Haematinic and Hepatoprotective Properties of Telfairia occidentalis Fruit Mesocarp on Phenylhydrazine-Induced Anaemia in Experimental Rats

The level and potential of iron contained in fluted pumpkin (Telfairia occidentalis) has been exploited as a blood tonic; however, the potentials of some other parts of the plant are unknown. The effect of T. occidentalis fruit mesocarp (aqueous extract) on phenylhydrazine (PHZ)-induced anaemia in experimental rats was investigated in a bid to determine its curative properties and potential in reversing haemolytic anaemia and protection of liver health. The LD50 of the fruit extract was determined using Lorke's method for the determination of acute toxicity. The study involved oral administration of varying doses of the extract to different groups of rats which were monitored for 24 hours. The test sample did not show any signs of toxicity at doses of 5000 mg/kg b.wt, which is the highest possible recommended dose for toxicity testing. For the evaluation of the effects of the fruit extract on haematological indices and biochemical enzyme markers in anaemic rats, 30 matured albino Wistar rats were used. The rats were divided into five groups of six rats each. Group 1 consisted of normal rats (control group), Group 2 consisted of anaemic untreated rats, and Group 3 consisted of anaemic rats treated with the standard drug Astymin, while Groups 4 and 5 were made up of anaemic rats given the extract at doses of 600 mg/kg b.wt and 1000 mg/kg b.wt, respectively. The fruit extract failed to show any significant effect in improving the haemoglobin (Hb), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphate (ALP) levels in anaemic rats but rather may have contributed to a reduction in Hb levels and an unhealthy increase in serum enzyme levels. This is indicative of the apparent inability of the aqueous extract of the T. occidentalis fruit mesocarp to reverse PHZ-induced haemolytic anaemia and may suggest a possible detrimental effect of high doses of the extract over a prolonged period.


Introduction
Anaemia is characterized by the reduction in red blood cell (RBC) count, haemoglobin (Hb) content, and packed cell volume (PCV).Generally, reduction in all three occurs due to decreased production of RBCs, increased destruction of RBCs, and excess loss of blood from the body [1].Studies reveal that anaemia may be caused by inherited disorders or environmental factors such as nutritional problems, infections, and exposure to certain drugs or toxins [2].Typically, gradual development of anaemia produces vague symptoms such as fatigue, weakness, shortness of breath, or a poor ability to exercise; however, abrupt development has more severe symptoms such as confusion, dizziness, loss of consciousness, or increased thirst.Furthermore, although anaemia is often characterized by paleness, it is only in signifcant cases [2].
Although the three main types of anaemia are identifed primarily due to either blood loss, decreased RBC production, or increased RBC breakdown, anaemia can also be classifed based on the morphological and etiological classifcation of anaemia.Tis includes macrocytic normochromic anaemia, macrocytic hypochromic anaemia, and nutrition defciency anaemia (like iron defciency) [2].According to various studies, treatment largely depends on the cause of the anaemia.A typical example is in the case of iron defciency anaemia, where a replacement iron therapy is usually prescribed.Te two categories of iron supplements are those containing the ferrous form of iron and those containing the ferric form of iron, with the former being the better absorbed of the two [3].Although iron supplements are widely used in treating cases of anaemia, certain chemical substances are capable of inducing or bringing about the onset of anaemia in otherwise living organisms, one of such being phenylhydrazine (PHZ).
Research has shown that PHZ is a particularly potent redox-active drug capable of inducing haemolytic anaemia, even in individuals without erythrocytic enzyme defciencies [3].Tis is a result of haemolysis caused by interaction with sulfhydryl groups, inhibition of various enzymes, immune mechanisms, and the fragmentation of erythrocytes as they pass through the platelet-fbrin mesh or by unknown or poorly defned mechanisms.Yeshoda's study [4] induced anaemia in rats following a single PHZ intraperitoneal administration at a dose of 20 mg/kg b.wt (aqueous solution).In this study, erythrocyte concentration lowered to about 50% and haemoglobin level to about 60% of normal values in the course of 4 days.Tis is consistent with various data that reveal that PHZ induces anaemia via the destruction of red blood cells by oxidation stress and many joint changes at cellular levels.It is worth mentioning, however, that although various studies highlight the ill effects of PHZ on RBCs, few studies have tested the efects of other substances (like medicinal plants) on PHZ-induced anaemia.Figure 1 shows the structure of phenylhydrazine.
Te medicinal application of plants is common in Africa as the efcient use of these plants has been well established [5].Ancient Egyptian medicine of 1000 BC was known to have used garlic, opium, castor oil, coriander, mint, indigo, and other herbs for medicinal purposes.In the seventh century AD, the Slavic people used Rosmarinus ofcinalis, Ocimum basilicum, and Allium sativum as a remedy against several injurious insects (such as lice, feas, moths, mosquitoes, and spiders) [6].However, in more recent years, the bark of willow trees which contains large amounts of salicylic acid (which is the active metabolite of aspirin) has been used as an efective pain reliever and fever reducer [7].
Telfairia occidentalis is a tropical vine grown in West Africa as a leaf vegetable and for its edible seeds.It is commonly known as futed gourd or futed pumpkin and is grown or planted in the forest zone of West and Central Africa, most frequently in the Benin Republic, Nigeria, and Cameroon [8].In a study by Anthony and Ojeifo [9], the result of phytochemical screening of T. occidentalis leaf, root, fruit mesocarp, and stem showed impressive amounts of favonoids, alkaloids, saponins, terpenoids, tannins, steroids, and glycosides, with the roots having more of these phytochemicals than other plant parts.Te fruit mesocarp also showed high levels of tannins and steroids.Generally, although the level and potential of iron contained in futed pumpkin has been exploited as a blood tonic which can be administered to weak patients [10], there is still uncertainty, however, about the benefts and potentials of other parts of the plant.In addition, the increase in malnutrition globally has prompted several studies on the impact of diferent foods on human health and well-being.Depending on their nutritional value, certain foods are considered healthier than others [11].According to FAO [12], the composition/quality of various foods depends on several factors: species, breeds, cultivars, ecological factors, postharvest handling, preservation, and storage techniques.Consequently, this study seeks to investigate the haematinic and hepatoprotective properties of futed pumpkin mesocarp (aqueous extract) on PHZ-induced anaemia in experimental rats.

Sample Collection and Preparation.
Fresh matured pods of Telfairia occidentalis fruit were obtained from Marian Market, Calabar Municipal, Cross River State.Te plants were taken to the Botany Department, University of Calabar, for identifcation and confrmation.Figures 2 and 3 show diagrams of T. occidentalis.
Te protocol for plant preparation and extraction was based on the work by Olorunfemi et al. [14].Te fruits were boiled for 45 minutes after which it was opened and the mesocarp scrapped of.Te mesocarp was then blended into a thick paste using an electric blender.Te resultant paste was macerated with distilled water for 72 hours to obtain the aqueous extract.Te extract was sieved with a cheese cloth and subsequently with a flter paper in order to obtain a fne extract.Te fltrate was then evaporated in a water bath.Te brownish thick paste obtained was stored in a refrigerator for laboratory analyses.

Acute Oral Toxicity
Testing.Twelve experimental mice of 330.6 ± 2.24 g body weight were purchased from the animal house, Department of Biochemistry, University of Calabar, Cross River State.Te animals were housed in transparent wire-gauzed plastic cages to acclimatize for 5 days.Te rats were fed with pelletized poultry grower's mesh feed and tap water ad libitum.Te extract was administered once, orally after the animals were left to fast for 24 hours.Lorke's method (a new method for determining 2 Biochemistry Research International acute toxicity) as described by Enegide et al. [15] was used to determine the acute oral toxicity of aqueous extract of Telfairia occidentalis fruit mesocarp using twelve mice in all.Tis method consisted of diferent stages (Table 1) with the result of each stage determining whether to proceed or terminate and go for the confrmatory test.A confrmatory or confdence test was used to validate any test sample which showed signs of toxicity or caused mortality.Te test sample was administered 50 mg/kg, 200 mg/kg, 400 mg/kg, and 800 mg/kg body weight in stage 1 which involved four animals: one animal each per dose; stage 2 involved three animals, each received 1000 mg/kg, 1500 mg/kg, and 2000 mg/kg body weight, respectively.Lastly, stage 3 also had three animals, and each received 3000 mg/kg, 4000 mg/kg, and 5000 mg/kg body weight, respectively.At each stage, animals were observed for any signs of toxicity or mortality for 24 hours at an interval of 10 minutes and, thereafter, after every 1-2 hours.If the test sample did not show any sign of toxicity or mortality at 5000 mg/kg body weight which is the highest possible recommended dose for the toxicity test [15], a confrmatory test was carried out using two animals to validate the test dose.For any dose with signs of toxicity or mortality at any stage of the test, two animals were used to confrm the dose.
Te formula for calculating the LD 50 after the toxicity test is given as follows: where D 0 is the highest dose of test sample that gave no mortality or showed any sign of toxicity and D 1 is the lowest dose of test sample that gave no mortality or showed any sign of toxicity.

Experimental
Rats.Tirty albino Wistar rats were purchased from the animal house of the College of Medical Sciences, University of Calabar, Calabar, Cross River State, Nigeria.Te rats (6-8 weeks of age at the time) of average body weight 300 g were acclimatized for 3 weeks.Te rats were allowed free access to water and food (growers mesh feed) under standard laboratory conditions of temperature, light, ventilation, and relative humidity.

Induction of Anaemia.
Phenylhydrazine chlorhydrate was used to induce haemolytic anaemia in the rats.DMSO solution was used to dissolve phenylhydrazine to one-tenth in distilled water.Te solution was then administered intraperitoneally (IP) to the rats at a dose of 40 mg/kg of body weight/day for two days (D0 and D1).

Experimental Protocol.
At day 0, rats were randomly classifed into the following 5 groups (6 rats per group): normal control group, phenylhydrazine (PHZ) group, PHZ + Astymin group, PHZ + extract (low dose) group, and PHZ + extract (high dose) group.Group 1 rats were not anaemic and served as control while the rats of other groups were anaemic.Group 3 rats were treated with Astymin.Groups 4 and 5 were treated with the extract 600 mg/kg of b.wt/day and 1000 mg/kg of b.wt/ day, respectively, from D2 to D8. Te extract and Astymin were administered by gavage using a gastric tube.Astymin is a blood tonic commonly prescribed for anaemic patients, in order to boost red blood cells production.Te detail of the protocol is shown in Table 2.

Blood Sample Collection.
Under chloroform anaesthesia, 2 rats from each group were dissected on days 3, 5, and 8. Tey were frst weighed and then anaesthetized by placing them in a closed jar containing cotton wool soaked with 5 ml of chloroform anaesthesia.Tey were then dissected using surgical scissors to expose the heart; two blood samples were immediately withdrawn from the vena cava of each rat via cardiac puncture.Te frst sample was collected into a vial containing disodium salt of ethylene diamine tetra acetic acid (EDTA) anticoagulant.Te second blood sample was collected into a plain sample vial without any anticoagulant.

Blood
Tests.Approximately 5 ml of blood samples was collected in both EDTA and plain sample vials.Blood samples collected were used in the determination of haematological indices and liver enzymes.Biochemistry Research International (i) Estimation of haematological indices: Haematological parameters such as haemoglobin (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC) and number of platelets were assessed by the standard haematological measurements using an automatic haematological assay analyser (Beckman Coulter, USA).(ii) Determination of liver enzymes: Blood serum enzyme levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphate (ALP) were determined using standard laboratory procedure at the University of Calabar Teaching Hospital Laboratory, chemical pathology unit.

Acute Oral Toxicity Test Result.
From acute oral toxicity tests carried out on mice in a bid to determine the LD 50 of the extract using Lorke's method, results obtained (Table 3) showed no mortality within 24 hours after treatment with the extract with doses of up to 5000 mg/kg b.wt.No death was recorded among all the dose groups throughout the experimental period, thus being indicative of LD 50 values of up to 5000 mg/kg b.wt and above.

Haematological and Liver Enzymes Analysis Results.
From Table 4, a signifcant diference is observed in the haemoglobin concentration when comparing G2 to G1.With G2 having a lower Hb concentration in comparison to the control group (G1), a signifcant diference was also found between G2 and G3, with G2 maintaining a lower Hb concentration than G3.While G4 and G5 showed a statistically signifcant decrease in haematological parameters such as WBC, PCV, MCV, MCH, and MCHC, a drop is further observed when comparing G2 to G1.An increase in WBC, MCV, and MHC levels of G3 is noticed when compared to G2.Also, a signifcant decrease in WBC in both G4 and G5 is observed when compared to those of G3.An accompanying drop in PCV and MCV levels of G4 and G5 occurs as compared to those of the control group, G1.MCV values of D0 � day the experiment was started; D1 � frst day after the start of the experiment; D2 � second day after the start of the experiment; D8 � eighth day after the start of the experiment (also the last day of the experiment). 4 Biochemistry Research International G4 were also found to statistically difer from those of G2 and G3.While the MCH of G4 difered from G1 and G3, both MCH and MCHC of G5 difer signifcantly from G1, G2, and G3.For enzyme parameters, AST, ALT, and ALP showed a signifcant increase when comparing G2 to G1.A contrary signifcant decrease in AST, ALT, and ALP levels is noticed when comparing G3 to G2 with a marked diference (increase) in AST, ALT, and ALP levels in G4 and G5 when compared to G1 and G3.However, a signifcant diference in ALT and ALP levels is noticed between G5 and G2.From Table 5, showing the result for day 5 of testing, a signifcant diference is observed in the PCV and MCV when comparing G2 and G3 to G1.A signifcant diference, however, still exists among these parameters when compared between the two groups, i.e., G3 and G2.A signifcant decrease in Hb level is observed when comparing G2 to G1 while G3 Hb level is signifcantly higher than G2.Signifcantly elevated levels of MCH and MCHC in G3 were noticed when compared to G1 and G2.Furthermore, a signifcant diference in Hb, PCV, and MCV levels in G4 is observed compared to G2 and G3.A signifcant diference in MCH and MCHC in G3 was noticed when compared to G1 and G2.Further diference in Hb, PCV, and MCV levels in G4 is observed compared to G2 and G3.A signifcant difference in MCH and MCHC levels of G4 occurs when compared to G1 while the MCHC level of G4 difers signifcantly from G3.In G5, RBC, Hb, PCV, and MCV levels signifcantly difer from both G1 and G3 while MCH and MCHC levels of G5 difer only from G3.For biochemical parameters, such as AST, ALT, and ALP, G2 levels are signifcantly higher when compared to G1 and G3.G4, AST, ALT, and ALP levels are also signifcantly lower compared to G2 while those of G5 tend to difer signifcantly from those of G1, G2, and G3.
On day 8 of testing, Table 6 shows there was no signifcant diference in haematological parameters when comparing among the three groups: G1, G2, and G3.However, Hb, PCV, and MCH showed statistically signifcant diferences when comparing their levels in G4 to those of G1, G2, and G3.Meanwhile in G2, AST, ALT, and ALP levels showed a signifcant increase when compared to those of G1 and G3.AST, ALT, and ALP levels of both G4 and G5 also showed signifcant increase compared to those of G1, G2, and G3.Figures 4 and 5 also give a pictorial representation of the haematological indices and liver enzymes at days 4, 6, and 8 after the extract administration.

Discussion
Similar to previous research results, values obtained on day 3 testing (Table 1), G2 which was administered phenylhydrazine (PHZ) showed a signifcant decrease in the haemoglobin (Hb) level compared to the control group G1.Tis decrease could be explained by the mechanism of action of PHZ, which induces haemolytic anaemia via the destruction of the red blood cells (RBCs) by oxidative stress which causes alterations to the RBCs proteins [3,16].Te destruction of RBCs led to a concomitant release of the liver enzymes ALT, AST, and ALP into the blood serum, leading to a spike in the serum levels of these enzymes as observed from Table 1.However, these efects were efectively reversed in the group (G3) given the standard drug Astymin.Te reversal of the debilitating efects of haemolytic anaemia and accompanying elevated liver enzyme levels is most likely due to the ability of Astymin to improve erythropoiesis, promote tissue repair, improve haemoglobin synthesis, protect the liver, and stimulate protein manufacture in the body [17][18][19].
In G4 and G5, fed with the low and high doses of the extract respectively, a signifcant decrease in Hb levels is observed with an accompanying increase in enzyme levels to values even higher than those of G2.Te fndings reported here suggest an inability of the fruit mesocarp extract to improve anaemic conditions.
From day 5 of testing (Table 2), the trend in decrease of Hb levels and elevation of liver enzyme levels in G2 is sustained.A possible explanation for this has been previously stated, which is due to the ongoing destruction of the RBCs [3,16].Additionally, G3 maintained healthy Hb and enzyme levels, thus lending credence to the potency of Astymin as a blood tonic and haematinic agent.In G4 and G5, an increase and decrease in Hb levels, respectively, are observed.Tis seems to be out of place with readings previously obtained in Table 1 where there was a uniform drop in Hb levels, and readings subsequently obtained in Table 3, where the Hb levels still fell signifcantly below the average healthy levels compared to the control (G1) and treated groups (G3).Tis inconsistency may be attributed to a myriad of reasons ranging from factors such as diferences in the weights of the rats to physiological variations which may inevitably cause some of the rats to respond in a way diferent from the experimentally anticipated or expected outcome.
In Table 3 showing results for the fnal day of testing, a notable increase in Hb levels of G2 is noticed, having almost the same levels as those of G1.Tis phenomenon can readily be explained due to the regenerative ability of the RBCs which enables it to regenerate/repair itself after the agent which caused the haemolysis has been removed.Tus, it is most likely that this ability is what may have caused the Hb level to climb back up to normal levels after the considerable time lapse since the administration of PHZ.However, in towing this line, the interesting question may arise as to why it is not the same case in G4 and G5, which also had time to recuperate from the damaging efects of haemolytic anaemia.A possible explanation for this taking into consideration the fact that, while G2 was administered PHZ only and left to take the natural course of progression, G4 and G5 were constantly fed the extracts at low and high doses, respectively.Terefore, from the results obtained so far, it could be inferred that the fruit mesocarp may not only Biochemistry Research International be inefective in ameliorating or improving anaemic conditions due to the absence of key minerals and vitamins necessary to mediate this action but might also tend to worsen the case in a diseased condition.Tese fndings seem to be in keeping with the research of Eseyin et al. [13], where it was suggested that the fruit extract may be unsafe for consumption due to its damaging efects on the rat liver and bones.

Conclusion
Te aqueous extract of the fruit mesocarp of futed pumpkin (Telfairia occidentalis) was discovered to be nontoxic at an acute level.Meanwhile, its efcacy as a haematinic agent in reversing PHZ-induced haemolytic anaemia and hepatoprotective ability is not proven and may on the contrary even pose a detrimental efect to health when ingested at high doses over a long period of time.Consequently, the leaves of the futed pumpkin and not the fruit mesocarp may be used to boost blood levels until otherwise established.
Approval.Ethical clearance for this study was applied for, and duly obtained from the Faculty Animal Research Ethics Committee (FAREC-FBMS) of the Faculty of Basic Medical Sciences, College of Medicine, University of Calabar with reference number: FAREC/GP/008/21.All ethical procedures/rules were followed.
2.6.Statistical Analysis.Te results were expressed as the mean ± standard error of mean (SEM) which were calculated from duplicate laboratory test values.Graphs were plotted using GraphPad Software.Diferences among each group were investigated using one-way analysis of variance (ANOVA) with post hoc test for least signifcant diference (LSD) assuming equal variances using the IBM SPSS statistic software version 22 (SPSS: Statistical Package for Social Sciences).Signifcant diference was accepted at p < 0.05.

Table 2 :
Detail of the experimental protocol and design.

Table 3 :
Acute toxicity efect of aqueous extract of fruit mesocarp of Telfairia occidentalis administered orally to albino mice.Experiment was conducted in four stages; each dose group of stages 1-3 made up of 1 mouse for each dose while the confrmatory test consisted of 2 mice.

Table 4 :
Haematological indices and liver enzymes for the various rat groups at day 3.Values are expressed as mean ± SEM. *

Table 5 :
Haematological indices and liver enzymes of the various rat groups at day 5.
#Values are expressed as mean ± SEM. *

Table 6 :
Haematological indices and liver enzymes for the various rat groups at day 8.
#Values are expressed as mean ± SEM. *