PRELIMINARY IN VITRO ANTISICKILNG PROPERTIES OF CRUDE JUICE EXTRACTS OF PERSIA AMERICANA , CITRUS SINENSIS , CARICA PAPAYA AND CIKLAVIT ®

The antisickling properties of crude juice extracts of the edible portions of three commonly consumed tropical fruits namely Persia americana, Citrus sinensis, and Carica papaya were investigated in vitro alongside a new drug preparation called Ciklavit® that has antisickling activity. Four different solvent extracts of the crude juice of each fruit including aqueous, acidic, alkaline and alcoholic extracts were prepared and their antisickling effects on sickle cell trait (HbAS) and sickle cell disease (HbSS) blood samples checked alongside Ciklavit®. Blood samples were stabilized using normal saline and the antisickling effects were checked by counting the number of sickle cells remaining after incubation of the blood samples with the crude fruit extracts and Ciklavit® for twentyfour hours. The results showed that Ciklavit® produced a sustained reduction in the number of sickle cells in both HbAS and HbSS blood samples. Also the alkaline and alcoholic extracts of P. americana and C. papaya produced significant reduction in the number of sickle cells.


Introduction
Sickle cell disease is encountered in many parts of the world but is predominant in people of African, Mediterranean, Indian and Middle Eastern descent as well as blacks and Hispanics in the Caribbean and Central America (Bownas, 2000).The disease is a genetic disorder associated with the synthesis of abnormal hemoglobin as a result of point mutation in the gene coding for its beta chain resulting in the substitution of glutamic acid with valine (Koch et al., 2000).The affected hemoglobin expresses gross changes in its chemical and physical properties especially shape and oxygen-carrying capacity.The distorted sickle shape of the Hemoglobin slows down its mobility through blood vessels resulting in intensely painful blockage that prevent vital oxygen and nutrients in the blood from reaching target organs and tissues thereby impairing their functions (Campbell et al., 1999).This results in a number of health problems such as brain damage, thrombosis, liver damage etc. (Enang, 1992).
Individuals can either be homozygous for the sickle cell hemoglobin (HbSS) or heterozygous (HbAS).The HbAS individuals are termed carriers and do not usually exhibit the grave health conditions associated with the HbSS individuals except when they are exposed for a long duration to conditions that support sickling (Koch et al., 2000).Certain conditions including low oxygen concentration, low pH and high temperature could cause a decrease in solubility of deoxy HbSS, leading to its polymerization into fibers that distort its shape and function (sickling).This culminates in symptoms and complications associated with sickle cell diseases including joint pain, anemia, fever, paleness, shortness of breath and jaundice (Bownas, 2000) Treatment of sickle cell disease has proved difficult and inefficient due to its genetic origin.Earlier therapies included use of liver-based extracts and diets, oxygen vasodilator, carbonic anhydrase inhibitors and spleenectomy.Other conventional therapeutic management involves the use of drugs such as piracetam, tucaresol, hydroxyurea as well as bone marrow transplantation and gene therapy (el-Hazmi et al., 1995;Krishnamurti et al., 2001,Walters, 2005, Bownas, 2000, Dean and Schechter,1978).Research is still going on to discover potential antisickling agents, especially from plants (Sofowora et al., 1975, Adesina, 2005).The antisickling properties of plants are due to their content of alkaloids, resins, phenolics and oils, (Ekong et al., 1975, Elujoba and Nagels, 1985, Sofowora et al., 1975, Ouattara et al., 2004).
Fruits such as Persia americana (Avocado pear); Citrus sinensis (Orange), and Carica papaya (Pawpaw) are commonly consumed in the tropics and have a wide array of biologically active substances which could possibly show antisickling properties comparable to that of a new drug preparation called Ciklavit® (Pizzorno and Murray, 1985, Ahmed and Bonner 1980, Daziel, 1955, Bean, 1958, Cummings and Schroeder, 1942,).Ciklavit® produced from extracts of Cajanus cajan has been shown to reverse sickling and clinically reduce the painful crises associated with sickle cell disease (Ogoda et al., 2002, Akunsulie et al., 2005).The possible benefits that can accrue from new therapies for sickle cell disease from local African fruits in terms of safety and affordability when compared to conventional therapies such as hydroxyurea cannot be overemphasized.

Materials and Methods
The edible portion of matured and ripe fruits of Persia americana , Citrus sinensis, and Carica papaya bought from a local market were used.Blood samples from consenting and confirmed HbAS and HbSS volunteers attending the University of Nigeria Teaching Hospital (UNTH), Enugu, Nigeria were used.
Each (100g) of the washed and peeled fruits was extracted in 100mls of water using a juice extractor.The crude paste obtained was then extracted with different solvents.
Each fruit juice (10mls) was extracted with 5mls of 0.1N HCl by gently shaking the mixture for 1 hr in a shaker.The mixture was then centrifuged for 5 mins at 5,000g and the supernatant was concentrated to 5mls at 60 0 C in a shaking water bath and retained as the acidic extract.
Each fruit juice (10mls) was extracted with 5mls of 0.1M NaOH by shaking the mixture gently for 1 hr in a shaker.The mixture was then centrifuged for 5 mins at 5, 000g and the supernatant concentrated to 5mls at 60 0 C in a shaking water bath and retained as the alkaline extract.
Each fruit juice (10mls) was extracted with 5mls of 95% ethanol by shaking the mixture and they were then centrifuged for 5 mins at 5,000g and the supernatant concentrated to 5mls at 60 0 C in a shaking water bath and retained as the alcoholic extract.
Each fruit juice (10mls) was extracted with 5mls of water by shaking gently for 1 hr in a shaker.The mixture was centrifuged for 5 mins at 5,000g and the supernatant concentrated to 5mls at 60 0 C in a shaking water bath and retained as the water extract.

Antisickling tests
Antisickling test were done as outlined by Elekwa et al., (2003).Blood samples of HbAA, HbAS and HbSS patients were used to check for sickling on grease-free slides.Each blood sample (0.2mls) and 0.2ml of 2% sodium metabisuphite solution were mixed on slides, covered and rimmed with molten wax.The slides were incubated for 24 hrs and viewed under x40 magnification with a binocular light microscope (Olympus model CHC-XS2-107BN) for sickling after which sickle cells were seen in HbAS and HbSS samples.The antisickling effects were investigated by incubating 0.2mls of the acidic, alkaline, alcoholic and water extracts of each of the fruit juice extracts and 0.2mls of Ciklavit® solution on covered slides rimmed with molten wax for 24 hrs.The control consisted of only HbAS and HbSS blood samples and 2% sodium metabisulphite solution.All the tests were done in triplicates.After incubation, the numbers of sickled cells remaining in all the tests were counted under a light microscope.

Statistical analysis
The student t-test was used and statistical significance was checked at 95% confidence interval.

Results
Results obtained were expressed as percentage of sickle cells remaining after incubation of HbAS and HbSS blood samples with the different fruit extracts and Ciklavit®, as shown in Tables 1-3.

Effect of Citrus sinensis on sickling
The effects of extracts of Citrus sinensis and Ciklavit® on sickling are shown in Table 1.The result indicates that Ciklavit® produced remarkable antisickling on HbAS and HbSS blood samples.Only the alkaline extract of C. sinensis showed a significant antisickling effect on HbAS blood sample.

Effect of Carica papaya on sickling
The antisickling effects of Ciklavit® and the extracts of Carica papaya on HbAS and HbSS blood are shown in Table 2. From the results, Ciklavit® produced a significant antisickling effects on both blood samples followed by the acidic ,alcoholic and alkaline extracts of C. papaya on HbAS and HbSS blood samples respectively.The effects of Persia americana and Ciklavit® on sickling in HbSS and HbAS are shown in Table 3. Results showed that Ciklavit® produced the highest level of antisickling effect, followed by the alkaline and alcoholic extracts of P. americana on HbAS and HbSS blood samples respectively.

Discussion
Sickle cell disease is a genetic disease that affects the hemoglobin causing it to assume a sickle shape that cannot effectively carry oxygen to tissues and organs.This abnormality results in crises and may manifest as symptoms including anemia, pain, fever, jaundice, leg ulcers etc (Koch et al., 2000).It has been difficult to find an efficacious cure for this disease because of its genetic origin, even though it can be managed by using some medications such as hydroxyurea (Bownas, 2000).Some tropical plants have been found to have antisickling properties and are employed in the management of sickle cell disease (Sofowora, 1974, Sofowora et al., 1975, Elekwa et al., 2003.Only recently an indigenous pharmaceutical company in Nigeria produced a nutritional preparation called Ciklavit® from extracts of Cajanus cajan which purportedly has antisickling properties and is already being used in the management of sickle cell disease (Akunsulie et al., 2005).The antisickling potential of tropical plants is due to their peculiar content of a wide variety of biologically active substances and amino acids capable of reversing sickling (Sofowora, 1975, Ekeke andShode, 1990) The result obtained from this preliminary study confirms the in vitro antisickling property of Ciklavit® as well as the potential antisickling activity inherent in some tropical fruits such as Persia americana, Citrus sinensis and Carica papaya.These tropical fruits elaborate active constituents including essential oils, flavonoids, carotenoids and nitrogenous substances which could express antisickling activities in addition to other properties (Iwu, 1985,Thomas and Ajani, 1987, Duke, 1992, Eskin and Tamir , 2006, Ogunyemi et al., 2008, Elzebrook and Wind, 2008).Further investigations are underway to specifically characterize these potential antisickling substances from the fruits.

Table 1 :
Antisickling effect of c. sinensis and ciklavit® on hbas and hbss blood

Table 2 :
Antisickling effect of c. papaya and ciklavit® on hbas and hbss blood.

Table 3 :
Antisickling of p. americana and ciklavit® on hbas and hbss blood.