Author’s Accepted Manuscript

Telfairia occidentalis Hook.f.-associated

haematopoietic effect is mediated by cytokines but
independent of testosterone: a preliminary report

Toyin Mohammed Salman, Isiaka Abdullateef

Alagbonsi, Abdul-Rahuf Aderemi Feyitimi, Peter O
Ajayi
www.elsevier.com/locate/jep

PII: S0378-8741(17)32146-3
DOI: https://doi.org/10.1016/j.jep.2018.01.018
Reference: JEP11194
To appear in: Journal of Ethnopharmacology
Received date: 3 June 2017
Revised date: 27 December 2017
Accepted date: 14 January 2018
Cite this article as: Toyin Mohammed Salman, Isiaka Abdullateef Alagbonsi,
Abdul-Rahuf Aderemi Feyitimi and Peter O Ajayi, Telfairia occidentalis
Hook.f.-associated haematopoietic effect is mediated by cytokines but
independent of testosterone: a preliminary report, Journal of
Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.01.018
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Telfairia occidentalis Hook.f.-associated haematopoietic effect is mediated by cytokines but

independent of testosterone: a preliminary report

Toyin Mohammed Salmana, Isiaka Abdullateef Alagbonsib*, Abdul-Rahuf Aderemi Feyitimia,

Peter O Ajayia
a
Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Kwara,

Nigeria.
b
Department of Physiology, Faculty of Medicine and Health Sciences, University of Gitwe,

Ruhango District, Southern Province, Republic of Rwanda.

*
Correspondending author. Department of Physiology, Faculty of Medicine and Health

Sciences, University of Gitwe, Gitwe, P.O. Box 1 Nyanza, Ruhango District, Southern Province,

Republic of Rwanda. Phone: +250725300424. easylat@gmail.com.

Abstract

Ethnopharmacological relevance:

Telfairia occidentalis Hook.f. (TO) is popular in Nigeria for the ethnopharmacological use of its

leaves to improve haematological parameters in normal and anaemic subjects. Cytokines are

well-known to regulate haematopoiesis. However, their involvement in TO-associated

haematopoietic effect is not known and necessitated this study.

Materials and Methods:

Twenty five (25) male rats were randomly divided into 3 oral treatment groups as follows: Group

1 (control, n=5) received 0.2 ml/kg normal saline for 14 days. Groups 2 and 3 (n= 10 each) were

subdivided into 2 (n=5) and received 100 mg/kg and 200 mg/kg of aqueous extract of TO

respectively for 7 or 14 days.

1
Results:

TO increased the haematological parameters in a duration-dependent manner. The increase were

insignificant (p>0.05) in the first week but significant (p<0.05) in the second week (except for

lymphocyte and platelets count). Moreover, TO significantly reduced the testosterone but

increased the interleukin-3 and erythropoietin concentrations.

Conclusion:

The haematopoietic effect of TO might be partly mediated by cytokines (interleukin-3 and

erythropoietin) but independent of testosterone.

Graphical abstract

Keywords: Cytokines; Haematological parameters; Telfairia occidentalis Hook.f.; Testosterone

1.0. Introduction

Normal haematopoiesis is a well-regulated process in which the generation of mature

blood elements occurs from a primitive pluripotent stem cells in an ordered sequence of

2
maturation and proliferation. Its regulation occurs at various levels that include but not limited to

the structure (stroma and cell-cell interactions), hormones and cytokines (e.g. erythropoietin,

interleukin-3) (Smith, 1990).

The use of herbal plants as ethnopharmacological intervention and prognosis to ailing

health has been on an increase in Africa, especially Nigeria. Telfairia occidentalis Hook.f. (TO)

(Oliver, 1871) is one of such plants often consumed as leafy vegetable on daily basis in many

homes due to its traditionally perceived haematinic property (Eseyin et al., 2014; Salman et al.,

2008). Many researchers have documented that the plant possesses anti-oxidant (Nwanna and

Oboh, 2007), anti-diabetic (Eseyin et al., 2010; Nwozo et al., 2004), hepatoprotective (Oboh,

2005), anti-plasmodial (Okokon et al., 2009), anti-microbial (Oyewole and Abalaka, 2012),

testiculoprotective (Akang et al., 2011; Ejike and Ezeanyika, 2013; Salman et al., 2008),

testiculotoxic (Adisa et al., 2014; Saalu et al., 2010), anti-inflammatory (Oluwole et al., 2005) as

well as haematinic (Eseyin et al., 2014) properties.

The traditionally-reported haematinic property of the leaf has been well-validated in the

scientific literatures. For instance, its aqueous extract has been shown to increase haematocrit

and reticulocyte count in rats (Lawal et al., 2015; Salman et al., 2008). Its

ethnomedicinal/ethnopharmacological value was shown in anaemic mice and gave dose-

dependent increase in haematological indices such as red blood cell (RBC) and white blood cell

(WBC) counts, as well as haematocrit and haemoglobin concentrations (Alada, 2000; Odede et

al., 2010). Its potential to inhibit and reverse the sickling feature of sickled red blood cell has

also been reported (Cyril- Olutayo et al., 2012). Although the high iron content of its leaf has

been implicated as a possible reason for its traditional use as blood tonic, there appears to be

3
other under-explored mechanisms which might be responsible for its reported haematinic

property.

Interleukin-3 (IL-3), a growth factor majorly produced by thymus (T) cells and natural

killer (NK) cells, has been known to act predominantly by inducing the proliferation and

differentiation of early myeloid progenitors of haematopoietic stem cells during haemopoiesis

(Smith, 1990). Likewise, testosterone-induced erythropoiesis via erythropoietin dependent and

independent mechanisms has been documented (Delev et al., 2016).

This study aimed at investigating the role of IL-3 and erythropoietin in TO-associated

haematopoietic effect.

2.0. Materials and methods

2.1. Extraction of the leaves of Telfairia occidentalis Hook.f. (TO)

Fresh leaves of Telfairia occidentalis Hook.f. (TO) were purchased from a farmer in

Yoruba road, Ilorin, Kwara state, Nigeria and authenticated by Mr. Bolu Ajayi in the Department

of Plant Biology, University of Ilorin, Ilorin, Nigeria. Its voucher number (UILH/001/959) was

deposited in the Herbarium. The leaves were air dried and reduced to powdery form by grinding.

One hundred and thirty-six grams (136 g) of its powder was soaked in 2 litres of distilled water

for 72 hours, after which it was sieved with a white cloth. The filtrate was then evaporated in

water bath at 40°C in order to obtain solid dried extract which was then dissolved in normal

saline and used for the study.

2.2. Experimental Animals

Twenty-five (25) males Wistar albino rats weighing between 120-150g were obtained

from the Animal House of the Department of Biochemistry, Faculty of Life Sciences, University

4
of Ilorin, Kwara State, Nigeria, housed at room temperature with free access to food and water

ad libitum, and were maintained on the daily light/dark cycle. “Principles of laboratory animal

care (NIH publication No. 85-23, revised 1985)” were followed. All experiments have been

examined and approved by our institutional ethics committee.

2.3. Animal Grouping and Treatments

Twenty-five (25) male rats were randomly divided into 3 oral treatment groups as

follows:

-Group 1 (n=5) was the control and received 0.2 ml/kg normal saline for 14 days.

-Group 2a animals (n=5) received 100 mg/kg of TO while group 2b animals (n=5) received 200

mg/kg of TO for 7 days.

-Group 3a animals (n=5) received 100 mg/kg of TO while group 3b animals (n=5) received 200

mg/kg of TO for 14 days.

The doses and durations of treatment used in this study have been previously reported by

us (Salman et al., 2013). A day after the last treatment, the rats were sacrificed by administering

0.2 ml/kg body weight of ketamine hydrochloride. Blood samples were then collected via retro-

orbital and cardiac puncture into heparinised bottles and centrifuged at 3000 rpm for10 minutes.

Plasma was collected into plain bottles and refrigerated at 4oC prior to testosterone, IL-3 and

erythropoietin analyses. Another blood sample collected in EDTA bottle was analysed

immediately for haematological parameters.

2.4. Determination of haematological parameters

The haematological indices including red blood cell count, haemoglobin concentration,

packed cell volume, total white blood cells, differential white blood cells count for lymphocytes

5
and neutrophils as well as platelets count were determined using the automated analyser

employing the methods described by Dacie and Lewis (2002).

2.5. Determination of plasma testosterone concentration

Determination of total testosterone concentration in the plasma was done

spectrophotometrically (Spectramax Plus microplate spectrophotometer; Molecular Device Co.,

Sunnyvale, CA, USA) using enzyme-linked immunosorbent assay (ELISA) [Calbiotech, Spring

Valley, California, USA; product code: TE-1875], following kit manufacturer’s procedure.

2.6. Determination of plasma Interleukin-3 concentration

Interleukin-3 (IL-3) assay was carried out spectrophotometrically (Spectramax Plus

microplate spectrophotometer; Molecular Device Co., Sunnyvale, CA, USA) using the rat IL-3

ELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China; product code: E-EL-RO556)

following the kit manufacturer’s procedure.

2.7. Determination of plasma erythropoietin (EPO) concentration

The concentration of plasma EPO was measured spectrophotometrically (Spectramax

Plus microplate spectrophotometer; Molecular Device Co., Sunnyvale, CA, USA) using

commercial ELISA kits (Elabscience Biotechnology Co., Ltd., Wuhan, China; product code: E-

EL-R0007) following the kit manufacturer’s procedures.

2.8. Data processing

Data were analysed using SPSS for windows (version 16.0). All values were expressed as

Mean±S.E.M of 5 rats. Significance was assessed by analysis of variance (ANOVA), followed

by Least Significance Difference (LSD) post-hoc test for multiple comparisons. p-Values of 0.05

or less were taken as statistically significant.

6
3.0. Results

3.1. Effects of aqueous extract of Telfairia occidentalis Hook.f. (TO) on haematological

parameters in male rats

Treatment with both 100 mg/kg (7.04 ± 0.25 106/µl) and 200 mg/kg (7.48 ± 0.20 106/µl) of TO

caused no significant change (p>0.05) in the RBC count of rats at 7 days but significantly

increased it (p<0.05) at 14 days (7.61 ± 0.32 106/µl; 8.48 ± 0.25 106/µl respectively) when

compared to the control (6.74 ± 0.13 106/µl) (Table 1).

Except for those that received 100 mg/kg of TO for 7 days (11.88 ± 0.86 g/dl), rats that

received its 100 mg/kg for 14 days (12.92 ± 0.49 g/dl) and those that received its 200 mg/kg for

7 days (12.90 ± 0.51 g/dl) and 14 days (14.54 ± 0.29 g/dl) had higher haemoglobin

concentrations than the control (10.53 ± 0.48 g/dl) (Table 1).

Furthermore, there were apparent increases in the PCV of TO-treated rats that was

significant (p<0.05) only in those that received its 200 mg/kg for 14 days (44.93 ± 0.59 g/dl)

when compared to the control (38.57 ± 0.33 g/dl) (Table 1).

The pattern of WBC is similar to that of the RBC. Treatment with both 100 mg/kg (8.48

± 0.19 103/μl) and 200 mg/kg (7.86 ± 0.41 103/μl) of TO caused no significant change (p>0.05)

in the RBC count of rats at 7 days but significantly increased it (p<0.05) at 14 days (15.03 ± 1.37

103/μl; 13.53 ± 0.93 103/μl respectively) by 80.43 % and 62.42 % respectively when compared to

the control (8.33 ± 0.28 103/μl) (Table 1).

Administration of TO to rats did not significantly affect the lymphocytes counts (Table

1).

7
 Furthermore, there were higher neutrophil counts in rats that received 100 mg/kg of TO

for 7 days (50.40 ± 3.32 %) and its 200 mg/kg for 14 days (43.78 ± 3.28 %) when compared to

the control (31.37 ± 4.09 %) (Table 1).

Administration of TO to rats caused no significant change to the platelets counts (Table

1).

3.2. Effects of TO on plasma testosterone concentration

There were significant reductions (p<0.05) in the plasma testosterone of rats that received

100 mg/kg and 200 mg/kg of TO for 7 days (30.00 ± 4.47 ng/ml; 55.00 ± 3.87 ng/ml

respectively) and 14 days (43.80 ± 8.57 ng/ml; 12.50 ± 1.44 ng/ml respectively) when compared

to control (75.00 ± 2.89 ng/ml) (Figure 1).

3.3. Effects of TO on plasma interleukin-3 (IL-3) concentration

The increase in IL-3 concentration was only significant (p<0.05) in rats that received 100

mg/kg of TO for 7 days (0.63 ± 0.09 pg/ml) but not significant (p>0.05) in others (0.41 ± 0.18

pg/ml), while there was an apparent insignificant decrease (p>0.05) in those that received 200

mg/kg of TO for 14 days (0.06 ± 0.01 pg/ml) when compared to control (0.23 ± 0.04 pg/ml)

(Figure 2).

3.4. Effects of TO on plasma erythropoietin (EPO) concentration

The pattern of erythropoietin is also similar to that of RBC. Treatment with both 100

mg/kg (1800.00 ± 100.00 mIU/ml) and 200 mg/kg (2266.70 ± 66.67 mIU/ml) of TO caused no

significant change (p>0.05) in the EPO concentration of rats at 7 days but significantly increased

it (p<0.05) at 14 days (2908.30 ± 200.17 mIU/ml; 2887.50 ± 162.46 mIU/ml respectively) when

compared to the control (1766.70 ± 133.33 mIU/ml) (Figure 3).

8
4.0. Discussion

Telfairia occidentalis hook.f. (TO) has been reported to enhance haematological

parameters in normal (Alada, 2000; Ifeanyi et al., 2014; Salman et al., 2008) and anaemic rats

(Toma et al.., 2015). The present study also supports the traditional and scientific claims that TO

has haematopoietic effect and additionally shows that this effect could be mediated by cytokines

(erythropoietin and interleukin-3).

Interleukin-3, primarily derived from activated CD4+ T-cells and mast cells, is a multi-

colony-stimulating factor that stimulates the development of varieties of haematopoietic

progenitor cells including erythrocytes, eosinophils, neutrophils, basophils, mast cells,

macrophages, dentritic cells, and megakaryocytes from the bone marrow in vitro (Broughton et

al., 2012) and in vivo (Ihle, 1992). In addition to promoting mast cell growth, differentiation and

mediator release (Gurish and Boyce, 2002; Hu et al., 2007), it also promotes basophil

development and stimulates IL-4, IL-6, and histamine release (Karasuyama et al., 2011; Lantz et

al., 2008; Voehringer, 2012) during allergic inflammation. Moreover, it promotes eosinophilic

secretion (Rothenberg et al., 1988), enhances monocyte/macrophage activation and phagocytosis

(Khwaja et al., 1994), modulates development of regulatory T cells (Srivastava et al., 2011), and

stimulates endothelial cells proliferation and migration (Dentelli et al., 1999). The present study

suggests that the TO-associated increase in IL-3 levels, possibly by increasing lymphocytes

concentration, might have contributed to its erythropoietic and leucopoietic effects. This is in

agreement with the previously reported improvement in CD4+, a marker of helper T-cells status

(Egba et al., 2014).

9
 The hormone erythropoietin, produced mainly in the kidney and liver in response to

oxygen tension, is required for the maintenance of a steady supply of red blood cells by

enhancing the proliferation and differentiation of erythrocytic progenitor cells in the bone

marrow. Hypoxia is known to be the main factor that induces erythropoietin mRNA expression

and protein secretion in the kidney and liver cells (Haase, 2013). Moreover, testosterone and its

metabolite, 5 α-dehydrotestosterone (5α-DHT), are known to stimulate erythropoietin production

in a hypoxia-dependent manner, though 5α-DHT is more potent than testosterone (Paulo et al.,

1974). For instance, pre-treatment of isolated perfused canine kidneys with testosterone

stimulated erythropoietin production under hypoxic condition, while exogenous addition of

testosterone to the perfusate of normal canine kidney did not (Malgor and Fisher, 1970).

Interestingly, hypoxia-induced depression of plasma testosterone has been previously reported,

while long-term oxygen therapy in male patients with respiratory failure increases plasma

testosterone level (Semple et al., 1980; Wang et al., 1996).

The reduction in testosterone in the presence of increasing erythropoietin concentration

observed in this study was unexpected. Our data suggest that the TO-associated constitutive

increase in erythropoietin secretion is not dependent on testosterone secretion, a claim that could

be explained from two perspectives. First, we rather speculate that the elevated red blood cells

that resulted from increased erythropoiesis following TO-associated increase in erythropoietin

secretion, in addition to the abundant iron present in TO, could have favoured high rate of

oxygen transport to the tissues and resulted in plasma ‘oxygen debt’. This resulting hypoxaemia

might have contributed to reduction in plasma testosterone and supports the contention that

hypoxia attenuates plasma testosterone levels (Semple et al., 1980; Wang et al., 1996). Second,

TO and certain fatty acids, including palmitic, stearic, myristic, oleic, linolenic and linoleic

10
acids, whose abundant presence in TO has been observed by us (unpublished data) and reported

by others (Esuoso et al., 1998), have been shown to possess inhibitory activity on 5 α-reductase

activity, thereby decreasing the formation of 5 α-DHT from testosterone (Abe et al., 2009;

Gossell-Willaims et al., 2006; Liu et al., 2009) and are therefore of therapeutic importance in the

management of acne, male pattern alopecia and benign prostatic hyperplasia (Cho et al., 2014;

Ejike and Ezeanyika, 2011). Though the present study is limited by non-availability of data on 5

α-reductase and 5α-DHT, it is however tempting to opine that the reduction in testosterone

following TO administration in this study might not be secondary to its conversion to 5α-DHT,

and that the TO-associated increase in erythropoietin is not dependent on androgen. This is in

agreement with previous observation that testosterone and erythropoietin do not depend on each

other to induce haematopoiesis (Maggio et al., 2013).

An enhancement in the biological functions of macrophages by erythropoietin has been

well-reported (Brines et al., 2000; Lifshitz et al., 2010; Siren et al., 2001). A decreased bone

marrow erythropoiesis and aggravated anaemia have been observed during Trypanosoma brucei

brucei infection in erythropoietin-deficient rats (Nishimura et al., 2011). Moreover,

erythropoietin has been used to prevent death of mice during Plasmodium berghei infection due

to its ability to ameliorate parasite-induced anaemia, decrease neuronal apoptosis, and reduce

TNF-α and interferon-γ production (Kaiser et al., 2006). However, exacerbated macrophages

activation and its associated excessive inflammation in erythropoietin-pre-treated mice have been

reported to increase mortality in Salmonella typhimurium (Nairz et al., 2011) and Histoplasma

capsulatum (Locachevic et al., 2015) infection. These suggest that macrophages (non-erythroid

cells) are also important target for erythropoietin.

11
 It is interesting to know that some important components of TO have haematological

implications. A GC-MS analysis of the organic compounds present in different fractions of TO

from our laboratory (unpublished data) and others (Esuoso et al., 1998) show that it has

appreciable amount of saturated (myristic, palmitic and stearic acids), monounsaturated (oleic

acid) and polyunsaturated (linoleic and α-linolenic acid) long chain fatty acids (FAs). TO has

also been known to be a rich source of iron (Esuoso et al., 1998), a widely known

haematopoietic metal that binds with plasma apotransferrin and forms iron-ferritin complex

which further binds with erythroblast’s receptors and activates reticuloendothelial cells in the

bone marrow. While the haeme component of the haemoglobin is synthesised in these cells, the

globin chains can also be formed by amino acids in TO (Ekpenyong et al., 2012). Moreover,

lymphocytes are enhanced by linoleic acid (Sierra et al., 2005) but depressed by oleic acid

(Llado et al., 2010; Verlengia et al., 2003). Palmitic acid increases the production of pro-

inflammatory cytokines (interleukins 1, 6 and 8 and TNF-α) (Joshi-Barve et al., 2007; Shirasuna

et al., 2016; Zhou et al., 2013) , which may lead to neutrophil infilteration and inflammation in

the hepatocyte. Contrarily, the anti-inflammatory activities of α-linolenic and linoleic acids by

decreasing interleukin 6 and increasing interleukin-1receptor antagonist have been reported (Lee

et al., 2009; Rallidis et al., 2003). It is hypothesised that the iron and fatty acids present in TO

might account for its haematopoietic action. It is also noteworthy that the possibility of

synergistic relationship between IL-3 and erythropoietin in the haematopoietic effect caused by

TO cannot be ruled out. For instance, their cooperation in the expansion of late erythropoiesis

has been previously observed in vivo as reticulocytes and late erythroid progenitors levels were

higher in primates given IL-3 and erythropoietin than in those given the later alone (Umemura et

al., 1989).

12
 The present study has some limitations. First, that metabolites of testosterone were not

measured could not avail us data to know what was actually responsible for the reduction in its

concentration. Second, how cytokines production was elicited by TO was not further

investigated. Third, it was not determined if TO could as well enhance the cytokines in anaemic

rats. The limitations are however offset by an important strength that this preliminary study is the

first to implicate cytokines in TO-associated haematopoiesis. This study is important as it gives

us a clue on the mechanisms involved in the already established TO-associated haematopoiesis.

Conflict of interest

None declared

Funding

None

Authors contributions

TMS and AAF conceived the study. AAF carried out the study. IAA and AOP analysed the data.

IAA interpreted the data and drafted the manuscript.

References

Abe, M., Ito, Y., Oyunzul, L., Oki-Fujino, T., Yamada, S., 2009. Pharmacologically relevant

receptor binding characteristics and 5α-reductase inhibitory activity of free fatty acids contained

in saw palmetto extract. Biol. Pharm. Bull. 32, 646-650.

Adisa, W.A., Okhiai, O., Bankole, J.K., Iyamu, O.A., Aigbe, O., 2014. Testicular damage in

Telfairia Occidentalis extract-treated Wistar rats. Am. J. Med. Biol. Res. 2, 37-45.

13
Akang, E., Oremosu, A., Dosumu, O., Ejiwunmi, A., 2011. Telfairia occidentalis, a prophylactic

medicine for alcohol's damaging effect on the testis. Macedon J. Med. Sci. 4, 380-387.

Alada, A., 2000. The haematological effect of Telferia occidentalis diet preparation. Afr. J.

Biomed. Res. 3, 185-186.

Brines, M.L., Ghezzi, P., Keenan, S., Agnello, D., de Lanerolle, N.C., Cerami, C., et al., 2000.

Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc.

Natl. Acad. Sci. USA. 97, 10526-10531.

Broughton, S.E., Dhagat, U., Hercus, T.R., Nero, T.L., Grimbaldeston, M.A., Bonder, C.S., et

al., 2012. The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation

of signalling. Immunol. Rev. 250, 277–302.

Cho, Y.H., Lee, S.Y., Jeong, D.W., Choi, E.J., Kim, Y.J., Lee, J.G., et al., 2014. Effect of

pumpkin seed oil on hair growth in men with androgenetic alopecia: a randomised, double-blind,

placebo-controlled trial. Evidence-based Compl. Altern. Med. Volume 2014, Article ID 549721,

7 pages

Cyril-Olutayo, C., Agbedahunsi, J., Elufioye, O., 2012. Anti-sickling properties of some

indigenous and exotic plant species in Nigeria. Planta Med. 78, I2-17.

Dacie, J.V., Lewis, S.M., 2001. Practical haematology. Eleventh ed. Longman Group.Ltd., Hong

Kong. pp 11-17.

Delev, D., Rangelov, A., Ubenova, D., Kostadinov, I., Zlatanova, H., Kostadinova, I., 2016.

Mechanism of action of androgens on erythropoiesis – a review. Int. J. Pharm. Clin. Res. 8,

1489-1492.

14
Dentelli, P., Sorbo, L.D., Rosso, A., Molinar, A., Garbarino, G., Camussi, G., et al., 1999.

Human IL-3 stimulates endothelial cell motility and promotes in vivo new vessel formation. J.

Immunol. 163, 2151-2159.

Egba, S.I., Njoku, O.U., Anaduaka, E.G., Okibe, C.O., 2014. Stimulation of haemopoiesis and

up-regulation of T-helper cells production in mice by different fractions of Telfairia occidentalis

Hook F and Tectona grandis Linn. J. Chem. Pharm. Res. 6, 486-492.

Ejike, C., Ezeanyika, L., 2013. A Telfairia occidentalis seed-incorporated diet may be useful in

inhibiting the induction of experimental andropause. Ann. Med. Health Sci. Res. 2, 41-45.

Ejike, C.E.E.C., Ezeanyika, L.U.S., 2011. Inhibition of the experimental induction of benign

prostatic hyperplasia: a possible role for fluted pumpkin (Telfairia occidentalis Hook f.) seeds.

Urol. Int. 87, 218-224.

Ekpenyong, C.E., Okon, U.A., Samson, T.K., Akpan, E.E., 2012. Bioactive constituents, acute

and sub-acute effect of Telfairia occidentalis Hook F (fluted pumpkin) leaves extract on some

kidney function indices in rat. Can. J. Pure Appl. Sci. 6, 2105-2112.

Eseyin, O., Ebong, P., Eyong, E., Awofisayo, O., Agboke, A., 2010. Effect of Telfairia

occidentalis on oral glucose tolerance in rats. Afr. J. Pharm. Pharmacol. 4, 368-372.

Eseyin, O.A., Sattar, M.A., Rathore, H.A., Ahmad, A., Afzal, S., Lazhari, M., et al., 2014.

Hypoglycaemic potential of polysaccharides of the leaf extract of Telfairia occidentalis. Annu.

Res. Rev. Biol. 4, 181-182.

Esuoso, K., Lutz, H., Kutubuddin, M., Bayer, E., 1998. Chemical composition and potential of

some underutilised tropical biomass. I: fluted pumpkin (Telfairia occidentalis). Food Chem. 61,

487-492.

15
Gossell-Willaims, M., Davis, A., O’Connor, N., 2006. Inhibition of testosterone-induced

hyperplasia of the prostate of Sprague-Dawley rats by pumpkin seed oil. J. Med. Food. 9, 284-

286.

Gurish, M., Boyce, J., 2002. Mast cell growth, differentiation, and death. Clin. Rev. Allergy

Immunol. 22, 107-118.

Haase, V.H., 2013. Regulation of erythropoiesis by hypoxia-inducible factors. Blood Rev. 27,

41-53.

Hu, Z., Zhao, W., Shimamura, T., 2007. Regulation of mast cell development by inflammatory

factors. Curr. Med. Chem. 14, 3044-3050.

Ifeanyi, O.E., Ifeoma, C., Ndubuisi, O.T., Nwakaego, O.B., Braxton, A.Q., 2014.

Haematological effects of fluted pumpkin (Telfairia occidentalis) leaves in rats. Int. J. Life Sci.

Biotechnol. Pharma Res. 3, 172-182.

Ihle, J., 1992. Interleukin-3 and haematopoiesis. Chem. Immunol. 51, 65-106.

Joshi-Barve, S., Barve, S.S., Amancherla, K., Gobejishvili, L., Hill, D., Cave, M., et al., 2007.

Palmitic acid induced production of pro-inflammatory cytokine interleukin-8 from hepatocytes.

Hepatol. 46, 823-830.

Kaiser, K., Texier, A., Ferrandiz, J., Buguet, A., Meiller, A., Latour, C., et al., 2006.

Recombinant human erythropoietin prevents the death of mice during cerebral malaria. J. Infect.

Diseases. 7, 987-995.

Karasuyama, H., Mukai, K., Obata, K., Tsujimura, Y., Wada, T., 2011. Non-redundant roles of

basophils in immunity. Ann. Rev. Immunol. 29, 45-69.

Khwaja, A., Addison, I., Johnson, B., Yong, K., Linch, D., 1994. Interleukin-3 administration

enhances human monocyte function in vivo. Br. J. Haematol. 88, 515-519.

16
Lantz, C., Min, B., Tsai, M., Chatterjea, D., Dranoff, G., Galli, S., 2008. IL-3 is required for

increases in blood basophils in nematode infection in mice and can enhance IgE-dependent IL-4

production by basophils in vitro. Lab. Inv. 88, 1134-1142.

Lawal, B., Shittu, O.K., Rotimi, A.A., Olalekan, I.A., Kamooru, A.A., Ossai, P.C., 2015. Effect

of methanol extract of Telfairia occidentalis on haematological parameters in Wistar rats. J.

Med. Sci. 15, 246-250.

Lee, Y., Thompson, J.T., Vandel Heuvel, J.P., 2009. 9E,11E-Conjugated linoleic acid increases

expression of the endogenous anti-inflammatory factor, interleukin-1 receptor antagonist, in raw

264.7 cells. J. Nutr. 139, 1861-1866.

Lifshitz, L., Tabak, G., Gassmann, M., Mittelman, M., Neumann, D., 2010. Macrophages as

novel target cells for erythropoietin. Haematol. 95, 1823-1831.

Liu, J., Shimizu, K., Kondo, R., 2009. Anti-androgenic activity of fatty acids. Chem. Biodivers.

6, 503-512.

Llado, V., Gutierrez, A., Martinez, J., Casas, J., Teres, S., Higuera, M., et al., 2010. Minerval

induces apoptosis in Jurkat and other cancer cells. J. Cell Mol. Med. 14, 659-670.

Locachevic, G.A., Pereira, P.A.T., Secatto, A., Fontanari, C., Galvao, A.F., Prado, M.K.B., et al.,

2015. Erythropoietin exacerbates inflammation and increases the mortality of Histoplasma

capsulatum-infected mice. Mediators Inflamm. Volume 2015, Article ID 786319, 11 pages.

Maggio, M., Snyder, P.J., Ceda, G.P., Milaneschi, Y., Luci, M., Cattabiani, C., et al., 2013. Is the

haematopoietic effect of testosterone mediated by erythropoietin? The results of a clinical trial in

older men. Androl. 1, 24-28.

17
Malgor, L.A., Fisher, J.W., 1970. Effects of testosterone on erythropoietin production in isolated

perfused kidneys. Am. J. Physiol. 218, 1732-1736.

Nairz, M., Schroll, A., Moschen, A.R., Sonnweber, T., Theurl, M., Theurl, I., et al., 2011.

Erythropoietin contrastingly affects bacterial infection and experimental colitis by inhibiting

nuclear factor-kappaB-inducible immune pathways. Immunity. 34, 61-74.

Nishimura, K., Nakaya, H., Nakagawa, H., Matsuo, S., Ohnishi, Y., Yamasaki, S., 2011. Effect

of Trypanosoma brucei brucei on erythropoiesis in infected rats,” J. Parasitol. 97, 88-93.

Nwanna, E.E., Oboh, G., 2007. Anti-oxidant and hepatoprotective properties of polyphenol

extracts from Telfairia occidentalis (Fluted Pumpkin) leaves on acetaminophen-induced liver

damage. Pak. J. Biol. Sci. 10, 2682-2687.

Nwozo, S.O., Adaramoye, O.A., Ajaiyeoba, E.O., 2004. Anti-diabetic and hypolipidemic studies

of Telfairia occidentalis on Alloxan-induced diabetic rats. Niger. J. Nat. Prod. Med. 8, 45-47.

Oboh, G., 2005. Hepatoprotective property of ethanolic and aqueous extracts of fluted pumpkin

(T. occidentalis) leaves against garlic-induced stress. J. Med. Food. 8, 560-563.

Odede, T., Ikusagba, B., Odetola, A., 2010. Effect of Telfairia occidentalis on erythrocyte

indices of rats following acute blood loss. Afr. J. Med. Med. Sci. 239, 117.

Okokon, J.E., Ekpo, A.J., Eseyin, O.A., 2009. Evaluation of in vivo anti-malarial activities of

ethanolic leaf and seed extracts of Telfairia occidentalis. J. Med. Food. 12, 649-653.

Oliver, D., 1871. Flora of tropical Africa. Dyer, T. (ed.), London: 1868-1900.

Oluwole, E., Falode, A., Ogundipe, O., 2005. Anti-inflammatory effect of some common

Nigerian vegetables. Niger. J. Physiol. Sci. 18, 35-38.

Oyewole, O., Abalaka, M., 2012. Anti-microbial activities of Telfairia occidentalis (fluted

pumpkins) leaf extract against selected intestinal pathogens. J. Health Sci. 2, 1-4.

18
Paulo, L.G., Fink, G.D., Roh, B.L., Fisher, J.W., 1974. Effects of several androgen and steroid

metabolites on erythropoietin production in the isolated perfused dog kidney. Blood. 43, 39-47.

Rallidis, L.S., Paschos, G., Liakos, G.K., Velissaridou, A.H., Anastasiadis, G., Zampelas, A.,

2003. Dietary α-linolenic acid decreases C-reactive protein, serum amyloid A and interleukin-6

in dyslipidaemic patients. Atherosclerosis. 167, 237-242.

Rothenberg, M., Owen, W., Silberstein, D., Woods, J., Soberman, R., Austen, K., et al., 1988.

Human eosinophils have prolonged survival, enhanced functional properties, and become

hypodense when exposed to human interleukin-3. J. Clin. Invest. 81, 1986-1992.

Saalu, L.C., Kpela, T., Benebo, A.S., Oyewopo, A.O., Anifowope, E.O., Oguntola, J.A., 2010.

The dose-dependent testiculoprotective and testiculotoxic potentials of Telfairia occidentalis

Hook f. leaves extract in rat. Int. J. Appl. Res. Nat. Prod., 3, 27-38.

Salman, T. M., Olayaki, L. A., Oyeyemi, W. A., 2008. Aqueous extract of Telfairia occidentalis

leaves reduces blood sugar and increases haematological and reproductive indices in male rats.

Afr. J. Biotechnol.. 7, 2299-2303.

Salman, T.M., Alagbonsi, I.A., Biliaminu, S.A., Ayandele, O.A., Oladejo, O.K., Adeosun, O.A.,

2013. Blood glucose-lowering effect of Telfairia Occidentalis: A preliminary study on the

underlying mechanism and responses. Biokemistri, 25, 133-139.

Semple, P.D., Beastall, G.H., Watson, W.S., Hume, R., 1980. Serum testosterone depression

associated with hypoxia in respiratory failure. Clin. Sci. 5, 105-106.

Shirasuna, K., Takano, H., Seno, K., Ohtsu, A., Karasawa, T., Takahashi, M., et al., 2016.

Palmitic acid induced interleukin 1 β secretion via NLRP3 inflammasomes and inflammatory

responses through ROS production in human placental cells. J. Reprod. Immunol., 116, 104-

112.

19
Sierra, S., Lara-Villoslada, F., Olivares, M., Jimenez, J., Boza, J., Xaus, J., 2005. Increased

immune response in mice consuming rice bran oil. Eur. J. Nutr. 44, 509-516.

Siren, A.L., Fratelli, M., Brines, M., Goemans, C., Casagrande, S., Lewczuk, P., et al., 2001.

Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc.

Natl. Acad. Sci. USA. 98, 4044-4049.

Smith, B.R., 1990. Regulation of Haematopoiesis. Yale J. Biol. Med. 63, 371-380

Srivastava, R.K., Tomar, G.B., Barhanpurkar, A.P., Gupta, N., Pote, S.T., Mishra, G.C., et al.,

2011. IL-3 attenuates collagen-induced arthritis by modulating the development of Foxp3

regulatory T cells. J. Immunol. 186, 2262-2272.

Toma, I., Victory, N.C., Kabir, Y., 2015. The effect of aqueous leaf extract of fluted pumpkin on

some haematological parameters and liver enzymes in 2,4-dinitrophenylhydrazine- induced

anaemic rats. Afr. J. Biochem. Res. 9, 95-98.

Umemura, T., Al-Khatti, A., Donahue, R.E., Papayannoupoulou, Th., Stamatoyannopoulos, G.,

1989. Effects of interleukin-3 and erythropoietin on in vivo erythropoiesis and F-Cell formation

in primates. Blood. 74, 1571-1576.

Verlengia, R., Gorjao, R., Kanunfre, C.C., Bordin, S., de Lima, T.M., Curi, R., 2003. Effect of

arachidonic acid on proliferation, cytokines production and pleiotropic genes expression in

Jurkat cells – a comparison with oleic acid. Life Sci. 73, 2939-2951.

Voehringer, D., 2012. Basophil modulation by cytokine instruction. Eur. J. Immunol. 42, 2544-

2550.

Wang, R.Y., Tsai, S.C., Lu, C.C., Shih, H.C., Chen, Y.H., Tung, Y.F., et al., 1996. Effect of

aging on erythropoietin secretion in male rats. J. Gerontol.: Biol. Sci. 51A, B434-B438.

20
Zhou, B., Zhang, J., Zhang, Q., Permatasari, F., Xu, Y., Wu, D., et al., 2013. Palmitic acid

induces production of pro-inflammatory cytokines interleukin-6, interleukin-1β, and tumour

necrosis factor-α via a NF-κB-dependent mechanism in HaCaT keratinocytes. Mediators

Inflamm. Volume 2013 (2013), Article ID 530429, 11 pages

100
Testosterone (ng/ml)

80

60 *
*
40 *
20 *
0
Control 100 mg/kg TO 7d 200 mg/kg TO 7d 100 mg/kg TO 14d 200 mg/kg TO 14d

Figure 1: Effect of aqueous extract of Telfairia occidentalis Hook.f. leaves on testosterone

concentration in male rats. TO = aqueous extract of Telfairia occidentalis Hook.f. leaves;
*
p<0.05 vs. control

0.8
*
Interleukin-3 (pg/ml)

0.6

0.4

0.2

0.0
Control 100 mg/kg TO 7d 200 mg/kg TO 7d 100 mg/kg TO 14d 200 mg/kg TO 14d

Figure 2: Effect of aqueous extract of Telfairia occidentalis Hook.f. leaves on interleukin-3

concentration in male rats. TO = aqueous extract of Telfairia occidentalis Hook.f. leaves;
*
p<0.05 vs. control

21
 Erythropoietin (mIU/ml) 4000

* *
3000

2000

1000

0
Control 100 mg/kg TO 7d 200 mg/kg TO 7d 100 mg/kg TO 14d 200 mg/kg TO 14d

Figure 3: Effect of aqueous extract of Telfairia occidentalis Hook.f. leaves on erythropoietin

concentration in male rats. TO = aqueous extract of Telfairia occidentalis Hook.f. leaves;
*
p<0.05 vs. Control

Table 1: Effect of aqueous extract of Telfairia occidentalis Hook.f. on haematological

parameters in male rats.

Control 100 mg/kg 200 mg/kg

100 mg/kg 200 mg/kg
TO for 7 days TO for 7 days
TO for 14 TO for 14
days days
6 *
RBC count (10 /µl) 6.74±0.13 7.04±0.25 7.48±0.20 7.61±0.32 8.48±0.25*
Hb (g/dl) 10.53±0.48 11.88±0.86 12.90±0.51* 12.92±0.49* 14.54±0.29*
PCV (g/dl) 38.57±0.33 41.52±1.94 43.52±1.92 42.68±1.80 44.93±0.59*
WBC (103/μL) 8.33±0.28 8.48±0.19 7.86±0.41 15.03±1.37* 13.53±0.93*
Lymphocytes (%) 68.63±4.09 55.60±6.86 75.74±3.55 67.78±3.61 62.55±7.12
Neutrophils (%) 31.37±4.09 50.40±3.32* 22.40±0.67 32.22±3.61 43.78±3.28*
Platelets (%) 528.33±81.25 781.25±93.25 587.00±10.47 575.20±33.12 633.25±92.74
RBC = red blood cells, Hb = haemoglobin, PCV = packed cells volume; WBC = white blood
cells; TO = aqueous extract of Telfairia occidentalis Hook.f. leaves; *p<0.05 vs. control

22