SEX VARIATION ON THE EFFECTS OF CARICA PAPAYA SEED EXTRACT ON RED BLOOD CELL, PACKED CELL VOLUME AND HAEMOGLOBIN IN MALE AND FEMALE WISTAR RATS
The effects of oral administration of different doses ofCaricapapayaseed extracts on haematological parameters specifically red blood cells (RBC), packed cell volume (PCV) and haemoglobin concentration in male and female wistar rats was examined. Previous studies have shown that Carica papaya has antisickling effects (Sofoworaet al., 1975), antifertility effects (Chinoyet al., 1997), membrane stabilization effects (Priyangaet al., 2012), nephroprotective effects (Olagunjuet al., 2009) and these effects tend to vary with sex. Other possible variations on the effects of Carica papaya seed extract due to sex is being investigated in this study.
Blood samples were taken by cardiac puncture from twenty (20) male and twenty (20) female wistar rats which were divided into four groups (n=5) each and graded dosages of Carica papaya seed extracts administered for twenty-one days (three weeks). The red blood cell mass, haemoglobin concentration and packed cell volume of the rats was determined by routine methods.
Two-way analysis of variance followed by Student’s T-test were performed on data and differences were considered statistically significant for p<0.05. There was an increase in parameters studied with response being higher in males than in females. Significant difference wereobserved between males and females only in red blood cell count though there were slight differences in the other parameters.
This study showed that Carica papaya seed extract has haemato-stimulatory effects on both males and females with higher response in males which is via another mechanism apart from its effects on sex hormones.
1.1 BACKGROUND STUDY
Blood is a special type of connective tissue composed of formed elements in a fluid matrix. The formed elements include red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (Bacha and Bacha, 2000). It is a bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells.
Assessment of haematological parameters can be quite informative about the health status of the individual and it is among the methods which may not be apparent during physical examination but may affect the fitness of the animals (Kronfeld et al., 1969). The red blood cell count, white blood cell counts, packed cell volume and haemoglobin concentration are regularly measured haematological parameters but this study is concerned with red blood cells, packed cell volume and haemoglobin.
Most of the haematological parameters are influenced by factors like sex, age, breed, seasonal variations, lactation, health and nutrition status (Aengwanich, 2002; Al-shami, 2007; Mohammed et al., 2007). Since it is a well-documented fact that there are variations due to age, sex, attitude etc. (Hawkins et al., 1954, Viteri et al.,1972, De Grunch et al.,1989), it is acknowledged that for comparisons between individuals and with reference data in a clinical diagnostic situation, it is necessary to consider normal variations due to sex, age and breed in order to increase diagnostic precision (Satue et al., 2009). It has however been observed that the modifications correlated with sex, age and taxonomic position are best shown by the erythrocytic counts (Duguy 1970).
SEX VARIATION IN RED BLOOD CELLS, PACKED CELL VOLUME AND HAEMOGLOBIN CONCENTRATION
Red blood cells are the most common type of blood cells and the vertebrates’ organism’s principal means of delivering oxygen to the body tissues via blood flow through circulatory system (Pierige et al., 2008). They are also called erythrocytes. They are biconcave and anucleated and there are about 5.2 million red cells per cubic millimeter of blood in the adult human (4.7 to 6.1 million in males and 4.2 to 5.4 million for females). They take up oxygen and release it while squeezing through the body’s capillaries. They are formed in the bone marrow and the average life cycle of a red blood cell is 120 days. Erythropoietin and oxygen levels are factors that regulate the production of red blood cells.
Haemoglobin is an iron-containing biomolecule found in red blood cell cytoplasm for transporting oxygen and carbon (iv) oxide. Iron can bind to oxygen and heme is responsible for the blood’s red colour. It also carries some of the waste products back and from the tissue. It is concerned with blood’s ability to carry oxygen and iron. Low iron could lead to morbidity and mortality. On the average, haemoglobin concentration for males is about 14 – 18g/dl and 12 -16g/dl.
The haematocrit is also known as packed cell volume or erythrocyte volume fraction. It is the volume percentage (%) of red blood cells in blood.it was coined in the year 1903. It comes from the Greek words ‘hema’ meaning blood and ‘krites’ meaning gage or judge. It is normally higher in males than in females and that is about 45% in men and 40% for women (Purveset al., 2004). It can be determined by centrifuging heparinized blood in a capillary tube at 10,000 RPM for five (5) minutes. The volume of packed red blood cells divided by the total volume of blood sample gives packed cell volume. It can also be calculated from automated analyzer.
Mean red cell mass and hematocrit levels are higher in men than women but the reason for this is yet to be discovered. It was reported that testosterone and other androgens have an erythropoietic stimulating effect that can cause polycythemia, which manifests as an increase in hemoglobin, hematocrit, or red blood cell count (Siddique et al., 2004, Dobs et al., 1999).Some studies however do not agree with this theory. It was reported that the increase of hemoglobin depending on testosterone is not along with the increase of erythropoietin. Hence another mechanism was proposed which stated that androgens increased the response of immature bone marrow cells to the effects of erythropoietin which in turn stimulates the production and regulation of red blood cells. However, other studies have shown that greater increase of hematocrit and hemoglobin observed in older males during testosteronetherapy is due to changes in erythropoietin rates (Alexanian, 1969). There is an important difference between male and female genders in testosterone and hematocrit rates but there is no important difference in their erythropoietin rates (Molinari, 1982). Then, androgens stimulate haematogenesis by a passage other than erythropoietin. Also the studies on patients with renal disease demonstrated that erythropoietin does not react to androgentherapy (Dobs et al., 1999). In contrast, studies have shown that estrogen can inhibit the production of red blood cells (Watanuki et al.,2002). Hence it can be seen that the sex of an individual is a determining factor on the haematological factors.
In the long history of the world, plants have been used medicinally. A large and increasing number of patients use medicinal herbs or seek the advice of their physician regarding their use (‘O’ Hara et al., 1998). It has been estimated roughly, that presently more than half of the total population of the world use herbal drugs (Chang, 1987). Increasing interest in medicinal herbs has increased scientific scrutiny of their therapeutic potentials and safety thereby providing physicians with data to help patients make wise decisions about their use (‘O’ Hara et al., 1998).
The papaya /pa’paia/ (Spanish name) also known as papaw or pawpaw is a fruit of the plant Carica papaya, the sole species in the genus carica of the plant family Caricaceae. The name papaya is widely recognized but it has been corrupted to kapaya, lapaya or tapaya in Southern Asia and the East indies. In French, it is papaya (the fruit) and papayer (the plant) or sometimes figuier des iles. Spanish speaking people employ the names melon zapote, payaya (fruit) and papayero (plant). Name in Brazil is mamao. It is known as pawpaw or papaw in both Africa and United Kingdom (Delbridgeet al., 1988, Watson 1997). It was nicknamed tree melon in Europe. It is a small, frost-tender, succulent broadleaf evergreen tree which is native to South America. It is best grown in topical or semi-tropical climates like Malaysia, the West Indies and Africa (Soforowa, 1996) and it is planted in well drained loams in full sun. It is properly called a giant herb because it never produces true woody tissue. Papaya is not a tree but an herbaceous succulent plant that possess self-supporting stems (Gross, 2003). Its fruits are large, fleshy and melon like. It is an erect fast growing tree measuring 7 – 8m tall, with copious latex and trunk of about 20cm in diameter (Duke, 1984). Its leaves are spirally arranged and confined to the top of the trunk.
The papaya has a rather complicated means of reproduction. The plants are male, hermaphrodite, or female (Bruce and Peter, 2008).The male trees are uncommon, but sometimes occur when homeowners collect their own seeds. Hermaphrodite trees (flowers with male and female parts) are the commercial standard, producing a pear shaped fruit. These plants are self - pollinated (Jari, 2009). Female flowers give way to smooth-skinned green pea-sized black seeds. Fruits and seeds are edible. The interior flesh of the fruit goes through colour changes from green (immature) to yellow (ripe and when it is overripe) (McGrath and Karahadian, 1994). It has been found t be available all through the year (Banerjee et al., 2006).
Phytochemical Components of Carica papaya
Papaya contains several unique protein-digesting proteolytic enzymes including papain and chymopapain (Aravind et al.,2013). Papain is similar to pepsin and it is also used to treat arthritis. Aside from its value as a remedy in kindred ailments, it has been utilized for the clarification of beer. The juice has been in use on dyspepsia meat to make it tender, (Wilson, 1994). Chymopapainis a more abundant proteolytic enzyme found in Carica papaya but papain is twice as potent (Morton 1987). Both papain and chymopapain can help lower inflammation and improve healing from burns.
This alkaloid occurs in all the green parts of the Carica papaya plant and in the seeds (Everette,1971). It slows the heart rate in humans and thus reduces blood pressure. Its action is similar to the drug prescribed for heart patients, digitalis. The alkaloid is reported to be able to kill worms and amoebas.
Lycopene is a bright red carotene and carotenoid pigment and phytochemical found in tomatoes and other red fruits and vegetables including papayas. Papaya has an abundance of cancer fighting lycopene. It is a key intermediate in the biosynthesis of many important carotenoids, such as beta-carotene and xanthophylls.
Another useful compound not readily found in the plant kingdom is Fibrin. It reduces the risk of blood clots and improves the quality of blood cells, optimizing the ability of blood to flow through the circulatory system. Fibrin is alsoimportant in preventing stroke.
Nutritional value of Carica papaya
The fruit of Carica papaya is not just delicious and healthy but whole plant parts, fruit, roots, bark peel, seeds and pulp are also known to have medicinal properties. The many benefits of papaya is due mainly to high content of vitamins A,B and C (Peterson et al., 1982). Proteolytic enzymes like papain and chymopapain which have antiviral, antifungal and antibacterial properties. The extracts of unripe Carica papaya contain terpenoids, alkaloids, flavonoids, carbohydrates, glycosides, saponins and steroids (Aravindet al., 2013).
Table 1: Papaya raw Nutritional value per 100g
Vitamin C 61.8mg
Folate (vit B9) 38mg
Vitamin B6 0.1mg
Niacin (vit. B3) 0.338mg
Riboflavin (vit. B2) 0.05mg
Thiamine (vit. B1) 0.04mg 0.04mg
Vitamin A 328mg
Dietary fibre 1.8mg
(Source: Aravindet al., 2013)
Actions of Carica papaya
A. Antisickling Effect of Carica papaya
Sickle cell disease is a genetic disease affecting the haemoglobin causing it to assume a sickle shape that cannot effectively carry oxygen to tissues and one of the many symptoms is anaemia (Koch et al., 2000). Some tropical plants like Carica papaya have been found to have antisickling properties and are employed in the management of sickle cell disease (Sofowora et al., 1975, Elekwa et al., 2003; Imaga et al., 2009). 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 and Shode, 1990). Antisickling activity of Carica papaya aqueous extract has been established and the antisickling agent was found to reside in the ethylacetate fraction of the methanol extract (Thomas et al., 1987). P-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid (protocatechuic acid,) and 4-hydroxy-3-methoxybenzoic acid (vanillic acid) are chemical constituents responsible for the antisickling activity of these plant and has also been used as a positive control in antisickling assays (Khadem et al., 2010). It has also been reported that Carica papaya oil extract is effective in enhancing sickle cell (Hbss) polymerization in only females but positively modulates peroxidase activity in both sickle cell sexes. It can hence be seen that the effectiveness of Carica papaya in managing sickle cell disease is sex dependent and this should be considered when administering the plant extract (Afolabiet al.,2012).
B. Membrane Stabilization Properties of Carica papaya
Extracts of papaya leaves have showed significant reduction in heat-induced hemolysis (Priyanga et al., 2012). This shows that Carica papaya has a therapeutic potential on disease processes causing destabilization of biological membranes. Carica papaya leaf extracts have also demonstrated a positive effect on increasing platelets counts in healthy mice (Sathasivam et al., 2009). Any compound or drug having a stabilization effect on the plasma membrane may effectively enhance survival of platelets with a potential morbidity and mortality benefits in patients with dengue viral infections. Mechanisms for this effect of Carica papaya is not yet known but a number of studies have revealed that flavonoids (David, 2007) and a host of other plant compounds (Jorge et al., 2004) exhibit analgesic and anti-inflammatory effects as a result of their membrane stabilizing ability in various experimental models.
Antiferility Effects of Carica papaya
Infertility is one of the adverse reactions from the consumption of Carica papaya. Aqueous extracts and benzene extracts given orally to female rats caused infertility and irregular oestrous cycles while in male rats given ethanol seed extracts orally or intramuscularly had decreased sperm motility. The oral doses also decreased testis mass and sperm count. The fertility of the male and female rats returned to normal within sixty (60) days after the treatments were discontinued (Harsha and Chinoy, 1996; Chinoy et al., 1997). Carica papaya seed extract and antifertility activities have also been discovered in langur monkeys and male humans (Lohiya et al., 2002). A study was carried out which revealed that alkaloid extracts of Carica papaya inhibited the serum level of androgen- testosterone which might result in infertility and reduce the effect of androgens to increase erythropoiesis (Udoh et al., 2009).
Nephroprotective properties of Carica papaya
In previous studies, it has been reported that pretreatment with Carica papaya seed extract prevented chloroform tetrachloride toxicity on the kidneys in both a time- and dose- dependent manner, an effect which could be mediated by any of the phytocomponents present in it via either antioxidant and/or free radical scavenging mechanism(s) (Olagunju et al., 2009). Another study also revealed that pretreatment with aqueous extract of Carica papaya L. significantly attenuated the physiological and histopathological alterations induced by gentamicin in the kidney and liver (Nale et al., 2012).The biochemical and histological findings obtained from these studies were suggestive of the fact that the extract may be mediating its protective effects by acting as a chain-breaking antioxidant for scavenging free radicals or by some other mechanisms depending on the condition of the kidney. In an earlier study, the presence of alkaloids, flavonoids, saponin, tannin, anthraquinones, and anthacyanosides in Carica papaya seed extract was reported (Adeneye and Olagunju, 2009). Other uses of Carica papaya include the following
Antioxidant Property of Carica papaya
Pawpaw fruits have a juicy taste rich in antioxidant nutrients like carotene, vitamin C, vitamin B, flavonoids, folate, panthotenic acids and minerals such as potassium and magnesium. The antiradical and antioxidant activities of Carica papaya leaf extract has been correlated with the polyphenolic components (Srikanth et al., 2010; Aiyegoro and Okon, 2009) hence it helps to prevent some forms of cancer.
Gastroprotective Properties of Carica Papaya
Papain belongs to a family of related proteins with a wide variety of activities, including endopeptidases, aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endopeptidase activity (Rawlings et al., 1994). Green or unripe papaya contains more papain than the fully ripened fruit hence green papaya is normally prescribed for acid indigestion and stomach ulcers.
Antihypertensive Actions of Carica Papaya
Hypertension is the most common cardiovascular disease and constitutes a major factor for several cardiovascular pathologies including atherosclerosis, coronary artery disease, myocardium infarction (oparil et al., 1999). Root extract of the Carica papaya plant gave similar effect of excretion of urinary electrolytes to that of the standard drugs. The determination of the osmolality of urine and electrolyte per unit time suggests that the extracts of Carica papaya exhibit such effect due to its high salt content (Sripanidhulchai et al., 1994). It has been reported that oral administration of papaya plant has anhypotensive effect (Arya et al., 2005). Proper standardization and clinical trials is needed to exploit therapeutic utility to combat cardiovascular diseases (Thakur et al., 2012).
Antidiabetic Effect of Carica Papaya
Some tropical herbs like Carica papaya offers both benefits as it has been recently informed in experimental models (Andrade-Cetto et al., 2005, Oliveira et al., 2009, Runnie et al., 2004). It has been observed that the aqueous extract of Carica papaya exerted a hypoglycemic effect. It also improved the lipid profile in diabetic rats (Isela et al., 2012).
Antibacterial and Antiparasitic Action of Carica papaya
Carica papaya fruit and seed extracts have been reported of possessing pronounced bactericidal activity against Staphylococcus aureus, Bacillus cereus, Escherischia coli, Pseudomonasaeruginosa and Shigellaflexnori (Emeruwa 1982). The pulverized seeds are also documented to possess anti-parasitic activities against Entamoebahystolica and Dirofilariaimmitis infections (Suhaila et al., 1994; Tona et al., 1998).The seed is used for intestinal worms when chewed.
Carica papaya L. leaf tea or extract has a reputation as a tumour-destroying agent (Walter Last, 2008). It appears to boost the production of key signaling molecules called Th1-type cytokines, which help regulate the immune system.
Papaya leaves are made into tea as a treatment for malaria (Titanjiet al., 2008). Antimalarial and antiplasmodial activity has been noted in some preparations of the plant, but the mechanism is not understood and not scientifically proven.
Rubbing the white pulp of raw papaya improves pimples as well as wrinkles. Papaya works as a good bleaching agent. It is an important ingredient in bath soaps, astringents, detergent bars and hand washes.
Protein enzymes including papain and chymopapain and antioxidant nutrients found in papaya; including vitamin C, vitamins E, and beta-carotene, reduce the severity of the conditions such as asthma, osteoarthritis, and rheumatoid arthritis (Elizabeth, 1994; Owoyele, 2008).
Also home applications of leaf and bark papaya extract is used to deal with mouth gums and toothaches which are being effectively practiced in many cultures around the world.
The black seeds of the papaya are edible and have a sharp, spicy taste. They are sometimes ground and used as a substitute for black pepper.
Carica papaya is also known to have nephroprotective (Olagunju, 2009) and neuroprotective function
SEX VARIATIONS AND THE EFFECTS OF CARICA PAPAYA
Evidently, it can be seen that Carica papaya mediates its effects on blood parameters directly or indirectly. Carica papaya high iron and vitamin B contents are contributing factors for stimulating erythropoiesis. It has however been observed that Carica papaya effects tend to vary with sex as reported by Afolabi (2013) in its antisickling and Lohiyaet al (2002) in its antifertility effects.
These variations of Carica papaya extract due to sex could be due to the direct effect of Carica papaya one sex hormones. It has been previously reported that the major cause of the difference in blood parameters in males and females is due to the stimulating effects by androgens in males (Siddique et al., 2004, Dobset al., 1999) and inhibitory effects of estrogen (Watanukiet al., 2002). However, there could be some other mechanisms that could cause sex variation in the effects of Carica papaya on haematological parameters.
To study the effects of Carica papaya seed extract on red blood cell, packed cell volume and haemoglobin.
To investigate the sex variation of the effects of Carica papaya seed extract on red blood cell, packed cell volume and haemoglobin.
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