Neuroprotective Effect of White Nelumbo nucifera Gaertn. Petal Tea in Rats Poisoned with Mancozeb

Nelumbo nucifera Gaertn. (N. nucifera) tea is used as food and folk medicine to reduce toxicity in Southeast Asia. Mancozeb (Mz) is used for controlling fungi in agriculture and contains heavy metals. This study aimed to examine the effect of white N. nucifera petal tea on cognitive behavior, hippocampus histology, oxidative stress, and amino acid metabolism in rats poisoned with mancozeb. Seventy-two male Wistar rats were divided into nine groups (n = 8 in each). Y-maze spontaneous alternation test was used to assess cognitive behavior, and amino acid metabolism was investigated by nuclear magnetic resonance spectroscopy (1H-NMR) from blood. There was a significant increase in relative brain weight in the Mz co-administered with the highest dose (2.20 mg/kg bw) of white N. nucifera group. The levels of tryptophan, kynurenine, picolinic acid, and serotonin in blood showed a significant decrease in the Mz group and a significant increase in the Mz co-administered with low dose (0.55 mg/kg bw) of white N. nucifera group. However, there was no significant difference in cognitive behavior, hippocampus histology, oxidative stress, and corticosterone. This study demonstrated that a low dose of white N. nucifera petal tea has a neuroprotective effect against mancozeb.


Experimental Design
The male Wistar rats weighed 200-250 g and were divided into nine groups (n = 8). Group one received distilled water orally one ml/day. Group two, group three and group four received white N. nucifera petals tea at doses of 0.55, 1.10, and 2.20 mg/kg bw, respectively. Group five received olive oil one mL/day as a vehicle group. Group six received mancozeb at dose of 500 mg/kg bw in olive oil. Group seven, group eight, and group nine received mancozeb at a dose of 500 mg/kg bw in olive oil 30 min after receiving white N. nucifera petals tea at doses of 0.55, 1.10, and 2.20 mg/kg bw, respectively, for 30 days consecutively. The learning and memory of all rats were assessed by the Y-maze spontaneous alternation test, which is a three-arm maze with equal angles between all arms [46] on the day after the last treatment (day 30). Then, the rats were sacrificed, followed by blood collection for hormonal assays and 1 H-NMR, and the histology and the antioxidant biomarkers of the hippocampi were studied.

Learning and Memory Behavior Test
The Y-maze spontaneous alternation test was used to assess learning and memory behavior test in this study on the day after the last treatment. The rats were initially placed in the center of the maze and allowed to freely explore for eight minutes. The number of alternations and the total of arm entries were noted by the researcher and video recorded. The percentage of alternation was calculated as (number of alternations/total arm entries − 2)) × 100. The number of alternations in which all three arms were denoted, i.e., ABC, ACB, CAB, CBA, BCA, or BAC (but not, for example, CAC) and was recorded as a measure of short-term memory. The Y-maze was cleaned with 70% alcohol and allowed to dry between use.

Hormonal Essay
After the treatment period, all rats were euthanized with isoflurane, immediately followed by blood collection via the left ventricle of the heart. Whole blood was centrifuged at 3000 rpm for 15 min and plasma levels of corticosterone were measured by the Veterinary Diagnostic Laboratory, Faculty of Veterinary Medicine, Chiang Mai University.

Histological Study
The brains were removed carefully. All brains were weighed, and the right cerebral hemispheres were fixed in a 4% paraformaldehyde solution, dehydrated in alcohol series, cleared in xylene, and then embedded in paraffin wax. The paraffin blocks were cut with a microtome at 3 µm. The sections were mounted on clean glass slides and dried in an oven at 60 • C for 45 min. After deparaffinization in down-grade ethyl alcohol (100%, 95%, and 80%), sections were processed for hematoxylin and eosin staining for observation and photomicrography using a light microscope. The alignment of pyramidal cells was investigated in the middle of the CA1 and CA3 regions, and the vesicular nuclei of pyramidal cells were counted in an area of 1 µm 2 of both the CA1 and CA3 regions under 40× magnification objective lens with 10× magnification eyepiece lens by using Image J software version 6.0 ( Figure 1).

Oxidative Stress Status 2.7.1. Lipid Peroxidase (LPO) Assay
The thiobarbituric acid-reactive species (TBARS) assay was used to assess 100 µL of supernatant of homogenized left cerebral hemisphere. This method followed Laoung-on et al. [11], and then, the mixture solution was measured by a microplate reader at 532 nm (Bio Tek Synergy H4 Hybrid Microplate Reader, BioTek Instruments, Winooski, VT, USA).

Lipid Peroxidase (LPO) Assay
The thiobarbituric acid-reactive species (TBARS) assay was used to assess 1 supernatant of homogenized left cerebral hemisphere. This method followed Lao et al. [11], and then, the mixture solution was measured by a microplate reader at (Bio Tek Synergy H4 Hybrid Microplate Reader, BioTek Instruments, Winooski, VT

Inhibition of Advance Oxidation Protein Products (AOPP) Formation
Briefly, one hundred microliters of supernatant of homogenized left cerebr sphere were added to 25 µL of 1.16 M potassium iodine (KI), flowed by 30 min of tion, after which 10 µL of absolute acetic acid was added. This method followed L on et al. [11]. The solution was measured by a microplate reader at 340 nm.

Inhibition of Advance Glycation End Products (AGEs) Formation
Two hundred microliters of supernatant of homogenized left cerebral hem were added into 96 well-plate following the method used and reported by Laou al. [11] and were measured by a microplate reader at excitation wavelength 360 emission wavelength 460 nm.

Amino Acid Metabolism Analysis by 1 H-NMR
Two mL of blood was collected from each rat, and the plasma samples were s −20 °C before analysis.
The samples were thawed. An 800 μ L aliquot was added to 2400 μ L of form/methanol 1:1 solution, centrifuged (1700 rpm, 4 °C, 15 min), and then the were dried by lyophilization. Afterward, the samples were resuspended in deuter ide (D2O) and trimethylsilyl propanoic acid (TSP), which was added to provide a nal reference for the chemical shift (0 ppm). Five hundred μL of the solution wa ferred to an NMR tube, and the NMR analysis was performed at the Central Scie

Inhibition of Advance Oxidation Protein Products (AOPP) Formation
Briefly, one hundred microliters of supernatant of homogenized left cerebral hemisphere were added to 25 µL of 1.16 M potassium iodine (KI), flowed by 30 min of incubation, after which 10 µL of absolute acetic acid was added. This method followed Laoung-on et al. [11]. The solution was measured by a microplate reader at 340 nm.

Inhibition of Advance Glycation End Products (AGEs) Formation
Two hundred microliters of supernatant of homogenized left cerebral hemisphere were added into 96 well-plate following the method used and reported by Laoung-on et al. [11] and were measured by a microplate reader at excitation wavelength 360 nm and emission wavelength 460 nm.

Amino Acid Metabolism Analysis by 1 H-NMR
Two mL of blood was collected from each rat, and the plasma samples were stored at −20 • C before analysis.
The samples were thawed. An 800 µL aliquot was added to 2400 µL of chloroform/methanol 1:1 solution, centrifuged (1700 rpm, 4 • C, 15 min), and then the samples were dried by lyophilization. Afterward, the samples were resuspended in deuterium oxide (D2O) and trimethylsilyl propanoic acid (TSP), which was added to provide an internal reference for the chemical shift (0 ppm). Five hundred µL of the solution was transferred to an NMR tube, and the NMR analysis was performed at the Central Science Laboratory, Faculty of Sciences, Chiang Mai University by the nuclear magnetic resonance spectrometer (Bruker NEO 500 MHz, Bruker Daltonics, Billerica, BA, USA).
MestReNova software version 14 was used to analyze the results. The chemical shifts were referred to the internal standard at 0 ppm. Tryptophan was referenced at 7.2 ppm, and kynurenine was denoted at 7.37 ppm, whereas kynurenic acid was denoted at 7.94 ppm and 7.95 ppm was the reference for picolinic acid. Moreover, serotonin and melatonin were found at 3.1 and 7.21 ppm, respectively, as shown in Figure 2. ppm and 7.95 ppm was the reference for picolinic acid. Moreover, serotonin and melatonin were found at 3.1 and 7.21 ppm, respectively, as shown in Figure 2.

Statistical Analysis
The data were presented as mean ± standard deviation (SD). Normal distributions were assessed by Kolmogorov-Smirnov test. The average of relative brain weight was statistically analyzed by one-way ANOVA followed by Tukey's test. The averages of other parameters were analyzed using the Kruskal-Wallis tests. The significance level was set at p < 0.05. Statistical analyses were performed using the SPSS program version 22.

Learning and Memory Behavior by Y-Maze Spontaneous Alternation Test
The percentage of alternation of the Y-maze spontaneous alternation test showed no significant difference when compared to the control group at p < 0.05 ( Figure 3). In contrast, a previous study showed male Wistar rats, after 28 days of exposure to methomyl (a type of pesticide), decreased the percentage of alternations of the Y-maze test due to alterations in synaptic plasticity [23]. Moreover, mancozeb was not reported to induce learning-memory behavior changes in adult rats at a dose of 500 mg/kg bw, but propylthiouracil (another fungicide) has been shown to impair learning and memory in rat offspring at doses as low as 1.6 mg/kg bw by disrupting brain development [47]. Manganese poisoning has been shown to cause behavioral alternations in the Y-maze test in rats [48]. A prior study showed red N. nucifera petals increased the percentage of alternations in mice with scopolamine-induced memory deficit [49]. In short, different pesticides and fungicides have differing effects on learning and memory behavior, and the current study showed no ill effects from mancozeb, perhaps because the dosage was too low.

Statistical Analysis
The data were presented as mean ± standard deviation (SD). Normal distributions were assessed by Kolmogorov-Smirnov test. The average of relative brain weight was statistically analyzed by one-way ANOVA followed by Tukey's test. The averages of other parameters were analyzed using the Kruskal-Wallis tests. The significance level was set at p < 0.05. Statistical analyses were performed using the SPSS program version 22.

Learning and Memory Behavior by Y-Maze Spontaneous Alternation Test
The percentage of alternation of the Y-maze spontaneous alternation test showed no significant difference when compared to the control group at p < 0.05 ( Figure 3). In contrast, a previous study showed male Wistar rats, after 28 days of exposure to methomyl (a type of pesticide), decreased the percentage of alternations of the Y-maze test due to alterations in synaptic plasticity [23]. Moreover, mancozeb was not reported to induce learning-memory behavior changes in adult rats at a dose of 500 mg/kg bw, but propylthiouracil (another fungicide) has been shown to impair learning and memory in rat offspring at doses as low as 1.6 mg/kg bw by disrupting brain development [47]. Manganese poisoning has been shown to cause behavioral alternations in the Y-maze test in rats [48]. A prior study showed red N. nucifera petals increased the percentage of alternations in mice with scopolamineinduced memory deficit [49]. In short, different pesticides and fungicides have differing effects on learning and memory behavior, and the current study showed no ill effects from mancozeb, perhaps because the dosage was too low.

Effect of White N. nucifera Gaertn. Petal Tea and Mancozeb on Relative Brain Weight
Relative brain weight showed a significant increase in Mz + NNE 2.20 mg/kg bw group when compared to the olive oil group, as presented in Figure 4. However, the Mz groups tend to increase relative brain weight when compared to the olive oil group (through not significant).
Similarly, a previous studies showed increased relative brain and liver weights in albino rats poisoned with mancozeb at a dose of 500 mg/kg bw for 30 days due to hypertrophy of both tissues [50,51]. Moreover, subchronic treatment with chlorpyrifos-methyl, a type of pesticide, also caused an increase in relative brain weight due to minor histopathological changes in the brain [52]. In contrast, other studies showed decreased relative brain weight in adult male rats after receiving cypermethrin (a pesticide) for 28 days consecutively [53] and a reduction of brain weight of adult rats on mancozeb exposure to a high dose of 1500 mg/kg/day for 360 days [26]. However, evidence shows that changes in relative brain weight are rarely to a neurotoxic level [54]. In short, mancozeb has been shown to induce relative brain weight increase in acute and subchronic periods and decrease relative brain weight at high doses in chronic time scales. . Relative brain weight is presented as mean ± standard deviation (SD), n = 8. # p < 0.05 significant difference when compared to the olive oil group (relative brain weights were analyzed by One-way ANOVA followed by Tukey's test).

Effect of White N. Nucifera Gaertn Petal Tea and Mancozeb on Relative Brain Weight
Relative brain weight showed a significant increase in Mz + NNE 2.20 mg/kg bw group when compared to the olive oil group, as presented in Figure 4. However, the Mz groups tend to increase relative brain weight when compared to the olive oil group (through not significant).

Effect of White N. nucifera Gaertn. Petal Tea and Mancozeb on Relative Brain Weight
Relative brain weight showed a significant increase in Mz + NNE 2.20 mg/kg bw group when compared to the olive oil group, as presented in Figure 4. However, the Mz groups tend to increase relative brain weight when compared to the olive oil group (through not significant).
Similarly, a previous studies showed increased relative brain and liver weights in albino rats poisoned with mancozeb at a dose of 500 mg/kg bw for 30 days due to hypertrophy of both tissues [50,51]. Moreover, subchronic treatment with chlorpyrifos-methyl, a type of pesticide, also caused an increase in relative brain weight due to minor histopathological changes in the brain [52]. In contrast, other studies showed decreased relative brain weight in adult male rats after receiving cypermethrin (a pesticide) for 28 days consecutively [53] and a reduction of brain weight of adult rats on mancozeb exposure to a high dose of 1500 mg/kg/day for 360 days [26]. However, evidence shows that changes in relative brain weight are rarely to a neurotoxic level [54]. In short, mancozeb has been shown to induce relative brain weight increase in acute and subchronic periods and decrease relative brain weight at high doses in chronic time scales. . Relative brain weight is presented as mean ± standard deviation (SD), n = 8. # p < 0.05 significant difference when compared to the olive oil group (relative brain weights were analyzed by One-way ANOVA followed by Tukey's test). . Relative brain weight is presented as mean ± standard deviation (SD), n = 8. # p < 0.05 significant difference when compared to the olive oil group (relative brain weights were analyzed by One-way ANOVA followed by Tukey's test).
Similarly, a previous studies showed increased relative brain and liver weights in albino rats poisoned with mancozeb at a dose of 500 mg/kg bw for 30 days due to hypertrophy of both tissues [50,51]. Moreover, subchronic treatment with chlorpyrifos-methyl, a type of pesticide, also caused an increase in relative brain weight due to minor histopathological changes in the brain [52]. In contrast, other studies showed decreased relative brain weight in adult male rats after receiving cypermethrin (a pesticide) for 28 days consecutively [53] and a reduction of brain weight of adult rats on mancozeb exposure to a high dose of 1500 mg/kg/day for 360 days [26]. However, evidence shows that changes in relative brain weight are rarely to a neurotoxic level [54]. In short, mancozeb has been shown to induce relative brain weight increase in acute and subchronic periods and decrease relative brain weight at high doses in chronic time scales.

Number of Pyramidal Cells and Histology of Hippocampus
The present study showed no significant difference in number of pyramidal neurons in both CA1 and CA3 regions when compared between groups (Table 1). Both the CA1 and CA3 regions in all groups showed shrinkage and degeneration (darkly stained neurons) of pyramidal neurons, as shown in Figures 5 and 6, and this is likely due to a natural phenomenon seen in pyramidal neurons in the adult neurogenesis of the brain [55].

Number of Pyramidal Cells and Histology of Hippocampus
The present study showed no significant difference in number of pyramidal neurons in both CA1 and CA3 regions when compared between groups (Table 1). Both the CA1 and CA3 regions in all groups showed shrinkage and degeneration (darkly stained neurons) of pyramidal neurons, as shown in Figures 5 and 6, and this is likely due to a natural phenomenon seen in pyramidal neurons in the adult neurogenesis of the brain [55].
Adult neurogenesis in the hippocampus plays a role in learning and memory, especially pyramidal neurons, which are large neuronal cells in the hippocampus [56]. Interestingly, a prior study on the effect of pesticides on the hippocampus structure of mice showed significantly decreased density and size of pyramidal cells after pesticide exposure [27]. Moreover, histopathological changes in the hippocampus of rats poisoned with chlorpyrifos and cypermethrin, especially pyramidal cells in CA1 and CA3 regions, were induced by stimulating ChE activity and impairing neuron conductivity [53]. Additionally, a recent study showed that white N. nucifera petal extract at a dose of 1.10 mg/kg bw [12] had no toxic effect on the hippocampus structure and learning-memory after oral administration for 30 consecutive days [57]. In view of these and the discussion of the cognitive behavior changes above, further research on the connection between histopathological changes and cognitive behaviors would be helpful in the evaluation of mancozeb and white N. nucifera petal extract effects on brain function.

Corticosterone Level
Corticosterone level was increased in the mancozeb group (though not significantly) when compared between groups. There was no significant difference between all groups when compared between groups, as presented in Figure 7.
Our study agreed with a prior study where mancozeb induced hypercorticosteronaemia in mice after ingestion by lactating mothers for 28 days [30], and is similar to a study in which fipronil pesticide-induced elevated serum corticosterone in adult male rats due to disruption of the hypothalamus-pituitary-adrenal (HPA) axis [58]. Elevated cortisol levels were also observed in Garra gotyla fish after exposure to manganese for a week [59]. In contrast, the N. nucifera flower tea at doses of 10, 100, and 200 mg/kg bw was reported to reduce the level of corticosterone in stress-induced rats [9]. Thus, the difference in these studies, including the present study, may be that the corticosterone effect of mancozeb is correlated to the age of rodents. Adult neurogenesis in the hippocampus plays a role in learning and memory, especially pyramidal neurons, which are large neuronal cells in the hippocampus [56]. Interestingly, a prior study on the effect of pesticides on the hippocampus structure of mice showed significantly decreased density and size of pyramidal cells after pesticide exposure [27]. Moreover, histopathological changes in the hippocampus of rats poisoned with chlorpyrifos and cypermethrin, especially pyramidal cells in CA1 and CA3 regions, were induced by stimulating ChE activity and impairing neuron conductivity [53]. Additionally, a recent study showed that white N. nucifera petal extract at a dose of 1.10 mg/kg bw [12] had no toxic effect on the hippocampus structure and learning-memory after oral administration for 30 consecutive days [57]. In view of these and the discussion of the cognitive behavior changes above, further research on the connection between histopathological changes and cognitive behaviors would be helpful in the evaluation of mancozeb and white N. nucifera petal extract effects on brain function.

Corticosterone Level
Corticosterone level was increased in the mancozeb group (though not significantly) when compared between groups. There was no significant difference between all groups when compared between groups, as presented in Figure 7.
Our study agreed with a prior study where mancozeb induced hypercorticosteronaemia in mice after ingestion by lactating mothers for 28 days [30], and is similar to a study in which fipronil pesticide-induced elevated serum corticosterone in adult male rats due to disruption of the hypothalamus-pituitary-adrenal (HPA) axis [58]. Elevated cortisol levels were also observed in Garra gotyla fish after exposure to manganese for a week [59]. In contrast, the N. nucifera flower tea at doses of 10, 100, and 200 mg/kg bw was reported to reduce the level of corticosterone in stress-induced rats [9]. Thus, the difference in these studies, including the present study, may be that the corticosterone effect of mancozeb is correlated to the age of rodents.

Effect of White N. nucifera Gaertn. Petal Tea Extract on Oxidative Stress Status
Regarding the antioxidant activity of white N. nucifera petal extract alone and coadministered with mancozeb, levels of lipid peroxidation (LPO), advanced oxidation protein products (AOPPs), and advanced glycation end products (AGEs) were measured but showed no significant difference (Table 2).
Antioxidants are substances that can prevent, inhibit, and reduce cell damage to DNA, lipids, and proteins of cells due to oxidative stress [60,61]. Free radicals are generated from endogenous sources, such as immune cells, inflammation, mental stress, and also by exogenous sources, including environmental pollutants, smoking, and alcohol [62]. Cell and organelle membranes are made from a double layer of a polar hydrophilic head and a non-polar hydrophobic tail. In most mammalian cell membranes, there are both straight-chained saturated fatty acids and unsaturated fatty acids [63]. Their polyunsaturated fatty acids (PUFAs) are sensitive to oxidative stress, which is known as lipid peroxidation (LPO) [64]. LPO is a process that involves the formation of lipid radicals [65]. In an in vivo study, white N. nucifera petal extract was shown to reduce LPO of PUFAs in Charolais cattle sperm exposed to mancozeb at low doses of 0.22 and 0.44 μg/mL [12].
Moreover, on the external surface of cell membranes, most of the proteins and some of the lipids are conjugated with short chains of carbohydrates [63]. The most common results of glycoxidation are increased advanced oxidation protein products (AOPPs) and advanced glycation end products (AGEs) [66]. AOPPs are specific markers of protein oxidation, which are made during oxidative stress by the interaction between protein plasma and chlorinated oxidants [63,67], while AGEs are lipids or proteins that become glycated after exposure to sugars [68]. Our present study is similar to an in vivo study of the effect of white N. nucifera petal extract on Charolais cattle sperm exposed to mancozeb at low doses of 0.22 and 0.44 μg/mL, which showed no significant differences in the AOPPs and AGEs [12]. Furthermore, a prior study showed white N. nucifera petal extract decreased the levels of oxidative stress markers (2-hydroxybutyric acid, 4-hydroxynonenal, l-tyrosine, pentosidine, and N6-carboxymethyllysine) on rat livers poisoned with mancozeb [50].

Effect of White N. nucifera Gaertn Petal Tea Extract on Oxidative Stress Status
Regarding the antioxidant activity of white N. nucifera petal extract alone and coadministered with mancozeb, levels of lipid peroxidation (LPO), advanced oxidation protein products (AOPPs), and advanced glycation end products (AGEs) were measured but showed no significant difference ( Table 2). Antioxidants are substances that can prevent, inhibit, and reduce cell damage to DNA, lipids, and proteins of cells due to oxidative stress [60,61]. Free radicals are generated from endogenous sources, such as immune cells, inflammation, mental stress, and also by exogenous sources, including environmental pollutants, smoking, and alcohol [62]. Cell and organelle membranes are made from a double layer of a polar hydrophilic head and a non-polar hydrophobic tail. In most mammalian cell membranes, there are both straightchained saturated fatty acids and unsaturated fatty acids [63]. Their polyunsaturated fatty acids (PUFAs) are sensitive to oxidative stress, which is known as lipid peroxidation (LPO) [64]. LPO is a process that involves the formation of lipid radicals [65]. In an in vivo study, white N. nucifera petal extract was shown to reduce LPO of PUFAs in Charolais cattle sperm exposed to mancozeb at low doses of 0.22 and 0.44 µg/mL [12].
Moreover, on the external surface of cell membranes, most of the proteins and some of the lipids are conjugated with short chains of carbohydrates [63]. The most common results of glycoxidation are increased advanced oxidation protein products (AOPPs) and advanced glycation end products (AGEs) [66]. AOPPs are specific markers of protein oxidation, which are made during oxidative stress by the interaction between protein plasma and chlorinated oxidants [63,67], while AGEs are lipids or proteins that become glycated after exposure to sugars [68]. Our present study is similar to an in vivo study of the effect of white N. nucifera petal extract on Charolais cattle sperm exposed to mancozeb at low doses of 0.22 and 0.44 µg/mL, which showed no significant differences in the AOPPs and AGEs [12]. Furthermore, a prior study showed white N. nucifera petal extract decreased the levels of oxidative stress markers (2-hydroxybutyric acid, 4-hydroxynonenal, l-tyrosine, pentosidine, and N6-carboxymethyllysine) on rat livers poisoned with mancozeb [50].
3.6. Effect of White N. nucifera Gaertn. Petal Tea and Mancozeb on Amino Acid Metabolism by 1

H-NMR
The results of the amino acid metabolism study of tryptophan, L-kynurenine, kynurenic acid, picolinic acid, serotonin, and melatonin are presented in Figure 8. Tryptophan showed a significant decrease in the Mz group when compared to the olive oil group ( Figure 8A). Tryptophan is an essential amino acid in all animals [69]. The main pathway of tryptophan metabolism is the kynurenine pathway, leading to bioactive molecules which are both neurotoxic and neuroprotective and antioxidative [41]. It has been reported that low circulating levels of tryptophan were found in elderly patients with neurodegenerative diseases [70,71]. Tryptophan plays a crucial role in the kynurenine pathway and may serve as a biomarker for monitoring neurodegenerative disease risk. Similarly, a previous study showed reduced tryptophan levels in mice exposed to maneb (a fungicide similar to Mz) [72]. The present study shows that Mz disrupts the level of tryptophan, while white NNE tends to ameliorate this effect.
The Mz group, as well as the co-administered of Mz/NNE groups, all showed a significant decrease in L-kynurenine levels when compared to the control group ( Figure 8B). Kynurenic acid is synthesized as tryptophan degrades via the kynurenine pathway [34]. The levels of kynurenic acid showed no significant difference when compared between groups at p < 0.05 ( Figure 8C). However, the NNE groups showed increased levels of kynurenic acid (though not significant) when compared to the control group, while the level of kynurenic acid in the Mz group was lower than in the olive oil group (though not significant). Likewise, picolinic acid level showed a significant decrease in the Mz group when compared to the olive oil group, while the co-administered Mz with NNE groups at doses of 0.55 and 2.20 mg/kg bw showed a significant increase when compared to the Mz group ( Figure 8D). Kynurenic acid and picolinic acid act as neuroprotectives in depression and various neurodegenerative diseases [73]. Previous studies showed depletion of plasma kynurenic acid in mice with multiple sclerosis induced by cuprizone, and in humans, a reduction of plasma picolinic acid was found in those who attempted suicide [74,75]. Depletion of kynurenic acid was also reported in deltamethrin and fenpropathrin poisoning in rat brain slices [32]. Therefore, Mz effects of reducing the level of picolinic acid, and white N. nucifera petal tea at low (0.55 mg/kg bw) and high (2.20 mg/kg bw) doses had a beneficial neuroprotective effect against Mz toxicity. Moreover, the picolinic acid showed a significant increase in the NNE 2.20 mg/kg bw group when compared to the control group. Thus, white N. nucifera petal tea has a beneficial effect on health promotion.
(NAE) have high contents of total phenolics, total tannins, and antioxidant activity [11], which have been reported to be protective in neurodegenerative diseases and depressive disorders [79,80]. Another study reported N. nucifera seeds have antidepressant effects and are closely related to serotonin [81]. In brief, white N. nucifera petal tea at a low dose protects serotonin levels after Mz exposure.
Melatonin levels were lower in all the Mz groups, though not at significant levels when compared between groups at p < 0.05 (Figure 8f). Figure 8. The mean ± SD of products of tryptophan metabolism: Tryptophan (A), L-kynurenine (B), kynurenic acid (C), picolinic acid (D), serotonin (E), melatonin (F). * p < 0.05 significant difference when compared to control group. # p < 0.005 significant difference when compared to the olive oil group. ## p < 0.05 significant difference when compared to the Mz group. Figure 8. The mean ± SD of products of tryptophan metabolism: Tryptophan (A), L-kynurenine (B), kynurenic acid (C), picolinic acid (D), serotonin (E), melatonin (F). * p < 0.05 significant difference when compared to control group. # p < 0.005 significant difference when compared to the olive oil group. ## p < 0.05 significant difference when compared to the Mz group.

Conclusions
In addition, the Mz-only group, as well as in the co-administered Mz/NNE groups at doses of 1.10 and 2.20 mg/kg bw, showed significant decreases in serotonin when compared to the olive oil group ( Figure 8E). The low-dose NNE appeared to prevent the drop in serotonin due to the Mz. Serotonin plays crucial roles in neural activities, mood, and behavior [45]. Previous studies have shown low levels of serotonin in people with psychosocial stresses such as depression and anxiety [76,77]. Similarly, organophosphate, which is an agricultural pesticide, was associated with anxiety and depression via the serotonin system disruption [78]. Thus, pesticides such as Mz have been repeatedly shown to reduce the levels of serotonin. Additionally, the white N. nucifera petal aqueous extracts (NAE) have high contents of total phenolics, total tannins, and antioxidant activity [11], which have been reported to be protective in neurodegenerative diseases and depressive disorders [79,80]. Another study reported N. nucifera seeds have antidepressant effects and are closely related to serotonin [81]. In brief, white N. nucifera petal tea at a low dose protects serotonin levels after Mz exposure.
Melatonin levels were lower in all the Mz groups, though not at significant levels when compared between groups at p < 0.05 ( Figure 8F).

Conclusions
In conclusion, white N. nucifera petal tea at doses of 0.55, 1.10, and 2.20 mg/kg bw did not affect relative brain weight, learning-memory behavior, number of pyramidal neurons,