POTENTIAL BENEFITS OF PAPAYA LEAVE AND GRAPE FRUIT JUICES FOR MANAGEMENT OF THROMBOCYTOPENIA
a) BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Herbs have been an integral part of society since the beginning of human civilization and are valued for their culinary and medicinal properties. With the development of patented medicines in the early part of the 20th century, herbal medicine lost ground to the new synthetic medicines touted by scientists and physicians to be more effective and reliable. Nevertheless, herbal remedies are still popular globally1.
Platelets (PLTs) are essentially tiny cells that are part of the anatomy of blood cells and play a significant role in the clotting of blood. PLTs are the first responders when the brain receives a signal that there is some blood loss as a result of some kind of injury or trauma. They utilize the help of proteins, minerals and vitamins to help stop the bleeding and clot the injury. The average lifespan of a single platelet is considered to be around eight to twelve days, after which a new batch of platelets is produced and takes over. Some individuals may have a lower than normal count of platelets while others may have a higher count. PLTs play a key role in hemostasis, clot stability and retraction as well as in vascular repair and anti-microbial host defense. Upon vessel wall damage, PLTs undergo a highly regulated set including adhesion, spreading, aggregation, release reactions as well as exposure of procoagulant surfaces to rapidly form a hemostatic plug that occludes the site of damage. When PLT function is impaired, the bleeding risk increases, but (hyperreactive) PLTs are also involved in many pathophysiological events like thrombosis, vessel constriction, atherogenesis, tumor growth and metastasis, inflammation including atherosclerosis and the subsequent formation of arterial thrombi resulting in stroke and myocardial infarction2. The body has a mechanism to bring the blood cells to normal. Healthy diets containing flavonoid could help us in maintaining normal homeostasis3-5.
Carica papaya L. belongs to the plant family Caricaceae. It is being cultivated widely for consumption as fresh fruit, dried and crystallised fruit as well as for use in drinks, jams and candies6. Green fruit, the leaves and flowers may also be used as a cooked vegetable 7. Nakasone and Paull 8 have shown that papaya is a good source of calcium and an excellent source of vitamins A and C. Papaya also has several industrial uses. Biochemically, its leaves and fruit are complex, containing several proteins and alkaloids with important pharmaceutical and industrial applications 9. Commonly, Carica papaya is used as food or as medication in folk medicine 10. Considerable work has been carried out on plant parts such as fruit, seed and root, indicating the presence of biologically active compounds. The quantity of the compounds differs in fruit, latex, leaves and roots, and varies with the extraction method, age of the plant part, the cultivation and the gender of the tree. In Malaysia, many people take papaya leave juice to increase platelet count when they get dengue fever. It’s a fact that, to date, there’s no known medical cure for dengue fever since it’s caused by a virus transmitted through the bite of the Aedes mosquito. However, it seems drinking papaya leaf extract helps to bring up the platelet count 11.
Purple grape (Vitis vinifera L) is not only very tasty and refreshing, but it also has healing properties. It acts as a natural body tonic, slows aging, prevents dementia and degenerative diseases, cleans the blood, removes the fat from the body, strengthens the heart and etc. Purple grapes contain lots of fruit sugar (18-20%), providing a high energy value, so 1 kg of grapes can replace 25 to 30% of daily energy needs. It contains plenty of vitamin C and smaller amounts of vitamin B1, B2, B6 and carotene. Also grape is rich in minerals - potassium, calcium, iron, phosphorus, magnesium and boron. Purple grapes contain phenolic substances that provide color and flavor, resveratrol, anthocyans and antioxidants (catechins, quercetin) that provide a healing effect. It has been demonstrated that 10 ml/kg of Purple Grape juice significantly inhibited in vivo platelet activity and experimental coronary thrombosis 12. Grapes and grape juice contain many of the same biologically active phenolic compounds such as catechines, quercetin, kaemferol and anthocyanin that are found in red wine 13.
As stated above, papaya leaves and purple grape juices have been used in folklore for ameliorating large number of ailments. Traditionally, one of the common remedial actions was for combating fall in platelet count. Even though modern method of treatment is successful in alleviating the pains and discomforts of human being, there is still dearth of discovery for effective management of thrombocytopenia. Corroborating with this, we have designed this study to fight against fall in platelet count in experimental models of animals with the help of nature’s gift of herbs and fruits. Hence the present study is designed to explore the role of papaya leaves and purple grape juices separately as well as in combination on platelet count during collagen/heparin induced thrombocytopenia in rats.
6.2 Review of literature:
Erwinia mallotivora was isolated from papaya infected with dieback disease showing the typical symptoms of greasy, water-soaked lesions and spots on leaves. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain belonged to the genus Erwinia and was united in a monophyletic group with E. mallotivora DSM 4565 (AJ233414). Current studies, through Koch's postulate, have confirmed that papaya dieback disease is caused by E. mallotivora. To our knowledge, this is the first new discovery of E. mallotivora as a causal agent of papaya dieback disease in Peninsular Malaysia. Previous reports have suggested that E. mallotivora causes leaf spot in Mallotus japonicus. However, this research confirms it also to be pathogenic to Carica papaya14.
The aim of one of the study was to examine the effect of aqueous-extracted Carica papaya leaf fraction on the growth of various tumor cell lines and on the anti-tumor effect of human lymphocytes. The effect of Carica papaya leaf extract on the proliferative responses of tumor cell lines and human peripheral blood mononuclear cells (PBMC), and cytotoxic activities of PBMC were assessed by [(3)H]-thymidine incorporation. Significant growth inhibitory activity of the Carica papaya extract on tumor cell lines were observed. In PBMC, the production of IL-2 and IL-4 was reduced following the addition of Carica papaya extract, whereas that of IL-12p40, IL-12p70, IFN-gamma and TNF-alpha was enhanced without growth inhibition. In addition, cytotoxicity of activated PBMC against K562 was enhanced by the addition of Carica papaya extract. Since Carica papaya leaf extract can mediate a Th1 type shift in human immune system, results suggest that the purple grape leaf extract may potentially provide the means for the treatment and prevention of selected human diseases such as cancer, various allergic disorders, and may also serve as immunoadjuvant for vaccine therapy15.
The effects of Carica papaya leaf (CPL) aqueous extract on alcohol induced acute gastric damage and the immediate blood oxidative stress level were studied in rats. The results showed that gastric ulcer index was significantly reduced in rats pretreated with CPL extract as compared with alcohol treated controls. The in vitro studies using 2,2-Diphenyl-1-Picryl-Hydrazyl (DPPH) assay showed strong antioxidant nature of CPL extract. Biochemical analysis indicated that the acute alcohol induced damage is reflected in the alterations of blood oxidative indices and CPL extract offered some protection with reduction in plasma lipid peroxidation level and increased erythrocyte glutathione peroxidase activity. Carica papaya leaf may potentially serve as a good therapeutic agent for protection against gastric ulcer and oxidative stress16.
The Nagano Purple grape (Vitis (V.) viniferaxV. labrusca) is a hybrid created by a cross between Kyoho (V. viniferaxV. labrusca) and Rosario Bianco (V. vinifera) grapes. The grape, including its skin, can be eaten and contains no seeds because of gibberellin treatment. The skins of various fruits have been shown to contain antioxidant activity. However, it is unclear whether the Nagano Purple grape contains antioxidant activity. The skins and dried fruits (including the skins) of the Nagano Purple grape was prepared, so as to assay for the presence of an antioxidant activity. The concentration of polyphenols in the grape were prepared and further assayed whether components in the grape inhibited the oxidation of low-density lipoprotein (LDL). The presence of cyanidin-3-glucoside (Cy-3-glc), five anthocyanidins and resveratrol in the skins were detected. A trace of resveratrol was detected in the pulp. LDL collected from human subjects 1 h following the consumption of the skins or dried fruits revealed significant inhibition of LDL oxidation compared to that observed in fasting venous blood samples17.
Oxidative damage to biomolecules occurs by the accumulation of molecular damage due to free radicals and/or a diminution of antioxidant protection. The aim of one of the study was to evaluate the protection of organic and conventional purple grape juices in brain, liver, and plasma from adult Wistar rats (7 months old) against the oxidative damage provoked by carbon tetrachloride (CCl4). Adult rats were divided into three groups (control, conventional purple grape juice, and organic purple grape juice). Half of the rats received CCl4, and the other half received the vehicle (vegetable oil). The chemical analytical determination showed that the highest levels of total phenolic, resveratrol, and catechins were seen in organic purple grape juices. Considering the treatment groups, it was observed that in all tissues (brain structures and liver) and plasma, CCl4 treatment increased the lipid peroxidation (LP) levels. Both grape juices were capable to reduce LP levels in cerebral cortex and hippocampus; however, in the striatum and substantia nigra only the organic grape juice reduced LP level. This implies that both grape juices have an important antigenotoxic activity18.
Grape juice is a source of polyphenols, as catechin, anthocyanidins, resveratrol, and others. Some health benefits have been attributed to these compounds (e.g., antioxidant and antitumorigenic properties). In one study, the possible antioxidant activity of two different grape juices: organic purple grape juice and conventional purple grape juice were investigated. The antioxidant activity of both grape juices was evaluated by an animal model of three groups: control and organic and conventional juices. After 30 days, all animals were sacrificed, and blood and liver were collected to evaluate lipid peroxidation level (thiobarbituric acid-reactive substances [TBARS] assay), protein oxidative level (carbonyl assay), and catalase (CAT) and superoxide dismutase (SOD) activities. The group treated with organic grape juice showed the highest SOD and CAT activities in both plasma and liver when compared with the conventional and control groups (P < .05). The grape juices were capable of reducing carbonyl and lipid peroxidation levels in plasma and liver. However, in plasma, the organic group showed lower carbonyl and TBARS levels when compared to the conventional grape juice group (P < .05)19.
6.3 Objective of study
The objective of the present research will be to explore the role of papaya leaves and purple grape fruit juices on platelet count in animals with thrombocytopenia.
SPECIFIC OBJECTIVES:
LEVEL 1
· To collect and authenticate the papaya leaves and purple grape fruit.
· To standardize the method of preparation of papaya leaves and purple grape fruit.
· To standardize the thrombocytopenic dose of collagen/heparin.
· To established the effective dose of papaya leaves and purple grape fruit under normal conditions for platelet count.
· To ascertain the different concentrations of papaya leaves and purple grape fruit individually as well as in combination for influencing the platelet count.
LEVEL 2
· To study the extent of platelet enhancement by papaya leaves and purple grape fruit individually in thrombocytopenic animals.
· To know the platelet count when papaya leaves and purple grape fruit are used in combination during thrombocytopenia.
LEVEL 3
· To prepare suitable formulation of mixture containing effective concentrations of papaya leaves and purple grape fruit juices.
· To carryout stability study of the prepared formulation.
· To periodically repeat the evaluation of formulation for platelet count.
b) MATERIALS AND METHODS:
7.1 Source of Data:
Source of Data:
Data will be obtained from laboratory based studies by using Sprague dawley rats of either sex weighing between 150-200 gms maintained at room temperature having free access to food (std pellet diet), tap water ad libitum. These studies will be carried out in serum of animal and the platelet count will be carried out in authenticated laboratories. Chemical reagents will be procured from standard companies.
7.2 Method of Collection of Data:
Chemicals and reagents will be procured from standard companies. Collagen or Heparin induced thrombocytopenia will be used as model to evaluate platelet changes. The data collected will be based on animal experimentation as per the parameters studied under each animal model.
Experimental protocol:
Multiple level studies will be carried out to ascertain the role of papaya leaves and purple grape fruit as platelet enhancer.
Level 1
Animals will be divided into four groups (n=10)
Group I: collagen/heparin administration
Group II: Papaya leaves juice administration
Group III: Purple grape juice administration
Group IV: Papaya leaves and purple grape fruit juices in combination.
Blood samples will be collected before administration as shown above, as well as at 30 minutes, 1, 2, 4 and 8 hours after administration from all animals.
Level 2
Animals will be divided into three groups (n=10)
Group I: Papaya leaves juice + collagen/heparin administration
Group II: Purple grape juice + collagen/heparin administration
Group III: Papaya leaves and purple grape fruit juices in combination + collagen/heparin administration
Blood samples will be collected before administration as shown above, as well as at 30 minutes, 1, 2, 4 and 8 hours after administration from all animals.
Level 3
Animals will be divided into three groups (n=10)
Group I: prepared formulation of papaya leaves and purple grape fruit + collagen/heparin administration
Group II: vehicle used for dissolving collagen/heparin + collagen/heparin administration
Group III: Excipients of the formulation + collagen/heparin administration