Effect of dietary inclusions of bitter kola seed on geotactic behavior and oxidative stress markers in Drosophila melanogaster

Abstract This study evaluated the effect of dietary inclusions of Garcinia kola (GK) seed on geotactic behavior and some oxidative stress markers in wildߚtype fruit flies (Drosophila melanogaster). Flies were raised on diet supplement with GK seed for 5 days. The negative geotactic behavior of flies which was used to evaluate their locomotor performance was thereafter evaluated. The flies were subsequently homogenized and the reactive oxygen species (ROS) level, acetylcholinesterase (AChE), catalase and glutathioneߚSߚtransferase (GST) activities, as well as nitric oxide (NO) and total thiol contents were assayed. The phytochemical constituents of GK seed were also determined. It was observed that higher dietary inclusions of GK seed reduced the survival rate of D. melanogaster more significantly compared to control flies. Also, higher dietary inclusions of GK seed significantly reduced locomotor performance and AChE activity, while the ROS level was increased compared to the control. Activities of GST and catalase were significantly increased in flies fed diet supplemented with higher GK seed inclusions but their NO content was significantly reduced compared to control. Phytochemical analysis of GK seed revealed abundance of saponin > glycosides > alkaloids > phenols > flavonoids. These results have shown that dietary inclusion of GK seed at higher concentrations reduced survival rate of D. melanogaster and impaired cholinergic system, with elevated activities of some antioxidant enzymes under acute exposure. These observations could be associated with bioactivities of predominant phytochemicals in GK seed such as saponin and glycosides which have been reportedly toxic at high concentration. Therefore, this study suggests that high consumption of GK seed could induce some toxicological effects and moderate consumption is hence recommended.


| INTRODUC TI ON
Edible plant seeds have been in existence for many years, Bitter kola (Garcinia kola) is one of the edible plant seeds that are well consumed in Nigeria and other parts of sub-Saharan Africa. The seeds of bitter kola are highly valued for its stimulating effects (Atawodi, Mende, Pfundstein, Preussmann, & Spiegelhalder, 1995).
Fruit fly (Drosophila melanogaster) is an arthropod, belonging to the family Drosophillidae. D. melanogaster has become a model for biological research since its introduction over 100 years ago, especially in genetics and molecular biology (Abolaji, Kamdem, Farombi, & Rocha, 2013). Drosophila is highly sensitivity to varying degrees of toxicants and is considered as a model for toxicity studies (Abolaji et al., 2013). It also serves as a useful model for evaluating biological actions of therapeutic agents against several human diseases (Adedara, Klimaczewski, 2015;Adedara, Rosemberg, 2015). Drosophila has unique physiological features similar to that of vertebrates (Baker & Thummel, 2007), and it has been certified to help reduce, refine, and replace higher animal usages such as rodents in biomedical research such as toxicity studies (Benford et al., 2000).
The awareness that medicinal plants can play a major role in ameliorating disease conditions is often overshadowed by the dosage intake of these medicinal plants. Garcinia kola (GK) seeds have long been consumed among various cultures of sub-Saharan Africa for various purposes including recreational and tradio-medicinal purposes. It is often consumed among various cultures of Nigeria especially for its stimulatory effects (Atawodi et al., 1995).
Although previous studies have reported various extracts and fractions of GK seed for their therapeutic uses in treating throat infections, bronchitis, cough, hepatitis, liver disorders (Farombi, Adepoju, Ola-Davies, & Emerole, 2005), the fact that consumption of whole seeds especially for long time produce stimulantlike effect could induce some toxicological effects which is worth investigating. This study therefore investigates the effects of GK seed on survival, locomotion, and oxidative stress markers in D. melanogaster.

| Experimental design
The flies (both gender, 3-5 days old) were divided into four groups containing 60 flies each. Group I was placed on normal diet alone while groups II-IV were placed on basal diet containing; GK seed at 0.1, 0.5 and 1.0% of diet (equivalent weight replacement) as shown thus; The flies were exposed to these treatments for 5 days, and the vials containing flies were maintained at room temperature. All experiments were carried out in triplicate (each experimental group was carried out in five independent vials).

| Survival study
A study was conducted to assess the effect of dietary inclusion of GK seed on survival rate of flies after 5 days of exposure. Flies (both gender, 3-5 days old) were divided into four groups containing 60 flies each. Each group was exposed to different dietary inclusions of GK seed (0.1%, 0.5%, and 1.0%). The flies were observed daily for the incidence of mortality, and the survival rate was determined by counting the number of dead flies for the first 5 days. The data were subsequently analyzed and plotted as cumulative mortality and percentage survival after the treatment period (Abolaji et al., 2014;Adedara, Abolaji, Rocha, & Farombi, 2016).

| Measurement of locomotor performance (negative geotaxis)
The negative geotaxis assay was used to evaluate the locomotor performance of flies (Le Bourg & Lints, 1992). In brief, after the treatment period of 5 days, the flies from each group were briefly immobilized in ice and transferred into a clean tube (11 cm in length 3.5 cm in diameter) labeled accordingly. The flies were initially allowed to recover from immobilization for 10 min and thereafter were tapped at the bottom of the tubes. Observations were made for total number flies that crossed the 6-cm line within a period of 6 s and recorded. The results are expressed as percentage of flies that escaped beyond a minimum distance of 6 cm in 6 s during three independent experiments.

| Preparation of tissue homogenate
The flies were immobilized in ice and homogenized in 0.1 M phosphate buffer, pH 7.4. The resulting homogenates were centrifuged at 10,000 × g, at 4°C for 10 min in a Kenxin refrigerated centrifuge Model KX3400C (KENXIN Intl. Co., Hong Kong). Subsequently, the supernatant was separated from the pellet into labeled Eppendorf tubes and used for the various biochemical assays.

| Reactive oxygen species (ROS) level
Reactive oxygen species level in the whole fly tissue homogenates was estimated as H 2 O 2 equivalent according to the method of Hayashi et al. (2007), with slight modifications. In brief, 50 μl of tissue homogenate and 1,400 μl sodium acetate buffer were transferred to a cuvette. After then, 1,000 μl of reagent mixture of nn-diethyl-para-phenylenediamine (DEPPD) (6 mg/ml of DEPPD with 4.37 μM of ferrous sulfate dissolve in 0.1 M sodium acetate pH 4.8) was added at 37°C incubated for 5 min. The absorbance was measured at 505 nm using a spectrophotometer. ROS levels was estimated from an H 2 O 2 standard calibration curve and expressed as unit/mg protein, where 1 unit = 1 mg H 2 0 2 /L.

| Determination of catalase (CAT) activity
Catalase activity in the homogenate samples was determined according to the method of Shina (1972). In brief, 0.1 ml of each tissue homogen-

| Determination of glutathione-Stransferase activity
This assay was carried out according to the method of Habig and Jakoby (1981). It involves the preincubation of reaction mixture containing 1.0 ml 100 mM phosphate buffer (pH 6.5), 30 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 0.7 ml 0f distilled water for 5 mins at 37°C. The reaction was started by the addition of 0.1 ml of the tissue homogenate and 0.1 ml 30 mM glutathione as substrate. The absorbance of the reaction mixture was monitored after 5 min at 340 nm in a spectrophotometer. Reaction mixture without enzyme was used as a blank. The activity of GST was calculated and expressed as unit of GST activity per mg protein.

| Determination of the total thiol content
Determination of the level of total thiol content in tissue homogenate was performed by the method of Ellman et al. (1959). The reaction mixture was made up of 270 μl of 0.1 M potassium phosphate buffer (pH 7.4), 20 μl of homogenate, and 10 μl of 10 mM DTNB. This was followed by 30-min incubation at room temperature, and the absorbance was measured at 412 nm. The total-thiol content was subsequently calculated and expressed as μmol/mg protein.

| Determination of nonprotein thiol content
Determination of nonprotein thiols (NPSH) content was carried out by the method of Ellman (1959). Aliquot amount of Drosophila homogenate was equally mixed with 10% trichloroacetic acid. This allowed for protein precipitation, which was centrifuged at 10,000 × g for 5 min at 4°C and the free sulfhydryl groups were determined in the supernatant. The reaction mixture consisting 50 μl of sample, 450 μl phosphate buffer, and 1.5 ml of 0.1 mM of 5,5-dithiobis 2-nitro benzoic acid was incubated for 10 min at 37°C. The absorbance was measured at 412 nm, and NPSH level was expressed as μmol/mg of protein.
After incubating at 37°C for 60 min, nitrite levels, which corresponds to an estimated of level of NO, were determined spectrophotomerically at 540 nm. (Miranda, Espay, & Wink, 2001). Nitrite and nitrate levels were calculated and expressed as nanomole of NO/milligram of protein.

| Acetylcholinesterase (AChE) activity assay
Acetylcholinesterase activity was assayed according to the method of Ellman et al. (1961). The reaction mixture was made up of 195 μl of distilled water, 20 μl of 60 mM potassium phosphate buffer (pH 8.0), 20 μl of 20 mM DTNB, 5 μl of homogenate, and 20 μl of 20 mM acetylthiocholine (as initiator). Thereafter, reaction was monitored for 3 min (30-s intervals) at 412 nm. The AChE activity was thereafter calculated and expressed as mmolAcSch/h/ mg protein.

| Determination of total protein
Total protein content of fly homogenates was measured by the Coomassie blue method according to Bradford (1976) using bovine serum albumin (BSA) as standard.

| Determination of saponin
Saponin was quantified as previously reported by Brunner (1994).
This involved 2 g of pulverized sample added to 100 ml of Isobutyl alcohol. The reaction mixture was shaking for 5 hr to ensure uniform mixing using orbital shaker. The reaction mixture was filtered with No 1 Whatman filter paper into a clean beaker, and 20 ml of 40% saturated solution of MgCO 3 was added. This was followed by another filtration to obtain a clean filtrate. Subsequently, 2 ml of 5% FeCl 3 was added to 1 ml of the filtrate and made up with distilled water.
The reaction was allowed to stand for 30 min for the color development and the absorbance against the blank was taken at 380 nm.
Saponin content was expressed in mg/g of sample.

| Preparation of aqueous extracts
Ten grams of the powdered samples was weighed and extracted with 50 ml distilled water in extraction bottle and shaken vigorously for 2 hr. The extract was filtered using Muslin cloth and further filtered with Whatman filter paper and further centrifuged to obtain clear supernatant.

| Determination of cardiac glycosides
Cardiac glycosides were determined as previously reported (Sofowora, 1995). An aliquot of the extract (10 ml) was added in a reaction flask to 50 ml chloroform, which vortexed for 1 hr. The mixture was thereafter filtered followed by addition of 10 ml pyridine and 2 ml of 29% sodium nitroprusside which was shaken thoroughly for 10 min. This was followed by addition of 3 ml of 20% NaOH to develop a brownish yellow color. Digitoxin was used as standard glycoside. The absorbance was read @ 510 nm. The glycoside content was expressed as mg/g of sample.

| Determination of total phenol content
The total phenol content of GK seed was determined according to the method of Singleton, Orthofor, and Lamuela-Raventos (1999) with some modifications (Oboh & Ademosun, 2012). Briefly, the reaction mixture consists of an aliquot of the extract, 2.5 ml 10% Folin-ciocalteau's reagent (v/v), and 2.0 ml of 7.5% Na 2 CO 3 . This was followed by incubation for 40 min at 45°C, and the absorbance was quantified at 765 nm in a spectrophotometer. The total phenol content was subsequently calculated as mg/g gallic acid equivalent.

| Determination of total flavonoid content
The total flavonoid contents of Gk seed was determined using the modified method of Meda, Lamien, Romito, Millogo, and Nacoulma (2005) as reported by Oboh and Ademosun (2012). Briefly, the reaction mixture consists of 0.5 ml of extract, 0.5 ml of methanol, 50 μl 10% aluminum chloride, 50 μl 1 M Potassium acetate, and 1.4 ml distilled water. The reaction was incubated at room temperature for 30 min. This was followed by absorbance quantification at 415 nm.
The total flavonoid content was subsequently calculated and expressed as mg/g quercetin equivalent.

| Data analysis
The results of replicate readings were pooled and expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to analyze the results followed by Bonferoni's post hoc test, with levels of significance accepted at p < 0.05, p < 0.01, and p < 0.001. All statistical analysis was carried out using the software Graph pad PRISM (V.5.0).
Specifically, there have been several reports implicating ROS generation and oxidative stress in reduced life span in D. melanogaster (Hyrsl, Büyükgüzel, & Büyükgüzel, 2007;Lozinsky, Lushchak, Storey, Storey, & Lushchak, 2012;Lozinsky et al., 2013). Therefore, this study investigated the possibility of ROS generation and oxidative stress as possible mechanisms behind the reduction in survival rate of Drosophila fed GK seed at higher percentage inclusions. It was observed that dietary inclusions of GK seeds at higher percentage inclusions caused significant elevation in ROS production in the flies.
Interestingly, the Pearson correlation analysis revealed that there was strong correlation between treated flies' survival rate and ROS level with r = 0.946 for flies treated with 1.0% GK seed. This showed that elevation in ROS level would have contributed to the decline in survival rate of flies experienced at higher (0.5% and 1.0%) GK seed inclusions.
Dietary inclusions of GK seed (0.5% and 1.0%) increased significantly the activities of Catalase and Glutathione S-transferase (GST). Indeed, cellular macromolecules are protected primarily from the insult of free radical species by endogenous antioxidant molecules including catalase, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase (Rand, 2010 (Abolaji, Olaiya, Oluwadahunsi, & Farombi, 2017). Therefore, the increase in these antioxidant enzymes could be associated with induction of ROS production in the flies by GK seeds inclusions.
The elevated ROS level in the flies signifies a state of redox imbalance and oxidative stress. This could explain the elevation in these antioxidant enzymes as an adaptive response to the oxidative assaults. Adaptive response, which is the ability of an organism to effectively counteract cellular damages induced by cytotoxic agents such as free radicals, has been identified in D. melanogaster . Such elevation in antioxidant enzymes' activities as an adaptive response to cytotoxic agents has been previously reported in D. melanogaster, and they are  Values represent mean ± SD. *Values are significantly different at p < 0.05; **Values are significantly different at p < 0.01. Key: as described for Figure 1 TA B L E 1 Pearson's correlation coefficient (r) between flies' survival rate and selected bioassays often accompanied by impairments in cellular thiol levels Bayliak et al., 2018;Perkhulyn et al., 2017). Thiols represent the major portion of cellular antioxidant systems, and they play a vital role in defense against ROS (Mungli, Shetty, Tilak, & Anwar, 2009). However, the overall response of thiols to oxidative cellular assaults is often a product of interactions between thiol oxidation and synthesis (Osburn et al., 2006). Therefore, the nonsignificant difference in thiol contents among the treatment groups in this study could be as a result of correlation between thiol consumption in response to the presence of ROS and replenishing as a result of adaptive response under acute exposure.
Furthermore, investigations into the basis of the reduction in survival rate of flies fed with the dietary inclusions were necessitated. Acetylcholinesterase (AChE) hydrolyses the neurotransmitter-acetylcholine that regulates locomotion and motor function (Day, Damsma, & Fibiger, 1991). Higher dietary inclusions of GK seeds (0.5% and 1.0%) showed a significant decrease in survival rate also induced a decrease in AChE activity.
The decrease in the AChE activity following dietary inclusions of GK seeds at 0.5% and 1.0% could be an increase in acetylcholine levels in the synaptic cleft and as a result induce cholinergic toxicity which could impair neuromuscular activities such as climbing abilities of flies (Akinyemi, Oboh, Ogunsuyi, Abolaji, & Udofia, 2017). It should also be noted that prolonged reduction in the activity of AChE in the flies could lead to oxidative stress which could also contribute to their reduced survival rate (Olney, Collins, & Sloviter, 1986). This is further corroborated by the strong correlation between the survival rate of flies and their AChE activities among the treated groups.
Nitric oxide is a diffusible signaling molecule, and its presence in both vertebrates and insects nervous system has been established (Müller, 1997). In D. melanogaster, NO has been well reported in cellular development during the developmental stages of the flies (Enikolopov, Banerji, & Kuzin, 1999;Jaszczak, Wolpe, Dao, & Halme, 2015). Studies have also shown that NO function in immune responses of the flies to pathogens and parasites (Eleftherianos et al., 2014;Nappi, Vass, Frey, & Carton, 2000).
Although there is dearth of information on the role of NO in adult fly physiology, nevertheless, the production of NO is reportedly more confined to the brain of adult flies (Enikolopov et al., 1999).
It was shown in this study that dietary inclusions of GK seeds at 0.5% and 1.0% showed a significant decrease in endogenous NO contents in the flies. This significant reduction in NO con-  (Smith, Kapoor, & Felts, 1999;Wink et al., 2001). NO's protective roles on mammalian cells are associated with such mechanisms as scavenging radical species, formation of iron-nitrosyl complexes that limits availability of iron as prooxidant, prevention of lipid peroxidation reactions, attenuation of enzymatic (xanthine oxidase), and nonenzymatic (H 2 O 2 )-mediated cell death, as well as up-regulating antioxidant enzymes' activities (Hasanuzzaman et al., 2017;Park, 1996;Wink et al., 2001). These observations suggest that a decrease in NO production may elicit oxidative responses that could culminate in increase ROS level and altered antioxidant enzyme activities.
Consequently, the observed high abundance of saponin in GK seed could be one of the major reasons for the mortality and impairment in locomotor performance in flies. Also high abundance of glycosides, which have been reported to be neurotoxic and cardiotoxic (David et al., 2013), could also contribute to the impairment in locomotor performance.

| CON CLUS ION
The results showed that dietary inclusions of GK seed especially at high percentage reduced survival rate and locomotor performance of the flies. It also produced some cholinergic impairment and increased ROS production. These observations could be attributed to the predominant phytochemicals in GK seed-saponins and glycosides that have been reported to be toxic at high concentration.
However, dietary inclusion of 0.1% of GK seed seems tolerable to flies and even produced some percentage increase in flies' survival rate, no significant effect on locomotor performance, and nonimpairment in AChE activity compared to the control. This study therefore suggests that high consumption of GK seed in D. melanogaster induces some toxicological effect, while moderate-low consumption could be beneficiary and offer the well reported medicinal effects attributed to GK seeds.

ACK N OWLED G M ENT
The authors wish to appreciate Dr Amos Abolaji of Department of Biochemistry, University of Ibadan, Nigeria for graciously supplying the Drosophila stock culture used for this research.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing that requires ethical approval.