Comparative effects of Telfairia occidentalis Hook. F. leaf extract and Ranferon-12 ® as dietary supplements on partially starved male Sprague-Dawley rats

This work examined the supplemental effects of Telfairia occidentalis Hook.F. leaf extract compared to a standard hematinic drug, Ranferon -12 ® , on the performance of thirty-six male Sprague-Dawley rats. The rats were randomly assigned into six groups of six rats each, and were fed standard rat ration at 80 % of ad lib within 24 hours. Access to clean water was allowed ad libitum. The leaf extract was administered orally at the dose of 200mg/kg, 400mg/kg, 600mg/kg, and 800mg/kg (for groups 1-4 respectively), and group 5 was given Ranferon 12 ® (at 0.3ml/kg), while group 6 (Control) received no extract but water. The extract was administered once daily for 21 days, while feed intake, weight gain, water intake, haematology, and some liver enzymes as well as cholesterol levels were the parameters measured. Results also revealed that weight gain, water intake and total WBC were significantly higher in group 3(600mg/kg) compared to other groups, while group 5 (Ranferon-12 ® ) had significantly higher Packed cell volume and total red blood cell counts. There were no significant differences (P>0.05) in the liver enzymes and cholesterol levels of rats in all groups. The results of this study have shown that supplementation with Telfairia occidentalis leaf extract was quite beneficial as it produced a significantly higher weight gain at 600mg/kg, and that neither increased dose of extract nor supplementation with Ranferon-12 ® yielded any special advantage.


INTRODUCTION
Telfairia occidentalis is a well-known f ood crop in West Af rica, which is also popular for provision of oil, f ibre, and medicines (Fern, 2020). Protein f rom plant leaves has been shown to be an extremely nutritious f ood and can be distinguished f rom ordinary leaf extract as they are made by pressing out the juice containing the proteins, vitamins, and minerals through mechanical means, thus separating them f rom the indigestible f iber.
According to Feedipedia (2020), the protein extracted f rom the leaf juice can be used f resh, or coagulated by heating, dried thereaf ter and stored f or f eature use. T. occidentalis also has been reported to be a supplement in micronutrient def iciencies (hidden hunger) for upgrading starchy crops and poor-quality food sources consumed by poor rural populations in sub-Saharan Af rica (Lawal et al., 2021).
Many programmes involving the use of plant leaves f or the production of alternative protein sources have been approved and f unded by many organizations such as United Nations, the European Economic Community, the British Overseas Development Agency, the Rotary Club International, and others (King et al. (1991). Leaf extracts have been shown to be very ef f ective in combating malnutrition, anaemia and vitamin A def iciency, which are common in the vulnerable groups (especially children and pregnant women) in some developing countries such as Mexico, India, Bolivia, Sri Lanka, Ghana, Nicaragua and Bangladesh, etc. (Cameron, and Yngve, 1983). Leaf extracts have also been shown to have huge economic advantage in human nutrition, and af ter the protein production, it has been suggested that the remnant f ibre residue can then be used to f eed cows, goats, sheep, horses, rabbits, or guinea pigs (Lowe, 2002;Fellows and Hampton,1992).Ranf eron-12 ® is a standard and very ef f ective hematinic commonly used f or replenishment, and f or the treatment of iron and f olic acid def iciency anaemia in humans (MIMS.com, 2022),although it is contra-indicated in patients suf f ering f rom hemochromatosis and hemosiderosis, including those who show hypersensitivity to iron, f olic acid, vitamin B12, vitamin C, Zinc, and Cobalt (Pillintrip.com, 2022).
Fluted pumpkin (Telfairia occidentialis) is a vegetable f ood crop of importance that belongs to the f amily Cucurbitaceae and is commonly f ound in the low land humid tropics of West Af rica with Nigeria, Ghana and Sierra Leone being the major producers (Nkang et al 2003).
It has dif f erent traditional names; among the Igbos (Ugu) (Akoroda, 1990); in Yoruba (Aporoko), and called "Ubong" in Ef ik (Badifu and Ogunsina, 1991;Amanke, 2003). According to Kayode et al. (2010), T. occidentalis Hoof is an important plant used in ethno-medical practice, whereby its supplemental ef f ects have been reported to improve haematological indices in man, thus preventing anaemia which occurs as a result of malnutrition. Its use has also been associated with signif icant increase in the weight gain of animals (Aregheore, 2007), as well as having both nutritional and medicinal values (Ntinya et al., 2019). Consequently, Emeka and Obidoa, (2009) also reported that the leaf extracts are rich in essential minerals, vitamins, proteins, in addition to antioxidant and antimicrobial properties.
This study investigated the ef f ects of graded doses of Telfairia occidentalis leave extract compared to a standard hematinic drug Ranf eron -12 ® on the perf ormance of partially starved male Sprague-Dawley rats.

Animal Management
Thirty-six male Sprague-Dawley rats weighing between 120 -140g were used f or the study. They were obtained f rom the Department of Animal Health and Production, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, f ed standard rat diet at 80 % ad lib, with clean drinking water provided ad libitum throughout the experiment. The animals were housed in metal cages, and were acclimatized f or two weeks bef ore the commencement of the experiment.

Preparation of T. occidentalis leaf extract
The leaves and stem of Telfairia occidentalis were bought f rom a local market at Orba, near the University, and was identif ied in the Department of Plant Science and Biotechnology of the University. Af ter botanical identif ication, two kilogram (2 kg) of f reshly cut T. occidentalis leaves with the stalk was separated f rom the stem, washed with clean water to remove dirt and soil, drained, and wilted f or thirty minutes.
The leaves were chopped, packed into a muslin bag and then squeezed with a mechanical screw press to obtain a homogenous extract of the f luted pumpkin leaves. The concentration of the leaf extract was determined by drying 2mls of the leaf extract in a hot air oven (using a watch glass) to constant weight, and thereaf ter, the actual weight of the extract was determined by dif f erence. The extract was stored at 4 0 C in the ref rigerator until f urther use. The calculated doses were administered per os (by mouth) in individual rats using gastric lavage.

Experimental design
The experimental animals were divided into six groups of six rats each and were acclimatized f or two weeks bef ore the commencement of the experiment. During the experiment, the rats were partially starved, as they were fed standard rat diet at 80% of ad lib, with constant availability of f resh water throughout the experiment.

Dosage and constituents of Ranferon-12 ®
Ranf eron-12 ® , a standard hematinic used for this study, was procured f rom a registered Patent medicine dealer, and was administered at 0.3ml/kg body weight which was based on the recommended dose of 15ml to 30ml daily f or an average adult human weighing approximately 60 kg body weight. Components of Ranf eron -12 ® include Iron f umarate 305 mg (equiv elemental Fe 100 mg), Folic acid 0.75 mg, Cyanocobalamin 5 mg, Ascorbic acid 75 mg, Zinc sulf ate 5 mg, (MIMS.com, 2022). Ranf eron-12 ® in made by Ranb ax y Nig eria Limit ed -a s ubsidiary c o mpany of Sun P harmac eut ical Ind us tries Lt d, Lag os

Determination of Proximate fraction (AOAC, 2012)
The proximate f ractions were determined according to the method of AOAC (2012). Crude protein content was obtained by multiplying the value obtained f rom Kjeldahl's nitrogen by a protein f actor of 5.3, a f actor recommended f or vegetable analysis.
Ether extract was quantif ied using the Soxhlet apparatus and petroleum ether (B.P. 60 o C-80 o C) as a solvent.
The moisture content of the leaves was determined by drying 5 g of the leaves (in triplicate) in an oven at 105 0 C until constant weight was attained.
Ash content was determined by burning sample in muf f le f urnace at 550 0 C until white ash was obtained.
At the end of the experiment, the parameters measured were weight gain, f ood and water intakes.
The animals were bled for haematological determinations using the orbital technique, into sample bottles containing ethylene-diamine-tetra-acetic acid (EDTA). The sample bottles were shaken gently to mix up the blood with EDTA to prevent clotting. Packed cell volume (PCV) was measured using microhematocrit tubes according to the method of Coles (1986), while the total red blood cells (TRBC), as well as the total white blood cells (TWBC) were determined using the method of Schalm et. al. (1975). Serum biochemical parameters such as Alanine Amino Transf eras e (ALT), Aspartate Amino Transf erase (AST), were measured as described by Cartwright (1979), while cholesterol levels were determined according to the method of Schalm et. al. (1975).

Statistical analysis
Statistical signif icance between groups was analyzed using One-way analysis of variance (ANOVA with computer sof tware (SPSS) version 20.0 (IBM). Values were expressed as mean ± Standard error of mean and separated using Duncan's multiple range test. The means were considered signif icant at P<0.05 (Steel and Torrie, 2000). Table 1 shows the components of the rat ration in addition to the constituents of the premix as well as the calculated values of Lysine and Methionine.  The weight gain of the various groups is presented in Figure 1 below. The result shows that Group 3 (600mg/kg) had a signif icantly higher weight compared to the Control(P<0.05). Figure 2 shows water consumption of dif ferent groups. The result shows that water consumption was signif icantly higher (P<0.05) in group 3(600mg/kg), which was closely f ollowed by the water consumption in groups 4 (800mg/kg) and group 5 (Ranf eron-12).

RESULTS
The f eed intake in Figure 3 shows that rats in group 3(600mg/kg) consumed a mean ration quantity of 116g by day 21, at the dose of 600mg/kg. This value was signif icantly higher (P<0.05) when compared to the Control and other groups.
The Packed cell volume and the Total red blood cell counts of the various groups are presented in Fig.4. Consequently, there was no signif icant dif f erence in the Packed cell volume of the groups. However, Group 5 (Ramf eron-12 ® ) had non-signif icantly higher values (P>0.05) in both Packed cell volume and the Total red blood cell counts compared to the rest of groups. The results in Table 3 shows signif icantly higher values (P<0.05) only in the Total white blood cell counts f or group 3 (600 mg/kg) compared to other groups except f or the Ranf eron -12 ® group.
There was no signif icant dif f erence(P>0.05) in the values of other parameters across dif f erent groups The result in Fig.5 shows that there was no signif icant dif f erence (P>0.05) in the liver enzymes and the cholesterol levels across the groups.

DISCUSSION
The leaf extract of Telfairia occidentalis Hook. F. which was used in this study as dietary supplement has shown unique nutritional attributes capable of maintaining physiological activities in partially starved murine animal model. The proximate f ractions of Telfairia occidentalis leaf extract f rom this study (crude protein 16.2% , crude f ibre 15.6%, ether extract 6.2% and ash 6.5%) were within the ranges reported by Taiga et al (2008) which were, 17.5% f or protein, ash (7.9%) ether (6.8%) and crude f ibre (16.2%).However, Aregheore (2007) reported higher crude protein of 21.9% , while f at (5. 9% ), c rud e f ib re (15. 9% ) and as h (12. 9% ) were wit hin o ur rang e. This is one important reason why samples must be analyzed in order to determine dif ferences in value. More so, the results on weight gain showed that all the supplemented groups had signif icantly higher (P<0.05) weight compared to the Control. However, the rats in group 3 (600mg/kg) had signif icantly highest weight gain, compared to those in group 4 (800mg/kg) and other groups, except group 5 which ranked closer. The growth increase observed in rats in this study could be attributed to a balance in the rich nutrient content of T. occidentalis in terms of its amino acids, f atty acids, minerals, and vitamins as reported by other reseachers such as Emeka and Obidoa (2009) 2021) reported on the palatability and acceptability of the leaf extracts when used to f ortify lownutrient diets f or human consumers. Water intake was also signif icantly higher (P<0.05) in the treated groups (though highest in Group 3(600mg/kg) compared to the Control. This increase in water intake could be attributed to higher protein content of the leaf extract. This f inding agrees with the observations of McDonald et al. (2011) who reported that consumption of protein diets provoke increased water intake, adding that water is necessary in protein digestion, as well as in the elimination of toxic products f rom protein metabolism.
Although the animals were f ed 80% ad lib of their rations, but Group 3(600mg/kg) had signif icantly higher (P<0.05) intake at day 21, compared to control and the other treated groups. Although there was no signif icant dif f erence (P>0.05) between Groups 4 (800mg/kg) and 5(Ranf eron-12). Consequently, this suggested that at 600mg/kg, the rats had better ef f iciency of nutrient utilization, hence better gain in weight. It was also an indication that T. occidentalis leaf extract was most benef icial at this level compared to the other doses as recorded in this study. The above observation supports the f indings of Ogunka-Nnoka et al. (2017) who reported best perf ormance of oral administration of T. occidentalis leaf extract at 500mg/kg in male adult Wistar rats. It is also possible that the leaf extract of T. occidentalis at 800mg/kg may likely contain more quantity of anti-nutrients such as tannins, saponins, etc., (Dube et al., 2001;Sof owora, 1996;Harborne, 1973), which may likely interf ere with intake, digestion and absorption of nutrients, and this may have af f ected their f eed intake weight gain compared to Group 3.However, increased intake and weight gain should be evidence of balanced nutrients, leading to improved f ood digestibility as well as enhanced f ood absorption. The benef icial ef f ects of leaf extracts were also documented by Lowe (2002) who reported better growth rate and overall perf ormance in malnourished Bolivian school children whose diets were supplemented with leaf extracts. Similarly, other evidence concerning the ef f ects of T. occidentalis on digestibility and conversion rate in animals have also been provided in the reports of Adedapo et al. (2008), while the use of the extract for biof ortification in human diets have also been documented according to Bouis. and Saltzman (2017). Haematological results indicated that the group supplemented with standard haematinic drug (Ranf eron-12 ® ) had signif icantly higher (P<0.05) Packed cell volume as well as Total red blood (TRB C) counts compared to the other groups, whereas a higher Total white blood cell (TWBC) count was observed in Group 3 (600mg/kg) in comparison to the other groups. The reason for the dif ferences in haematology could be explained based on the reports of Alada (2000) and Agbede et al. (2008), which emphasized that the haematological result of T. occidentalis leaf extracts may not necessarily f ollow any regular pattern, even though it has been repeatedly shown to be protective against anaemia. The cholesterol levels in the treated groups were not signif icantly higher (P>0.05) compared to the Control, while no signif icant changes (P>0.05) were also observed in the cardinal liver enzymes, Alanine amino transaminase (ALT), aspartate transaminas e (AST). These non-signif icant changes may suggest some level of saf ety f or the animals, particularly the liver, as well as other vital organs of their body. This observation also corroborates the f indings of Oboth (2005) and Emeka and Obidoa (2009), in addition to the observations of Aderibigbe et al. (2008) concerning the saf ety of Telfairia occidentalis leaf extract.

Conclusion
The results of this study suggested that supplementation with Telfairia occidentalis leaf extract was quite benef icial as it produced a signif icantly higher weight gain at 600mg/kg, and no signs of toxicity in the liver and blood. Consequently, this study has shown that Telfairia occidentalis leaf extract was most benef icial at 600mg/kg, such that neither increased dose of nor supplementation with Ramf eron-12 ® yielded special advantage in the partially starved rats.