Salvia hispanica (Chia) seeds afford hepatoprotection against Isoniazid and Rifampicin induced toxicity in a murine model

 

Nayak Apoorva¹, Rao Rashmi R¹, Shenoy Preethi J¹, H Sindhu², S Teerthanath³, Bhuvaneshwari⁴

¹Department of Pharmacology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India-575001.

²Department of Biochemistry, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India-575001.

³Department of Pathology, K S Hegde Medical Academy, Nitte University, Deralakatte, Mangalore.

⁴Department of Pharmacology, Karpagam Faculty of Medical Sciences and Research, Coimbatore, Tamil Nadu.

 

ABSTRACT:

Salvia hispanica L. (chia) seeds are a rich source of omega-3 fatty acid and polyphenolic compounds with documented antioxidant property protecting against oxidative stress, which is implicated in the pathophysiology of drug-induced hepatotoxicity. Hence, the present study evaluates the effects of Salvia hispanica seeds in an animal model of antitubercular drug induced hepatic damage. Adult wistar rats were divided into six groups; normal control, hepatotoxic control, test groups treated orally with ground Salvia hispanica seeds at doses 250,500 and 1000mg/day respectively, standard control receiving silymarin at a dose of 50mg/kg orally. Hepatotoxicity was induced by administering isoniazid with rifampicin at a dose of 100mg/kg each intraperitoneal. After administration of drugs for a period of 21 days, the blood samples were evaluated for estimation of liver enzymes (AST, ALT, and ALP), total protein, albumin and total bilirubin and the liver was dissected and sent for histopathological evaluation. The hepatotoxic group showed a significant increase in liver enzymes and total bilirubin compared to normal control. There was a significant decrease in liver enzymes and total bilirubin in the drug treated and silymarin treated groups when compared to the control. However, there was no significant difference in protein and albumin between groups. Histopathological evaluation of the liver further confirmed the hepatoprotective potential of the seeds of Salvia hispanica. Salvia hispanica seeds protect against drug induced liver injury in a murine model and the underlying mechanism can be accorded to its antioxidant activity.

 

 

 

INTRODUCTION:

Drug-induced damage to the liver is a very common occurrence as it is the main site of drug metabolism contributing to various adverse drug reactions¹. Hepatotoxicity is a serious concern with the use of Isoniazid (INH) and rifampicin (RIF) which comprise of the main drugs in the armamentarium of antitubercular regimens advocated currently in India. It has also been implicated in reducing the effectiveness and compliance of antitubercular therapy².

 

Hepatotoxicity induced by these drugs could be attributed to a direct noxious effect of the principle compound/metabolite, or an immune mediated reaction involving the liver vasculature, hepatocytes and biliary epithelial cells³. The hepatotoxicity by INH is due to conversion of its metabolite into a toxic metabolite which binds covalently to microsomal proteins and induces damage to the hepatocytes⁴. RIF induces the cytochrome P450 enzymes thereby producing more active noxious metabolites of INH and contributes to increased hepatic damage⁵. Reactive oxygen species (ROS) generated further stimulate lipid peroxidation and propagates cell damage by altering mitochondrial permeability and inducing apoptosis⁶. Therefore the use of antioxidants signifies a rational strategy to prevent and cure hepatic damage involving oxidative stress.

Salvia hispanica L. also known as chia is widely used because of its massive nutritional and therapeutic potential. The chia seed is a rich source of dietary fibre, proteins, essential fatty acids (including polyunsaturated fatty acids like Omega 3 alpha-linolenic acid and Omega 6 linoleic acid), vitamins, minerals, polyphenolic compounds and a high amount of antioxidants⁷. Omega 3 fatty acids have been implicated in modulating various physiological functions. Its cellular antioxidant property protects against hepatotoxicity and apoptosis, reduces lipid peroxidation and improves the body’s antioxidant system⁸. Determination of phytochemicals in chia seed showed that it contains polyphenolic compounds such as rosmarinic acid, kaemferol, myricetin, quercetin, flavonol glycosides, caffeic acid, and chlorogenic acid⁹. These compounds have proven antioxidant actions, ascribed to their free-radical scavenging property, metal chelation¹⁰, antioxidant enzymes and glutathione elevation and a diminishing lipid peroxidation in the hepatocytes¹¹.

 

Phytochemicals in herbal products have been extensively evaluated due to their potential and efficacy in drug induced hepatotoxicity and the underlying basis for the protection is hypothesized to be their antioxidant property¹². Since there was no scientific evidence on hepatoprotection afforded by Salvia hispanica, the current study was designed to analyse the actions of Salvia hispanica seeds in a murine model of INH and RIF induced hepatic damage.

 

MATERIALS AND METHODS:

The study was conducted in accordance with the recommendations set by the committee for the purpose of control and supervision of experiments on animals and the necessary ethical approval was obtained from the institutional animal ethics committee.

 

Experimental Animals:

Adult wistar albino rats of either sex, weighing 150-200 grams, inbred in the institutional animal house were used for the study. Animals were housed in clean polypropylene cages under standard environmental conditions (22± 3⁰C, 55 ± 5% humidity and a 12 h light/ dark cycle) and fed with standard rodent diet and water ad libitum. A period of one week was set aside for acclimatization of the rodents prior to the experiment.

 

Drugs:

Isoniazid was obtained from Sigma Aldrich Co. and rifampicin from Sisco research laboratories Pvt. Ltd. Silymarin tablets were procured from Signova Co. and served as a standard control. Normal saline was used as a control. The drugs were suspended in a suitable vehicle and administered either oral (p.o.) or intraperitoneal (i. p.).

 

Plant material and preparation of extract:

A local botanist authenticated Salvia hispanica seeds obtained locally. The voucher specimen of the plant (F.1721514) has been conserved in the local herbarium. The seeds were dried in an incubator for 2 days at 40^oC, levigated and dissolved in distilled water. It was then administered through the oral route. The dose of Salvia hispanica seed is extrapolated from previous human trials and literature^13-15.

 

Evaluation of hepatoprotective activity:

The animals were divided into six groups, comprising of six animals each (n=6). The animals were treated with intraperitoneal injections of isoniazid co-administered with rifampicin to induce hepatotoxicity at a dose of 100 mg/kg body weight each for 21 days¹⁶. Silymarin (50 mg/kg body weight) administered orally was used as a reference drug in this study¹⁷. The test drug ground Salvia hispanica seeds dissolved in distilled water was administered orally at doses 250, 500 and 1000 mg per day respectively for 21 days^13-15. The treatment protocol is depicted in Table 1.

 

Table 1. Treatment protocol

Groups (n=6)	Treatment received
Group 1 Normal control	Normal saline (10ml/kg), p.o.
Group 2 Hepatotoxic control	INH+RIF (100 mg/kg body weight each) i.p. once daily for 21 days
Group 3 Test group	INH+RIF (100 mg/kg body weight each) i.p. once daily for 21 days+ Salvia hispanica250 mg/day p.o. for 21 days
Group 4 Test group	INH+RIF (100 mg/kg body weight each) i.p. once daily for 21 days + Salvia hispanica500 mg/day p.o. for 21 days
Group 5 Test group	INH+RIF (100 mg/kg body weight each) i.p. once daily for 21 days + Salvia hispanica1000 mg/ day p.o. for 21 days
Group 6 Standard control	INH+RIF (100 mg/kg body weight each) i.p. once daily for 21 days + Silymarin 50mg/kg body weight p.o. for 21 days

n-number,INH- Isoniazid, RIF-Rifampicin, p.o- per oral, i.p-intraperitoneal

 

On day 21, one hour following drug administration, the blood samples were collected by cardiac puncture under ketamine-xylazine anesthesia¹⁸, for biochemical estimation. These blood samples were centrifuged for 10 minutes at 3000 rpm to separate the serum. The rats were then sacrificed and the liver dissected out for histopathological evaluation.

 

Biochemical parameters assessed:

The levels of total protein, albumin and bilirubin were estimated from the serum, which serve as indicators of liver function. The activities of serum hepatic enzymes namely aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were analyzed. All the above parameters are estimated using commercially available kits from Diatek healthcare Pvt. Ltd.

 

Histopathological investigation:

The liver tissues dissected out were preserved in 10% formaldehyde, and later embedded into paraffin blocks. Thin sections (5μM) were then prepared using rotary microtome. Sections were stained with hematoxylin and eosin and then observed under a microscope for degeneration, necrotic changes, fatty changes, inflammation, fibrosis and evidence of hepatotoxicity.

 

Statistical analysis:

The results were expressed as Mean ± SEM. The data was analyzed using One-way ANOVA followed by Tukey’s test. The SPSS version 20 was used to conduct the statistical analysis. A p value <0.05 was considered significant.

 

RESULTS:

Effect on serum biochemical parameters:

In the disease control, a significant elevation of biochemical enzyme markers AST, ALT and ALP was observed when compared to the normal control indicating liver damage as shown in table 2. Pre-treatment with Salvia hispanica at doses 250, 500and 1000mg per day showed a decrease in these enzyme markers when compared to hepatotoxic group signifying significant hepatoprotection afforded by Salvia hispanica.The group treated with silymarin 50mg/kgbody weight also showed a statistically significant decrease in the hepatic enzymes as depicted in Table 2.The decrease in serum hepatic enzymes in the treatment group was not significant when compared to silymarin.Total bilirubin significantly increased in the hepatotoxic group compared to normal control. Salvia hispanica at doses 250, 500 and 1000 mg per day and silymarin 50mg/kg body weight showed a significant hepatoprotection as evidenced by a decrease in total bilirubin compared to hepatotoxic group. (Table 3)

 

Table 2: Effect of Salvia hispanica on liver enzymes in isoniazid and rifampicin induced hepatotoxicity in rats

Group	AST(IU/L)	ALT(IU/L)	ALP(IU/L)
Normal control Normal saline (10ml/kg)	95.36±1.98	31.58±0.49	90.80±2.73
Hepatotoxic control INH+ RIF(100 mg/kg )	480.46±54.36	95.62±1.73	546.98±74.62
Standard control Silymarin (50mg/kg)	196.45±2.18$	47.23±1.08$	129.21±6.64$
Salvia hispanica 250mg/day	336.35±10.20*	64.75±1.02*	234.05±9.42*
Salvia hispanica 500mg/day	333.95±18.81*	66.98±1.26*	197.48±3.75*
Salvia hispanica 1000mg/day	219.11±4.31*	53.33±0.91*	169.46±14.31*

Values are expressed as Mean± SEM, ^$ p < 0.05 when compared to hepatotoxic group, ^* p < 0.05 when compared to hepatotoxic group, INH- Isoniazid, RIF-Rifampicin, AST- aspartate aminotransferase (AST), ALT-alanine aminotransferase, ALP- alkaline phosphatase

 

Table 3: Effect of Salvia hispanica on Total protein, Albumin, Total Bilirubin in isoniazid and rifampicin induced hepatotoxicity in rats

Group	Total protein (g/dl )	Albumin (g/dl )	Total bilirubin (mg/dl)
Normal control Normal saline (10 ml/kg)	7.20±0.20	3.31±0.04	0.68±0.26
Hepatotoxic control INH+ RIF(100 mg/kg)	7.00±0.47	3.22±0.66	1.72±0.20
Standard control Silymarin (50mg/kg)	7.60±0.23	3.25±0.10	0.21±0.62$
Salvia hispanica 250mg/day	7.80±0.23	3.31±0.10	0.59±0.28*
Salvia hispanica 500mg/day	7.68±0.43	3,06±0.21	0.27±0.19*
Salvia hispanica 1000mg/day	6.95±0.22	3.16±0.12	0.23±0.12*

Values are expressed as Mean± SEM, ^$ p < 0.05 when compared to hepatotoxic group, ^* p < 0.05 when compared to hepatotoxic group, INH- Isoniazid, RIF-Rifampicin

 

Histopathological evaluation:

The control group showed a normal liver architecture with no evidence of hepatic damage. (Figure 1). The liver section treated with isoniazid and rifampicin showed hepatocellular disintegration with portal inflammation, hepatocyte necrosis, fibrosis, fatty degeneration, dilated sinusoids with proteinaceous material (Figure 2). Treatment with Salvia hispanica seeds at doses 250, 500 and 1000mg per day showed minimal portal infiltration, necrosis, and focal cell death and fatty changes (Figure 3, 4, 5). At doses 500 and 1000mg of Salvia hispanica histological changes were significant with the presence of regenerating atypia illustrating protection against the isoniazid and rifampicin induced damage The silymarin treated group also showed maintenance of normal architecture of the liver signifying hepatoprotection. (Figure 6). Table 4 depicts the above findings.

 

 

 

Table 4: Histological changes observed in hepatic tissue sections of different groups

Groups	Fatty degeneration	Necrosis	Portal inflammation	Fibrosis	Regeneration Atypia	Others
1 Normal control	--	--	--	--	--	--
2 Hepatotoxic control	+++	+++	F +++L, N	F++	--	DS
3 Salvia hispanica 250mg	+++	+	++L, N	F++	++	--
4 Salvia hispanica 500 mg	F+	--	+L, OCC N	+	++	--
5 Salvia hispanica 1000mg	F+	--	SCT L, N	+	++	--
6 Silymarin 50mg	OCC	SCT N	--	--	+	--

F-Focal, DS-Dilated sinusoids, OCC- Occasional, SCT-Scattered, L-Lymphocytes, N-Neutrophils, + Positive, -Absent.

 

DISCUSSION:

Combined administration of INH and RIF produces morphological and metabolic changes in the hepatocytes as the liver is their main detoxifying site. INH + RIF in response to stress generates the noxious metabolite hydrazine, which causes a alteration of mitochondrial functions and induces programmed cell death. RIF, a strong inducer of cytochrome P450 2E1 (Cyp2E1) and INH hydrolase potentiates the toxic effects of INH¹⁹. INH and RIF have been commonly employed in animal models to elucidate cellular mechanisms and generate evidence for therapeutic utility of various drugs and dietary antioxidants. Evidence suggests that oxidative stress leading to lipid peroxidation is crucial for cytotoxic action of INH and RIF. Increased peroxidative processes lead to impairment of circulating antioxidants and intracellular scavenging enzymes altering various cellular defence mechanism culminating in hepatic injury. Previous studies have shown that the drugs opposing oxidative stress are likely to show a significant role in hepatoprotection^20,21.

 

Elevation of the hepatocellular enzymes which act as biomarkers concentrated mainly in the liver is speculative of drug induced necrosis of the hepatocyte²². Serum bilirubin estimation illustrates the normality of hepatic function. An abnormal increase might be suggestive of a severe impairment of hepatocellular functions²³. The results of this study revealed significant alteration in biochemical parameters as shown by an increase in the liver enzymes and bilirubin in the hepatotoxic group indicating hepatic damage caused by INH+RIF. This is re-enforced by the histological changes of the liver that include loss of normal liver architecture, fatty degeneration, necrosis, fibrosis, portal inflammation, and dilated sinusoids. The histology and biochemical analysis in INH+RIF treated group showcased in the present study is in concordance with earlier studies^16,17,24.

 

Treatment with ground seeds of Salvia hispanica at doses 250, 500 and 1000mg per day provided marked protection against INH+RIF induced hepatotoxicity as revealed by a significant lowering of the liver enzymes and bilirubin. This was further evidenced in the histological evaluation; concurrent administration of Salvia hispanica appeared to diminish the gravity of the INH+RIF induced liver necrosis, fatty degeneration and inflammation resulting in the preservation of the liver architecture. The signs of regeneration atypia were also seen with two doses administered. Restoration of hepatic structure and function in Salvia hispanica treated group provides unswerving evidence for hepatoprotective action of this product. The standard control silymarin also illustrated protective effects by decreasing the biochemical markers and the preservation of normal histology. The changes observed in total protein and albumin levels failed to show significance in all the treatment groups.

 

Since free radical induced oxidative stress signifies to be the culprit in causing liver damage, natural antioxidants used as medicine or dietary supplement might accord relief and help to maintain vitality. Plants contain a wide variety of bioactive molecules exhibiting an extensive array of pharmacological properties. Salvia hispanica with its excellent documented antioxidant action and also being a vital source of omega-3 fatty acid and polyphenolic compounds might offer significant protection against the damage induced by highly reactive oxygen species^10,25. These compounds are also believed to protect against cancer, aging and cardiotoxicity. Chia seeds have been proved to be superior to its other natural counterparts in terms of free radical scavenging activity¹³ as documented by various researchers like S.C. Oliveira-Alves et al. The phenolic compounds in chia seeds have been analyzed and expressed to provide protection against oxidative stress²⁶. Scapin et al has shown that phenolic compounds and flavonoids are the active principles according antioxidant capacity to chia seed extracts²⁷. Corroborative evidence by in vitro antioxidant activity studies of chia seed provided by Marineli et al have proved that it reduces lipid peroxidation in diet-induced obese rats^28,29. Rodrigues et al also reported an increase in resistance against free radical and reduced lipid peroxidation with chia seed extract in an animal model using nematode²⁵. Salvia hispanica can protect biomolecules in membranes and also prevent their oxidation due to the above properties.

 

Therefore we propose that the hepatoprotection shown by Salvia hispanica in INH+RIF induced hepatotoxicity may probably be due to its potent antioxidant effect as shown by earlier studies. Further studies can shed more light on the association between the antioxidant effect and hepatoprotection afforded by Salvia hispanica and improve our understanding of the mechanisms involved in attenuating INH+RIF induced hepatic damage. To conclude, the present study is an attempt to provide scientific evidence for hepatoprotective effects of orally administered ground seeds of Salvia hispanica in INH+RIF induced hepatic damage. It further suggests that antioxidant potential may be interlinked with the observed hepatoprotective effects of Salvia hispanica.

 

SOURCE(S) OF SUPPORT:

ICMR- STS grant.

 

ACKNOWLEDGEMENTS:

The authors acknowledge with gratitude the funds received for the project from the ICMR as Short Term Student (STS) grant. The authors also acknowledge the assistance of Dr K V Nagalakshamma, Head of the Department of Botany, St Aloysius College, Mangalore in authentication of the plant seeds.

 

CONFLICTS OF INTEREST:

None

 

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Received on 28.10.2019           Modified on 22.12.2019

Accepted on 13.02.2020         © RJPT All right reserved