Evaluation of some metabolic activities and immuno-stimulatory potential of methanolic seed extract of Garcinia kola ( Heckel ) in female albino Wistar rats

Evaluation of some metabolic activities and immuno-stimulatory potential of the methanolic seed extract of Garcinia kola (Heckel) in female albino Wistar rats was carried out using standard methods. Animals in treated groups were administered 125, 250 and 500 mg/kg body weight, respectively of the extract through the oral route for 21 days. The control group received normal saline (0.5 ml/100 g body weight) for the same period during which food and water intakes were monitored. At the end of the treatment period, complete and differential blood counts were evaluated for each group. Results show that the extract at the doses of 125 and 250 mg/kg caused significant increase in body weight of treated animals compared with those of control (p < 0.05). Similarly, there was significant increase in food and water intake as well as faecal and urine output in groups treated with 250 and 500 mg/kg (p < 0.05). Results of the complete blood count showed that the extract caused significant increase in total white blood cell count (WBC) (p < 0.05) but a non-significant increase in haemoglobin (p > 0.05). There was significant increase in lymphocytes in groups that received the extract at a dose of 125 mg/kg compared with control (p < 0.05). These results suggest that the methanolic seed extract of G. kola possesses immuno-stimulatory potential that may justify the ethno-medicinal claims of the efficacy of G. kola in treatment of some immune disorders.


INTRODUCTION
Garcinia kola (Heckel) is a tropical forest tree that grows between 15 and 17 m high (Burkill, 1985).The fruits and seeds are edible but have strong bitter taste (Ehiem and Eke, 2014).The seeds have many social and medicinal uses in traditional settings of many communities in the West and Central African sub-region (Adesuyi et al., 2012).It is traditionally used for the management of different ailments in such communities.Various scientific investigations have been carried out to confirm or refute such traditional claims.Interestingly, it has been found *Corresponding author.E-mail: bukata33@yahoo.com.Tel: +2348036183979.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Values are mean ± SEM; *p < 0.05; n = 5; control = normal saline; 0.5 ml/100 g.
This study evaluated the immuno-stimulatory potential of the methanolic seed extract of G. kola in female albino rats.This is premised on some recent ethno-medicinal claims that the seed alone or in combination with other herbs may be beneficial in the management of some disorders of the immune system such as those arising from human immune deficiency virus (HIV) and Ebola.

Animals
Twenty five female albino rats weighing between 115 and 123 g were purchased from the Animal House Unit of the Department of Pharmacology, University of Jos following approval by the Committee on use of experimental animals.The animals were handled under ethical conditions and guidelines for the use and care of laboratory animals (NIH, 2011).They were housed in metal cages padded with sawdust placed on metal shelves in a wellventilated room at controlled ambient temperature.The animals had access to standard solid nutritional pellets and water ad libitum.

Preparation of extract
G. kola seeds were purchased from Forestry Research Institute of Nigeria (FRIN), Ibadan, Oyo state, Nigeria and re-authenticated at the Federal College of Forestry, Jos in Plateau State.A herbarium voucher specimen (number FHJ 202) was prepared.The seeds were washed, de-husked and carefully cut into small pieces with a clean sharp knife to enhance drying, then dried under shade in the laboratory.
Thereafter, they were ground to powder with mortar and pestle and extracted as described by Adaramoye (2010).In the procedure, 150 g of the powdered seed was extracted continuously with methanol (99.5%) in a Soxhlet extractor for 72 h.The extract was evaporated to dryness in a vacuum evaporator maintained at 40°C until a constant yield of 73.7 g (49.13%) following repeated weighing was obtained.The brownish colored extract was stored in an airtight container in the refrigerator and reconstituted in distilled water for the purpose of the experiment.

Sub-chronic administration of Garcinia kola
The rats were divided into 4 groups of 5 rats each.Group I (control) was administered normal saline (0.5 ml/100 g body weight) while groups II, III and IV were administered the extract in daily oral doses of 125, 250 and 500 mg/kg body weight, respectively for 21 days.Concentrations of the extracts were prepared such that final volumes used for each administration of the extract were not more than 1 ml.On day 22, animals were sacrificed by exsanguination and the blood collected into EDTA bottles for haematological evaluations.

Food and water intake
The quantity of food and water intake per day together with the amount of faecal and urine outputs were measured throughout the 21 days of extract administration.The body weights of the rats were also determined weekly.The mean food/water intake and faecal/urine output were each calculated at the end of the treatment.

Complete blood count
At the end of the three weeks the rats were anaesthetized with phenobarbitone (60 mg/kg) and their blood collected from the femoral vein into EDTA bottles for determination of haemoglobin (Hb), packed cell volume (PCV), total white blood cell (TWBC) and differential count of the white blood cells using Haematology Auto-Analyzer (Mindray BC 3200, Shenzen, China).Differential cell counts were done on a thin slide, prepared with a smearing blood sample using Wright-Giemsa's stain.The absolute number of each white cell type was calculated by multiplying the number of each cell counted (expressed as a decimal fraction) by the total white blood cell (TWBC) count.

Effect of Garcinia kola seed extract on some metabolic parameters
The effect of the extract on some metabolic parameters in female albino Wistar rats is shown in Table 1.The extract at the doses of 125 and 250 mg/kg caused significant increase in body weights of treated animals compared with those of control (p < 0.05).However, at the dose of 500 mg/kg, the increase in body weight of the treated animals was not significantly different from those of the control (p > 0.05).There was a significant increase in both food and water intakes in treated animals at doses of 250 and 500 mg/kg (p < 0.05).However, while there was a non-significant increase (p > 0.05) in food intake at the dose of 500 mg/kg, there was nonsignificant decrease (p > 0.05) in water intake with the same dose.
The extract caused increase in faecal output at doses of 250 and 500 mg/kg.However, while the increase was significant at 500 mg/kg (p < 0.05), it was not so at 250 mg/kg (p > 0.05).On the other hand, the extract at the dose of 125 mg/kg caused decrease in faecal output but this was not significantly different compared with control (p > 0.05).The extract generally caused decrease in urine output.The decrease in urine output was significant at the dose of 250 mg/kg (p < 0.05) but not significant at 125 and 500 mg/kg (p > 0.05) compared with control.

Effect of Garcinia kola seed extract on full blood count
The results indicate that the extract increased haemoglobin level of treated animals at all the dose levels (Table 2).However, this was found not to be significantly different compared with the control (p > 0.05).The result of the effect of the extract on packed cell volume showed that the extract caused increase in PCV of animals in all treated groups.However, while this was observed to be significant with animals in the groups that received 250 and 500 mg/kg (p < 0.05), it was not significant with those in the group that received 125 mg/kg (p > 0.05).There was significant increase in total white blood cell count (Table 2) of animals in groups treated with 125 and 250 mg/kg of the extract (p < 0.05) while in the group treated with 500 mg/kg, the increase was not significant compared with the +control (p > 0.05).The results showed that the extract caused increase in platelet counts at all the dose levels.The increase was particularly significant at the dose of 125 mg/kg (p < 0.05) while at higher doses of 250 and 500 mg/kg, the increase was not significantly different compared with control (p > 0.05).
The extract significantly (p < 0.05) increased lymphocytes count at the dose of 125 mg/kg compared with control (Table 3).However, at the higher doses of 250 and 500 mg/kg, the extract caused non-significant (p > 0.05) decreases in lymphocyte counts compared with the control.The result for the neutrophil count showed that the extract caused significant decrease in count in the group treated with 125 mg/kg compared with control (p < 0.05).However, at the higher doses of 250 and 500 mg/kg the extract caused non-significant increase in count compared with control (p > 0.05).The extract increased eosinophils count of animals at all doses used in this experiment.The results for the effect of the extract on monocyte count indicated that the extract had no effect on the count, except for the non-increase at the highest dose.

DISCUSSION
The methanolic seed extract of G. kola caused increase in body weight of treated animals.This observation is consistent with the increase in food intake in treated groups.Similarly, the extract increased water intake at the dose of 125 and 250 mg/kg but a non-significant decrease at the dose of 500 mg/kg.There was increased faecal output groups treated with 250 and 500 mg/kg but a decrease output at the lower dose of 125 mg/kg.Urine output was observed to decrease in all groups of rats treated with the extract.
Various reports have been established to show the fact that food and water consumption in the rat is coordinated by the hypothalamus (Strominger, 1947;Bruce and Kennedy, 1951).Nonetheless, there is significant interplay between neural and chemical processes in regulation of food and water intake.This is because neural mechanisms are apparently mediated by chemical regulators, especially orexin, the stimulatory hormone for appetite (Norton et al., 1993;Ida et al., 1999;Baird et al., 2000).Nutrition has been noted as playing an important role in stimulating and modulating the immune functions.This is because the immune system needs adequate supply of nutrients to function properly.A variety of mechanisms have been highlighted on the interaction between the immune system and some foods and food components in terms of modulation of the immune functions (El-Gamal et al., 2011).The effect of the extract on the metabolic behavior of treated rats perhaps suggests its immuno-stimulatory potential in the rat model which could justify its traditional use in some immune disorders.
There was a non-significant increase in haemoglobin levels in all treated groups but a significant increase in packed cell volume at 250 and 500 mg/kg.Atsukwei et al. (2015) reported a similar finding in ethanolic seed extract of G. kola in male wistar rats.This supports the relationship between haemoglobin and packed cell volume and suggests that the extract may improve erythropoiesis through mechanisms yet to be determined (Smith and Bidlack, 1980;Naidu, 2003).The extract increased WBC, lymphocytes, neutrophils and platelet counts which were particularly significant (p < 0.05) at the dose of 125 mg/kg.Given the roles of these cellular components in immune function, the results suggest an immuno-stimulatory potential of the extract in the rat model.This observation agrees with the findings of Nworu et al. (2007Nworu et al. ( , 2008) ) who used the mice model.Findings from this evaluation suggest that the methanolic seed extract of G. kola possesses immuno-stimulatory potential that authenticates its claimed ethno-medicinal use in management of some immune disorders.of the extract.We are also grateful to Mr. Luka Dabum of Physiology Department, University of Jos, for some of the haematological analysis and to Topsy Computers, Murtala Mohammed Way, Jos, for their clerical assistance.

Table 1 .
Effect of Garcinia kola extract on some metabolic parameters in female albino rats.

Table 2 .
Effect of Garcinia kola seed extract on complete blood in female albino rats.

Table 3 .
Effect of Garcinia kola seed extract on differential WBC counts in female albino rats.