaves may be due to alkaloid. Additionally, no visible toxicity or any adverse effect was observed in animals, The study of Gamulle (2012)11 reiterated that the mature leaf Carica papaya concentrate (MLCC), has the potential to be developed as a plant based therapeutic agent for thrombocytopenia. It was also observed that the MLCC prepared by the mature leaves of Carica papaya can be orally administered, is safe (non-toxic), effectively increasing platelet, WBC and RBC counts in normal (nonthrombocytopenic) and thrombocytopenic rats. Thrombocytopenic parameter of papaya leaf extract is studied with crude extract of water, followed by methanolic and ethanolic extract up to some extent. The current study detailed study of action of five different extracts of papaya leaf from polar to non-polar solvents.

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MATERIALS AND METHODS Sample preparation: The authenticated, disease free sapling of papaya plant of breed “Taiwan 786” was purchased from “Ram Biotech limited”, Nashirabad, Maharashtra, India. These saplings were given organic manure with fifteen days interval. Fifteen days old leaves were shade dried, powdered and used for extraction. 25 grams powdered plant materials (leaves) were soaked separately with 250 ml of each of the ethanol, methanol, n-hexane and acetone, and extracted in a Soxhlet apparatus for 6 hours at boiling point until complete extraction of the materials. Extracts were concentrated at room temperature in order to reduce the volume. In distilled water the same was soaked separately with 250 ml and macerated for 6 hours with some intervals. At the end of 6 hours, each extract was filtered through Whatman No.1 filter paper and filtrates were concentrated at room temperature in order to reduce the volume. These extracts were used for further analyses. Animal experiments: Experimental Design: Phase-I: Standardization of carboplatin doze: The dose of carboplatin was standardized to check the toxicity and lethalness of drug. Thrombocytopenia was induced in experimental Sprague dawley rats by a single intraperitoneal injection of carboplatin (Fresenius Kabi Oncology Ltd). The dose of carboplatin was given in 50 mg/kg body weight, 100 mg/kg body weight, 150 mg/kg body weight concentrations to 12 Sprague dawley rats of either sex. The dose of 50 mg/kg body weight of carboplatin was finalized as this dose was found to be suitable for induction of thrombocytopenia with no or very little toxic effects and non-lethal; while other two doses were found to be lethal. Phase-II: Standardization of extracts doze: Group of 06 animals (Sprague dawley rats) of either sex set was experimented to decide the doze of extract for 05 different solvents in 02 different concentrations of 100 mg/kg body weight and 200 mg/kg body weight, a set of standard

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drug prednisilon (Intervet India Pvt Ltd). These results concluded without any rise in platelet count. So the new preclinical experiment was designed with double dose that was 400 mg/kg body weight. This doze gave results as per hypothesis showing rise in platelet count. Phase-III: Animal trials: A set of48 Sprague dawley rats was divided into seven groups of 06 (03 Males and 03 females) animals each. After inducing thrombocytopenia in all animals, on 6th day, the control group (group 0) was given 0.3 ml distilled water and experimental groups were orally treated with 400 mg/kg body weight of extracts (group 1 to 6). This was continued every alternate day from day 06 to 27. (Group1: hexane extract, 2: acetone extract, 3: ethanol extract, 4: methanol extract, 5: distilled water extract, 6: standard). Blood Sample Collection: Retero-orbitally blood sample was drawn on day 6, 15, 25 for platelet count rise. Platelet Examination: Induction of thrombocytopenia was established by determining platelet counts with the standard protocols as given in book name12.

like toxicity, nausea and vomiting13. Carboplatin causes a rapid decrease in circulating platelet number14 and it affects multi-lineage hemopoietic cell which are mature in nature and does not affect the stem cells15. After induction of thrombocytopenia with the help of carboplatin, the platelet count was reduced by 71.46%, 74.22%, 73.53%, 77.89%, 79.03%, 75.51%, and 75.10 % in control, n-hexane, acetone, ethanol, methanol and distilled water extract and standard respectively on day 06. From day 06 onwards on every alternate day till the end of the experiment, extract and standard were given as per decided doze and schedule. On 15th day the platelet count rise was observed by 18.19%, 42.49%, 42.26%, 49.92%, 60.10%, 70.97%, 139.94% in case of control, n-hexane, acetone, ethanol, methanol and distilled water extract and standard groups respectively. On 25th day increase in platelet count was found to be 126.52%, 166.36%, 178.94%, 181.20%, 190.36%, 216.96%, 303.66% in case of control, n-hexane, acetone, ethanol, methanol and distilled water extract and standard groups respectively. Fig. 1 and Table 1 represents percentage increased in platelet count and the number of platelets observed.

Histopathological studies: At the end of experiment animals were sacrificed for histopathological examinations of Liver, lungs, kidney, spleen, heart and intestine to check effect of extract on organs and its toxicity with the standard protocols12. Result and discussion: Animal trials were conducted on Sprague dawley rats to check the rise in platelet count as a result of extract treatment, after inducing thrombocytopenia with the help of carboplatin. Reteroorbitally blood sample was drawn, from rats of each group on day 0, on 6th, 15th, and 25th day of the experiment. Papaya leaf extracts of various solvents prevented fall in platelet count throughout the study period. Before induction of thrombocytopenia in rats (day 0), the platelet count in control, n-hexane, acetone, ethanol, methanol and distill water extract and standard were found to be 8.83±103, 8.99 ±97, 8.90±104, 9.18±139, 9.40±129, 8.90±104, 9.68±145 × 105/ cmm respectively. Carboplatin has been used to induce thrombocytopenia. It is a derivative of Cis-platin. It is a second generation platinum drug that is clinically effective in malignant tumors. It was chosen because it causes lesser side effects

Fig.1: Percentage increase in the platelet count Study reveals that difference between male and female animal platelet count was not significant. Table1: Platelet Count × 105/ cmm Mean ± SD observed in various extracts treated rat groups. Group name Hexane Acetone Ethanol Methanol Distilled water Control Standard

Day 0

Day 6

Day 15

Day 25

8.99 ±97 8.90±104 9.18±139 9.40±129 8.90±104

2.31±63 2.35±84 2.03±112 1.97±86 2.18±89

3.30±54 3.35±78 3.04±95 3.15±96 3.73±84

6.17.5±73 6.57±64 5.70±52 6.00±68 6.91±85

8.83±103 2.52±75 2.97±84 5.70±28 9.68±145 2.41±62 5.78±112 9.73±130


Histopathological examination of the liver, lungs, kidney, intestine, heart and spleen of control and treated groups did not reveal any morphological differences or toxicity even after 27 days of treatment. This suggests that Carica papaya leaf extracts are safe and have no side effects as well as toxic effects. Figure 1 shows the representative histopathological images of the various organs.

Kidney

Heart

Intestine

Spleen

Liver Lungs Fig. 2: Histopathological images of the various organs. Platelet count was decreased after administration of carboplatin injection (50mg/Kg body weight). Maximum effect of the carboplatin was observed on 7th day and then increase in platelet count was observed. Carica papaya leaf extracts prevented fall in platelet count throughout study period. Platelet count was found to be at higher level on day 7th, 15th and 27th in all experimental groups as compared to the same group at day 1 (Fig. 2). From the observations it is clear that not only fall in platelet count was prevented but count was also increased significantly above baseline. When platelet count in experimental groups was compared to the standard (Prednisolon) and control (Natural recovery), it was observed that increase in platelet count in standard group was rapid and at higher level as compared to experimental groups while platelet count in control group was found to be at lower level. Genderwise difference in platelet count was not significant in all groups.

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One animal study investigating effect of papaya leaf juice on platelet count on 5 healthy mice showed increased platelet count within hours, suggesting increase of platelets from bone marrow16. Gammulle et al (2012)17 studied effect of papaya leaf juice on hydroxyl urea induced thrombocytopenia in rats. 7.2 ml/ kg of juice given for three consecutive days increased platelet count by 76.5% as compared with control. Tahir et al (2014)4 studied difference in effect of male and female Carica papaya tree leaf juice on platelet count. Difference between male and female plant was not significant while higher dose (10 ml/kg) produced significantly high response as compared to low dose (5 ml/kg). This is in concordance with our study. Multiple mechanisms mediated by many active principles in papaya leaf juice may be responsible for increasing the blood cell counts. Papaya leaves contain active compounds papain, chymopapain, alkaloids, flavonoids, flavonaols, tannins and benzylglucosinolate. These compounds stimulate and/or improve the megakaryocytes to produce sufficient numbers of platelets to maintain a suitable platelet count in mammals, in particular during chemotherapy4. Biologically active compounds present in leaf are proteolytic enzymes, for example papain and chymopapain. As proplatelet formation is regulated by a caspase (protease) activation, protein digestion by these enzymes may increase platelet count18. The flavonols (such as kaempferol, quercetin, myricetin and fisetin), and flavonoids are used as ingredients in many pharmaceuticals composition. They are shown to have anabolic effect19, which may be responsible for stimulant effect on blood cell production. Tannins are large polyphenolic compounds containing sufficient hydroxyls and other groups (such as carboxyl) to form strong complexes with proteins and other macromolecules. As retraction events release individual pro-platelets18, the complex – forming properties of tannins may contribute to the positive effects on platelet count in blood. At the end of experiment animals were sacrificed for histopathological examinations of liver, lungs, kidney, spleen, heart and intestine to check effect of extract on organs and its toxicity by the protocols given by Godkar and Godkar (2003)12. From histopathological studies it is clear that all extracts had no toxic effects on major organs like liver, lungs, kidney, spleen, heart and intestine. Hence these extracts are safe by oral route for increasing platelet count.

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CONCLUSION Papaya leaf extracts not only prevents fall in platelet count induced by carboplatin but also increases platelet count in a dose dependent manner without any toxic effects. On other hematological parameters, effect of extracts was insignificant and there was no significant effect of extracts on male and female rats. From this it is very much clear that Carica papaya extracts contain potential component(s) which is responsible to increase platelet count and so further studies on isolation, purification and characterization of such component(s) is required on urgent basis. However, this is a primary study and more work is needed to isolate and identify the active bio-components of Carica papaya leaves to create a suitable formulation responsible for production of the platelets.

ACKNOWLEDGEMENT The authors are very grateful to the Vice Chancellor Research Motivation Scheme (VCRMS), North Maharashtra University, Jalgaon and Assistance for S&T application through university systems of RGS&TC, government of Maharashtra, through North Maharashtra University, Jalgaon for funding the current research project. The authors are also grateful to Dr. D.P. Chaudhari, Unique bio-diagnostics enterprises, Mumbai, who guided us performing animal trials as and when required.

2. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, Bussel JB, Cines DB, Chong BH, Cooper N, Godeau B. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood. 2009 Mar 12;113(11):2386-93.

3. Sheikh N, Papadakis M, Ghani S, Zaidi A, Gati S, Adami P, Carré F, Schnell F, Avila P, Wilson M, McKenna W. Comparison of ECG criteria for the detection of cardiac abnormalities in elite black and white athletes. Circulation. 2014 Mar 11:CIRCULATIONAHA-113.

4. Tahir N, Zaheer Z, Kausar S, Chiragh S. Prevention of fall in platelet count by Carica papaya leaf juice in carboplatin induced thrombocytopaenia in mice. Biomedica. 2014 Mar 31;30(1).

5. Stasi R. How to approach thrombocytopenia. ASH Education Program Book. 2012 Dec 8;2012(1):191-7.

6. Chakravarty S, Reddy BR, Sudhakar SR, Saxena S, Das T, Meghah V, Swamy CV, Kumar A, Idris MM. Chronic unpredictable stress (CUS)-induced anxiety and related mood disorders in a zebrafish model: altered brain proteome profile implicates mitochondrial dysfunction. PloS one. 2013 May 14;8(5):e63302.

7. Imaga NO, Gbenle GO, Okochi VI, Akanbi SO, Edeoghon SO, Oigbochie V, Kehinde MO, Bamiro SB. Antisickling property of Carica papaya leaf extract. African Journal of Biochemistry Research. 2009 Apr 30;3(4):102-6.

8. Subenthiran Soobitha, Tan Chwee Choon, Kee Chee Cheong, (2013); “Carica papaya Leaves Juice Significantly Accelerates the Rate of Increase in Platelet Count among Patients with Dengue Fever and Dengue Hemorrhagic Fever,” Evidence-Based Complementary and Alternative Medicine, 10,1155.

9. Kathiresan S, Surash R, Sharif M, Mas Rosemal N, Walther H (2009). Thrombocyte counts in spraguedawley rats after the administration of papaya leaf suspension. Wien. Klin. Wochenschr. 121(3): 19-22

Ethical clearance: Taken from Ethical Committee of Veterinary College, Mumbai (MVC/IAEC/2016). Source of funding: Rajiv Gandhi Science & Technology Commission (RGSTC), NMU Centre and Vice Chancellor Research Motivation Scheme (VCRMS), North Maharashtra University, Jalgaon. Conflict of Interest: Nil

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