Neuroprotective properties of UV-C treated Bacopa floribunda leaves: in vitro and in vivo studies

Abstract

This study investigated the effect of UV-C irradiation on antioxidant, cholinergic and neuroprotective properties of Bacopa floribunda leaves in scopolamine-induced rats. In vitro study was done by the determination of phenolics, flavonoid, total antioxidant capacity, 2, 2-diphenyl-1-picrylhydrazyl scavenging activity, inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Loss of memory was induced in Wistar rats pre-treated with extracts of Bacopa floribunda using scopolamine (2 mg/kg) administered intraperitoneally. Behavioural studies using elevated plus maze (EPM) and biochemical analysis on the were carried out. Exposure to UV-C radiation increased the phenolics, flavonoid, antioxidant and inhibitory properties against AChE and BChE of the Bacopa floribunda leaves. The administration of scopolamine caused significant increase in the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, but decrease in the activities of antioxidant enzymes and enhanced generation of malondialdehyde and nitric oxide in vivo. The loss of memory and Biochemical analysis in scopolamine-induced rats was ameliorated by treatment with UV-C treated Bacopa floribunda (TBF) extracts.

Treatment with TBF improved memory retention and ameliorated oxidative damage in hippocampus of scopolamine treated rats.

KEYWORDS:

• Bacopa floribunda
• elevate plus maze
• scopolamine
• lipid peroxidation
• acetylcholinesterase
• butyrylcholinesterase

1. Introduction

Recent advances in the technology of ultraviolet (UV) irradiation have demonstrated that UV treatment holds considerable promise for enhancing the secondary metabolites of fruits and vegetables [1]. The use of low doses of ultraviolet C (UV-C) postharvest treatment to enhance bioactive compounds with antioxidant potentials such as phenolics, tetraterpenoids and flavonoids has been well established in vegetables [2,3]. Apart from the enhancement of secondary metabolites, it is also used to preserve or extends the shelf life of fruits and vegetables due to its ability to inactivate bacteria and viruses. UV-C radiation is an abiotic stressor that when used cannot cause ecological problems and are not harmful to humans; the equipment is not expensive and is easy to use [4]. UV-C radiation has been widely used for surface sanitation of fruits, delaying of softening and senescence of fruits and in treatment of water [5].

Neurodegenerative diseases are group of disease that is characterized by loss of neuron and memory retention in the brain and they have been reported to affect more than 50 million people globally [6]. Due to increased life expectancy in human, age-related neurodegenerative diseases are significant contributors to the number [7]. Oxidative stress and enhanced activities of cholinergic enzymes have been implicated in the progression of neurodegenerative diseases. Deleterious effect of oxidative stress includes activation of glia cell, misfolding of protein and loss of mitochondrial functions while cholinergic enzyme dysfunction causes a decline in acetylcholine and butyrylcholine levels thereby contributes to pathogenesis of neurodegenerative diseases [8]. Presently, there are concern about safety, availability and cost of most orthodox drugs available for the treatment of neurodegenerative diseases. Due to these and misconception that traditional medicines are “safer”, people tend to use medicinal plants for their treatment [9]. One of the prominent plants used locally in Nigeria for the treatment of neurodegenerative disease is Bacopa floribunda.

Bacopa floribunda (B. floribunda) belongs to Scrophuliaceae family with 146 aquatic herbal species. Bacopa floribunda leaf has been in use as herb for a very long time by the Yoruba people of Nigeria for the treatment and management of cognitive malfunctioning and for enhancing memory [10]. Ethnobotanical survey conducted by Olatunji et al. [11] on plants used as memory enhancer in Nigeria revealed that Bacopa floribunda had the highest frequency. B. floribunda has proven to inhibit enzymes like β-secretase and cholinesterases linked to Alzheimer's disease (AD) [10,12]. It has also been reported to ameliorate scopolamine-induced memory loss in rodents and used to manage beta-amyloid (1-42) induced AD in Wistar rats [13–15]. The mean lethal dose (above 5000 mg/kg) and safety of oral acute administration of non-irradiated and irradiated leaves of B. floribunda in experimental rats have been reported [16].

With the promising results obtained with the use of UV-C radiation on the enhancement of bioactive components and biological activities of some medicinal plants. There is call to extend the technology to other medicinal plants. Therefore, this study was designed to evaluate possible effects of postharvest UV-C irradiation on the neuroprotective properties of Bacopa floribunda leaves on scopolamine-induced amnesia and oxidative damage in rats.

2. Materials and methods

2.1. Chemical reagents

Scopolamine, acetylthiocholine iodide, butyrylthiocholine iodide, 5’5’ dithiobis 2-nitrobenzoic acid (DTNB), epinephrine, glutathione, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) were purchased from AKScientific, USA. Acetylcholinesterase and butyrylcholinesterase were purchased from Sigma Aldrich, USA. All other chemicals were in high analytical grade.

2.2. Sample collection

Freshly harvested leaves of B. floribunda were collected in the month of March 2022, from the bank of river Oluwa, Okitipupa, Ondo State, Nigeria. It was identified and authenticated (OAUSTECH/H/720) at the Biological Sciences Department, OAUSTECH. The leaves were sorted and rinsed in a running water to remove dirt. The selected fresh leaves were categorized into two: one part was irradiated for 20 min and the other part (control) was not irradiated.

2.3. Radiation treatment

Radiation of the fresh leaves was carried out in the radiation chamber at room temperature with an average of 2.217 J/m² UV-C radiation doses and 210 nm wavelength. Irradiation intensity was measured using a photo-radiometer [2].

2.4. Preparation of extracts

The treated and untreated leaves were air-dried for 28 days and ground into powdery form. One hundred and fifty gram (150 g) each of UV-C treated B. floribunda (TBF) and untreated B. floribunda (UTBF) were soaked in 1.5 L of distilled water separately for 48 h and the mixtures were shaken intermittently. After 48 h, the slurry was filtered and the filtrates were concentrated on rotary evaporator at 40°C.

2.5. HPLC analysis of phenolics

HPLC–MS analysis was done under gradient conditions at room temperature according to the method [17]. The obtained UV spectra of the chromatographic peaks and retention time (Rt) was confirmed by comparing with reference standards.

2.6. In vitro biochemical analysis

2.6.1. Bioactive compounds determination

Total phenolic determination was done using the method of Kim et al. [18] while total flavonoids determination was carried out with the method of Park et al. [19].

2.6.2. Antioxidant assays

To determine the reducing power, Oyaizu method was used [20]. ABTS scavenging ability was done according to Re et al. method [21]. For DPPH radical scavenging activity, Gyamfi et al. method [22] was used and Panda et al. method were adopted for the determination of nitric oxide scavenging ability [23].

2.6.3. Cholinesterase (AChE and BChE) activity

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were determined using Ellman method as described by Perry et al. [24]. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates for AChE and BChE activities, respectively.

2.6.4. Lipid peroxidation in brain homogenate (ex vivo)

Two rats were anaesthetized with sodium pentobarbitone and sacrificed; whole brain removed, weighed on ice and centrifuged (at 5000 rpm for 10 min) to obtain the low speed supernatant (SI) used for the Lipid peroxidation assay. Lipid peroxidation was induced using 250 µm ferrous sulphate and assay was done according to the method described by Adetuyi et al. [25]. Absorbance was taken at 532 nm and malondialdehyde (MDA) was calculated from MDA standard curve [25].

2.7. In vivo study

2.7.1. Experimental animals

Healthy Wistar albino rats weighing 125–140 g were obtained from the animal house of the Department of Physiology, College of Medicine, University of Ibadan. They were fed with standard pellet diet and water was given ad libitum. They were kept under a constant 12-h light and dark cycle. The animals were acclimatized for 2 weeks. Forty-two animals were divided into 7 groups of 6 animals each. Group 1: control distilled water; Group 2: Scopolamine 2 mg/kg (Scop); Group 3: Scopolamine + TBF 100 mg/kg (Scop + TBF1); Group 4: Scopolamine + TBF 200 mg/kg (Scop + TBF11); Group 5: Scopolamine + UTBF 100 mg/kg (Scop + UTBF1); Group 6: Scopolamine + UTBF 200 mg/kg (Scop + UTBF11) and Group 7: Scopolamine + donepezil 2 mg/kg (Scop + DPZ).

The extracts were administered early in the morning (7–8 am) every day for 14 days, after the 14th day the animals were administered scopolamine (2 mg/kg) and behavioural assessment was conducted 30 min later. Twenty-four hours after the completion of behavioural assessment, animals were sacrificed and hippocampus section of the brain (placed on ice) was carefully removed for biochemical analysis.

2.7.2. Behavioural assessment

Behavioural assessment was conducted using elevated plus maze (EPM) apparatus as described by Ojo and Mouzon [26]. The apparatus consists of two open arms with each arm having a dimension of 50 cm by 10 cm (length to width) and two covered arms, each having similar dimension with open arm and walls of 40 cm, forming the close arm. Each arm is extended from a central platform by 5 cm and they are above the floor with an elevated height of 50 cm.

Prior the behavioural experiments, rats from each group were habituated to the maze for 10 min by allowing free exploration across the EPM arms. Thereafter, animals in group 2–7 were administered 2 mg scopolamine/kg body weight of rats intraperitoneally and allowed to stand 30 min before the commencement of the EPM test. After the treatment, each animal was placed singly at the centre of the EPM apparatus facing an open arm and given access to freely explore the maze for 5 min. Parameters that were recorded during experiment include transfer latency time (TLT), central zone time (CZT), central zone count (CZC), close arm entry count (CAEC), open arm entry count (OAEC), open arm time (OAT), close arm time (CLAT) and rearing.

2.7.3. Preparation of homogenate

Excised hippocampus section of the brain was homogenized in cold 0.1 M phosphate buffer (pH = 7.4) and centrifuged at 5000×g for 10 min at 4°C. The supernatant obtained was used for the determination of activities of cholinergic enzymes, oxidative stress markers and antioxidant status. The protein content of the supernatant was assayed using the method of Bradford [27].

2.7.4. Determination of activities of cholinergic enzymes

Acetylcholinesterase and butyrylcholinesterase activities of the brain homogenate were determined using the method of Ellman et al. [28].

2.7.5. Determination of markers of oxidative stress

Lipid peroxidation was evaluated using the method of Farombi et al. [29]. Nitric oxide was determined using Griess reagent according to the method of Green et al. [30]. Reduced glutathione (GSH) level was determined by measuring the procedure of Jollow et al. [31]. Glutathione peroxidase (GPx) activity was measured using the method of Rotruck et al. [32]. Superoxide dismutase (SOD) activity was determined according to the method of Misra and Fridovich [33]. Catalase (CAT) activity was determined according to the method of Clairborne [34].

2.7.6. Statistical analysis

Results were expressed as mean ± standard error and data obtained were analysed by one-way analysis of variance followed by least square difference post hoc test using Graph Pad Prism 6 software. The level of significance was considered at P < 0.05.

3. Results

3.1. Effect of UV-C treatment on the phytochemical, antioxidant and inhibitory properties of B. floribunda

The results in Table 1 and Figure 1(A and B) revealed that UV-C irradiation treatment caused a significant (P ˂ 0.05) increase in the content of gallic acid (0.13–0.16 mg/g), caffeic acid (0.02–0.10 mg/g), quercetin (0.15–0.19 mg/g), isoquercitirin (0.13–0.17mg/g) and kaempferol (0.49–0.58 mg/g). Irradiation has no significant (P ˂ 0.05) influence on the content of chlorogenic, rutin, apigenin, quercitirin, isoquercitirin and luteolin of B. floribunda.

Figure 1. Representative of HPLC profile of untreated (A) and UV-C treated (B) B. floribunda leaves.

Table 1. HPLC phenolic composition of UV-C treated and untreated B. floribunda leaves in mg/g.

Phytochemical	Untreated	UV-C treated
Chlorogenic acid	0.66 a	0.65 a
Gallic acid	0.13 b	0.16 a
Caffeic acid	0.02 b	0.10 a
Quercetin	0.15 b	0.19 a
Rutin	0.01 a	0.01 a
Apigenin	0.03 a	0.03 a
Quercitirin	0.01 a	0.01 a
Isoquercitirin	0.13 b	0.17 a
Kaempferol	0.49 b	0.58 a
Luteolin	0.10 a	0.09 a

Values = mean triplicate readings. Values with the same letter on the same row are not significantly (P < 0.05) different.

Total phenolics, total flavonoids and reducing power of B. floribunda leaves significantly increased as a result of UV-C irradiation treatment (Table 2), total phenolics from 23.29 to 30.20 mg GAE/g, total flavonoids from 11.48 to 14.46 mg QE/g and reducing power from 21.27 to 23.98 mg AAE/g. There was a non-significant (P ˂ 0.05) increase in ABTS (mmol TEAC/g) properties of B. floribunda as a result of UV-C irradiation treatment.

Table 2. Bioactive compound, antioxidant and IC₅₀ values of UV-C treated and untreated B. floribunda leaf.

Bioactive compound and antioxidant capacity	Untreated	UV-C treated
Total phenol (mg GAE/g)	23.29 b	30.20 a
Total flavonoid (mg QE/g)	11.48 b	14.46 a
Non flavonoid (mg QE/g)	11.81 b	15.74 a
Reducing power (mg AAE/g)	21.73 b	23.98a
ABTS (mmol TEAC/g)	1.03 a	1.18 a
MDA	0.50 a	0.45 b
IC50 values (µg/ml)
DPPH	599.35 a	518.55 b
NO	387.56 a	328.20 b
AChE	19.11 a	16.69 b
BChE	26.74a	20.54 b

Values = mean of three determinations. Values with the same letter on the same row are not significantly (P < 0.05).

The result (Figure 2A and B) showed that B. floribunda (UV-C treated and untreated) scavenged DPPH and NO radicals as the concentration increases. The scavenging ability of UV-C treated B. floribunda leaves extract (IC₅₀ = 518.55 μg/ml DPPH, IC₅₀ = 328.20 μg/ml NO) is significantly higher than untreated sample (IC₅₀ =599.35 μg/ml DPPH, IC₅₀ = 387.56 μg/ml NO) (Table 2).

Figure 2. DPPH (A) and NO (B) scavenging abilities of untreated and UV-C treated B. floribunda leaves extract. Values = mean of three determinations.

Cholinesterase enzyme (AChE and BChE) activities (Figures 3A and B) were significantly inhibited in a dose-dependent manner by the extract (UV-C treated and untreated B. floribunda) considering the IC₅₀ values (Table 2). The UV-C treated sample has lower IC_50.

Figure 3. AChE (A) and BChE (B) inhibition ability and inhibition of Fe²⁺-induced MDA production in rat brain (C) of UV-C treated B. floribunda leave extract. Values = mean of three determinations.

Protection capacity of B. floribunda leaves extracts against Fe²⁺ induced lipid peroxidation in isolated rat brain was investigated and expressed in Figure 3(C). The MDA content of the brain homogenates increased to 160% when incubated with 250 μM FeSO₄ solution. The addition of B. floribunda (UV-C treated and untreated) leaves extracts caused a decrease in the MDA content as the concentration of the added extract increases (Figure 3C). Table 2 showed the IC₅₀ value of UV-C treated B. floribunda (0.45 mg/ml) having a significantly (P < 0.05) higher inhibitions than untreated B. floribunda (0.50 mg/ml) (Table 2).

3.2. Effect of UV-C treated B. floribunda on the memory function of scopolamine-induced amnesia rats

The result of transfer latency time and central zone time following the EPM task is presented in Figure 4. Relative to control and group that was administered with scopolamine, groups that received UV-C treated (TBF) and untreated (UTBF) B. floribunda demonstrated statistical (P < 0.05) decline in transfer latency time. Only group that received high doses (200 mg/kg) of TBF and UTBF as well as donepezil group showed significant increase in central zone time compared to control and scopolamine group.

Figure 4. Behavioural index of Scopolamine-induced amnesic rats administered untreated and UV-C treated B. floribunda leaves extract. Scop = scopolamine, TBFI = UV-C treated B. floribunda I (100 mg/kg), TBFII = UV-C treated B. floribunda II (200 mg/kg), UTBFI = untreated B. floribunda I (100 mg/kg), UTBFII = untreated B. floribunda II (200 mg/kg) and DPZ = Donepezil.

The central zone count (CZC), open arm entry count (OAEC) and close arm entry count (CAEC) of animals in the different experimental group are presented in Figure 4. There was similar trend in central zone entry count and open arm entry count of animals that received donepezil and varying dose of TBF and UBTF with only animals treated with 200 mg/kg dose showing significant (P < 0.05) increment compared to the control and group administered with scopolamine. There is a concentration dependent decrease in CAEC of rat administered with varying dose of TBF and UTBF with only TBFII showing significant decrease (P < 0.05) in close arm entry count compared to group treated with scopolamine.

The result of percentage time spent in open arm and close arm are presented in Figure 4. Only rats that received the highest dose of treated B. floribunda spent more time in open arms as demonstrated by the significant increase (P < 0.05) relative to scopolamine group. Besides the groups that were administered 200 mg/kg UTBF that showed significant decrease in percentage time spent in close arm relative to scopolamine and control group, all other treated groups including donepezil were insignificant compared to the control.

The number of rearing (tested animals stand on their hind legs in the enclose arms) is presented in Figure 4. Animals treated with only scopolamine demonstrated significant increase (P < 0.05) in number of rearing compared to control, donepezil (standard drug), BTF and UBTF treated rats. However, only group treated with 200 mg/kg treated B. floribunda showed a more pronounced effect with indistinguishable difference relative to the control.

3.3. Effect of UV-C treated B. floribunda on the AChE and BChE activities of scopolamine-induced amnesia rats

Figure 5 presented the effect of pre-treatment with TBF and UTBF on AChE and BChE activities on the hippocampus of scopolamine-induced amnesia in rats. The induction with scopolamine led to increase in AChE and BChE activities in the rats. These activities were, however, decreased in pre-treated rats administered UV-C treated (TBF) and untreated Bacopa floribunda (UTBF) leaves extracts. The decrease in pre-treated rats is significant (p < 0.05) at the two doses used and corresponds with the increase in dosage. There was also a significant difference when compared rats administered UV-C treated and untreated B. floribunda at the dose of 100 mg/kg. The same trend was observed for BChE activity in amnesia rats upon treatment with the samples.

Figure 5. Acetylcholinesterase and butyrylcholinesterase activities of scopolamine-induced amnesic rats administered untreated and UV-C treated B. floribunda leaves extract. Scop = scopolamine, TBFI = UV-C treated Bacopa floribunda I (100 mg/kg), TBFII = UV-C treated Bacopa floribunda II (200 mg/kg), UTBFI = untreated B. floribunda I (100 mg/kg), UTBFII = untreated Bacopa floribunda II (200 mg/kg) and DPZ = Donepezil.

3.4. Effect of UV-C treated B. floribunda on the oxidative stress and antioxidant activities of scopolamine-induced amnesia rats

Figure 6 presented the level of MDA, NO and GSH in the hippocampus of scopolamine-induced amnesia in rats. The levels of MDA and NO were significantly increased in scopolamine treated rats. The increase was significantly (p < 0.05) lowered in rats pre-treated with TBF, UTBF and donepezil. There was no significant (p < 0.05) difference in MDA level of those treated with TBF and UTBF but there was significant (p < 0.05) difference in NO level. Unlike MDA and NO, the level of GSH was significantly decreased in scopolamine treated rats. Pre-treatment with the extracts (TBF and UTBF) was able to mitigate the reduction significantly (p < 0.05). There was no significant difference (p < 0.05) in the GSH level of rats pre-treated with TBF and UTBF.

Figure 6. Lipid peroxidation (LPO), nitric oxide (NO) and glutathione (GSH) contents of scopolamine-induced amnesic rats administered untreated and UV-C treated B. floribunda leaves extract. Scop = scopolamine, TBFI = UV-C treated B. floribunda I (100 mg/kg), TBFII = UV-C treated B. floribunda II (200 mg/kg), UTBFI = untreated B. floribunda I (100 mg/kg), UTBFII = untreated B. floribunda II (200 mg/kg) and DPZ = Donepezil.

Figure 7 presented the activities of SOD, CAT and GPx in the hippocampus of scopolamine-induced amnesia in rats. There was significant (p < 0.05) decrease in the activities of these enzymes upon administration of scopolamine. Rats pre-treated with TBF, UTBF and donepezil exhibited significantly (p < 0.05) higher activities than those that were not treated. When compared between TBF and UTBF, there was significant difference in the activity of SOD of rats pre-treated at 100 mg/kg.

Figure 7. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities of scopolamine-induced amnesic rats administered untreated and UV-C treated B. floribunda leaves extract. Scop = scopolamine, TBFI = UV-C treated B. floribunda I (100 mg/kg), TBFII = UV-C treated B. floribunda II (200 mg/kg), UTBFI = untreated B. floribunda I (100 mg/kg), UTBFII = untreated B. floribunda II (200 mg/kg) and DPZ = Donepezil.

4. Discussion

The phenolics identified in B. floribunda have diverse biological activities. Gallic acid, caffeic acid, quercetin, isoquercitirin and kaempferol were positively affected by UV-C irradiation. Yildirim [1] in the study of Echium orientale observed that UV irradiation caused an increase in quercetin, chlorogenic acid and rosmarinic acid content [1]. The quantified phenolics in this study have been acknowledged to be potential inhibitors of lipid peroxidation, nutraceutical and antioxidants [17].

Phenolics in plants protect the body cells against free radicals and neurodegenerative diseases [35]. The increase in total flavonoid and total phenol of B. floribunda leave due to UV-C irradiation could be as a result of the enhancement of the activities of the enzymes phenylalanine ammonia lyase (PAL) and peroxidase responsible for the biosynthesis of phenolic compounds in tissues of plants to combat the produced reactive oxygen species (ROS) due to UV-C irradiation [36].

The effect of UV-C irradiation on the in vitro antioxidant capacity of B. floribunda leave extracts was assessed using reducing power, ABTS, DPPH and NO radical scavenging abilities because these antioxidant assays have their advantages and disadvantages. UV-C irradiation significantly affects the reducing power, DPPH and NO radical scavenging abilities of B. floribunda in the positive but the effect was not significant on ABTS assay. It has been reported by Perkins et al. and Adetuyi et al. that UV-C irradiation increased the antioxidant abilities of Vaccinium corymbosum and Clerodendrum volubile extracts respectively [2,37]. The increase in antioxidant capacity may be attributed to the synthesis of phenols, flavonoids or non-phenolic compounds such as enzymes [36]. There is positive relationship between phenolics of medicinal plants and its antioxidant capacity [38]. Phenolics and flavonoids have the ability to donate electrons or hydrogen to free radicals in other to stabilize them [2].

The symptoms of Alzheimer’s disease (AD) may be reversed temporarily provided there is increase in brain neurotransmitter acetylcholine concentrations which enhance communication between nerve cells [39]. UV-C irradiation increased the ability of B. floribunda leave extract to inhibit AChE and BChE enzymes. This observation can be related to the increase in phenolics and flavonoids content of UV-C treated sample. Medicinal plants with higher phenolic content have been observed to inhibit AChE activity in vitro and when AChE is inhibited, it becomes difficult for acetylcholine to be degraded in the brain [17].

One of the effects of oxidative stress is that free radicals initiate lipid peroxidation through polyunsaturated fatty acids (PUFA) oxidation in biomembranes which is catalysed by Fe²⁺ [40]. The extracts of UV-C treated B. floribunda showed a higher inhibition against lipid peroxidation in the brain. The higher inhibition ability of UV-C treated B. floribunda could be due to its high total phenols and flavonoids. Phenolics in foods react with Fe²⁺ forming structural complexes, thereby chelating Fe²⁺ from starting the lipid peroxidation reaction [40]. Plant phenolic extracts have been shown to have the capacity to inhibit lipid peroxidation induced by Fe²⁺ [41].

Neurodegenerative diseases like Parkinson and Alzheimer, which are associated with loss of memory and cognitive function, are becoming prevalent these days [42]. They are managed and treated using different medicinal herbs because the orthodox drugs have failed to provide required efficacy and safety over time. Also, the inability to get the drugs when required coupled with their high cost have promoted the use of easily-available medicinal plants with marginal toxicity to treat the diseases [43]. Many experimental evidences have shown that ultraviolet-C radiation enhances phytochemicals and subsequently biological functions of plants [2,44]. Bacopa floribunda, one of the prominent herbs used to treat neurodegenerative diseases as memory enhancer is usually prepared as decoction in folk medicine. Scopolamine, a muscarinic cholinergic antagonist, is a well-documented model used to induce loss of memory and cognitive functions in experimental rodents [45]. The significant increase in transfer latency time obtained for scopolamine group could be as a result of cognitive impairment and psychological disturbance hence the longer time taken to make decision on action to take. Corroborating with the study of Yadang et al., on neuroprotective effects of Carissa edulis in scopolamine-induced mice, the decreased latency time in group administered with UV-C treated and untreated B. floribunda leaves extracts could suggest improved reference memory [46]. Extended time spent in central zone by animals that were administered with high dose of TBF and UTBF extracts might be as a result of enhanced cholinergic neurotransmission system in the rat’s hippocampus. Further, the increased duration in central zone could also suggest decision making hence the frequent head dipping over the edges of the central zone that was observed in this study [47].

There is dose-dependent increase in number of entries into central zone and open arm by animals that received TBF and UTBF with group that received the highest dose of UV-C treated B. floribunda leaves extracts showing more entries. In consonance with previous study, a more anxious experimental animal(s) is less likely to explore open arm owing to fear of falling off from the open arm but dwells more in the closed arm [48]. The administration of UV-C treated B. floribunda leaves further reversed the effects of scopolamine by enhancing locomotors activity of the hippocampus and the cerebrum cortex. These effects could presumably be attributed to enhanced antioxidants activities and anxiolytic agents such as terpenes. Previous research has revealed that several biologically active compounds such as terpenoids could mediate anxiolysis through interaction with gamma-amino butyric acid (GABA), glutamate, serotonin receptors [49]. In vice versa, while scopolamine caused increase number of entry and duration in the closed arms, groups that were treated with B. floribunda leaves extracts entirely demonstrated opposite behaviours as observed by the decrease in number of entry as well as less duration in the close arm.

Steady increase in the percentage time spent in open arm of animals that were administered with graded doses of TBF and UTBF could indicate the presence of anxiolytic agent in the extract. Previous studies have shown that anxiogenic agents specifically decrease the number of entries into the open arms and the time spent while anxiolytic agents enhance the number of entries and total time spent in open arm [48].

Findings from the rearing count could suggest effective reversal of scopolamine in B. floribunda treated rat, frequent exploration of the EPM, improved recognition of the maze and balanced reactivity to new stimuli [50].

In neurodegenerative diseases, cholinergic systems are adversely affected and it is one of the earlier hypotheses proposed to explain Alzheimer’s disease (AD). This has led to the development of AChE and BChE inhibitors as treatment strategy for AD [51]. In agreement with previous studies, the administration of scopolamine (2 mg/kg) led to increase in activities of AChE and BChE enzymes. The enhanced AChE and BChE activities upon scopolamine administration were reduced in experimental rats pre-treated with TBF, UTBF or standard donepezil. This might be attributed to the ability of the extracts to inhibit AChE and BChE enzymes, which further confirm the result obtained in in vitro study. The improved therapeutic activity of TBF might be attributed to the ability of UV-C to enhance secondary metabolites in plants at low doses [2].

Also, AD is usually associated with the elevation of oxidative stress in the brain and many studies have confirmed that in scopolamine-induced rats [52]. The enhancement of biomarkers of oxidative stress in the brain can create memory deficits in hippocampus region through impairment of synaptic plasticity [53,54]. Oxidative stress, which is defined as imbalance between the generation of free radicals and scavenging ability by antioxidants, is evaluated by lipid peroxidation (MDA), generation of nitric oxide (NO) and glutathione (GSH) content in the present study. The generation of lesser lipid peroxides and nitric oxides in rats pre-treated with TBF and UTBF might be due to reinforced antioxidant potential of the rats by the phytochemicals present in the extracts. Also, it may be attributed to the probable neuroprotective ability of the extracts against scopolamine-induced brain damage. Glutathione, an intracellular thiol protein, which has been implicated in regulation of redox balance in the cell, was also decreased with scopolamine injection. The decrease in GSH level might be due to mobilization to quench the effect of excessive generation of lipid peroxides and nitric oxide. The enhanced GSH level in pre-treated rats relative to scopolamine-induced rats further confirmed the probable neuroprotective and antioxidant abilities.

5. Conclusion

These results demonstrate that UV-C irradiation as a postharvest treatment for B. floribunda could be used to enhance total phenolic, total flavonoid amount, antioxidant abilities and cholinesterase enzyme inhibition activities of B. floribunda. This study has demonstrated that UV-C treated B. floribunda leaves extracts possibly contain several biologically active compounds that are capable of mitigating the neurologic effects of scopolamine. Further study is, however, encouraged to isolate and identify agents that are responsible for the enhanced neurological effects.

Acknowledgements

The authors would like to appreciate Tertiary Education Trust Fund (TETFUND) of the Federal Government of Nigeria for supporting this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

The protocol for this research was approved by the Research Ethics Committee of Olusegun Agagu University of Science and Technology (OAUSTECH/ETHC-BCH/2022/001).

Additional information

Funding

This research work was funded by Tertiary Education Trust Fund (TETFUND) under grant YEAR(S) 2020 (MERGED) TETFUND INTERVENTION IN RESEARCH PROJECT (RP). OAUSTECH/TETFund/VOL.1/001.