Antioxidant Potential of Solanum nigrum Aqueous Leaves Extract in Modulating Restraint Stress-Induced Changes in Rat's Liver.

INTRODUCTION: This study was carried out to evaluate the antioxidant potential of crude extract of Solanum nigrum leaves and its active constituents as treatment against restraint stress in rat's liver.
METHODS: For this purpose, male albino Wistar rats were treated with crude extract of leaves and its alkaloid and flavonoid fractions both before and after 6 h of acute restraint stress. Prooxidant status of rat liver was assessed by determining the levels of thiobarbituric acid reactive substances, reduced glutathione, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, and the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST).
RESULTS: Six hours of restraint stress generated oxidative stress in rat's liver resulted in a significant rise in the level of the aforementioned liver enzymes. On the other hand, SOD, CAT, and GST enzymatic activities showed a significant decline in their level. The administration of crude leaves extract, both before and after stress exposure, significantly prevented the rise in the level of liver enzymes and reverted the activities of studied biochemical parameters toward their normal control values. However, the reversion was found to be more prominent in after-stress group.
CONCLUSION: The aforementioned results highlight the significant antioxidant potential of S. nigrum extracts. On the basis of our study, we suggest the possible use of S. nigrum leaves extract as a nutritional supplement for combating oxidative stress induced damage.

INTRODUCTION

There has been an upsurge in the clinical use of ethnic drugs, such as herbal plants, against a diverse pathophysiological states.[12] Solanum nigrum is a weed of wasteland, up to 90 cm tall with dark green leaves, and is found abundantly in most parts of India and Southern Europe. S. nigrum flowers are white with a small pedicel with five widely spread petals. It is traditionally used as food and also has some pharmacology effects such as fever reduction, diuretic action, liver protection, eyesight improvement, blood pressure control, and glucose tolerance.[3] S. nigrum extract contains high amount of polyphenols showing its antioxidant and antitumor properties. Some of the other beneficial uses of S. nigrum extract include its action against microbial infections, cure of skin diseases, and as a hypoglycemic and antiulcerogenic agent.[345] Earlier studies highlighted the antiatherogenic and hepatoprotective properties of S. nigrum, therefore this study was conducted to examine whether S. nigrum extract can reduce the liver oxidative stress induced by acute restraint stress.

Stress is a collective occurrence that is drawing cumulative attention as a cofactor in the severity and development of numerous diseases.[67] Stress plays an important role in aggravating several ailments, particularly hepatic inflammation.[89] The key outcome of stress on the liver is exclusively linked to changes in the hepatic blood stream[10] with centrilobular hypoxia, subsequent vasospasm, and eventually liver damage.[11] Hepatosis is a vigorous and extremely unified cellular response that prolonged liver injury.[812] Cirrhosis is characterized by fibrosis and nodule formation of the liver. It is escorted by albumin, altered prothrombin, insulin resistance, and cholesterol synthesis.[13] The mild grade of liver injury that is frequently present in cirrhosis may be accountable for an upsurge in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as lactate dehydrogenase 5.[14] Oxidative stress has been concerned in the growth of both experimental and clinical liver fibrosis.[151617] Restraint stress is a valuable and investigational animal model for studying oxidative stress cohort.[21819] In restraint stress model, there is a decrease in free radical-scavenging enzyme activities along with an increase in lipid peroxidation, which may assist cellular and tissue proceedings and is accountable for chronic liver damage.[2021]

There are numerous first-line defense against stress in the animal system, namely, vitamin E (tocopherol), A (retinol), and C (ascorbic acid), and glutathione (GSH).[2223] The levels of GSH content is solely linked to nutritional status, particularly the cysteine-containing nutrition, which does not disturb the enzymes involved in its production.[24] Stress has been also reported for a significant role in the evolution of cirrhosis and hepatic fibrosis. Therefore, we examined the antioxidant potential of crude leaves extract of S. nigrum on the restraint stress-induced oxidant/prooxidant status of rat hepatic tissues. The plant leaves extract was given to rats both before and after the restraint stress and the results were inferred in terms of modulation in the liver antioxidant enzyme activities, marker enzymes, and levels of total GSH and thiobarbituric acid reactive substances (TBARS). We believe that the results of this study would increase our understanding toward potential antioxidant property of S. nigrum leaves to alleviate/prevent stress-induced ailments linked with oxidative injury to liver cellular constituents.

MATERIALS AND METHODS

Reagents and chemicals

Bovine serum albumin, 1-chloro-2,4-dinitrobenzene (CDNB), and thiobarbituric acid were purchased from Sigma (St Louis, Missouri). Pyrogallol, hydrogen peroxide, and 5-5’-dithiobis-2-nitrobenzoic acid (DTNB) were procured from E-Merck (Darmstadt, Germany). All additional chemicals used in this study were of analytical grade and purchased from various commercial sources.

Animal stress procedure and treatments

Albino Wistar strain male rats (180–200 g) were used in this study of approximately 3 months. The rats were housed and preserved under 12-h light/12-h dark cycles at 24°C for 1 week (for adaptation) and during the experiment. The rats were on rat feed pellets diets (H-Liver, Bombay, India) and water ad libitum. The animals were kept in polypropylene cages conferring to the investigational protocols, the wire mesh cages, and aseptic bed of husk. All the experimental protocols adhered to guidelines of the animal welfare committee of the university.

The acute restraint stress was induced by restraining individual rats inside the cylindrical wire mesh cages (approximately 8 cm diameter and 18 cm length) attached to a wooden board. Control rats were left freely walking in their respective cages.

A pilot study was performed with 50, 100, 150, and 200 mg/kg of crude leaves extract of S. nigrum using three animals per dose to find out the optimum therapeutic dose of extract that can modulate deranged free radical metabolism (results not shown). On the basis of optimized dose treatments, we observed that the extract at 100 mg/kg body weight (BW) showed the best antioxidant effect on oxidative stress in liver. Hence, this optimized dose was selected for evaluating the antioxidative potential of crude extract.

Experimental protocol

To examine the antioxidant potential of S. nigrum extract on restraint stress rat’s liver, environment acclimatized 70 rats (male, 10 weeks old, 180–200 g) were randomly selected and distributed into five groups (n = 14 per group). The groups were: Group I: without any treatment (CON-group); Group II: this group was given 6 h of restraint stress deprived of any other treatments (STR-group). Animals in this group were sacrificed 1 h after removal of restraint stress to account for the effect of possible natural relaxation after the stress. Group III: normal rats treated with a single oral dose of 100 mg/kg of S. nigrum extract to obtain the baseline data of extract per se effect (S.NIG-group). These rats were sacrificed 7 h after dose administration along with stress-induced posttreatment animals. Group IV: this set of animals was pretreated with a single dose of 100 mg/kg BW of S. nigrum extract, 1 h before they were subjected to restraint stress (Pre-S.NIG + STR-group) and Group V: a group that was treated with extract after 6 h of stress (STR + Post-S.NIG-group). Rats were exposed to acute restraint stress for 6 h, between 7 AM and 1 PM, by engaging animals into a cage of wire mesh of their size, the cage was attached to a board as reported earlier.[25] The animals were deprived of food and water through out the experiment. The rats were treated with a single dose (100 mg/kg) of liquid extract via oral route with the help of gavage needle. After 1 h completion of stress treatments, animals were given pentobarbital intraperitoneally (50 mg/kg BW) and then sacrificed. Control non-stressed rats (with or without S. nigrum extract) and animals treated with extract before restraint stress were handled at the same time similar to stressed groups.

Preparation of homogenate

All the animals were carefully dissected and the liver tissues were cleared of adhering connective tissues, weighed, and homogenized with sterile (ice cold) 0.1 M sodium phosphate buffer (pH 7.4). The homogenate was then centrifuged at 3000g for 15 min at 4°C on Beckman Coulter centrifuge (rotor radius: 20.4 cm) to eliminate cellular wreckage. The supernatant was aliquoted and stored in −20°C for biochemical assays.

Superoxide dismutase assay

The superoxide dismutase (SOD) activity in the liver samples was measured by the method of Marklund and Marklund.[26] The activity of SOD depends on the pyrogallol autoxidation in the existence of Tris succinate buffer (0.05 M, pH 8.2). The SOD activity was calculated as the inhibition of pyrogallol autoxidation at 412 nm.

Glutathione S-transferase assay

The glutathione S-transferase (GST) assay in the liver samples was performed by the method of Habig et al.[27] The substrate CDNB was used in this assay. The CDNB-GSH conjugate generated is measured by the increase in absorbance at 340 nm.

Lipid peroxidation assay

The method by Halliwell and Chirico[28] was followed to measure the liver tissue’s levels of lipid peroxidation. The pink color appeared was spectrophotometrically measured with an extinction coefficient of 156 mM/cm at 532 nm.

Catalase assay

The catalase (CAT) activity in the liver samples was assayed based on the method by Beers and Sizer[29] with hydrogen peroxide (30 mM) as the substrate.

Total glutathione assay

The procedure by Sedlak and Lindsay[30] was used to quantify the liver GSH levels. The procedure is based on the reduction of 0.01 M DTNB by sulfhydryl groups of GSH to form 2-nitro-5-mercaptobenzoic acid per moles of GSH.

Alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity

The activities of ALT, AST, and alkaline phosphatase (ALP) were measured by using specific kits from Reckson Diagnostic (Delhi, India).

Protein estimation

Protein in the liver tissue homogenate was quantified by the method by Lowry et al.[31] by using bovine serum albumin as standard.

STATISTICAL ANALYSIS

All the data are expressed as mean ± standard deviation (SD) of triplicate values. Statistical analyses were performed using GraphPad Prism, version 4.0, software (GraphPad Software, La Jolla, California). One-way analysis of variance (ANOVA) was also used to compare control group versus the stressed group and stress + pre- and post-extract-group, using Dunnett’s test. P value < 0.05 was considered as statistically significant.

RESULTS

This study showed that 6 h of restraint stress resulted in a significant decline in the activities of SOD, CAT, GST, and GSH along with a significant increase in TBARS, AST, ALT, and ALP compared with both CON-group (untreated and non-stressed rats) and S. nigrum group (extract treated but non-stressed) [Figures 1 and 2]. Thus, indicating that restraint stress significantly induced oxidative stress in rats (STR-group). A single dose of S. nigrum extract (100 mg/kg BW) did not result in any significant change in general behavior, food intake, or BW of animals, and the aforementioned biochemical parameters. However, posttreatment with crude extract markedly neutralized restraint stress in the studied antioxidant enzymes and the levels of GSH, ALP, AST, and ALT. Administrations of aqueous extract both before and after the restraint stress resulted in significant modulations in liver and antioxidant enzyme levels. However, the post-stress oral administration of extract (100 mg/kg BW) was found to be more effective in restricting stress-induced decline in SOD, CAT, GST, and GSH and increase in levels of TBARS, AST, ALT, and ALP than prestress extract treatment [Figures 1 and 2].

Figure 1

Effect of crude Solanum nigrum leaves extract treatment on immobilization stress-induced changes in liver’s superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione (GSH). Significant decrease in antioxidant enzymes activities and glutathione level were observed after immobilization stress. The pre- and post-stress extract treatment revert the deranged free radical system toward their normal values with a relative dominance by latter. (*P values compared with controls, #P values compared with stressed rats, where * <0.05 and # <0.05)

Figure 2

Effect of crude extract of Solanum nigrum leaves on restraint stress-induced changes in liver’s glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), and malondialdehyde (MDA). Significant increase in liver markers enzymes along with a significant increase in MDA levels were observed after restraint stress. The pre- and post-stress extract treatment revert the deranged free radical system toward their normal values with a relative dominance by latter. (*P values compared with controls, #P values compared with stressed rats, where * <0.05 and # <0.05)

Intragastric administration of active constituents resulted in no significant changes in the antioxidant enzyme activities and other biochemical parameters. However, the treatment of these active constituents both before and after the restraint stress resulted in a significant reversion of values toward their control values but a relative dominance by latter [Table 1].

Table 1

Effect of crude Solanum nigrum leaves extract and its active constituents treatment on restraint stress induced changes in liver’s superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), MDA, glutathione (GSH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). The pre- and post-stress extract treatment revert the deranged free radical system toward their normal values with a relative dominance by latter

Groups (6)	SOD (U/mg protein)	GST (U/mg protein)	CAT (U/mg protein)	MDA (nmol/mg protein)	GSH (mmol/mg protein)	AST (units)	ALT (units)	ALP (nmol/min/ mg protein)
Control (6)	14.152 ± 3.21	145.541 ± 6.21	14.216 ± 2.31	15.485 ± 1.41	12.584 ± 0.21	60.125 ± 3.15	40.254 ± 6.21	1.752 ± 0.21
Stressed (6)	6.124 ± 1.21d	80.548 ± 8.21c	5.652 ± 2.14d	25.016 ± 2.45d	6.214 ± 2.10d	110.564 ± 5.32d	75.541 ± 4.21b	2.985 ± 0.42a
Crude extract (6)	15.214 ± 1.25	140.486 ± 9.48	15.014 ± 1.58	16.521 ± 1.44	11.254 ± 2.65	58.511 ± 2.65	35.452 ± 1.58	1.521 ± 0.32
Alkaloid Fraction (6)	13.354 ± 2.15	135.214 ± 10.21	12.524 ± 2.48	14.598 ± 1.65	11.965 ± 1.15	59.555 ± 1.21	39.247 ± 5.47	1.421 ± 0.12
Prestressed alkaloid treatment (6)	11.254 ± 1.54c’	100.247 ± 8.41b’	8.541 ± 2.15a’	20.121 ± 2.65a	8.254 ± 1.85a’	85.214 ± 5.32a’	61.145 ± 5.62b’	2.248 ± 0.52b’
Post-stressed alkaloid treatment (6)	11.248 ± 1.98a’	121.294 ± 12.14b’	12.547 ± 2.45b’	22.154 ± 3.85c’	11.214 ± 2.36b’	95.214 ± 9.32a’	50.249 ± 1.54b’	2.014 ± 0.41d’
Flavonoid (6)	16.254 ± 3.25	154.965 ± 16.21	15.278 ± 1.98	16.852 ± 2.74	13.524 ± 1.35	65.214 ± 5.41	41.541 ± 5.48	1.772 ± 0.21
Prestressed flavonoid treatment (6)	11.214 ± 2.65a’	129.521 ± 10.45a’	12.154 ± 2.56b’	13.541 ± 2.16a’	12.325 ± 3.65a’	75.541 ± 4.32b’	55.744 ± 5.45c’	2.054 ± 0.11c’
Post-stressed flavonoid treatment (6)	15.264 ± 1.21a’	139.547 ± 11.14b’	16.524 ± 3.12c’	11.521 ± 3.54c’	16.521 ± 1.65d’	59.149 ± 4.21d’	43.145 ± 5.3c’	1.693 ± 0.11c’

aP < 0.05, bP < 0.02, cP < 0.01, and dP < 0.001 as compared with control rats

a’P < 0.05, b’P < 0.02, c’P < 0.01, and d’P < 0.001 as compared with stress alone. Number of animals i.e n = 6

DISCUSSION

In the living systems, stress is defined as an imbalance between the antioxidant and prooxidant, which is very detrimental to cells.[1032] Oxidative stress defense solely depends on an orchestrated synergism between the exogenous and endogenous antioxidant. Peroxidation of polyunsaturated fatty acid membrane leads to liver damage or injury, which results in the production of malondialdehyde.[2433] The decrease in the fluidity of the biomembrane due to lipid peroxidation may impair major metabolic functions and depends upon membrane structure and integrity.[3435]

Restraint stress is a convenient and easy method of persuading both physical and psychological stress, which results in aggression and restricted mobility.[8] Restraint stress has also been reported to bring out the antioxidant defense changes in rat liver.[36] SOD, CAT, and GST play a vital role in scavenging oxy-radicals and their products.[2237] The stability of living organism must be maintained by its balance between oxidative and antioxidant defense. In this study, 6 h of restraint stress resulted in the production of reactive oxygen species (ROS) in rat’s liver, indicated by decline in GSH level and free radical-scavenging enzymes along with increase in lipid peroxidation [Figures 1 and 2]. The generated ROS can attack biomolecules directly, with consequential increases in membrane lipid peroxidation (LPO), which could be the major mechanism of liver cell injury.[383940] Moreover],[ increase in lipid peroxidation of liver has also been revealed to be associated with decrease in endogenous GSH.[4142]

Tissues such as liver, brain, and heart challenged an increase in the TBARS levels because of short- and long-term stress[4344] accompanied by a decline in hepatic GSH.[4445] Decreased activities of antioxidant enzymes such as CAT, GST, and SOD might lead to a significant depletion in hepatic GSH. Thus, decreased GSH level in restraint stressed rats may be associated with enhanced vulnerability of the plasma membrane to peroxide outbreak. In the last one decade, a number of plant origin drugs have been examined for their valuable effect in humans. Lately, S. nigrum has been a subject of considerable contemporary research. S. nigrum is widely used in many traditional systems of medicine worldwide for disparate ailments, but has not gathered attention for modern therapeutic use. The anti-stress potential of this plant has not been clearly outlined as yet and in this study, we observed some biochemical modulations by S. nigrum, which explain their adaptive role against free radical damage.[4647] This study showed the beneficial effect of crude extract of S. nigrum, which is confirmed by the deterioration of stress change in ALT and AST marker enzymes toward their normal values [Figure 2].

This study clearly showed that intragastric administration of crude extract of S. nigrum leaves or alkaloid/flavonoid significantly modulates the circulating activities of CAT, GST, SOD, LPO, GSH, AST, ALT, and ALP [Table 1]. Various studies suggested anti-inflammatory potential of flavonoids[48] and identified them as an antioxidant or free radical scavengers.[4950] Flavonoid fraction of S. nigrum leaves showed an antioxidant consequence by enhancing the activities of SOD, GST, CAT, and GSH. Other flavonoids from dissimilar origins are also reported to enhance CAT and GST activities.[4647484950]

S. nigrum possesses various compounds that are responsible for diverse activities. The major active components are alkaloids/flavonoids, glycoprotein, and polysaccharides. It also contains polyphenolic compounds such as gallic acid, catechin, protocatechuic acid, epicatechin, rutin, and naringenin. It comprises 95% of the total alkaloid concentration present in the plant and is found naturally in any part. It is one of the plant’s major natural defenses as it is toxic even in small quantities.

On the basis of previous studies, the extract of S. nigrum in this study was observed to prevent and normalize oxidative stress deranged free radical metabolism, which was evident by the return of the deranged activities of GST, CAT, and SOD and the levels of TBARS, GSH, ALT, AST, and ALP toward their normal values, as compared to either untreated controls or stress-alone treated groups. The extract of S. nigrum served as a free radical scavenger that enhances the activities of CAT, SOD, GST, and GSH. The treatment of S. nigrum extract before the stress exposure resulted in a significant resistance toward the deranged free radical metabolism induced by restraint stress, though the posttreatment of extract of S. nigrum was found to be more effective in restoring the aforementioned biochemical parameters toward their normal values. S. nigrum is testified to act as an active antioxidant of major rank against diseases and worsening process caused by oxidative stress.[5152] Several studies reported that S. nigrum contains several polyphenolic compounds, mainly steroids and flavonoids along with various other constituents, namely, nicotinic acid, riboflavin, β-carotene, citric acid, oils, and vitamin C. The antioxidant property of this plant is believed to be due to the presence of β-carotene, polyphenolic compounds, and vitamin C.[52]

Financial support and sponsorship

This study was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.

Conflicts of interest

There are no conflicts of interest.