Comparative studies on antisickling properties of brown and green leaves of Carica papaya

Sickle Cell Disease (SCD) is characterised by non under hypoxia conditions and this promotes red blood cell sickling. Inhibition of sickle cell haemoglobin polymerization is one of the areas of focus in the manage Carica papaya anaemia; without specifying if it is the dried green or brown leaf. This study was aimed at verifying the antisickling activity of the crude aqueous extract, crude methanol extract and fractions of dried brown and green leaves of The method used was the s expe used as a deoxygenating agent while isotonic saline (0.9% NaCl) was used as the negative control and phenylalanine as a positive control. aqueous extracts of both the sickled haemoglobin (HbSS) polymerization at200 mg/ml (97.76% and 93.25% ), 100 mg/ml(95.89% and 97.93%) also, 50 mg/ml(97.89% and95.84%) respectively which c favourably and significantly (p<0.05) with that of phenylalanine. The summary of the antisickling activity of the crude extracts and fractions of both the green leaves and brown leaves of Butanol> potential of inhibiting polymerization of sickle cell haemoglobin thus would be beneficial in the management of sickle cell disease.


Introduction
Sickle Cell Disease (SCD) is a multi-system disease associated with episodes of acute illness and progressive organ damage and it is one of the most common severe monogenic disorders worldwide [1]. It is a genetic disease caused by abnormal haemoglobin called sickle haemoglobin (HbSS), which polymerizes under deoxygenated condition and deforms the red blood cells into a 'sickle' shape [2]. SCD is characterized by premature breakdown of the red blood cells causing constant anaemia and occlusion of small blood vessels leading to excruciating body pains and other manifestations [3]. The treatment of SCD using orthodox approach has proved difficult and inefficient due to the genetic origin of the disease. Inhibition of sickle cell haemoglobin polymerization is one of the areas of focus in the management of SCA.
Carica papaya Linn. (Caricaceae) is an herbaceous succulent plant that possesses self-supporting stems [4]. The latex from the leaves has been used as antihelminths, antibacterial and for the DOI: 10 Carica papaya leaves are used in traditional herbal remedies for the management of sickle cell anaemia; without specifying if it is the dried green or brown leaf. This study was aimed at verifying the antisickling activity of the crude aqueous extract, crude methanol extract and fractions of dried brown and green leaves of The method used was the sickle cell haemoglobin polymerization inhibition experimentmeasured with the Ultra Violet (UV) spectrophotometer. Sodium metabisulphite was used as a deoxygenating agent while isotonic saline (0.9% NaCl) was used as the negative control and phenylalanine as a positive control. The results obtained showed tha aqueous extracts of both the green and brown leaves exhibited high level of inhibition of sickled haemoglobin (HbSS) polymerization at200 mg/ml (97.76% and 93.25% ), 100 mg/ml(95.89% and 97.93%) also, 50 mg/ml(97.89% and95.84%) respectively which c favourably and significantly (p<0.05) with that of phenylalanine. The summary of the antisickling activity of the crude extracts and fractions of both the green leaves and brown leaves of C. papaya is Crude aqueous>Crude aqueous methanol> Butanol>Chloroform>Ethyl acetate>Aqueous. This study showed that the extracts exhibited the potential of inhibiting polymerization of sickle cell haemoglobin thus would be beneficial in the management of sickle cell disease.
Keywords: Carica papaya, Sickle cell disease, crude aqueous extract, methanol extract and fractions. system disease associated with episodes of acute illness and progressive organ damage and ne of the most common severe monogenic disorders . It is a genetic disease caused by abnormal haemoglobin called sickle haemoglobin (HbSS), which polymerizes under deoxygenated condition and deforms the red blood cells into [2]. SCD is characterized by premature breakdown of the red blood cells causing constant anaemia and occlusion of small blood vessels leading to excruciating body pains and other manifestations [3]. The treatment of SCD using orthodox approach ifficult and inefficient due to the genetic origin of the . Inhibition of sickle cell haemoglobin polymerization is one of the areas of focus in the management of SCA.
Linn. (Caricaceae) is an herbaceous succulent plant The latex from the leaves has been used as antihelminths, antibacterial and for the production of papain which is used in food, textile and pharmaceutical industries [5]. An infusion of the mature leaves is also taken as an antidote for fever and malaria [6]. In Ivory Coast and Nigeria, water in which the young leaves have been crushed and squeezed is drunk three times daily for the treatment of hernia and urogenital infections [7]. Traditionally, the leaf extract was used as a tonic for the heart, analgesia and treatment of stomach ache [8]. C. papaya leaf methanolic extract has been reported to possess antisickling properties as they contain compounds that are capable of inhibiting and reversing the sickling of the red blood cel [9,10].

Plant Material
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. ickle cell haemoglobin polymerization inhibition rimentmeasured with the Ultra Violet (UV) spectrophotometer. Sodium metabisulphite was used as a deoxygenating agent while isotonic saline (0.9% NaCl) was used as the negative The results obtained showed that crude green and brown leaves exhibited high level of inhibition of sickled haemoglobin (HbSS) polymerization at200 mg/ml (97.76% and 93.25% ), 100 mg/ml(95.89% and 97.93%) also, 50 mg/ml(97.89% and95.84%) respectively which compared The summary of the antisickling activity of the crude extracts and fractions of both the green is Crude aqueous>Crude aqueous methanol> Chloroform>Ethyl acetate>Aqueous. This study showed that the extracts exhibited the potential of inhibiting polymerization of sickle cell haemoglobin thus would be beneficial in the sease, crude aqueous extract, crude aqueous production of papain which is used in food, textile and ies [5]. An infusion of the mature leaves is also taken as an antidote for fever and malaria [6]. In Ivory Coast and Nigeria, water in which the young leaves have been crushed and squeezed is drunk three times daily for the treatment of hernia al infections [7]. Traditionally, the leaf extract was used as a tonic for the heart, analgesia and treatment of stomach ache leaf methanolic extract has been reported to possess antisickling properties as they contain compounds that are able of inhibiting and reversing the sickling of the red blood cells . Following careful siphoning of the plasma with Pasteur pipette, the erythrocytes were washed three times with a volume of isotonic saline (0.9%) equivalent to the siphoned plasma. The samples were then centrifuged each time at a speed of 2000 rpm to remove the supernatant. The resulting erythrocytes were then suspended in a volume of isotonic saline equivalent to the siphoned plasma. The erythrocyte suspension was then frozen at 0 o C, and subsequently thawed before the experiment to produce haemolysates.

Sickle cell haemoglobin polymerization inhibition experiment
The underlying principle is that HbSS undergoes polymerization when deprived of oxygen, transiting to deoxyHbSS molecules; sodium metabisulphite was used as a deoxygenating agent [11]. 4.8 ml of freshly prepared 2% sodium metabisulphite (Na 2 S 2 O 5 ), 0.1 ml of HbSShaemolysate and 0.1 ml of isotonic saline (0.9% NaCl) the negative control (NC) was added into a test tube. The mixture was mixed thoroughly on a votex mixer, transferred into a cuvette and optical density measured using the spectrophotometer. Absorbance (O.D) values were taken at one (1) minute interval for 10 mins at 700 nm to determine the rate of polymerization; this served as the negative control. For the test assay, 0.1 ml of the test compound (C. papaya of different concentrations, 200 mg/ml, 100 mg/ml, 50 mg/ml of the extracts and fractions) was used in place of 0.1 ml isotonic saline and the procedure repeated as above. 10 mM of the antisickling amino acid, phenylalanine (Phe) was used as a positive control (PC) in place of the test compound [12] and the test conducted accordingly.

Statistical Analysis
The rates of haemoglobin polymerization for the extracts and fractions were estimated by calculating the tangent of a plot of average change in extinction or change in optical density (∆OD 700 nm) versus time in minutes using the Graph pad prism software 5.0. The rates were equally expressed as percentages with respect to control. This gives the relative percent (%) inhibition. Results were considered to be statistically significant at p<0.05

Discussion
Inhibition of sickle cell haemoglobin polymerization is one of the areas of focus in the management of SCD thus, it has been hypothesized that antisickling drug or agent should significantly inhibit polymerization of the abnormal sickle haemoglobin HbS [13]. For the green leaves, the CAE exhibited high level of inhibition of HbS polymerization of 97.76%, 93.25% and 95.89% at all concentrations -200mg/ml, 100mg/ml, 50mg/ml respectively which compared favorably and significantly (p<0.05) with that of Phe (Table 2), a well-researched standard antisickling agent [14,15]. The CAME also exhibited a high level of inhibition of HbS polymerization of 90.88% and 93.65% at 200mg/ml and 100mg/ml respectively while only the lowest concentration -50mg/ml of the BF exhibited high level of inhibition-93.08% which compared favorably and significantly (p<0.05) with that of Phe (  (Table 2). These results, coupled with the over 50% inhibition potential (although significantly, p<0.05, lower than that of Phe) observed in the CF of both the fresh green and the brown leaves may be due to the polar nature of these fractions and their ability to diffuse into the haemoglobin molecule to bind at the heme pocket, thereby obstructing the 'sticky patches' of the sickle cell Hb molecules [11]. This will prevent polymerization of Hb molecules into long fibers that would have caused deformation into sickle shapes of the normal disc biconcave shape of RBCs [16]. On the other hand, the AF of both the green and brown leaves at 50mg/ml exhibited a significantly (p<0.05) low percent inhibition of polymerization in comparison with that of the positive control. This may be attributed to the fact that the antisickling activity is concentration-dependent.
The 200mg/ml concentration of C. papaya leaf fractions were more effective in inhibiting sickling than other concentrations. C. papaya leaf crude extracts exhibited the highest level of inhibition of HbS polymerization compared to the other extract fractions and positive control used. This finding is comparable to previous studies which have found plant crude extracts to be more effective than its various fractions [17]. The antisickling properties of the leaf could be concentrated in the polar and phenolic constituents of C. papaya as evidenced by the potent antisickling activities of the crude aqueous extract, crude aqueous methanol extract and butanol fraction. The summary of the antisickling activity of the crude extracts and fractions of the green leaves and dead leaves of C. papaya is CAE>CAME>BF>CF>EAF>AF. This study also supports the claims of previous studies on antisickling activity of phytomedicines which showed that aqueous methanol forms of extraction contained the active constituents responsible for their observed activity [11,18,19]. The use of sodium metabisulphite to induce sickling is probably a more drastic approach than what actually happens in the vascular system of humans [20]. It is therefore expected that the extracts may achieve more efficient sickle inhibition in vivo. The high level of inhibition of HbS polymerization exhibited by C. papaya leaf indicates that the extract may be used to attenuate SS cell sickling and establish their abilities to inhibit sickling under hypoxic conditions thus justifying their use in folklore medicine for SCD management.

Conclusion
The results obtained in this study showed that the extracts exhibited the potential of inhibiting polymerization of sickle cell haemoglobin thus they would be very beneficial in the management of sickle cell disease. Funding support This study received no external financial support.

Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication.