According to WHO, Dengue is one of the most rapidly increasing mosquito-borne viral diseases rampant around the world especially in tropical and sub-tropical regions. It is transmitted by the mosquitoes of the genus Aedes with Aedesaegypti as the most common specie causing the disease. Other types of the Aedesmosquito species include A. albopictus, A. polynesiansis and A. scutellaris.
Its incidence has grown dramatically around the world in recent decades. The actual numbers of dengue cases are underreported and many cases are misclassified. One recent estimate indicates 390 million dengue infections per year (95% credible interval 284’528 million), of which 96 million (67’136 million) manifest clinically (with any severity of disease). Another study of the prevalence of dengue, estimates that 3900 million people, in 128 countries, are at risk of infection with dengue viruses.
Dengue hemorrhagic fever is the most serious form of dengue fever. Symptoms include hypotension, increased vascular permeability, thrombocytopenia and hemorrhagic manifestations. Thrombocytopenia is rare blood disorder that affects the platelets of the blood.
As of now, there are no specific treatments available for dengue, only support care and the prevention of the transmission of the dengue virus. Of the available methods for treating thrombocytopenia, the treatment method mainly depends on the disease severity. Blood transfusions, steroid treatment, splenectomy, immunosuppressive therapy, intravenous gamma-globulin therapy can be used to correct thrombocytopenia. Nevertheless, due to certain side effects and the costs involved, the availability of treatment for thrombocytopenia is limited.
In lieu to this and the limited supportive treatment available, the search for alternative drugs becomes more imperative than ever which lead to the discovery of Carica papaya as an anti-dengue plant.
Recent studies disclosed platelet-increasing activity of Carica papaya making a breakthrough in the treatment and management of dengue. The discovery all started with a folkloric tale of its use, the Papaya leaf juice extract being given orally to patients with dengue. The papaya leaf juice, however, is very bitter and unpalatable to the taste buds making it difficult to ingest orally.
Though oral route of administration is said to be the most important method of administering drugs for systemic effects mainly because it is convenient, safe, and less expensive, an important consideration should be made into the oral dosage form. Usually, patient acceptance of a liquid dosage form largely depends on its palatability.
As such, designing of oral formulation proves to be a challenge in modern pharmaceutics until now. In the study, the researchers attempted to develop a syrup formulation out of the leaves of papaya (Carica papaya L.) and evaluate its properties as well as the capability to increase platelet.
Review of related literature:
This chapter presents various literatures and studies that are in some ways similar to the study at hand. The different points of these collected articles provided the researchers insight and concepts that help prove the thoughts presented in this research work.
Plant name and Taxonomic classification
The papaya, Carica papaya L., is a member of the small family Caricaceae allied to the Passifloraceae. As a dual- or multi-purpose, early-bearing, space-conserving, herbaceous crop, it is widely acclaimed, despite its susceptibility to natural enemies.
In some parts of the world, especially Australia and some islands of the West Indies, it is known as papaw, or pawpaw, names which are better limited to the very different, mainly wild AsiminatrilobaDunal, belonging to the Annonaceae. While the name papaya is widely recognized, it has been corrupted to kapaya, kepaya, lapaya or tapaya in southern Asia and the East Indies. In French, it is papaye (the fruit) and papayer (the plant), or sometimes figuier des Iles. Spanish-speaking people employ the names mel”nzapote, lechosa, payaya (fruit), papayo or papayero (the plant), frutabomba, mam”n or mamona, depending on the country. In Brazil, the usual name is mamao.
When first encountered by Europeans it was quite naturally nicknamed "tree melon".
Plant Description
Commonly and erroneously referred to as a "tree", the plant is properly a large herb growing at the rate of 6 to 10 ft. (1.8-3 m) the first year and reaching 20 or even 30 ft. (6-9 m) in height, with a hollow green or deep-purple stem becoming 12 to 16 in (30-40 cm) or more thick at the base and roughened by leaf scars. The leaves emerge directly from the upper part of the stem in a spiral on nearly horizontal petioles 1 to 3 1/2 ft. (30-105 cm) long, hollow, succulent, green or more or less dark purple. The blade, deeply divided into 5 to 9 main segments, each irregularly subdivided, varies from 1 to 2 ft. (30-60 cm) in width and has prominent yellowish ribs and veins. The life of a leaf is 4 to 6 months. Both the stem and leaves contain copious white milky latex.
The 5-petalled flowers are fleshy, waxy and slightly fragrant. Some plants bear only short-stalked pistil late (female) flowers, waxy and ivory-white; or hermaphrodite (perfect) flowers (having female and male organs), ivory-white with bright-yellow anthers and borne on short stalks; while others may bear only staminate (male) flowers, clustered on panicles to 5 or 6 ft. (1.5-1.8 m) long. There may even be monoecious plants having both male and female flowers. Some plants at certain seasons produce short-stalked male flowers, at other times perfect flowers. This change of sex may occur temporarily during high temperatures in midsummer. Some "all-male" plants occasionally bear, at the tip of the spray, small flowers with perfect pistils and these produce abnormally slender fruits. Male or hermaphrodite plants may change completely to female plants after being beheaded.
Generally, the fruit is melon-like, oval to nearly round, somewhat pyriform, or elongated club-shaped, 6 to 20 in (15-50 cm) long and 4 to 8 in (10-20 cm) thick; weighing up to 20 lbs. (9 kg). Semi-wild (naturalized) plants bear miniature fruits 1 to 6 in (2.5-15 cm) long. The skin is waxy and thin but fairly tough. When the fruit is green and hard it is rich in white latex. As it ripens, it becomes light- or deep-yellow externally and the thick wall of succulent flesh becomes aromatic, yellow, orange or various shades of salmon or red. It is then juicy, sweetish and somewhat like a cantaloupe in flavor; in some types quite musky. Attached lightly to the wall by soft, white, fibrous tissue, are usually numerous small, black, ovoid, corrugated, peppery seeds about 3/16 in (5 mm) long, each coated with a transparent, gelatinous aril.
Origin and Distribution
Though the exact area of origin is unknown, the papaya is believed native to tropical America, perhaps in southern Mexico and neighboring Central America. It is recorded that seeds were taken to Panama and then the Dominican Republic before 1525 and cultivation spread to warm elevations throughout South and Central America, southern Mexico, the West Indies and Bahamas and to Bermuda in 1616. Spaniards carried seeds to the Philippines about 1550 and the papaya traveled from there to Malacca and India. Seeds were sent from India to Naples in 1626. Now the papaya is familiar in nearly all tropical regions of the Old World and the Pacific Islands and has become naturalized in many areas. Seeds were probably brought to Florida from the Bahamas. Up to about 1959, the papaya was commonly grown in southern and central Florida in home gardens and on a small commercial scale. Thereafter, natural enemies seriously reduced the plantings. There was a similar decline in Puerto Rico about 10 years prior to the setback of the industry in Florida. While isolated plants and a few commercial plots may be fruitful and long-lived, plants in some fields may reach 5 or 6 ft., yield one picking of undersized and misshapen fruits and then are so affected by virus and other diseases that they must be destroyed.
In the 1950's an Italian entrepreneur, Albert Santo, imported papayas into Miami by air from Santa Marta, Colombia, Puerto Rico and Cuba for sale locally as well as shipping fresh to New York, and he also processed quantities into juice or preserves in his own Miami factory. Since there is no longer such importation, there is a severe shortage of papayas in Florida. The influx of Latin American residents has increased the demand and new growers are trying to fill it with relatively virus-resistant strains selected by the University of Florida Agricultural Research and Education Center in Homestead.
Successful commercial production today is primarily in Hawaii, tropical Africa, the Philippines, India, Ceylon, Malaya and Australia, apart from the widespread but smaller scale production in South Africa, and Latin America.
Annual papaya consumption in Hawaii is 15 lbs. (6.8 kg) per capita, yet 26 million lbs. (11,838,700 kg) of fresh fruits were shipped by air freight to mainland USA in 1974, mainly direct from Hilo or via Honolulu.
Puerto Rican production does not meet the local demand and fruits are imported from the Dominican Republic for processing.The papaya is one of the leading fruits of southern Mexico and 40% of that country's crop is produced in the state of Veracruz on 14,800 acres (6,000 ha) yielding 120,000 tons annually.
Fruits from bisexual plants are usually cylindrical or pyriform with small seed cavity and thick wall of firm flesh which stands handling and shipping well. In contrast, fruits from female flowers are nearly round or oval and thin-walled. In some areas, bisexual types are in greatest demand. In South Africa, round or oval papayas are preferred. (https://www.hort.purdue.edu/newcrop/morton/papaya_ars.html)
Traditional and Medicinal Uses of Carica papaya
Papaya is a powerhouse of nutrients and is available throughout the year. It is a rich source of threes powerful antioxidant vitamin C, vitamin A and vitamin E; the minerals, magnesium and potassium; the B vitamin pantothenic acid and foliate and fiber. In addition to all this, it contains a digestive enzyme-papainthat effectively treats causes of trauma, allergies and sports injuries. All the nutrients of papaya as a whole improve cardiovascular system, protect against heart diseases, heart attacks, strokes and prevent colon cancer. The fruit is an excellent source of beta carotene that prevents damage caused by free radicals that may cause some forms of cancer. It is reported that it helps in the prevention of diabetic heart disease.
Papaya lowers high cholesterol levels as it is a good source of fiber. Papaya effectively treats and improves all types of digestive and abdominal disorders. It is a medicine for dyspepsia, hyperacidity, dysentery and constipation. Papaya helps in the digestion of proteins as it is a rich source of proteolytic enzymes. Even papain-a digestive enzyme found in papaya is extracted, dried as a powder and used as an aid in indigestion. Ripe fruit consumed regularly helps in habitual constipation. It is also reported that papaya prevents premature aging. It may be that it works because a poor digestion does not provide enough nutrients to our body.
The fruit is regarded as a remedy for abdominal disorders. The skin of papaya works as a best medicine for wounds. Even you can use the pulp left after extracting the juice from papaya as poultice on the wounds. The enzymes papain and chymopapain and antioxidant nutrients found in papaya have been found helpful in lowering inflammation and healing burns. That is why people with diseases (such as asthma, rheumatoid arthritis, and osteoarthritis) that are worsened by inflammation, find relief as the severity of the condition reduces after taking all these nutrients. Papaya contributes to a healthy immune system by increasing your resistance to coughs and colds because of its vitamin A and C contents.
Papaya included in your diet ensures a good supply of vitamin A and C that are highly essential for maintaining a good health. Carica papaya constituents exhibit alkaline combination, as with borax or potassium carbonate and they have showed good results in treatment of warts, corns, sinuses, eczema, cutaneous tubercles and other hardness of the skin, and also injected into indolent glandular tumors to promote their absorption. Green fruits of papaya are used to treat high blood pressure, dyspepsia, constipation, amenorrhea, general debility, expel worms and stimulate reproductive organs.
The many benefits of papaya owed due to high content of Vitamins A, B and C, proteolytic enzymes like papain and chymopapain which have antiviral, antifungal and antibacterial properties. Carica papaya can be used for treatment of a numerous diseases like warts, corns, sinuses, eczema, cutaneous tubercles, glandular tumors, blood pressure, dyspepsia, constipation, amenorrhea, general debility, expel worms and stimulate reproductive organs and many, as a result Carica papaya can be regarded as a Neutraceutical. Carica papaya contains an enzyme known as papain which is present in the bark, leaves and fruit. The milky juice is extracted, dried and used as a chewing gum for digestive problems, toothpaste and meat tenderizers.
It also contains many biological active compounds including chymopapain and papain which is the ingredient that aids digestive system, and again used in treatment of arthritis (Aravind. G et al., 2013).
Recently, Carica papaya leaves have been successfully employed in folk medicine for the treatment of dengue infections with haemorrhagic manifestations, using suspensions of powdered leaves in palm oil.
Carica papaya leaves contain various phytoconstituents like saponins, tannins, cardiac glycosides and alkaloids. The alkaloids present include carpaine, pseudocarpaine and dehydrocarpaine I and II. These constituents can act on the bone marrow, prevent its destruction and enhance its ability to produce platelets. Moreover, it can also prevent platelet destruction in the blood and thereby increase the life of the platelet in circulation (Patil S. et al., 2013).The leaves also contain cardiac glycosides, anthraquinones, carpaine, pseudocarpaine, phenolic compounds (Owoyele et al., 2008; Zunjar et al., 2011).Flavonoids and other phenolic compounds present in papaya leaf extracts were responsible for the observed membrane stabilizing property and thereby prevent the internal bleeding in the blood vessels (Ranasinghe et al., 2012). A recent study showed that flavonoids present in Carica papaya inhibits NS2B-NS3 protease and thereby prevent the DEN-2 Virus assembly (Senthilvel et al., 2013). Recent studies have also shown that papaya leaf juice significantly increases the platelet count (Subenthiran et al., 2013; Dharmarathna et al., 2013).
Papaya leaf contains anti-oxidant vitamins and minerals which may help to increase the haemoglobin, hematocrit, Red blood cells, thrombocytes and total protein contents (Kathiresan et al., 2009; Halim et al., 2011). Vitamin A keeps bile production normal and Vitamin B9 helps in blood DNA synthesis, cell growth and development. Vitamin B12 helps in maintaining the normal count of thrombocytes and helps to fight against thrombocytopenia (Kathiresan et al., 2009; Betty et al., 2009). Vitamin C may act as anti-oxidant to scavenge the oxygen radicals (superoxide, hydroxyl, peroxyl) sulphur radicals and nitrogen – oxygen radicals (Sebastian et al., 2003). Minerals present in papaya leaves play an important role in fighting DENV infection. Calcium ions helps in the proliferation of lymphocyte cells, play key role in platelet aggregation when combine with Vitamin D and prevents thrombocytopenia (Cabrera-Cortina et al., 2008; Emilio et al., 2009). Magnesium ions improves erythrocyte hydration. Sodium ions helps in maintaining electrolyte balance and prevents hyponatremia during dengue infection (Jutrat et al., 2005). Potassium ions maintains body potassium level and helps to prevent Acute hypokalaemicquadriparesis during dengue infection ( Sanjeev and Ansari., 2010; Amitava et al., 2011; Harmanjith et al., 2012).
C. papaya leaf has been used traditionally in the treatment of Dengue Fever. The leaf has been investigated for its potential against DF. The aqueous extract of leaves of this plant exhibited potential activity against DF by increasing the platelet (PLT) count, white blood cells (WBC) and neutrophils (NEUT) in blood samples of a 45-year-oldpatient bitten by carrier mosquitoes.
After 5 days of oral administration of 25 mL aqueous extract of C. papaya leaves to the patient twice daily, the PLT count increased from 55 x 10^3/”L to 168 x 103/”L, WBC from 3.7 x 103/”L to 7.7103/l” and NEUT from 46.0 to 78.3 %. Increased platelets could lead to reduced bleeding, thus avoiding progression to the severe illness of DHF. (Kadir et al., 2013)
A significant increase in the platelet counts has been observed after oral administration of freshly prepared, mature leaf concentrate of C. papaya. Both mature and immature leaves of C. papaya have the potential to be developed as a plant based therapeutic agent for thrombocytopenia (Gammulle et al., 2012). Juice consumption during the course of dengue infection had the potential to induce the rapid production of platelets (Subenthiran et al., 2013).
Commencing on studies of Dr. SanathHettige, who conducted the research on 70 dengue fever patients; said papaya leaf juice helps increase white blood cells and platelets, normalizes clotting, and repairs the liver. (Aravind. G et al., 2013
Papaya and related studies
In a recent study on platelet augmentation activity of selected Philippine plants, plants in the Philippines that are currently being used to increase platelet counts in thrombocytopenic disorders including dengue hemorrhagic fever were investigated. These plants include Carica papaya L. (Family- Caricaceae), Ipomeabatatas (L.) Lam (Family-Convolvulaceae), Althernantherasessilis (L.) R. Brown ex De Candolle (Family-Amaranthaceae), Euphorbia hirta(L.)(Family-Euphorbiaceae) and Momordicacharantia L. (Family-Cucurbitaceae).
The plant extracts were screened for phytochemical constituents. Platelet reduction on Sprague-Dawley rats was induced by oral administration of 0.083 mg/kg body weight of anagrelide. A solution of the lyophilized aqueous plant samples were administered for 9 days. Pre-and post-treatment blood samples for platelet counts were taken on the tenth day. Results showed that all the plant extracts tested were positive for glycosides. Extracts from Carica papaya (p=0.0002), Ipomeabatatas violet variety (p=0.0070) and green variety (p=0.0000), Alternantherasessilis (p=0.0001) and Euphorbia hirta (p=0.0489) have significant platelet augmentation (p<0.05) activity. Only Momordicacharantia extract failed to show significant platelet increasing activity (p=0.1014). The percentage increase of mean platelet counts after reduction with anagrelide were as follows: Carica papaya group (125.87%), Ipomeabatatas green variety group (107.88%), Ipomeabatatas violet variety group (106.07%), Althernantherasessilis group (93.17%) and Euphorbia hirta group (80.92%). These results suggest that extracts from Carica papaya, and Ipomeabatatasgreen and violet variety, Althernantherasessilis and Euphorbia hirtamay be used as potential supportive treatment for thrombocytopenic disorders. (Arollado et al., 2013)
Studies on toxicity of papaya leaf extract
Herbal medicines are used because of the fact that plants contain natural ingredients that can promote health and alleviate illness (Komal et al., 2010). They make an enormous contribution to primary health care and have shown great potential in modern phytomedicine against numerous infirmities and the complex diseases and ailments of the modern world. There will always be risks when appropriate regulations do not handle the appropriate formulation of the remedies or when self-medication fosters abuse (Mukesh et al., 2010).
The CP leaf extract was well tolerated by rats showing no overt signs of stress, aversive behavior or behavioral changes. Previous observations have shown no adverse effects or mortality under acute toxic condition due to orally administered CP leaf extract at 2000 mg/kg body weight (Afzan et al., 2012).An earlier study categorized Carica papaya leaf as non toxic (LD50 >15g per Kg body weight) (Kardono et al.,2003).
In one previous study, (Gammulle et al., 2012) conducted an acute toxicity study of mature leaf papaya concentrate (MLCC) to confirm its safety for oral administration over a period of 3 days. It was shown that the MLCC was well tolerated by rats showing no overt signs of toxicity, stress, aversive behavior or behavioral changes. Also, hepatotoxicity, renal toxicity, haematotoxicity, neurotoxicity were also ruled out.
Pathophysiology of dengue fever
Dengue infection is caused by bites of the female Aegypti mosquito carrying Flavivirus. After a person is bitten, the virus incubation period varies between 3 and 14 days, after which the person may experience early symptoms such as fever, headache, rash, nausea, and joint and musculoskeletal pain. This classic DF records temperatures between 39 and 40 _C and usually lasts 5’7 days. During this period, the virus may get into the peripheral bloodstream and, if left untreated, can damage blood vessels and lymph nodes resulting in DHF with symptoms such as bleeding from the nose, gums or under the skin. DHF patients also have difficulty in breathing and severe development can lead to Dengue Shock Syndrome (DSS). DSS can result in death if proper treatment is not provided.
Aedes mosquitoes are small and black with white markings on the body and legs. Female mosquitoes need blood from biting humans or animals to produce live eggs. It takes 2’3 days for egg development. The principal vector of dengue (Ae. aegypti) has adapted well to the urban environment and always breeds in stagnant containers. Eggs need moist conditions, and mature in 24’72 h. Mosquito bites are the only route of DENV spread. The transmission of DENV is often from human to human through domestic mosquitoes. An outbreak starts after a mosquito sucks the blood of a patient with DF/ DHF. After being transmitted to a new human host by infected mosquitoes, the virus replicates in the lymph nodes and spreads through the lymph and blood to other tissues. To identify a potential antiviral treatment for DENV, it is necessary to understand the life cycle of the virus. The dengue virion is a small particle with a lipoprotein envelope and an icosahedral nucleocapsid containing a positive single-stranded RNA genome. Virus infection of the cell begins with binding to the host cell surface. It enters the cell by receptor-mediated endocytosis, with the cell membrane forming a sac-like structure known as an endosome. In the endosome, the virus penetrates deep into the cell until the endosome membrane acquires a negative charge, which allows it to fuse with the endosomal membrane to open a port for release of genetic material. At this point, the virus in the cell fluid starts to reproduce. Changes in the acidity of the secretory pathway during this viral journey travel play an important role in its maturation.
Transmission
The Aedesaegypti mosquito is the primary vector of dengue. The virus is transmitted to humans through the bites of infected female mosquitoes. After virus incubation for 4’10 days, an infected mosquito is capable of transmitting the virus for the rest of its life.Infected humans are the main carriers and multipliers of the virus, serving as a source of the virus for uninfected mosquitoes. Patients who are already infected with the dengue virus can transmit the infection (for 4’5 days; maximum 12) via Aedesmosquitoes after their first symptoms appear.
The Aedesaegypti mosquito lives in urban habitats and breeds mostly in man-made containers. Unlike other mosquitoes Ae.aegypti is a day-time feeder; its peak biting periods are early in the morning and in the evening before dusk. FemaleAe.aegypti bites multiple people during each feeding period.
Aedesalbopictus, a secondary dengue vector in Asia, has spread to North America and Europe largely due to the international trade in used tyres (a breeding habitat) and other goods (e.g. lucky bamboo). Ae.albopictus is highly adaptive and, therefore, can survive in cooler temperate regions of Europe. Its spread is due to its tolerance to temperatures below freezing, hibernation, and ability to shelter in microhabitats.(http://www.who.int/mediacentre/factsheets/fs117/en/)
Characteristics
Dengue fever is a severe, flu-like illness that affects infants, young children and adults, but seldom causes death. Dengue should be suspected when a high fever (40”C/104”F) is accompanied by 2 of the following symptoms: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash. Symptoms usually last for 2’7 days, after an incubation period of 4’10 days after the bite from an infected mosquito.
Severe dengue is a potentially deadly complication due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, or organ impairment. Warning signs occur 3’7 days after the first symptoms in conjunction with a decrease in temperature (below 38”C/100”F) and include: severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, fatigue, restlessness and blood in vomit. The next 24’48 hours of the critical stage can be lethal; proper medical care is needed to avoid complications and risk of death. (http://www.who.int/mediacentre/factsheets/fs117/en/)
Patients with Dengue Hemorrhagic Fever (DHF) usually have platelet counts less than 100 ” 109 /L. Thrombocytopenia is most prominent during the toxic stage. The mechanisms of thrombocytopenia include decreased platelet production and increased peripheral destruction. Na Nakorn et al. studied the bone marrow of patients with DHF during the acute febrile stage and found marked hypocellularity with a decrease in megakaryocytes, erythroblasts and myeloid precursors. The finding was later explained by the direct dengue virus infection of hematopoietic progenitor cells and stromal cells. Additionally, the increased peripheral destruction is markedly prominent during 2 days before defervescence. The bone marrow then revealed hypercellularity with an increase in the megakaryocyte, erythroblast and myeloid precursors. Hemophagocytosis of young and mature erythroid and myeloid cells, lymphocytes and platelets was observed. Survival of patients’ and transfused platelets was markedly decreased because of immune-mediated injury of platelets. In 1987 Funaharaet al. demonstrated interaction in vitro between platelets and dengue virus-infected endothelial cells inducing platelet aggregation and subsequent lysis that resulted in thrombocytopenia. Subsequently, the number of platelets is rapidly increased in the convalescent stage and reaches the normal level within 7’10 days after the defervescence. Platelet dysfunction as evidenced by the absence of adenosine diphosphate (ADP) release was initially demonstrated in patients with DHF during the convalescent stage by Mitrakul et al. in 1977. The subsequent study during the febrile and early convalescent stages by Srichaikul et al. in 1989 also demonstrated the impaired platelet aggregation response to ADP that returned to a normal response 2’3 weeks later. An increase in plasma ”-thromboglobulin and platelet factor 4, indicating increased platelet secretory activity, was observed. The platelet dysfunction might be the result of exhaustion from platelet activation triggered by immune complexes containing dengue antigen.(http://www.niceindia.net/knowledge_base/Dengue/Chuansumrit_2007.pdf)
Treatment
There are currently no specific treatments for dengue fever. Only standard treatment for management of fever is given, i.e., nursing care, fluid balance, electrolytes and blood clotting parameters. Patients with dengue fever will be treated symptomatically, for example, sponging,acetaminophen, bed rest and oral rehydration therapy, and if signs of dehydration or bleeding occur the patients are usually hospitalized. Aspirin should be avoided because it may cause bleeding. Platelet count and Hematocrit should be measured daily from the suspected day of illness until 1’2 days after defervescence. Current prevention of dengue by potential dengue vaccine and vector control is highly cost effective. In addition, mosquito control programs are the most important preventive method. However, these are difficult to implement and maintain.
Development of a vaccine for dengue is difficult since there are four closely related, but antigenically distinct, serotypes of the virus that can cause disease. Infection by one serotype does not ensure protection of the patient from infection by the other three serotypes. Therefore, if vaccine were produced for only one or two serotypes, the other serotypes would increase the risk of more serious illness. Ribavirin has shown significant in vivo activity against RNA viruses; however, it exhibited only very weak activity against Flaviviruses. A possible strategy in the treatment of dengue is to use chimeric tetravalent vaccines that show high neutralizing antibody against all dengue serotypes. Studies on the development of tetravalent vaccines are ongoing in Thailand and these should be available in the near future. In addition, recombinant vaccines against capsid, premembrane and envelope genes of DENV-1, -2 and -3 inserted into a copy of a DNA infectious clone of DENV-2 are being developed and are currently undergoing clinical trials.(Kadir et al, 2013)
Corticosteroids are potent anti-inflammatory agents that have a wide range of effects on the immunological processes. Although corticosteroids are not mentioned in the WHO guidelines on the management of dengue, clinicians use corticosteroids empirically, based on the presumed immunological basis of the complications of dengue, particularly in the South East Asian countries.(Kularatne et al, 2003).
To determine the mechanism by which platelet counts increase after corticosteroid therapy for human immune thrombocytopenic purpura (ITP), we studied the platelet kinetics using prednisolone (PDN)-treated ITP-prone mice, (NZW x BXSB) F1 (W/B F1). An increase in platelet counts was observed in W/B F1 mice (n = 10, mean +/- SD, 1,202 +/- 202 x 10(3)/microL) 4 weeks after treatment with PDN (2 mg/kg/d); no increase occurred in nontreated W/B F1 mice (n = 5,651 +/- 126, P less than .005). Prolonged platelet life-spans (PLSs) were observed in treated W/B F1 mice (1.29 +/- 0.40 days), but not in nontreated controls (0.60 +/- 0.24 days, P less than .01). No increase in platelet production (platelet turnover) was found in PDN-treated W/B F1 mice, but significant decreases in platelet-associated antibodies (PAAs) and platelet-bindable serum antibodies (PBAs) were noted. Studies on organ localization of radiolabeled platelets showed that hepatic uptake significantly decreased in the treated W/B F1 mice, but not in nontreated W/B F1 mice. To elucidate the effect of PDN on the reticulo-endothelial phagocytic activity in W/B F1 mice, we studied in vivo clearance of IgG-sensitized, 51Cr-labeled autologous erythrocytes. W/B F1 mice treated with PDN showed a marked impairment of their ability to clear these cells, although PDN had little effect on the number of splenic or hepatic macrophage Fc gamma receptors. These results and our previous findings of splenectomy suggest that PDN improves platelet counts not only by suppressing systemic reticulo-endothelial phagocytic function but also by reducing antibody production. (http://www.ncbi.nlm.nih.gov/pubmed/1737103)
Corticosteroids are man-made drugs that closely resemble cortisol, a hormone that your adrenal glands produce naturally. Corticosteroids are often referred to by the shortened term "steroids." Corticosteroids are different from the male hormone-related steroid compounds that some athletes abuse. Some corticosteroid medicines include cortisone, prednisone, and methylprednisolone. Prednisone is the most commonly used type of steroid to treat certain rheumatologic diseases and is often the first-line of treatment for idiopathic thrombocytopenia purpura. (http://my.clevelandclinic.org/drugs/corticosteroids/hic_corticosteroids.aspx)
In animal studies, Hydrocortisone, a naturally occurring corticosteroid was used as a basis to compare and evaluate the platelet-increasing activity of papaya leaf juice. In a similar study, prednisone was used as a positive control to evaluate the platelet increasing activity of tawa-tawa (Euphorbia hirta).
QUININE
Quinine is a drug which is made from the bark of the Cinchona tree. A number of various other chemicals can also be synthesised from Cinchona, and these include cinchonine, cinchonidine and quinidine. Quinine is the cause of the most widely studied drug-induced immune thrombocytopenia (DIT). In most cases of DIT, antibodies bind to the platelet membrane glycoprotein (GP) Ib-IX complex in a drug-dependent fashion and bring about increased platelet clearance by the reticuloendothelial system resulting in thrombocytopenia. Here, we report the characterization of the quinine-dependent antibody activity of sera from 13 patients with quinine-induced thrombocytopenia. In our series of patients, GPIX was the most prevalent target of quinine-dependent antibodies. To identify the structural determinants of GPIX recognized by quinine-dependent antibodies, 4 chimeric mouse/human GPIX constructs and stable Chinese hamster ovary (CHO) cell lines that expressed the chimeras in association with GPIbalpha and GPIbbeta were produced. The analysis of 6 patient sera with the chimeric cell lines provided evidence for localization of the anti-GPIX quinine-dependent antibody binding site to the C-ext region (amino acid [aa] 64-135) of human GPIX. Further characterization of the C-ext region of the GPIX indicated that replacement of the Arg110 and Gln115 of the human GPIX with the corresponding residues from mouse (Gln and Glu, respectively) resulted in a significant reduction in the binding of GPIX antibodies in our series of patients, with Arg110Gln, giving a more pronounced effect than Gln115Glu. Hence, these 2 residues, particularly Arg110, play an important role in the structure of the antigenic site on GPIX recognized by anti-GPIX antibodies. (http://www.ncbi.nlm.nih.gov/pubmed/12738668)
Quinine is an oral prescription medication for the treatment of uncomplicated cases of malaria, according to Drugs.com. The adult dose is 648 mg taken with food every eight hours over the course of seven days. The condition develops because the immune system attacks and destroys platelets after quinine binds to them. In some instances, quinine-induced thrombocytopenia has been fatal. The patient must stop taking quinine because continued use of the drug can result in a fatal hemorrhage. (http://www.livestrong.com/article/122277-drugs-cause-low-blood-platelet/)
Quinine-induced thrombocytopenia has been recognized for more than 100 years. The antibody induced by drugs of this type interacts with platelets only in the presence of the drug. Many drugs can induce such antibodies, but quinine, quinidine, and sulphonamide derivatives do so more often than other drugs. When antibody production has begun, the platelet count falls rapidly and often may be less than 10,000 ”L. Patients may have abrupt onset of bleeding symptoms. If this type of drug-induced thrombocytopenia develops in a pregnant woman, she and her fetus may be affected. Quinine previously was used to facilitate labor but is no longer used for this purpose. (Price, Wilson, 2006)
The hallmark of thrombocytopenia induced by quinine and many other drugs is a remarkable antibody that binds tightly to normal platelets only in the presence of the sensitizing drug. The epitopes targeted by these antibodies usually reside on glycoprotein IIb/IIIa or Ib/V/IX complexes, the major platelet receptors for fibrinogen and von Willebrand factor, respectively.
SODIUM BENZOATE
Sodium benzoate is a food preservative that has been in use for years. As early as 1909, the ‘harmlessness’ of sodium benzoate as a food preservative was extensively verified in actual human feeding studies performed by three independent research organizations under the direction of the Secretary of Agriculture. A summary of these studies was published in a 784 Page book titled Report No. 88 of the US Dept. of Agriculture. This report verified that sodium benzoate, when mixed with food in the quantities specified, was not injurious to the general health nor found to adversely affect or impair the quality or nutritive value of such food.
For a long time, sodium benzoate has been generally recognized as safe (GRAS) as a direct food additive and recently this status was reaffirmed (21 CFR ”184.1733) for use as an antimicrobial agent, as defined in 21 CFR ”170.3(o)(2), and as a flavoring agent and adjuvant, as defined in 21 CFR ”170.3(o)(12). Sodium benzoate may be used in food at levels not to exceed good manufacturing practice. Current usage results in a maximum level of 0.1 % in food.
Sodium benzoate, N.F./F.C.C. is widely used in carbonated and still beverages, syrups, cider, salted margarine, olives, sauces, relishes, jellies, jams, preserves, pastry and pie fillings, low fat salad dressing, fruit salads, prepared salads, and in storage of vegetables. In syrups, concentrated sugar solutions are somewhat resistant to fermentation under ideal conditions but may be subject to quality deterioration in non-ideal circumstances. (http://doc.ccc-group.com/spec/800910.pdf