Fiber isolated from discarded fruits and vegetables reduces valproic acid-induced hepatotoxicity in rats

Abstract

In this work, a methodology was developed to obtain dietary fiber from agro-industrial fruit and vegetable waste. The resulting raw material was named DF. The physicochemical analysis revealed a high total dietary fiber content, along with an appropriate soluble to insoluble fiber ratio and excellent water and fat retention capacity. Subsequently, its potential protective effect against hepatotoxicity induced by valproic acid (VPA) was investigated in Wistar rats, both as preventive and curative treatments. For this purpose, two different trials were conducted. In the preventive trial, VPA (250 mg/kg/day; oral) was administered concomitantly with DF (0.3 and 0.15 mg/kg/day) for 14 days. In the curative trial, VPA was administered for 14 days, followed by DF for an additional 14 days. The results demonstrated that DF supplementation normalized body weight, liver biomarkers and attenuated VPA-induced tissue damage, while normal liver architecture was preserved. These findings suggest that DF obtained from agro-industrial fruit and vegetable waste materials may serve as a functional feedstock to counteract the harmful effects associated with prolonged VPA treatment.

Keywords:
Circular economy; Fiber; Waste; Hepatotoxicity; Sodium valproate

INTRODUCTION

Wholesale produce markets play a crucial role in the fruit and vegetable marketing and distribution chain, spanning from harvest to the consumer’s table. However, within this chain, large waste volumes are generated due to products failing to meet consumer quality standards. Disposing of these discards not only poses environmental challenges but also results in substantial economic losses, encompassing both the discarded products and the cost associated with their transportation to landfills (Clementz et al., 2019). This issue is further repeated in fruit and vegetable cleaning and packing facilities, which highlights the urgent need for technological solutions that provide both economic viability and environmental sustainability. Moreover, these waste materials represent a source of valuable and health-promoting bioactive compounds that can be recovered and utilized as raw materials for other products, in line with the principles of sustainability and circular economy (Lucarini et al., 2021).

Interestingly, among these compounds are fibers that are inherent components of fruits and vegetables. Their consumption is inversely correlated with the risk of chronic diseases, including non-alcoholic fatty liver disease (NAFLD), which is currently of public health concern (Chen et al., 2020). Among the variety of causes that can induce NAFLD, it has been estimated that a high percentage of cases are caused by the administration of medications (Liebe et al., 2021). Some of the medications that can potentially induce NAFLD include antiarrhythmic, antineoplastic, antihypertensive, antimicrobial and antiepileptic medicines (Di Pasqua et al., 2022). Valproic acid (VPA) belongs to the family of antiepileptic drugs and is also utilized to treat neuropathic pain, migraine, and bipolar disorder. It was estimated that around one million people worldwide consume VPA daily (Shnayder et al., 2023).

Furthermore, effective long-term therapy with VPA can result in numerous adverse effects, including weight gain and hepatotoxicity (Guo et al., 2019). Hepatotoxicity associated with VPA is characterized by mitochondrial injury, oxidative stress, and microvesicular steatosis, often accompanied by varying degrees of inflammation and cholestasis (Shnayder et al., 2023). Despite significant progress in comprehending VPA-induced hepatotoxicity, there is no preventive treatment for this condition currently available. At present, among the most widely used therapeutic approaches to improve human health are combined programs involving diet and aerobic exercise (Maciejewska-Markiewicz et al., 2022).

According to the dietary guidelines, it is recommended to reduce the consumption of saturated fat-rich food while increasing fiber intake (Abdel-Dayem et al., 2014). In this regard, it has been reported that the fermentation of dietary fiber by intestinal bacteria produces postbiotic butyrate, a short-chain fatty acid that significantly enhances intestinal permeability, reduces serum endotoxin levels and mitigates the progression of inflammation and liver damage (Pirozzi et al., 2020; Wu et al., 2017). To date, to the best of our knowledge, no study has ascertained the potential of isolated and purified fiber supplementation to counteract VPA hepatotoxicity in animals.

However, it has been reported that fiber intake (14 g) does not affect the pharmacokinetics of VPA (500 mg; single dose) in healthy individuals (Issy et al., 1997). As a consequence, we hypothesized that fiber supplementation could protect the liver from the damage induced by prolonged administration of VPA, both with preventive and curative purposes. Therefore, the aim of this work was to develop a new methodology to isolate and purify dietary fiber from agro-industrial fruit and vegetable waste, and to study its potential protective effects on valproic acid-induced liver damage in rats.

MATERIAL AND METHODS

Raw materials

Discarded fruits and vegetables were obtained from the wholesale produce market for fruits and vegetables of the city of Santa Fe, province of Santa Fe, Argentina. They were separated into three groups according to their characteristics: fruits and vegetables of color (G1), tubers (G2) and green leaves (G3). The present work focuses on the valorization of the discarded fruits and vegetables in the G1 group; they were obtained in April 2023. The proportion in percentage of each fruit and vegetable is shown in Table I. As regards handling and storage, the method used consisted in removing the areas attacked by microorganisms and then storing the discards at 4°C until use (Ramirez, Manuale, Yori, 2023).

TABLE I
Proportion of discarded fruits and vegetables in group 1 (G1)

Fruits/Vegetablesa	Content (%)
Pineapple	0.75
Eggplant	0.46
Plum	1.18
Peach	13.58
Kiwi	0.88
Lemon	5.37
Tangerine	5.39
Mango	0.42
Apple	5.33
Melon	6.42
Orange	3.27
Pear	13.65
Grapefruit	10.49
Tomato	0.75
Grape	11.04
Carrot	18.12
Zucchini	1.71
Pumpkin	1.18

• ^aThe discarded materials were obtained in April 2023.

Preparation of dietary fiber from food waste

Discarded food waste of the G1 group was processed in a juice extractor. At the end of the process, two products were obtained: bagasse and must, which were separated by filtration. Bagasse was fed to a stirred-tank extractor provided with temperature and stirring speed control. Water was used as the extraction solvent at a temperature of 80°C in a 2: 1 mass ratio with respect to the bagasse, with a stirring speed of 100 rpm. Contact time was 15 minutes for each extraction step. Four extraction steps were used in order to efficiently extract the sugars present in the bagasse. Then, the resulting solid was dried in a stove at 110°C overnight and milled in a micronizer into a fine powder (Ramirez, Manuale, Yori, 2023). The solid was called treated dietary fiber (DF).

Analytical methods

For this study, contents of moisture, ash, fats and total dietary fiber (soluble and insoluble) were determined employing AOAC standard methods 934.01, 942.05, 922.06, and 991.43, respectively (AOAC, 2000). The nitrogen content was determined by the Kjeldahl method 2001.11 (AOAC, 2000). Protein content was estimated as the nitrogen content multiplied by 6.25. The contents of phosphorus, iron and heavy metals (Hg, Pb, As, Cd) were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES, in a Perkin Elmer, Optima 2100 DV) after digestion in an acid solution of 1: 5 (V/V) nitric: perchloric acid. The water-holding capacity (WHC) and the oil-holding capacity (OHC) of DF were determined following the methodology described by Ramirez, Manuale, Yori (2023). The density of DF was determined by a technique of weight: volume using a graduate test tube. The microbiological characterization of DF was made using a bacteriological diagnostic kit provided by ZT Lab of Argentina. Total plate count, total coliforms and Salmonella were determined following the methodology described by Ramirez et al., (2022).

Animals

The research protocol was approved (Res. No. 0017) by the Animal Research Ethics Committee of the Faculty of Medicine of Universidad Nacional del Nordeste (Corrientes, Argentina). Thirty-six adult male Wistar rats weighing 162-175 g (seven weeks old) from the laboratory animal facility at Universidad Nacional del Litoral, Santa Fe, Argentina, were used. The animals were housed under controlled environmental conditions (23±2°C temperature, 60±10% humidity, 12/12 hrs. light/dark cycle). They received food (Ganave-Argentina) and water ad libitum. The centesimal composition of the feed was: Protein (minimum): 24%. Ether extract (minimum): 6%. Fiber (maximum): 7%. Calcium (minimum): 1%. Calcium (maximum): 1.2%. Phosphorus (minimum): 0.5%. Phosphorus (maximum): 0.9%. Total minerals (maximum): 8%. Humidity (maximum): 13%.

In order to induce hepatotoxicity, VPA (Teva-Argentina) was administered (250 mg/kg/day) for 14 days in accordance with the work of Abdelkader et al., (2020). To assess the possible protective effect of DF, one group received a dose of 0.15 mg/kg body weight, while the other group received a dose of 0.3 mg/kg body weight in accordance with previous studies that showed that supplementation with these doses improved certain metabolic parameters, which are of interest for this study (Ramirez, Manuale, Yori, 2023). DF was administered orally; to this end, the pellet of the standard diet was crushed and mixed with the said fiber (in established amounts), and then the feed was reconstituted.

Experimental design

• 1. In the preventive trial, VPA was administered concomitantly with DF for 14 days.

At the start of the experiment, 20 animals were randomly divided into five groups.

• Control group (n=4): animals received oral saline solution for 14 days.

• DF (n=4): animals were treated with DF (0.3 mg/kg/day) for 14 days and sacrificed after the end of treatment.

• VPA (n=4): animals were treated with VPA (250 mg/kg/day) for 14 days and sacrificed after the end of treatment (to corroborate hepatotoxicity).

• VPA+DF LD (n=4): animals received VPA (250 mg/kg/day) and DF (0.15 mg/kg/day) for 14 days and then they were sacrificed.

• VPA+DF HD (n=4): animals received VPA (250 mg/kg/day) and DF (0.3 mg/kg/day) for 14 days and then they were sacrificed.

• 2. In the curative trial, VPA was administered for 14 days, followed by DF for an additional 14 days. At the start of the experiment, 16 animals were randomly divided into four groups.

• Control group (n=4): animals received oral saline solution for 14 days.

• VPA (n=4): animals were treated with VPA (250 mg/kg/day) for 14 days and sacrificed 14 days after the end of treatment (to check if liver damage persists).

• VPA+DF LD (n=4): animals received VPA (250 mg/kg/day) for 14 days, and after that period, they were supplemented with the lower dose of DF (0.15 mg/kg/day) for 14 days and then sacrificed.

• VPA+DF HD (n=4): animals received VPA (250 mg/kg/day) for 14 days, and after that period, they were supplemented with the higher dose of DF (0.3 mg/kg/day) for 14 days and then sacrificed.

Biochemical analysis

At the time of euthanasia (24 hrs. after the end of treatment and 8 hrs. after fasting), blood was collected from all anaesthetized animals (ketamine and xylazine, 75-100 mg/kg). The samples were centrifuged at 4000 x g for 15 min to obtain plasma for the assessment of liver function parameters.

Plasma determinations

The parameters assessed in each case were alkaline phosphatase (ALP), alanine amino transferase (ALT), aspartate amino transferase (AST), total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), and total triglycerides (TG) (Ramirez, Manuale, Yori, 2023). The procedures were performed according to the manufacturer’s instructions and as described below.

Alkaline phosphatase

In the presence of magnesium and zinc ions, p-nitrophenylphosphate is cleaved by phosphatases into phosphate and p-nitrophenol. The p-nitrophenol released is directly proportional to the catalytic activity of ALP (409 nm).

Alanine aminotransferase

ALT catalyzes the reaction between L-alanine and 2-oxoglutarate. The pyruvate formed is reduced by NADH in a reaction catalyzed by the enzyme lactate dehydrogenase to form L-lactate and NAD+. Thus, the rate of NADH oxidation is directly proportional to the catalytic activity of ALT (340 nm).

Aspartate aminotransferase

AST in the sample catalyzes the transfer of an amino group between L-aspartate and 2-oxoglutarate to yield oxaloacetate and L-glutamate. In the presence of malate dehydrogenase, oxaloacetate reacts with NADH to form NAD+. The rate of NADH oxidation is directly proportional to the catalytic activity of AST (340 nm).

Total cholesterol

Under the action of the enzyme cholesterol esterase, cholesterol esters are cleaved into free cholesterol and fatty acids. Cholesterol oxidase enzyme then catalyzes the oxidation of cholesterol to form cholest-4-ene-3-one and hydrogen peroxide (H₂O₂). In the presence of peroxidase, the H₂O₂ formed results in the oxidative coupling of phenol and 4-amino antipyrine to form a red quinoneimine dye. The color intensity of the dye formed is directly proportional to the cholesterol concentration (512 nm).

HDL cholesterol

The HDL cholesterol test is based on the adsorption of synthetic polyanions on the surface of lipoproteins. The combined action of polyanions and detergent solubilizes HDL cholesterol, excluding LDL, VLDL and chylomicrons. The consecutive action of cholesterol esterase and cholesterol oxidase enzymes catalyzes the oxidation of cholesterol in solution. In the presence of P peroxidase, the H₂O₂ formed reacts with N, N-bis(4-sulfobutyl)-m-toluidine and 4-aminoantipyrine to produce a quinoneimine red dye; the color intensity of which is directly proportional to the HDL cholesterol concentration (552 nm).

LDL cholesterol

HDL, VLDL and chylomicrons are hydrolyzed with the detergent. Cholesterol released from these lipoproteins is immediately reacted by the action of the enzymes cholesteryl esterase and cholesterol oxidase to generate hydrogen peroxide. This is consumed by peroxidase in the presence of 4-aminoantipyrine, and a colorless product is generated. The LDL cholesterol reaction is initiated by the addition of the second detergent, together with N, N bis(4-sulfobutyl)-m-toluidine as a conjugating agent. This releases cholesterol from the LDL particles, which undergo an enzymatic reaction with the conjugating agent to produce a chromatic compound (quinoneimine red), which is directly proportional to the LDL cholesterol concentration (552 nm).

Triglycerides

Triglycerides are hydrolyzed by the enzyme lipoprotein lipase to fatty acids and glycerol. The resulting glycerol is phosphorylated to glycerol-3-phosphate by ATP in a reaction catalyzed by the enzyme glycerol kinase. Oxidation of glycerol-3-phosphate is catalyzed by the enzyme glycerol phosphate oxidase to form dihydroxyacetone phosphate and hydrogen peroxide. In the presence of peroxidase enzyme activity, hydrogen peroxide carries out the oxidative coupling of 4-chlorophenol and 4-aminophenazone, and forms a quinoneimine red dye that is measured at 512 nm.

Histological studies

Organs such as the liver and the kidneys were then removed, cleaned of adherent tissue, washed with ice-cold saline and weighed. Liver and kidney from each group were fixed by immersion in 10% buffered formalin for 24 hours. After this period, the samples were dehydrated in isopropyl alcohol of increasing strength, then rinsed with xylol and finally embedded in paraffin, and blocks were obtained and cut with a microtome. These histological sections were deparaffinized and stained with haematoxylin and eosin (H&E) to evaluate histopathological alterations. The stained sections were examined under a light microscope (Ramirez, Manuale, Yori, 2023).

Statistical analysis

The data were expressed as mean±standard deviation, from three separate experiments performed in triplicate, and the significance of the data between groups was tested by Infostat software (Argentina version 2020). Statistical analyses were carried out using Tukey’s multiple comparisons test (after ANOVA). In all cases, the probability level of 95% was considered significant (p<0.05).

RESULTS

Physicochemical analysis

As it is shown in Table II, the moisture content in the DF obtained through the extraction method herein developed was relatively low, suggesting that the physical properties of the material, its usability and the quality of the product were preserved throughout the process of obtaining the fiber (Figure 1). The ash content fell within the range of values reported by other researchers in publications on similar discards process, and may be associated with the presence of iron and phosphorus, which are essential nutrients in human nutrition. The analysis of the heavy metal content revealed that the concentrations were within the permissible limits set by current legislation. This is important because they are generally toxic to humans.

FIGURE 1
Raw material obtained from the revalorization process of discarded fruits and vegetables.

TABLE II
Composition of dietary fiber obtained from discarded materials

General characterization	DF
Texture	Powder
Appearance/color	Pale yellow
Taste	Neutral
Chemical composition
Water, %	10.0±0.10
Soluble fiber, %	29.0±1.21
Insoluble fiber, %	38.0±0.52
Total fiber, %	67.0±0.22
Proteins, %	7.0±0.25
Fats, %	0.2±0.02
Ash, %	2.2±0.30
Iron, ppm	80.0±0.50
Phosphorous, ppm	700±0.50
Heavy metals, ppm	<10
Physical properties
Water-holding capacity (WHC), g.g-1	8.3±0.40
Oil-holding capacity (OHC), g.g-1	9.7±0.25
Density, g.cm-3	320±10.0
Microbiological characteristic
Total plate count, UFC.g-1	<1, 000
Salmonella	Negative
Total coliforms, UFC.g-1	<10

• ^aValues are means±standard deviations (n=3). Some results were expressed as % dry matter.

The total dietary fiber content was 67.0±0.22 % dry sample. The raw material analysis showed that 38.0±0.5 % corresponds to insoluble dietary fiber (IDF), and the remaining 29.0±1.2 % to soluble dietary fiber (SDF). The SDF/IDF ratio is important for physiological properties as well as for structural and sensory properties. Likewise, it is recognized that fiber sources suitable for use as food ingredient applications should have an SDF/IDF ratio close to 1: 2. In this study, the SDF/IDF ratio was 1: 1.31. It is acknowledged that a typical function of IDF is the ability to absorb and retain water. Water Holding Capacity (WHC) is defined as the amount of water bound to the fibers under conditions where no external force, except gravity and atmospheric pressure, is exerted. In this study, the WHC value obtained from DF was 8.3±0.40 g/g, which is high for a natural fiber. Moreover, DF swells both in cold and hot conditions, eliminating the need of any technological treatment (Lucas-Gonzalez et al., 2017).

Regarding the Oil Holding Capacity (OHC), the value obtained from DF was 9.7±0.25 g fat/g; this OHC value suggests the possibility of using DF for stabilizing fat-rich food products and emulsions (Ramirez, Manuale, Yori, 2023). The results of the microbiological analyses to which DF was subjected indicated that the method used for extraction ensures its safety, rendering it suitable for both human and animal consumption. Taken together, these results suggest that DF could serve as a promising source of dietary fiber or a low-calorie bulk ingredient or additive, with potential applications in the development of functional foods.

Animal assays

Although pharmacokinetic studies were not performed in this work, it is possible to suggest that DF supplementation did not alter serum VPA concentrations. In order to guarantee an adequate therapeutic effect and minimize the possibility of adverse effects, DF was administered 10 hours after VPA (one-time). In this regards, rats treated with either VPA or DF showed no mortality during both trials. In general, the animals showed no signs of disease. In addition, no abnormalities were observed in macroscopic evaluations of the hair, eyes, and behavioral pattern. Food and water intake was normal in all experimental groups in both trials.

Effect of treatments on body weight

In the preventive trial Table III, a significant increase (p<0.05) in body weight was observed in the VPA group compared to the control group (203.10±7.43; 235.08±9.22). DF treatment did not change (p>0.05) body weight in healthy animals (205.11±8.31), but both doses attenuated the effect of VPA on body weight to values close to those of the DF-treated control group (210.52±7.79; 214.81±6.63, respectively).

TABLE III
Evolution of body weight (g) during the study period in the different experimental groups

Groups	Initial BW	Final BW
Control	171.03±6.25	203.10±7.43
VPA	168.95±8.13	235.08±9.22a
DF	173.67±9.04	205.11±8.31b
VPA+DF LD	170.31±6.75	210.52±7.79b
VPA+DF HD	169.73±7.24	214.81±6.63b

• Data are given as mean±SD (n=4). VPA: Valproic acid (250 mg/kg/day). DF: group supplemented with the higher dose of DF (0.3 mg/kg) VPA+DF LD: group supplemented with the lower dose of DF (0.15 mg/kg/day). VPA+DF HD: group supplemented with the higher dose of DF (0.3 mg/kg/day). ^aindicates significantly different as compared with animals in the control group (p<0.05). ^bindicates significantly different as compared with VPA -treated group (p<0.05).

In the curative trial Table IV, a significant (p<0.05) increase in body weight was observed in the VPA group compared to the control group (223.57±6.82; 251.41±7.29). Treatment with both doses of DF modified the effect of VPA on body weight to values close to those of the control group (220.92±9.04; 221.51±8.01). However, a non-significant (p>0.05) decrease in relative liver, kidney and heart weights was observed in the VPA-treated animals compared to the control group (data not shown).

TABLE IV
Evolution of body weight (g) during the study period in the different experimental groups

Groups	Initial BW	Final BW
Control	199.21±7.90	223.57±6.82
VPA	240.42±8.32	251.41±7.29a
VPA+DF LD	239.06±9.11	220.92±9.04b
VPA+DF HD	237.53±6.76	221.51±8.01b

• Data are given as mean±SD (n=4). VPA: Valproic acid (250 mg/kg/day). VPA+DF LD: group pre-treated with VPA and subsequently supplemented with the lower dose of DF (0.15 mg/kg/day). VPA+DF HD: group pre-treated with VPA and subsequently supplemented with the higher dose of DF (0.3 mg/kg/day). ^aindicates significantly different as compared with animals in the control group (p<0.05). ^bindicates significantly different as compared with VPA-treated group (p<0.05).

Effect of treatments on plasma markers of liver damage

Preventive study

The results in Table V show that VPA administration resulted in a significant elevation (p<0.05) of serum levels of TC (70.08±3.37, 91.53±5.06), LDL-C (21.18±1.49, 33.10±2.98) and TG (154.27±6.05, 208.90±9.58), as well as a reduction in HDL-C levels (29.03±1.98, 18.37±1.91) compared to the control group. DF supplementation also produced an elevation of TC levels (70.08±3.37, 93.07±4.11), but induced a reduction in both LDL-C (21.18±1.49, 14.51±1.08) and HDL-C (29.03±1.98, 21.23±1.01), as well as a decrease in TG levels (154.27±6.05, 113.06±4.51) (p<0.05) compared to the control group.

TABLE V
Effects of valproic acid and/or DF treatments on the concentrations of some blood biochemical parameters in rats

Variable	Control	DF	VPA	VPA+DF LD	VPA+DF HD
TC (mg/dL)	70.08±3.37	93.07±4.11a	91.53±5.06a	78.52±4.03b	55.43±3.72b
HDL-C (mg/dL)	29.03±1.98	21.23±1.01a	18.37±1.91a	19.13±1.92a	33.27±1.90b
LDL-C (mg/dL)	21.18±1.49	14.51±1.08a	33.10±2.98a	32.04±1.24a	14.36±1.43b
TG (mg/dL)	154.27±6.05	113.06±4.51a	208.90±9.58a	89.02±3.47a, b	94.74±3.02a, b
AST (U/mL)	65.23±7.85	70.14±6.04	84.67±8.97a	60.22±7.23b	52.51±6.03a, b
ALT (U/mL)	34.05±7.12	36.27±6.12	100.67±7.49a	39.34±7.28b	42.73±6.02ª, b
ALP (U/mL)	311.16±39.21	334.21±45.87b	435.05±54.61a	282.52±35.23b	288.04±30.31b

• Data are given as mean±SD (n=4). TC=Total cholesterol. TG=Triglycerides. HDL-C=HDL cholesterol. LDL-C=LDL cholesterol. AST=aspartate aminotransaminase. ALT=alanine aminotransaminase. ALP=alkaline phosphatase. VPA: valproic acid (250 mg/kg/day). DF: group supplemented with DF (0.3 mg/kg/day). VPA+DF LD: group administered with both VPA and the lower dose of DF (0.15 mg/kg/day). VPA+DF HD: group administered with both VPA and the higher dose of DF (0.3 mg/kg/day). ^aindicates significantly different as compared with animals in the control group (p<0.05). ^bindicates significantly different as compared with VPA-treated group (p<0.05).

Furthermore, DF supplementation significantly reduced TC (78.52±4.03, 55.43±3.72) and TG (89.02±3.47, 94.74±3.02) levels in the VPA-treated animals, although there was an increase in HDL-C levels (33.27±1.90) and a reduction in LDL-C levels (14.36±1.43) in the rats supplemented with the higher dose of DF compared to the VPA group. AST, ALT and ALP activity was significantly higher (p<0.05) in VPA-treated animals in relation to the control group (65.23±7.85, 84.67±8.97; 34.05±7.12, 100.67±7.49; 311.16±39.21, 435.05±54.61) respectively. However, the results show no significant changes (p>0.05) in ALT, AST and ALP activity in the DF supplemented group compared to the control group.

On the other hand, DF supplementation significantly reduced (p<0.05) AST, ALT and ALP activity in the VPA-treated animals compared to the VPA group (84.67±8.97, 100.67±7.49, 435.05±54.61) (60.22±7.23, 39.34±7.28, 282.52±35.23) (52.51±6.03, 42.73±6.02, 288.04±30.31) respectively.

Curative study

The results in Table VI show that VPA administration resulted in a significant elevation (p<0.05) of serum levels of TC (80.05±4.51, 100.31±5.06), and TG (160.21±5.03, 247.72±5.38), as well as a reduction in HDL-C levels (31.37±0.83, 21.06±0.76) compared to the control group. In contrast, DF supplementation significantly reduced TC (90.28±4.31, 89.03±3.78) and TG (190.04±3.56, 140.31±4.42) levels in the VPA pre-treated animals.

TABLE VI
Effects of valproic acid and/or DF treatments on the concentrations of some blood biochemical parameters in rats

Variable	Control	VPA	VPA+DF LD	VPA+DF HD
TC (mg/dL)	80.05±4.51	100.31±5.06a	90.28±4.31a, b	89.03±3.78a, b
HDL-C (mg/dL)	31.37±0.83	21.06±0.76a	21.57±1.63a	21.79±1.43a
LDL-C (mg/dL)	19.22±1.95	19.12±1.75	13.11±2.23b	17.04±1.61
TG (mg/dL)	160.21±5.03	247.72±5.38a	190.04±3.56b	140.31±4.42ª, b
AST (U/mL)	76.09±7.24	73.28±6.07	53.61±5.32ª, b	50.92±5.03ª, b
ALT (U/mL)	48.24±5.09	76.42±6.86a	35.17±8.37ª, b	36.07±4.12ª, b
ALP (U/mL)	349.51±39.43	390.06±53.99	239.61±35.81a, b	322.16±42.94b

• Data are given as mean±SD (n=4). TC=Total cholesterol. TG=Triglycerides. HDL-C=HDL cholesterol. LDL-C=LDL cholesterol. AST=aspartate aminotransaminase. ALT=alanine aminotransaminase. ALP=alkaline phosphatase. VPA: valproic acid (250 mg/kg/day). VPA+DF LD: group pre-treated with VPA and subsequently with the lower dose of DF (0.15 mg/kg/day). VPA+DF HD: group pre-treated with VPA and subsequently with the higher dose of DF (0.3 mg/kg/day). ^aindicates significantly different as compared with animals in the control group (p<0.05). ^bindicates significantly different as compared with VPA-treated group (p<0.05).

ALT and ALP enzyme activity was significantly higher (p<0.05) in VPA-treated animals in relation to the control group (48.24±5.09, 349.51±39.43; 76.42±6.86, 390.06±53.99) respectively. However, DF supplementation significantly (p<0.05) reduced ALT and ALP activity in VPA pre-treated animals compared to the VPA-treated group (76.42±6.86; 390.06±53.99) (35.17±8.37; 239.61±35.81) (36.07±4.12; 322.16±42.94) respectively.

Effect of DF on liver histopathology

Macroscopically, changes in the color and size of livers were seen on VPA-treated animals, relative to those of the control group; this is likely attributable to the presence of lipids in the organ. Although there was a tendency towards an increase in the relative weight of this organ, no significant differences were found among the different experimental groups (data not shown). The results of the histopathological examination of the organs of rats in the control and treated groups are shown in Figures 2 and 3. Moderate hepatic fatty metamorphosis (steatosis), with small cytoplasmic vacuoles in the hepatocytes and some signet ring cells, was observed in the liver of VPA-administered rats (Figure 2A and 2B). In contrast, histopathological results showed a liver with preserved architecture, without hepatocellular damage, in both the untreated control group and the DF-administered group (Figure 3A and 3B). Hepatocytes were found to be neatly arranged to form a hepatic cord around the central vein. Therefore, DF-supplemented animals showed a conserved histological structure, similar to the control group. The results of the histopathological examination of the groups receiving VPA plus DF are also shown in Figure 3 (C-F). Under light microscope, there were no obvious abnormalities in the liver tissue structure in relation to the untreated control group, in both preventive and curative studies. Likewise, the effects of DF on several metabolic parameters correlated with liver histology, since both doses were effective in reducing lipid accumulation, in both preventive (Figure 3C and 3D) and curative trials (Figure 3E and 3F).

FIGURE 2
Effects of treatments on the liver tissue. (A and B) Photomicrographs of liver sections from: (A) VPA group showing the distorted hepatic lobules associated with edema and inflammatory cells infiltration (black arrow); (B) VPA group showing progressive incidence of liver damage (H&E×40).

FIGURE 3
Effects of treatments on the liver tissue. (A-F) Photomicrographs of liver sections from: (A) control and (B) DF groups, showing intact hepatic lobular architecture; (C and D) VPA plus DF (0.15 and 0.3 mg/kg/day), showing a preserved hepatic architecture without signs of degeneration; and from (E and F) VPA plus DF (0.15 and 0.3 mg/kg/day), showing restoration of the histological structure of the liver tissue. (H&E×40). VPA: Valproic acid. DF: Dietary fiber.

As regards the heart and kidney, no obvious microscopically abnormalities were observed in the tissue structure when compared to the control group (data not shown). Consequently, the results obtained in this study suggest that repeated administration of VPA does not compromise the integrity of major organs and therefore does not adversely affect their normal functions.

DISCUSSION

Dietary fiber has been the focus of numerous experimental and epidemiological studies, most of which have demonstrated an inverse relationship between fiber intake and degenerative diseases such as cardiovascular disease, hypertension, and non-alcoholic fatty liver disease (NAFLD) (Maciejewska-Markiewicz et al., 2022). It is now recognized that a significant percentage of NAFLD cases are induced by the administration of drugs that primarily cause mitochondrial damage. As mentioned before, the administration of valproic acid, as anticonvulsant medication in NAFDL patients, is a clear example of that toxic interaction (Di pasqua et al., 2022). Our results regarding the study of possible protective effects of DF to neutralize VPA-induced hepatotoxicity in rats, are shown in Table III. The significant increase in body weight observed in the group of rats treated with VPA compared to the rats in the control group is in agreement with previous reports (Abdelkader et al., 2020). In animals exposed to the preventive trial, supplementation with both doses of DF attenuated the effect of VPA on body weight. The mechanism by which the dietary fiber reduces body weight has been the subject of numerous investigations, and various mechanisms have been proposed, as follows: Dietary fiber increases satiety, decreases energy intake, and/or slows down nutrient absorption (Maciejewska-Markiewicz et al., 2022). Considering the adverse reactions associated with VPA treatment, it is plausible to suggest that the observed effect in this study is due to the ability of DF to increase fecal bulk and stool frequency, thereby promoting lipid excretion (Pérez-Montes de Oca et al., 2020).

On the other hand, DF supplementation did not modify body weight in healthy animals compared to the untreated control group. However, it increased TC concentrations compared to the control group. The reason for this finding is not fully established at present. The possible explanations could be the physicochemical characteristics of the fiber (molecular weight, water solubility, and viscosity), the physiological state of the rats, or the relatively short administration time in this study, which may not have been sufficient to reduce body weight and dampen TC synthesis to baseline level (Jovanovski et al., 2019). Indeed, it has been reported that the effects of increasing dietary fiber intake are more pronounced in individuals with obesity (Cantero et al., 2017).

As shown in Table IV, 14 days after the end of the VPA treatment, a progressive increase in body weight was observed in the unsupplemented animals throughout the study period. In contrast, a significant decrease in body weight was detected in animals supplemented with both doses of DF, indicating that after 14 days of treatment with VPA, supplementation with DF was capable of reversing the effects produced by the drug on body weight. This finding is in agreement with previous studies that demonstrated a more pronounced effect of fiber intake on body weight in obese individuals (Cantero et al., 2017).

Regarding the plasma markers of liver damage, it was observed that the administration of VPA increased the plasma activity of transaminases and ALP, compared to the control group Table V. The observed increase in plasma levels of these enzymes may be a consequence of the hepatocellular damage induced by the agent, resulting in the leakage of membrane components into the extracellular fluid (Guo et al., 2019). In these animals, plasma concentrations of TC, LDL-C and TG were significantly elevated, while those of HDL-C decreased compared to the untreated control group. This finding is consistent with previous studies (Shnayder et al., 2023). In contrast, DF supplementation decreased ALT, AST, and ALP levels, and modulated the lipid profile, significantly reducing TC and TG levels Table V. It is known that hepatic accumulation of TG can exacerbate existing hepatic cell damage. Therefore, the decrease in plasma levels of the mentioned enzymes and TG in supplemented rats suggests that the tested DF has a lipotropic effect. Through this effect, DF prevents lipid accumulation and stabilizes the plasma membrane, thereby preventing lysis and the release of cellular material, and maintaining a healthy bile flow (Zhao et al., 2020).

Meanwhile, in animals supplemented with the higher dose of DF, LDL-C concentrations were also modified and the reduction in TC was more pronounced. These results suggest that the action of DF on TC and LDL-C concentration is dependent on the dose used and the physiological state of the animal, especially because an increase in TC levels at baseline was detected in the DF control group that received the higher dose of 0.3 mg/kg (described above), compared to the untreated group. These findings are consistent with the findings of Jovanovski et al., (2019).

Similarly, as shown in Table VI, 14 days after VPA treatment, rats showed an increase in plasma concentration of TC and TG; besides, an increase in the activities of ALT and ALP was seen but without changes in the plasma concentrations of AST. This finding may be due to the fact that ALT is more liver-specific than AST, with limited concentration in other organs. This enzyme is known to have a longer half-life (37-57 hrs) than AST (12-24 hrs); consequently, ALT elevation persists longer after liver damage has ceased, whereas AST levels increase in response to liver damage but rapidly decrease once liver damage ceases (Cantero et al., 2017).

In contrast, DF supplementation reduced ALT, AST and ALP levels Table VI and modulated the lipid profile, significantly reducing TC and TG levels; however, only the lower dose decreased LDL-C levels. Some fibers, particularly soluble fibers, increase the viscosity of the stomach and digestive tract contents, which interferes with the absorption of bile acids from the ileum. Consequently, LDL-C is eliminated from the blood, and the liver converts it into bile acids to replace the bile acids lost in the feces. This alteration in the composition of the bile acid pool dampens cholesterol synthesis.

Therefore, slowing the synthesis could have a favorable effect on blood cholesterol concentrations, similar to statins (Kopecky et al., 2022). The reduction in TG was more pronounced in animals receiving the higher dose of DF, indicating that the effect on TG is dependent on the dose used. This could be the result of lower fat absorption in the small intestine, as well as preservation of the gastrointestinal tract due to the prebiotic activity of the fiber (Jovanovski et al., 2019).

However, it was observed that the administration of the higher dose of DF significantly increased plasma ALP activity, compared to the group supplemented with the lower dose of DF. Inflammation and hepatic TG accumulation may promote or exacerbate oxidative stress and liver cell damage (Pirozzi et al., 2020). Natural fibers may promote excretion of accumulated fat in hepatocytes; however, a high dose of DF could be detrimental to tissue cells intensely affected by VPA administration, leading to leakage of membrane components into the extracellular fluid. Nevertheless, this increase in ALP was not accompanied by histological changes. As shown in Figure 3 (E and F), histopathological examination of liver tissue from these animals revealed preserved tissue architecture.

Histopathological analysis revealed distorted lobular architecture, accumulation of hepatic lipid droplets and inflammatory cell infiltration in the liver of the VPA-treated rats (Figure 2A and 2B). Similar results have been found in previous studies that also reported distorted lobular architecture of the liver at the same doses (Abdelkader et al., 2020). In contrast, as it is shown in Figure 3 (A and B), examination of liver samples from both control (untreated) and DF-supplemented rats revealed preserved hepatic lobular architecture. The laminae, sinusoids and portal channels of the liver of these rats exhibited clear differentiation.

Interestingly, both doses of DF administered in both preventive and curative trials were effective in reducing the hepatic hepatotoxicity induced by prolonged VPA treatment, as depicted in Figure 3 (C-F). DF supplementation counteracted the tissue damage induced by VPA, resulting in minimal histopathological changes and restoration of normal liver architecture. In this regard, it is known that dietary fiber is fermented by intestinal microorganisms to produce short-chain fatty acids (propionic acid, butyric acid, etc.), which regulate hepatic lipid metabolism (Pérez-Montes de Oca et al., 2020). Less fermentable components can contribute significantly to fecal volume, ease and frequency of bowel movements (Jovanovski et al., 2019).

This effect could favor the excretion of toxic metabolites derived from VPA (4-PA, 4-en-VPA and 2, 4-dien-VPA), in particular valproyl-CoA (Guo et al., 2019; Shnayder et al., 2023). This molecule is not metabolized, and is capable of inhibiting the hepatic carnitine palmitoyltransferase 1A enzyme, which regulates β-oxidation of mitochondrial fatty acids and has been associated with drug-induced hepatotoxic mechanisms and weight gain, commonly observed in patients or animals administered with VPA (Di Pasqua et al., 2022). However, further studies are needed to corroborate this hypothesis.

CONCLUSION

In vivo studies confirmed that DF supplementation prevents and counteracts VPA-induced tissue damage, resulting in minimal histopathological changes and restoration of normal liver architecture. Furthermore, it was also demonstrated in this study that the process developed to obtain DF preserves the physicochemical quality and beneficial properties of the dietary fiber. Consequently, it can be affirmed that the methodology is feasible for revalorizing discarded fruits and vegetables, which could serve as raw material for obtaining dietary fiber that could be used for the production of supplements or food ingredients to treat or prevent drug-induced NAFLD, as well as to address the global obesity epidemic.

ACKNOWLEDGMENTS

Thanks are due to colleagues and friends Natalia and Carlos from the University of Washington (USA) for their collaboration in the preparation of this article.

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Edited by

• Associated Editor:
  Silvya Stuchi Maria-Engler.

Publication Dates

• Publication in this collection
  20 Jan 2025
• Date of issue
  2025

History

• Received
  12 Oct 2023
• Accepted
  17 July 2024