Drug and Chemical Toxicology ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/idct20 Evaluating Water bitter leaf (Struchium sparganophora) and Scent Leaf (Ocimum gratissimum) extracts as sources of nutraceuticals against manganese-induced toxicity in fruit fly model Adedayo Oluwaseun Ademiluyi, Opeyemi Babatunde Ogunsuyi, Josephine Oluwaseun Akinduro, Olayemi Philemon Aro & Ganiyu Oboh To cite this article: Adedayo Oluwaseun Ademiluyi, Opeyemi Babatunde Ogunsuyi, Josephine Oluwaseun Akinduro, Olayemi Philemon Aro & Ganiyu Oboh (2022): Evaluating Water bitter leaf (Struchium�sparganophora) and Scent Leaf (Ocimum�gratissimum) extracts as sources of nutraceuticals against manganese-induced toxicity in fruit fly model, Drug and Chemical Toxicology, DOI: 10.1080/01480545.2021.2021928 To link to this article: https://doi.org/10.1080/01480545.2021.2021928 Published online: 22 Mar 2022. Submit your article to this journal Article views: 22 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=idct20 DRUG AND CHEMICAL TOXICOLOGY https://doi.org/10.1080/01480545.2021.2021928 RESEARCH ARTICLE Evaluating Water bitter leaf (Struchium sparganophora) and Scent Leaf (Ocimum gratissimum) extracts as sources of nutraceuticals against manganese-induced toxicity in fruit fly model Adedayo Oluwaseun Ademiluyia , Opeyemi Babatunde Ogunsuyia,,b Olayemi Philemon Aroa and Ganiyu Oboha , Josephine Oluwaseun Akinduroa, a Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology, Akure, Nigeria; bDepartment of Biomedical Technology, Federal University of Technology, Akure, Nigeria ABSTRACT ARTICLE HISTORY Tropical vegetables remain one of the major sources of functional foods and nutraceuticals, while their constituent phytochemicals, especially alkaloids, have been reported to exhibit neuroprotective properties. Here, the protective effect of alkaloid extracts from Scent leaf (Ocimum gratissimum) and Water bitter leaf (Struchium sparganophora) on manganese (Mn)- induced toxicity in wild type fruit fly (Drosophila melanogaster) model was investigated. Flies were exposed to 30 mM of Mn, the alkaloid extracts (20 and 200 mg/g) and co-treatment of Mn plus extracts, respectively. The survival rate and locomotor performance of the flies were assessed 7 days post-treatment, after which the flies were homogenized and assayed for activities of acetylcholinesterase (AChE), monoamine oxidase (MAO), glutathione-S transferase (GST), catalase, superoxide dismutase SOD), as well as total thiol, reactive oxygen species (ROS) and neural L-DOPA levels. Results showed that the extract significantly reversed Mninduced reduction in the survival rate and locomotor performance of the flies. Furthermore, both extracts counteracted the Mn-induced elevation in AChE and MAO activities, as well as reduced antioxidant enzyme activities, with a concomitant mitigation of Mn-induced elevated ROS and neural L-DOPA level. The HPLC characterization of the extracts revealed the presence of N-propylamine, Vernomine and Piperidine as predominant in Water bitter leaf extract, while 2, 6-dimethylpyrazine and sesbanimide were found in scent leaf extract. Therefore, the alkaloid extract of these leaves may thus be sources of useful nutraceuticals for the management of pathological conditions associated with manganese toxicity. Received 28 June 2021 Revised 24 September 2021 Accepted 26 September 2021 Introduction Manganese (Mn), an essential metal is involved in a variety of physiological processes that occur in the body (Pfalzer and Bowman 2017). However, excessive Mn exposure is known to be associated with a number of adverse neurological effects (Siokas et al. 2021). A number of studies have demonstrated the significant association between Mn exposure and neurodegenerative diseases (Bowman et al. 2011). Also, a significantly higher circulating Mn levels have been identified in patients with Parkinson’s disease (Du et al. 2018). Mn neurotoxicity is also known to affect neurodevelopment (Lucchini et al. 2017). Specifically, maternal or early-life Mn exposure was shown to be associated with poorer cognitive and behavioral performance in children under 6 years old (Liu et al. 2020) The fruit fly (Drosophila melanogaster), is one of the most studied eukaryotic organisms and has made fundamental contributions to different areas of biology. It has also gained appreciation as a useful animal model of human diseases. Comparative genomic studies estimate that up to 75% of the Functional food; manganese toxicity; oxidative stress; LDOPA; alkaloids human genes implicated in diseases are expressed in Drosophila (Ugur et al. 2016). The similarity between human and Drosophila genomes is not limited to only genetic, but also numerous conserved biological mechanisms. The Drosophila genome is smaller in size and has a smaller number of genes compared to the human genome, which facilitates genetic studies. The fruit fly Drosophila melanogaster has proven to be a powerful platform with plenty of amenable genetic techniques to investigate the mechanism of human neurodegenerative diseases (Bilen and Bonini 2005). Medicinal plants are rich resources of nutraceuticals, food supplements, pharmaceutical intermediates and chemical entities (Hammer et al. 1999). The plant Ocimum gratissimum is one of those plants widely known and used for both medicinal and nutritional purposes. It is a perennial plant that is widely distributed in the tropics of Africa and Asia. It belongs to the Family Labiatae and it is the most abundant of the genus Ocimum. The common names of the plant are Basil Fever plant or Tea bush and vernacular names include Daidoya tagida (Northern), Nichonwu (East), and Efinrin (Southwest) (Abdullahi et al. 2003, Idris et al. 2011). The plant CONTACT Adedayo Oluwaseun Ademiluyi ademiluyidayo@yahoo.co.uk, aoademiluyi@futa.edu.ng Biochemistry, Federal University of Technology, Akure, Nigeria ß 2022 Informa UK Limited, trading as Taylor & Francis Group KEYWORDS Functional Foods and Nutraceuticals Unit, Department of 2 A. O. ADEMILUYI ET AL. is also believed to keep the baby’s cord and wound surface sterile from bacterial infection. It is used in the treatment of fungal infections, fever, cold and catarrh and also in West Africa it serves as an antimalarial and anti-convulsant drug. The crushed leaf juice is used in the treatment of convulsion, stomach pain and catarrh. Oil from the leaves have been found to possess antiseptics, antibacterial and antifungal activities (Sofowara 1984, Edeoga and Eriata 2001). The phytochemical evaluation of Ocimum gratissimum shows that it is rich in alkaloids, tannins, phytates, flavonoids and Oligosaccharides (Ijeh et al. 2004). Water bitter leaf (Struchium sparganophora) is a culinary herb found in most African countries. It belongs to the family of Asteraceae. The plant is found predominantly in Southwestern Nigeria and is known as ’Ewuro-od’. It is used in the preparation of soups in that locality. It is a shrub that normally grows near the water side and usually has a tap root, it is greatly branched and fibrous, and grows deep into the soil. Considering the rich bioactive phytochemicals in these vegetables which has been erstwhile reported for their neuroprotective properties, this study therefore, seek to investigate the neuroprotective properties of alkaloid extracts of scent leaf and water bitter leaf in manganese-induced toxicity in D. melanogaster. Materials and methods Materials Sample collection Fresh samples of Struchium sparganophora (Water Bitter Leaf) and Ocimum gratissimum (Scent leaf) were obtained from the south-western part of Nigeria, precisely in Akure, Ondo State. The samples were authenticated in the Department of Crop Soil and Pest management, Federal University of Technology, Akure. The leaves were washed, sliced, air dried and blended to fine powder using a stainless steel grinder. The pulverized samples were stored in an air-tight container prior to the extraction of their alkaloids. Drosophila melanogaster stock culture Wild type Drosophila melanogaster (Oregon strain) stock culture was obtained from Drosophila Research Lab, Functional Foods and Nutraceuticals Unit of The Federal University of Technology, Akure. The flies were maintained and reared on normal diet made up of cornmeal medium containing 1% w/ v brewer’s yeast and 0.08% v/w nipargin at constant temperature and humidity (25 ± 1  C; 60% relative humidity respectively) under 12 h dark/light cycle conditions. All the experiments were carried out with the same D. melanogaster strain. from Sigma Al-drich, Chemie GmbH (Steinheim, Germany), hydrogen peroxide, methanol, acetic acid, hydrochloric acid, manganese chloride, potassium acetate, sodium dodecyl sulfate, Iron (II) sulfate, potassium ferrycyanide and ferric chloride were sourced from BDH Chemicals Ltd., (Poole, England). Ascorbic acid and starch were products of Merck (Darmstadt, Germany). Except stated otherwise, all other chemicals and reagents were of analytical grades and the water was glass distilled. Methods Preparation of alkaloid extract Alkaloid extracts of samples were prepared according to the method of Harborne (1998), with slight modifications as described by Ademiluyi et al. (2016). Briefly, 100 g of the pulverized sample was defatted with N-hexane for 24 h. Thereafter, 200 mL of 10% acetic acid in ethanol was added to the defatted samples and covered in a 500 mL beaker. These were vigorously shaken, venting the mounted pressure and allowed to stand for 24 h to allow for sufficient extraction. The mixtures were thereafter, filtered first using muslin cloth and then filter paper (Whatman No. 1) to obtain a clear filtrate which was concentrated under vacuum using rotary evaporator (Laborota 4000 Efficient, Heidolph, Germany) at 45  C. Concentrated ammonium hydroxide was subsequently added in a drop wise manner to the concentrated filtrate until there was a good precipitation. The whole solution was allowed to settle and the precipitate was collected and rinsed with dilute ammonium hydroxide to obtain the alkaloid extracts. The extracts were collected and stored in the refrigerator at 4  C for further analysis Preparation of tissue homogenate for biochemical assays Following the negative geotaxis assay, the flies from control and the treatment groups were anesthetized in ice and weighed. The flies were homogenized in 10 volumes 0.1 M phosphate buffer, pH 7.4, and centrifuged at 10,000  g, 4  C for 10 min in a Kenxin refrigerated centrifuge Model KX3400C (KENXIN Intl. Co., Hong Kong). The supernatants were collected into labeled Eppendorf tubes and subsequently used for the determination of biochemical parameters. The protein concentration of head homogenates was determined using the Lowry method (Lowry et al. 1951). All biochemical determinations were performed in duplicates in three independent experiments. In vitro analysis Chemical and reagents Chemical reagents such as acetylthiocholine iodide, sulfanilamide, reduced glutathione, n-n-dimethyl-para-phenylenediamine (DEPPD), ferrous sulfate, semicarbazide were procured from Sigma Al-drich Co. (St Louis, Missouri, USA). Trichloroacetic acid (TCA) and sodium acetate was sourced Determination of 2, 2-diphenyl -1- picrylhydrazyl (DPPH) radical scavenging ability The free radical scavenging ability of the extracts against DPPH free radical was evaluated as described by Gyamfi et al. (1999). Briefly, appropriate dilutions of the extracts (1 mL) were mixed with 1 mL 0.4 mM DPPH radicals dissolved DRUG AND CHEMICAL TOXICOLOGY in methanol. The mixture was left in the dark for 30 min, and the absorbance was taken at 516 nm. The experiment was controlled by using a 2 mL DPPH solution without the test samples. The DPPH free radical scavenging ability was subsequently calculated as a percentage of the control. Determination of 2, 2-Azinobis (3-ethylbenzo-thiazoline-6sulfonate) (ABTS) radical scavenging ability ABTS scavenging ability of both extracts was determined according to the method described by Re et al. (1999) with slight modification. ABTS was generated by reacting an ABTS aqueous solution (7 mmol/L) with K 2S 2 O8 (2.45 mmol/ L, final concentration) in the dark for 16 h and adjusting the Absorbance to 0.700 at 734 nm with ethanol. Thereafter, 0.1 mL of appropriate dilution of each extract was added to 2.0 mL ABTS solution and absorbance was measured at 734 nm after 15 min incubation in the dark. The trolox equivalent antioxidant capacity (TEAC) was subsequently calculated. 3 stopped by the addition of 0.5 mL of 2.8% trichloroacetic acid. This was followed by addition of 0.4 mL of 0.6% thiobarbituric acid solution for color development. The tubes were subsequently incubated in boiling water for 20 min. The absorbance was measured at 532 nm in the UV-visible spectrophotometer. The percentage OH scavenging ability was subsequently calculated as percentage of control. Nitric oxide scavenging activity Nitric oxide scavenging assay was performed according to a previously reported method (Marcocci, 1994), where in 0.3 mL of sodium nitroprusside (5 mM) was added to 1 mL each of various concentrations of the extracts. The test tubes were then incubated at 25  C for 150 min. After 150 min, 0.5 mL of Griess reagent (equal volume of 1% sulfanilamide on 5% ortho-phosphoric acid and 0.01% naphthylethylenediamine in distilled water, used after 12 h of preparation) were added. The absorbance was measured at 546 nm. Lipid peroxidation and thiobarbituric acid reactions Determination erty (FRAP) of ferric reducing antioxidant prop- The reducing ability properties of the extracts were determined by assessing the ability of both extracts to reduce FeCl3 solution as described by Oyaizu (1986). 2.5 mL aliquot was mixed with 2.5 mL 200 mM sodium phosphate buffer (pH 6.6) and 2.5 Ml 1% potassium ferricyanide. The mixture was centrifuged at 650 rpm for 10 min. 5 mL of the supernatant was mixed with an equal volume of water and 1 mL 0.1% ferric chloride. The absorbance was measured at 700 nm in the UV-Visible spectrophotometer. The ferric reducing antioxidant property was subsequently calculated as ascorbic acid equivalent. Fe2þ chelation assay The Fe2þ chelating ability of both extracts were determined by using a modified method of Minotti and Aust (1987) with a slight modification by Puntel et al. (2005). Freshly prepared 500 mM FeSO4 (150 mL) was added to a reaction mixture containing 168 mL 0.1 M Tris-HCl (pH 7.4), 218 mL saline and each of the extracts (0–25 mL). The reaction mixtures were incubated for 5 min, before the addition of 13 mL 0.25% 1, 10phenanthroline (w/v). The absorbances were subsequently measured at 510 nm in the UV-Visible spectrophotometer. The Fe (II) chelating ability was subsequently calculated as percentage of control. Fenton reaction (degradation of deoxyribose) The method of Halliwell and Gutteridge (1981) was used to determine the ability of the extract to prevent Fe2þ/hydrogen peroxide (H2O2)-induced decomposition of deoxyribose. The extracts (0–100 lL) was added to a reaction mixture containing 120 lL of 20 mM deoxyribose, 400 lL of 0.1 M phosphate buffer, and 40 lL of 500 lM FeSO4, and the volume was made up to 800 lL with distilled water. The reaction mixture was incubated at 37  C for 30 min and the reaction was then The lipid peroxidation assay was carried out using modified method of Ohkawa et al. (1979), 100 mL of fly tissue homogenate was mixed with a reaction mixture containing 30 mL of 0.1 M Tris- HCl buffer (pH 7.4), extracts (0–100 mL) and 30 mL freshly prepared FeSO4 (the procedure was also carried out using 5 mM sodium nitroprusside and 15 mM Quilinonic acid). The volume was made up to 300 mLwith distilled water before incubation at 37  C for 1 hr. The color was developed by adding 300 mL, 8.1% sodium dodecyl sulfate (SDS) to the reaction; this was subsequently followed by the addition of 500 mL of acetic acid/HCl buffer (pH 3.4) and 500 mL, 0.8% thiobarbituric acid (TBA). This mixture was incubated at 100  C for 1 hr. Thiobarbituric reactive species (TBARS) produced were measured at 532 nm and the absorbance was compared with that of the standard curve using malondialdehyde (MDA). Acetylcholinesterase inhibition assay Inhibition of acetylcholinesterase (AChE) was assessed by a modified colorimetric method of Ellman (Perry et al. 2000). The AChE activity was determined in a reaction mixture containing 200 mL of fly tissue homogenate in 0.1 M phosphate buffer, pH 8.0, 100 mL of a solution of 5, 50 -dithio-bis (2- nitrobenzoic) acid (DTNB 3.3 mM in 0.1 M phosphate buffered solution, pH 7.0, containing NaHCO3 6 mM), alkaloid extracts (0–100 mL) and 500 mL of phosphate buffer, pH 8.0. After incubation for 20 min at 25  C, acetylthiocholine iodide (100 mL of 0.05 mM solution) was added as the substrate, and AChE activity was determined by UV spectrophotometry from the absorbance changes at 412 nm for 3.0 min at 25  C. The AChE inhibitory activity was expressed as percentage inhibition. Bioassay Survival study A study was conducted to assess the effect of alkaloid extracts of scent leaf and water bitter leaf on survival rate of 4 A. O. ADEMILUYI ET AL. flies after seven days of exposure. Flies (both gender, 3–5 days old) were divided into five groups containing 40 flies each. Groups I was placed on a basal diet (without alkaloid extracts) while groups 2–5 were placed on diet plus the alkaloid extracts (20–20,000 mg/mL). The flies were observed daily for the incidence of mortality and the survival rate were determined by counting the number of dead flies for the first seven days. The data were subsequently analyzed and plotted as cumulative mortality and percentage of living flies after the treatment period (Abolaji et al. 2014, Adedara et al. 2016). HPLC characterization of constituent alkaloids An aliquot of the alkaloid extracts (1 lL) was injected in splitless mode at an injection temperature of 110  C. Purge flow was 3 mL/min with a total flow of 11.762 mL/min; gas saver mode was switched on. Oven was initially programmed at 110  C (2 min) then ramped at 10  C/min to 200  C (2 min) then 5  C/min to 280  C (9 min). Run time was 38 min with a 3 min solvent delay. The mass spectrometer was operated in electron ionization mode with ionization energy of 70 eV with ion source temperature of 230  C, quadrupole temperature of 150  C, and transfer line temperature of 280  C. Prior to analysis, the MS was autotuned to perfluorotributylamine (PFTBA) using already established criteria to check the abundance of m/z 69, 219, 502, and other instrument optimal and sensitivity conditions. Analysis validation was conducted by running replicate samples in order to see the consistency of the constituent compound name, respective retention time, and molecular weight. These abundances were outputs from the NIST 11 library search report of the extracted constituents. Each compound identified via the NIST library search report has a corresponding mass spectrum showing the abundance of the possible numerous m/z peaks per compound. Experimental design The flies (both gender, 3–5 days old) were divided into 10 groups containing 40 flies each. Group 1 was placed on basal diet alone, group 2 was placed on diet treated with 30 mM MnCl2, groups 3 and 4 were placed on diets treated with 20 and 200 mg/g of scent leaf extracts respectively, groups 5 and 6 were placed on diets treated with 20 and 200 mg/g of water better leaf extracts respectively, groups 7 and 8 were MnCl2 exposed flies fed diets treated with 20 and 200 mg/g of scent leaf extracts respectively, while groups 9 and 10 were MnCl2 exposed flies fed diets treated with 20 and 200 mg/g of water bitter leaf extracts respectively. The choice of concentrations of the sample was based on the survival study which showed that the selected concentrations caused no significant mortality in flies. The specific dose used for Manganese chloride was based on previously published data on the toxicity of Manganese in D. melanogaster (Oboh et al. 2018). The flies were exposed to these treatments for 7 days and the vials containing flies were maintained at room temperature. All experiments were carried out in triplicate (n ¼ 6). The control and treated flies were observed daily for the incidence of mortality throughout the experiment. The survival rate was determined by counting the number of dead flies. The data were subsequently analyzed and plotted as cumulative mortality and percentage of lliving flies after the treatment period. Measurement of locomotor performance by negative geotaxis assay Evaluation of locomotor performance of the control and treated flies was carried out after the treatment period using the negative geotaxis assay (Adedara et al. 2016). The surviving flies from control and the treated groups were separately immobilized in ice and transferred into a labeled sterilized tube (11 cm in length 3.5 cm in diameter). Following a 10 min of recovery, the flies were tapped at the bottom of the tube and the number of flies that crossed the 6 cm line within 30 s was recorded. Normally, flies without locomotor deficiency move very fast to the top, whereas those with motor defect are slow in movement and may remain near the bottom. The climbing scores denote the average percentage of flies that crossed the 6 cm line among the total number of flies per experiment. The results are expressed as percentage of flies that escaped beyond a minimum distance of 6 cm in 30 s during three independent experiments. Biochemical assays Determination of catalase activity Catalase activity in the homogenate samples was determined according to the method of Sinha et al. (1972). In brief, 0.1 mL of each tissue homogenate sample was reacted with 0.4 mL 2 M H2O2 in the presence of 1.0 mL 0.01 M phosphate buffer (pH 7.0). The reaction was stopped by the addition of 2.0 mL dichromate acetic acid. The absorbance of the reaction mixture was taken at 620 nm in a spectrophotometer. A standard curve was prepared by reacting 0.4 mol of 2 M H2O2 with 2 mL dichromate acetic acid in the presence of 1.0 mL 0.01 M sodium phosphate buffer (pH 7.0). The catalase activity was thereafter calculated and expressed as unit/mg protein, where 1 unit ¼ mmol H2O2 consumed/min. Determination of the total thiol content Determination of the level of total thiol content in tissue homogenate was done by the method of Ellman (1959). The reaction mixture was made up of 270 lL of 0.1 M potassium phosphate buffer (pH 7.4), 20 lL of homogenate, and 10 lL 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 (mmol.GSH/mg protein). Estimation of Glutathione-S-Transferase (GST) activity Glutathione-S-transferase activity was assayed according to the method of Habig and Jakoby (1981) with slight modifications (Abolaji et al. 2014) using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The reaction mixture consisted of 270 mL of a solution A (20 mL of 0.25 M potassium phosphate DRUG AND CHEMICAL TOXICOLOGY buffer, pH 7.0, 10.5 mL of distilled water, and 500 mL of 0.1 M GSH at 25  C), 20 mL of sample (1:5 dilution), and 10 mL of 25 mM CDNB. The reaction was analyzed for 5 min (30 s intervals) at 340 nm in a SpectraMax plate reader (Molecular Devices, CA, USA) and GST activity expressed as mmol/min/ mg protein 5 filters and injected into the chromatographic system (Kulkarni and Dhir 2010). Determination of total protein Total Protein content of fly homogenates were measured by the Coomassie blue method according to Bradford (1976) using bovine serum albumin (BSA) as standard. Determination of superoxide dismutase activity Superoxide dismutase (SOD) activity was determined by the method of Alia et al. (Alıa et al. 2003) , in which 0.05 mL of tissue homogenate was treated with 0.1 mL of 50 mM carbonate buffer (pH 10.2) and 0.05 mL of adrenaline (0.06 mg/ mL). The absorbance was read at 480 nm in spectrophotometer for 2 min at 15 sec intervals. SOD activity was expressed as mmol/min//mg protein Reactive oxygen species (ROS) level Reactive Oxygen Species level was performed by the method of (Hayashi et al. 2007). 50 mL of tissue homogenate and 1400 mL sodium acetate buffer were transferred to a cuvette. After that, 1000 mL of reagent mixture of n-n-diethyl-paraphenylenediamine (DEPPD). (6 mg/mL of DEPPD with 4.37 mM of ferrous sulfate dissolve in 0.1 M sodium acetate pH 4.8) was added at 37 C incubated for 5mins. The absorbance was measured at 505 nm using a spectrophotometer. ROS levels from the tissue were calculated from H2O2 standard and expressed as mmol H2O2 produced/mg protein. Acetylcholinesterase (AChE) activity assay Acetylcholinesterase (AChE) activity was assayed according to the method of Ellman as previously reported (Perry et al. 2000). The reaction mixture was made up of 180 lL of distilled water, 20 lL of 100 mM sodium phosphate buffer (pH 7.4), 30 lL of 8 mM DTNB, 15 lL of homogenate, and 30 lL of 8 mM acetylthiocholine iodide as substrate. Thereafter, reaction was monitored for 5 mins (30-second intervals) at 412 nm. The AChE activity was thereafter calculated and expressed as mmolAcSch/h/mg protein. Monoamine oxidase inhibitory assay The method described by McEwen (1965) was used to estimate the monoamine oxidase activity. The assay mixture consist of 400 mL of 0.1 M phosphate buffer (pH 7.4.), 1300 mL of distilled water, 100 mL of benzylamine hydrochloride and 200 mL of tissue homogenate. The assay mixture was incubated for 30 min at room temperature and then 1 mL of 10% perchloric acid was added and centrifuged at 1500 g for 10 min. The absorbance was read at 280 nm in a UV/Visible Spectrophotometer. The MAO activity was thereafter calculated and expressed as mmol/min/mg protein. Quantification of L-DOPA content in fly head Head samples were homogenized with 0.1 M perchloric acid and were centrifuged at 43,000 rpm for 15 min. The supernatant was separated and filtered through 0.25 mm nylon Data analysis The results of replicate readings were pooled and expressed as mean ± standard deviation (S.D). One-way Analysis of Variance (ANOVA) was used to analyze the results followed by Turkey’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). Results The HPLC characterization of water bitter leaf (WB) and scent leaf (SL) are presented in Tables 1 and 2. Characterization of SL leaf extract (Table 1) revealed the presence of 2, 6-dimethylpyrazine and sesbanimide on the other hand, for WB extract, (Table 2), N-propylamine, piperidine, vernomine, among others were found. The results of the DPPH and ABTS radical scavenging abilities of both alkaloid extracts of WB and SL are presented in Figure 1(a,b). The results revealed that both extracts scavenged DPPH radical in a dose-dependent manner; however, SL alkaloid extract had a significantly (p < 0.05) higher DPPH Table 1. HPLC Characterization of constituent alkaloids in Scent leaf extract. Compound 2,6-dimthylpyrazine Sesbanimide RT (min) Amount (mg/100 g) 9.69 18.77 349.83 279.20 Table 2. HPLC Characterization of constituent alkaloids in Water bitter leaf extract. Compound N-propylamine Augustamine Oxoassoanine Dihydro-oxo-demethoxyhaemanthamine Peperidine Bupahanidrine Piperine Ephedrine Vernomine Powelline Undulatine 1,9 Octadecenamide Lactucin 1,6-Hydroxybuphanidrine Acronycine Monocrotaline 1,6 Hydroxypowelline Lactucopicrin Crinamidine 1,1 beta, 2beta-Epoxyambelline 6-Hydroxyundulatine 1 Epoxy-3, 7-dimethoxycrinane-11-one Akuammidine Echitamine Mitraphylline RT (min) 13.29 14.93 15.04 15.50 16.04 16.46 17.23 17.86 18.13 18.67 18.76 19.17 19.52 20.60 21.26 21.33 21.82 22.69 24.03 24.42 24.79 25.62 26.95 27.43 27.48 Amount (mg/100 g) 693.11 2.39 0.46 0.37 270.81 0.13 9.81 28.51 506.26 1.56 1.10 2.57 57.54 7.70 0.25 2.72 0.57 25.26 3.58 1.83 2.25 34.75 4.17 3.14 65.73 6 A. O. ADEMILUYI ET AL. Figure 1. (A) DPPH radical scavenging ability; (B) ABTS radical scavenging ability; (C) Ferric Reducing Antioxidant Property (FRAP); (D) Iron chelating; (E) Hydroxyl radical (OH) scavenging; (F) Nitric oxide (NO) scavenging abilities of by alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL). Values represent mean ± standard deviation. Mean values with different letters are significantly different at p < 0.05. and ABTS radical scavenging abilities than WB alkaloid extract. The result of the ferric reducing antioxidant property (FRAP) of SL and WB extracts was determined, expressed as ascorbic acid equivalents and presented in Figure 1(c). The result revealed that SL had higher reducing power than WB. The Fe2þ chelating ability of WB and SL alkaloid extracts is presented in Figure 1(d). The result shows the ability of both alkaloid extracts to chelate Fe2þ in a dose dependent manner. The hydroxyl radical (
OH) scavenging ability of the alkaloid extracts (WB and SL) is presented in Figure 1(e). As shown in the figure, the alkaloid extracts scavenged
OH in concentration dependent manner; with SL alkaloid extract having a significantly (p < 0.05) higher
OH scavenging ability than WB alkaloid extract. Also, nitric oxide radical (NO䊉) scavenging ability of alkaloid extracts of WB and SL were assessed. Figure 1(f) revealed that both alkaloid extracts scavenged NO in a dose-dependent manner with SL alkaloid extract having a significantly (P < 0.05) higher scavenging ability than WB alkaloid extract. Incubation of fly homogenate with 250 mM Fe2SO4 resulted in a significant (p < 0.05) increase in homogenate’s thiobarbituric acid reactive substance (TBARS) content as shown in Figure 2(a). In another experiment, the ability of the alkaloid extracts to inhibit quinolinic acid (QA) induced lipid peroxidation was assessed and presented in Figure 2(b). However, the introduction of the extracts caused a significant dose-dependent decrease (p < 0.05) in TBARS contents. Nevertheless, SL had a higher inhibitory effect on TBARS production in the flies than WB extract. Furthermore, incubation of the fly homogenates in the presence of sodium nitroprusside (SNP) caused a significant (p < 0.05) increase in the homogenate’s TBARS content; however, the alkaloid extracts caused a significant (p < 0.05) decrease in the homogenate’s TBARS content in a dose-dependent manner as shown in Figure 2(c). Furthermore, the ability of the alkaloid extracts to inhibit acetylcholinesterase (AChE) activity in vitro was investigated. The result in Figure 2(d) shows that both alkaloid extracts inhibited acetylcholinesterase (AChE) in a dosedependent manner, however, SL alkaloid extract had a significantly (p < 0.05) higher inhibitory effect on acetylcholinesterase activity than WB alkaloid extract. A prelimenary feeding experiment was first conducted to monitor the survival rate of flies in order to determine the concentration of alkaloid extracts and duration of exposure of alkaloid extracts to be used in the experiment. After seven (7) days exposure of the flies to the diet, the survival rates of flies were determined across the various alkaloid extracts concentration. The data were analyzed and presented as percentage fly survival, as shown in Figure 3(a,b). From the result gotten, no significant fly mortality were observed up till 200 mg/g, therefore 20 and 200 mg/g were selected for further bioassays. The effect of SL and WB extracts on the locomotor performance of D. melanogaster treated with Mn is shown in Figure 4. There was significant amelioration (p < 0.05) in the reduced locomotor activity in Mn treated flies fed diets containing the extracts of SL and WB when compared to Mntreated flies alone. The results of the endogenous antioxidant enzymes (catalase, GST and SOD) are shown in Figure 5(a–c) respectively. The result revealed that in Mn-treated flies fed diets containing SL and WB extracts, the reduced enzyme activities were significantly mitigated (p < 0.05) compared to Mn-treated fly group. Also, as presented in Figure 5(d and e), Mn-treated flies exhibited significantly (p < 0.05) reduced total thiol content and elevated ROS levels, which were DRUG AND CHEMICAL TOXICOLOGY 7 Figure 2. Inhibition of (A) FeSO4–induced (B) Quinolinic acid–induced; (C) Sodium nitropusside–induced lipid peroxidation in Drosophila melanogaster by alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL). (D) In vitro acetylcholinesterase inhibitory effect of alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL) in Drosophila melanogaster. Values represent mean ± standard deviation. #mean values are significantly different from basal group; ,,, mean values are significantly different from induced group at p < 0.05, 0.01, 0.001 and 0.0001 respectively. Figure 3. Effect of alkaloid extract of (A) Water bitter leaf (WB) and (B) Scent leaf (SL) on day 7 survival rate in Drosophila melanogaster treated with MnCl2. Bars represent mean ± standard deviation. a,b: mean values are significantly different in relation to the control group (p < 0.05). significantly improved in (p < 0.05) in Mn-treated flies fed diets containing SL and WB extracts. The modulatory effect of SL and WB alkaloid extracts on AChE and MAO activities in vivo is shown in Figure 6(a,b). Mntreated flies exhibited significantly higher AChE and MAO activities, which were significantly ameliorated in Mn-treated flies fed diets containing SL and WB extracts. Also, L-DOPA level was found significantly elevated (p < 0.05) in Mn-treated fly heads (Figure 7) which was ameliorated significantly (p < 0.05) in Mn-treated flies fed diets containing both extracts. 8 A. O. ADEMILUYI ET AL. Discussion Mn exposure is known to be associated with a number of adverse neurological effects (Siokas et al. 2021). A number of studies have demonstrated the significant association between Mn exposure and neurodegenerative diseases especially PD (Bowman et al. 2011, Du et al. 2018). In response to Figure 4. Effect of alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL) on locomotor performance (climbing ability) in Drosophila melanogaster treated with MnCl2. Bars represent mean ± standard deviation. #mean values are significantly different in relation to the control group (p < 0.05; , treatment groups are significantly different from Mn-group at p < 0.05 and p < 0.01 respectively. the effects of Mn exposure, the mechanisms of Mn-induced neurotoxicity have also been studied. These mechanisms include oxidative stress (Adedara et al. 2016), neuro-inflammation, impaired calcium homeostasis (Ijomone et al. 2019), dysregulation of mitochondrial function and redox homeostasis (Martinez-Finley et al. 2013), altered proteostasis (Harischandra et al. 2019), and altered neurotransmitter metabolism (Soares et al. 2020), among others. A considerable amount of natural products, most especially from plant extracts, have been reported to be used in traditional medicine for neuroprotective, memory enhancing and anti-aging purposes. Scent leaf (Ocimum gratissimum) and Water bitter leaf (Struchium sparganophora) have been preiously reported to be rich in alkaloid, tannins, phytates, flavonoids and oligosaccharides (Ijeh et al. 2004). However, there is still dearth of adequate scientific information on the biochemical mechanisms by which these plants elicit their neuroprotective This study showed that both extracts (SL and WB) exhibited AChE inhibitory effect (in vitro) in a concentration dependent manner and also progressively ameliorated the increase in the activity of AChE induced by Mn (in vivo) in Drosophila. AChE catalyzes the hydrolysis of acetylcholine to acetate and choline, bringing about the negative regulation of the cholinergic system neuronal functions (Valko et al. 2007). Acetylcholine as a neurotransmitter is essential to regulate cognitive function, learning/memory, motor function, and locomotion (Peres et al. 2016). Neurotoxic levels of Mn have been reported to bring about an impairment in the cholinergic system which is strongly associated with Mninduced impairments of motor function, locomotion, and Figure 5. Effect of alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL) on (A) catalase activity; (B) GST activity; (C) SOD activity; and (D) Total Thiol content in Drosophila melanogaster treated with MnCl2. Bars represent mean ± standard deviation. #mean values are significantly different in relation to the control group (p < 0.05; , treatment groups are significantly different from Mn-group at p < 0.05 and p < 0.01 respectively. DRUG AND CHEMICAL TOXICOLOGY 9 Figure 6. Effect of alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL) on (A) AChE activity; and (B) MAO activity in Drosophila melanogaster treated with MnCl2. Bars represent mean ± standard deviation. #mean values are significantly different in relation to the control group (p < 0.05; , treatment groups are significantly different from Mn-group at p < 0.05 and p < 0.01 respectively. Figure 7. Effect of alkaloid extracts of Water bitter leaf (WB) and Scent leaf (SL) L-DOPA content in head of Drosophila melanogaster treated with MnCl2. Bars represent mean ± standard deviation. #Mean values are significantly different from control group at p < 0.05. Mean values are significantly different from MnCl2 group at p < 0.05. cognitive dysfunction (Peres et al. 2016). Therefore, as demonstrated in this study, an increase in the activity of AChE after seven days exposure of the flies to Mn initiated a significant decrease in their climbing abilities. The impairment observed in the climbing ability of the flies could be due to a reduction in the bioavailability of acetylcholine for cholinergic neurotransmission at the neuromuscular junctions (Adedara et al. 2016). This condition is predominantly a major risk factor in the development and progression of dementia (Valko et al. 2007). The ability of SL and WB alkaloid extract to modulate the effect of Mn-induced elevated levels of AChE activity in flies, as well as bringing about an increase in their climbing ability could suggest one of the mechanisms of action behind the protective effect of the extract against Mn-induced toxicity. In addition, plant derived alkaloids such as berberine, caffeine, evodiamine, isorhynchophylline, tetramethylpyrazine, and trigonelline have also been reported to show neuroprotective properties in various experimental models and are often associated with their anti-cholinesterase, antioxidant, and anti-inflammatory properties (Kulkarni and Dhir 2010, Zhou et al. 2012, Zhou and Zhou 2012, Huang et al. 2014, Kim et al. 2014, Oboh et al. 2014). Therefore, SL and WB alkaloid extracts also show possible promise as a source of potent neuroprotective alkaloids. Generally, plant alkaloids are being advertised as drug substitutes for the management of neurodegenerative diseases owing to a number of their therapeutic mechanisms. Results from this study revealed that SL and WB alkaloid extracts significantly reversed the elevation levels of MAO activity in Mn-induced D melanogaster in a dose-dependent manner. Impairment of the monoaminergic system of neurotransmission resulting from elevated MAO activity contributes significantly to the pathogenesis and progression of several neurodegenerative diseases including AD and PD (Nwanna et al. 2016). Elevation levels of MAO activity results in the rapid breakdown of both inhibitory and excitatory monoamine neurotransmitters such as adrenaline, dopamine, and serotonin, which are crucial in anxiety and mood disorders (Thomas 2000, Oboh et al. 2016). In the clinical management of neurodegenerative diseases, especially AD and PD, MAO inhibitors are often useful therapies, especially as antidepressants (Thomas 2000, Oboh et al. 2016). Furthermore, several experimental studies have revealed that plant extracts such as phenolic rich and alkaloid rich extracts showed significant MAO inhibitory effects in both in vitro (Ademiluyi et al. 2016, Nwanna et al. 2016) and in vivo (Yu et al. 2002) studies. Therefore, the ability of the alkaloid extracts from SL and WB leaves to reverse elevation levels in MAO activities in the flies treated with Mn could therefore be attributed to the constituent bioactive properties present in the vegetable. As also observed (in vitro), both alkaloid extracts used in this study were able to inhibit MAO in a dose dependent manner with SL having the highest level of inhibitory effect than WB. The elevated MAO activity could be a pointer to the elevated 10 A. O. ADEMILUYI ET AL. L-DOPA level in the head of flies treated with Mn. L-DOPA is the precursor of dopamine and its elevation could be as a result of its excessive breakdown due to elevated MAO activity. Oxidative stress is one of the major risk factors for the pathogenesis and progression of several neurodegenerative diseases, through mechanisms such as free radical-induced mitochondrial dysfunction and neuronal cell death (Lin and Beal 2006, Bhat et al. 2015). A condition of oxidative stress occurs when more free radicals exist than all the antioxidants present can neutralize. Elevation in MAO activities have been linked to free radical-induced oxidative stress. This is because pro-oxidants such as hydrogen peroxide are by-product of MAO hydrolysis of biogenic amines (Cadenas and Davies 2000). In the presence of Fe2þ, the H2O2 produced participates in Fenton reaction to form a highly reactive hydroxyl (O H) radical which causes the oxidation of biomolecules including lipids, proteins, and DNA (Yamaguchi et al. 2000). Earlier findings have imbibed oxidative stress as a result of Mn-induced neurotoxicity. Oxidative stress occurs as a result of ROS overloads which cannot be effectively counteracted by the antioxidant defense mechanisms of the body (Valko et al. 2007). Results from this study showed that flies exposed to Mn exhibited elevation in ROS level which could indicate a state of oxidative stress. This is in agreement with earlier report on elevation of ROS level following Mn-induced toxicity in Drosophila (Adedara et al. 2016). However, co-treatment with SL and WB alkaloid extract significantly ameliorated ROS level in the flies. This observation could be corroborated by the in vitro antioxidant properties of SL and WB alkaloid extract as typified by their free radical scavenging abilities, metal chelating ability, reducing properties and inhibition of lipid peroxidation reaction. This study revealed that there was a significant increase in ROS (as quantified as H2O2 equivalent) content in Mn treated flies. However, the dietary inclusions of SL and WB leaves alkaloid extracts significantly prevented the elevation in ROS contents. One of the justifications for this observation could be as a result of the ability of dietary inclusions of SL and WB leaves to significantly reduce MAO activities in Mn treated flies as earlier discussed. Secondly, the antioxidant properties of SL and WB leaves could have also contributed to their abilities to reverse elevation in ROS content in Mn assaulted flies. Previous studies (Oboh et al. 2014, Ademiluyi et al. 2015) have shown that green leafy vegetables that are rich in antioxidant phytochemicals are capable of inhibiting lipid peroxidation chain reactions. Impairments in endogenous antioxidant defense systems such as catalase, glutathione (GSH), superoxide dismutase (SOD), and glutathione-S-transferase (GST) are possible consequences of oxidative stress. To further support the antioxidant properties of SL and WB extracts, it was also observed that there was increase in activities of catalase and GST in flies fed diets containing the extracts alone. Conclusion The result showed that dietary inclusion of MnCl2, induced toxicity in Drosophila melanogaster due to the changes in the antioxidant status, enzyme activities and neural L-DOPA level. Interestingly, the result of this study revealed that SL and WB alkaloid extracts improved the antioxidant status of the flies and mitigate against impaired enzyme activates resulting from Mn-toxicity This study therefore, suggests that these vegetables as sources of nutraceuticals which could mitigate manganese-induced toxicity in flies and associated neurotoxicity. Disclosure statement No potential conflict of interest was reported by the author(s). Funding This research was funded by The World Academy of Sciences (TWAS) Grant No: 16–500 RG/CHE/AF/AC_G – FR3240293300. ORCID Adedayo Oluwaseun Ademiluyi http://orcid.org/0000-0001-8325-1304 Opeyemi Babatunde Ogunsuyi http://orcid.org/0000-0003-3075-1086 Olayemi Philemon Aro http://orcid.org/0000-0001-5167-9779 References Abdullahi, M., Muhammed, G., and Abdulkadir, N. U., 2003. Medicinal and Economic plants of Nupeland. 1st ed. Nigeria: Jube Evans publisher. Abolaji, O.A., et al., 2014. 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