Effect of Chronic Administration of Hydromethanol Leaf Extract of Helianthus annuus on Erythrocytic Profile in Normal Rats

: Effect of chronic administration of hydromethanol leaf extract of Helianthus annuus on erythrocytic profile in normal rat was investigated. The extract was prepared using cold maceration method and concentrated at 40˚C. It was incorporated in feed at 2.5, 5 and 10 mg per 100 g feed. Albino rats were randomly assigned to 4 (A – D) groups of 14 rats each. Group A received standard feed while groups B – D received feed incorporated with H. annuus at 2.5, 5 and 10 mg/10 g feed, respectively. They were fed 10% of their body weight daily for 90 consecutive days. Blood samples were collected via retroorbital venous plexus from four rats selected at random from each group without replacement on day 30, 60 and 90 for the determination of erythrocytic profile. There was no significant (p > 0.05) change on the erythrocytic profile of the various groups on day 30 and 60 while the packed cell volume and hemoglobin concentration of H. annuus treated groups were significantly (p < 0.05) lower than group A rats on day 90. This study suggests that the chronic use of Helianthus annuus for up to 90 days in rat may lead to anemia and should be avoided.


INTRODUCTION
Helianthus annuus (sunflower) is relatively a primitive member of the family Asteraceae. It has worldwide distribution and usually found along water ways due to its mode of dispersal. It is used as an ornamental plant and the seed are widely used as sources of vegetable oil while seed meal serve as feedstuff for farm animals [1]. The plant is extensively used in ethnomedical practices of several cultures. Helianthus annuus leaves are used in folkloric medicine as diuretic, antidiabetic, expectorant, gastrointestinal stimulant, antimicrobial, analgesic agents etc [2][3][4]. Poultice of H. annuus leaves is applied on wound, snakebite and swelling [5]. Preliminary phytochemical studies on H. annuus have reported the presence of saponins, terpenes, flavonoids, tannins, alkaloids and glycoside [4,6]. The presence of several essential oil in the leaves of H. annuus; 80% monoterpenes and other sesquiterpenes have been reported [2,7]. The antispermatogenic [3], analgesic and anti-inflammatory [5], antidiabetic and antioxidant [4], antifertility [8], antidiarrheal and antihistaminic [9], antilithiatic [10] and antimicrobial [6] activities of H. annuus have been reported.
Helianthus annuus leaves contain high proportion of saponins and terpenes [4,6] and its consumption for a *Address correspondence to this author at the Department of Veterinary Physiology, Pharmacology, Biochemistry and Animal Health and Production, Michael Okpara University of Agriculture, Umudike, Nigeria; Tel: +2348030613032; E-mail: samonreal@yahoo.com, onoja.samuel@mouau.edu.ng, samuelonoja19@yahoo.com long period of time especially in diabetes mellitus management call for concern about its possible erythrocytic toxicity. Saponins causes hemolysis of red blood cell in vivo [11] This study investigated the effects of hydromethanol extract of H. annuus on erythrocytic profile in normal rat.

Plant Collection and Extract Preparation
The leaf of Helianthus annuus was collected from the wild in Nsukka, Enugu state, Nigeria and identified by plant taxonomist. The leaves were dried under shed at ambient temperature (25 -27˚C). Hydromethanol extract of H. annuus was prepared using cold maceration method as described by Onoja and Anaga [4] and was referred to as hydromethanol leaf extract of H. annuus (HLEHA).

Animals
Fifty six (56) female albino Wistar rats were used for the study. They were housed in aluminum cages in a well-ventilated room at ambient temperature (25 -27˚C) and natural light/darkness cycle. The rats were acclimatized for 2 weeks and the experimental protocol was approved by the Michael Okpara University of Agriculture, animal ethical committee.

Feed Formulation
The hydromethanol extract of H. annuus was incorporated in the feed at the rate of 2.5, 5 and 10 mg per 10 g feed. The rate of incorporation was based on the percentage yield of the extract and doses used in previous studies [4]. Briefly, 500, 1000 and 2000 mg of the extract were weighed and dissolved in 2 litre of water each and later mixed with 2 kg of feed respectively. The resulting moist feed mixture was manually pelleted with the aid of the barrel of 5 ml syringe. The pellets were dried under mild sunlight for 2 days in harmattan condition. The pelleted feeds were used within 2 weeks to avoid mold growth.

Experimental Design
The rats were randomly assigned to 4 (A -D) groups of 14 rats each and treated as follow: group A rats received standard pelleted feed while groups B -D received feed incorporated with HLEHA at 2.5, 5 and 10 mg/10 g feed, respectively. They were fed 10% of their body weight daily for 90 consecutive days. They finished their feed each day and a deep feeding troughs were used to avoid feed wastage. On weekly bases, the rats were weighed and feeding was adjusted to accommodate the weight change. The hematological profiles of the rats were determined on day 30, 60 and 90 of treatment. Four animals were randomly selected from each group at the timed interval and blood samples were collected from the retroorbital venous plexus with the aid of heparinized capillary tube into ethylenediaminetetraacetic acid (EDTA) bottle. The parameters were determined within 24 h of blood sample collection.
The method of Dacie and Lewis [12] was employed in red blood cell (RBC) count; hematocrit method [13] was used in packed cell volume (PCV) estimation while hemoglobin (HB) concentration was estimated with cyanomethemoglobin method using Drabkin's reagent [13]. The mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) was calculated as described by [14].

Data Analysis
The data were analyzed using one-way analysis of variance (ANOVA), variant means were separated least significant difference (LSD) and significance was accepted at p < 0.05 with statistical package for social sciences (SPSS) version 21.0

RESULTS
Generally there was no significant (p > 0.05) change in the value of all the parameters determined on day 30 and 60 except for MCHC on day 30. There was no significant (p > 0.05) difference in the RBC values of the treatment groups ( Table 1). At day 90, the PCV of Groups C and D were significantly (p < 0.05) lower than the value of group A ( Table 2). The Hb concentration of HLEHA -treated groups were significantly (p < 0.05) reduced compared to the rats that received standard feed on day 90 ( Table 3). The MCV of group B was significantly (p < 0.05) lower while  group C and D were not significant (p > 0.05) compared to group A on day 90 (Table 4). Also, on day 90, MCH of groups B and C were decreases significantly (p < 0.05) compared to group A ( Table 5).
The MCHC of group D was significantly higher (p < 0.05) compared to group A on day 30 while the was no significant (p > 0.05) difference in the various treatment groups on days 60 and 90 ( Table 6).

DISCUSSION
The choice of incorporating HLEHA in the feed was to avoid stress of daily oral administration of the extract throughout the duration of the study. The study showed hematological changes associated with chronic administration of HLEHA to rats. Feeding of the rats with extract-treated feed did not produce a significant (p > 0.05) hematological change on the first 60 days of     [15] reported that the physiological, biochemical and pharmacological activities of medicinal plants is related to their phytoconstituents. Helianthus annuus has been reported to contain terpenes, alkaloids, saponins and other phytoconstituents [16]. Some of the terpenes and saponins isolated from H. annuus leaves and oil include; D-limonene, α-pinene, menthol, sabinene, germacrene D, camphene, β-pinene, isobornyl acetate, α-terpineol etc [2]. Mendanha et al. [17], reported the in vitro hemolytic activity of terpenes; nerolidol, 1,8cineole, α-terpineol, pulegone, α-limonene etc, against normal human erythrocyte. The hemolytic activities of saponins have been proposed by researchers [18]. The reported hemolytic activities of saponins and terpenes shorten the life span of erythrocyte and predispose the affected animal to hemolytic anemia [19]. The mild anemia recorded in this study may be due to the low dose of H. annuus that was used in the study. High dose may cause a more severe anemia even within shorter period [20]. The absence of significant difference in the red blood cell count may be due to regenerative response of the erythropoeitic tissue [14].
This study suggests that the chronic use of H. annuus leaf for up to 90 days in rat may lead to hemolytic anemia.

ACKNOWLEDGEMENT
The authors are grateful to Mr. A. O. Ozioko, a taxonomist with Bioresource Development and Conservation programme (BDCP) Enugu state, Nigeria for identification of the plant sample. The authors are also grateful to the management of Department of Veterinary Physiology, Pharmacology, Biochemistry and Animal Health and Production, Michael Okpara University of Agriculture, Umudike, Nigeria, for granting us access to the laboratory facilities for this work.

DECLARATION OF CONFLICT OF INTEREST
The authors declare no conflict of interest.

FUNDING
There was no external funding for this project.