Research Article

Hepatotoxicity Induced by the therapeutic dose of Chlorpromazine and Ameliorative for Saussurea Costus Roots Extract and Effect of Physiological and Histological Liver of Albino Rats

Nbaa Mutea Abid AL-Alh1, Nuha Hatem Khalaf1, Nagam Khudhair1*, Ahmed Khalid2

1Department of Biology, Education College for Women, University of Anbar, Ramadi, Iraq; 2Department of Animal Production, College of Agriculture, Tikrit University, Iraq.

Received | June 01, 2024; Accepted | September 03, 2024; Published | November 01, 2024

*Correspondence | Nagam Khudhair, Department of Biology, Education College for Women, University of Anbar, Ramadi, Iraq; Email: edw.nagam1980_2005@uoanbar.edu.iq

Citation | AL-Alh NMA, Khalaf NH, Khudhair N, Khalid A (2024). Hepatotoxicity induced by the therapeutic dose of chlorpromazine and ameliorative for Saussurea Costus roots extract and effect of physiological and histological liver of albino rats. Adv. Anim. Vet. Sci. 12(12): 2540-2548.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.12.2540.2548

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Drug-induced liver damage is a significant medical concern in clinical settings. The liver plays a crucial role in the selective absorption, metabolism, and elimination of drugs, xenobiotics, and environmental pollutants. Chlorpromazine (CPZ), a phenothiazine derivative, has been utilized for over 60 years in psychiatry, particularly for the treatment of schizophrenia and bipolar disorders (Boyd-Kimball et al., 2019). However, its therapeutic use is accompanied by potential hepatotoxicity, which should not be overlooked (Derby et al., 1993). The hepatotoxicity induced by CPZ may be linked to the inflammatory process mediated by the continuous activation of c-Jun N-terminal kinase (JNK). In a study by Gandhi et al. (2013) it was shown that inflammation-mediated hepatotoxicity of CPZ depends on Toll-interleukin 1 receptor domain containing adapter protein, and involves prolonged JNK activation in vivo. Thus, TIRAP-based pathways may be targeted for prediction of idiosyncratic adverse drug reactions mediated by inflammation and their prevention. Furthermore, CPZ can inhibit bile flow in vivo, resulting in cholestasis. Substantial evidence suggests that CPZ serves as an effective model for studying drug-induced liver damage and is commonly used to mimic drug-induced cholestasis (Uzbekova et al., 2002).

Despite proposed mechanisms for CPZ-induced liver injury, the process is not fully understood due to the involvement of numerous factors affecting the liver adversely, also Seeman (2021) indicates the mechanism of CPZ effect by the post-synaptic blockade at the D2 receptors in the mesolimbic pathway. However, blocking D2 receptors in the nigrostriatal pathway is responsible for its extrapyramidal side effects.

CPZ impairs bile secretion in a dose-dependent manner and alters the fluidity of hepatocyte and canalicular membranes, thereby compromising their functional integrity (Samuels and Carey, 1978; Keefe et al., 1980).

For millennia, medicinal plants have been a primary source of bioactive substances, used either as pure compounds or raw materials to treat various illnesses. Of the estimated 250,000–500,000 plant species on Earth, humans utilize only 1–10%. Many contemporary medications have been derived from traditional medicinal plants, following ethnobotanical pathways from indigenous remedies used by traditional medical systems, thereby playing a pivotal role in the development and advancement of modern studies (Hayta et al., 2014).

Natural products are abundant in active antioxidant compounds, including flavonoids and polyphenolic chemicals (Enseleit et al., 2012). Saussurea costus, commonly known as Indian costus, is renowned for its medicinal properties (Singh et al., 2017). It has been used in modern medicine to treat various conditions, including ulcers, gastrointestinal issues, inflammatory diseases, and asthma (Amara et al., 2017). Saussurea costus is noted for its high antioxidant content, particularly sesquiterpene lactones such as dihydrocostose lactone, cinnarobicrin, and costunolide, which contribute to its pharmacological effects, including anti-inflammatory, immunomodulatory, hypoglycemic, anti-hepatotoxic, hypolipidemic, anti-parasitic, antiviral, and anti-cancer properties (Saleem et al., 2013; Zahara et al., 2014).

This study aims to investigate the effects of CPZ administration on liver function in rats and to evaluate the protective effects of Indian costus root extract on liver enzymes and the histological structure of the liver.

MATERIALS AND METHODS

Animals and Treatment

Thirty adult male albino rats (weighing 150- 220 g, aged 12-14 week) were obtained from animal house of College of Agriculture at Baghdad University. In the College of Education for Women /Anbar University’s animal home, animals were placed in plastic animal cages. The study designs and protocols confirm the requirements of the ethics considered by our institute in accordance with the St andard Guide for the Care and Use of Laboratory Animals. The cages were covered with sawdust, with attention to the cleanliness of the cages, and the sawdust was changed three times a week. Six rats were placed in each cage when rats were distributed. The rats were acclimatized at suitable conditions in laboratories for ventilation, temperature (23±2 C°) with controlled humidity conditions (50-55 %) at 12 h light and dark cycle, and a constant supply of water and a st andard dietary requirement. The drug CPZ (trade name “Lagactil” in the dose 100 mg) used is manufactured by “Aventis” (Oubari pharma). The experimental rats were divided into 5 groups (treated orally with one daily dose for 30 days) as follows:

G1 group, A negative control group was given water only. G2 group A positive control group was given oral chlorpromazine doses of 2 mg/kg of body weight as a single dose once a day (Al-Mushhadani et al., 2020) While the groups of Costus root extract (prophylactic groups) were distributed according to the concentration of the extract into three groups G3, G4 and G5: each one was given a concentration of 1.5, 3 and 5 mg/kg respectively, In conjunction with chlorpromazine (2mg/kg). In the study, groups G3, G4, and G5 served as a prophylaxis group dosed 1 hour after CPZ administration.

The Herbal Remedy

Botanists scientists recognized and verified the costus (Saussurea costus) roots that were collected from Al-Ramadi city local markets. An electric grinder is used to dry and crush, then stored in tightly sealed bottles for use in the extraction process later on. Regarding 100 g of the produced powder, it was steeped for 72 hours at room temperature in 1000 mL of 70% ethanol. This solution passed through Whatman grade-1 filter paper in a funnel under vacuum. The filtrate was next dried and evaporated under lower pressure in a rotary evaporator. S. Costus root crude ethanolic extract was gathered and then lyophilized to a dry powder (Abd El-Rahman et al., 2020). Until needed (the yield of the extract was 2gm), the samples were finally kept at 4 oC.

Blood and Tissue Sampling (Histopathology)

Rats were weighed after the treatment and then starved for the night. The animals received ether anesthetic so that capillary tubes could be used to extract 5 ml of blood from their eye sockets. The blood was then split into laboratory tubes without the use of anticoagulant. After whole blood was put into a st andard disposable tube and let to coagulate in a 37°C water bath for 15 to 30 minutes, the serum was separated by centrifugation at 3000 rpm. The biochemical parameters involved—AST, ALT, and ALP—were measured with sera. Animals were given up for autopsy and liver removal after blood was drawn under anesthesia by intramuscular injection of 50 mg/kg ketamine hydrochloride. After dissecting and cleaning the liver with tap water, the liver of each specimen was taken out and immediately fixed in 10% buffered formalin solution for 72 hours. Fixated tissues were dehydrated in aggraded series of alcohols, cleared in xylene, embedded in paraffin wax and cut with microtome at 6 to 7 µm. Sections were mounted on glass slides, deparaffinized and stained by Hematoxylin and Eosin (H and E) (Drury and Wallington, 1980) and then examined under olumpis light microscopy in Japan. Digital camera (sony-japa 14 migapixill) is used for the photos.

Biochemical Determinations

Alanine amino transferase (ALT) activities were measured in serum using the modified kinetic method of (Hafkenscheid, 1979). Aspartate amino transferase (AST) activities were measured in serum using the modified kinetic method of Henary (1974), serum alkaline phosphatase (ALP) was carried out according to the method of (Belfied and Goldberg, 1971).

Statistical Analysis

The statistical analysis of the data in this study was conducted by following the steps of the linear model of the statistical program (CoStat), and the significant differences (ANOVA) between the means were compared by SPSS programming difference were deemed significant at p≤ 0.05 after the data were presented as mean ± st andard error (Sciences, 2012).

RESULTS AND DISCUSSION

The liver is the main target of poisoning for a number of compounds, as most substances pass through metabolism. As a result, it becomes an organ of great importance for studying the effects of different chemicals excreted in the body, so the present work aims to study the potential modifying effects of Costus extract against hepatotoxicity. Drug-induced hepatotoxicity is a major concern in clinical practice. Although relatively uncommon, drug-induced liver injury is the leading cause of acute liver failure in the world and a major reason for liver transplantation (Reuben et al., 2010). In addition, drugs used in psychiatry and neurology are the second most important group of drugs causing hepatotoxicity, after anti-infective drugs. After discontinuing feeding for 30 days of experiment for all animals, the G2 (CPZ group) gained weight at faster rate (286.250 ±13.750 g Body Weight) (10.525 ± 0.217 g Liver Weight), while the G1 or control group gained weight at slower rate (231.250 ±21.250 g Body Weight) (8.160 ± 0.628 g Liver Weight)., the prophylactic group (G3, G4 and G5) gradually began to lose weight (260 ±10.607, 242.500 ±16.008 g and 241.250 ±6.575 g respectively of Body Weight) (10.163± 0.545 g , 9.050 ± 0.674 g and 8.725 ± 0.837 g respectively of Liver Weight). The results observed in Table 1, There was no significant change (p>0.05) in liver and body weight of animals among CPZ group, control group and prophylactic group.

 

Table 1: Comparision of Body and liver Weight of rats in control and experimental groups after administration of Saussurea costus.

Groups	Body Weight	Liver Weight
	Mean ± St. Error	Mean ± St. Error
(G1) Control negative group	231.250 ± 21.250	8.160 ±0.628
(G2) Chlorpromazine mg/ kg	286.250 ± 13.750	10.525 ±0.217
(G3) Antioxidant 1.5 mg/kg	260.000 ±10.607	10.163 ±0.545
(G4) Antioxidant 3 mg/kg	242.500 ±16.008	9.050 ±0.674
(G5) Antioxidant 5 mg/kg	241.250 ±6.575	8.725 ±0.837
LSD	43.74	1.855
P-Value	0.11533	0.078
C.V.	11.504	13.201

 

There are several theories put out to explain why antipsychotics tend to make people gain weight. Treatment of psychosis patients is plagued by antipsychotic-induced weight gain (AIWG); antipsychotics are also known to lower glucose metabolism, increase cholesterol and triglyceride levels, and cause arterial hypertension which leads to metabolic syndrome (De Hert et al., 2011). Interest in antipsychotics resulting in weight gain was aroused after the landmark study by Allison et al. (1999), the first meta-analysis on the subject. Over ten weeks, the study evaluated the weight gain caused by first- and second-generation antipsychotics (FGAs and SGAs, respectively). Molindone has been associated to a mean weight drop of 0.39 kg, clozapine, olanzapine, thioridazine, sertinazole, chlorpromidine, and risperidone have all been proven to cause significant weight increase between 4.45 and 2.19 kg. These medications’ effects at serotonin 5-HT2A and 5-HT2C, dopamine D2 and D3, histamine H1, and muscarinic M3 receptors have been connected to the high likelihood of weight gain (Fleischhacker et al., 2010).

Another research pointed out that steatosis is a different kind of lesion. This response is often chronic and results from the liver gradually and increasingly accumulating lipids, particularly triglycerides (Goncalves et al., 2015), which can be brought on by a number of illnesses, including the use of specific drugs. Sometimes benign macrovacuolar steatosis can progress to steatohepatitis and cirrhosis in drug-induced steatosis, which is nearly always reversible (Zhang et al., 2013).

Adipokines made in white adipose tissue, leptin and adiponectin, have been linked to weight gain; elevated leptin levels and decreased adiponectin levels (Ak et al., 2013). Antipsychotics also alter ghrelin, which stimulates food intake and deposits fat tissue by acting on the arcuate nucleus of the hypothalamus (Lu et al., 2015).

The ethanolic extract of S. Costus was found to decrease the body and liver weights of rats. According to several studies, the extracts of S. costus are the best inhibitors of pancreatic lipase and α glucosidase. Interestingly, the ethanolic extract of S. Costus showed notable levels of antioxidant activity in DPPH free radical scavenging assay. Moreover, flavonoids lessen the absorption of triacylglycerol by preventing alpha-glucosidase activity and the formation of micelle in the small intestine. Therefore, these studies clarified that reducing the gastrointestinal adverse effects linked to undigested starch reaching the colon would be possible by maintaining the balance between α-amylase and α-glucosidase inhibitors. Strongly positive correlations were observed between the antioxidant, antidiabetic, and anti-obesity potential of phenolic and flavonoid compounds with the inhibition activity of enzymes (alpha-amylase, alpha-glucosidase, and pancreatic lipase) and free radical scavenging/antioxidant activity (Naseer et al., 2022; Kumar et al., 2023).

Biochemical Results

In the current study details of findings observed in Table 2 revealed the treatment of rats that treated with CPZ or G2 group (2mg / day orally) for 30 days resulted in a high significant rise (p<0.001) in serum of liver enzymes (AST, ALT and ALP) compared with G1 (control group) and showed a gradually significant decreasing in the level of enzymes in the prophylactic group (G3, G4 and G5) respectively.

 

Table 2: Effect of ethanolic extract of Saussurea costus on AST, ALT and ALP in rat treated with Chlorpromazine.

Groups	AST (U/L)	ALT (U/L)	ALP (IU\L)
	Mean ± St. Error	Mean ± St. Error	Mean ± St. Error
(G1) Control negative group	147.74 ± 7.75 C	70.77 ± 4.23 D	78.83 ± 3.47 C
(G2) Chlorpromazine mg/ kg	251.28 ± 10.58 A	169.27 ± 9.04 A	116.42 ± 3.82 A
(G3) Antioxidant 1.5 mg/kg	200.09 ± 10.09 B	127.39 ± 7.39 B	94.72 ± 2.66 B
(G4) Antioxidant 3 mg/kg	170.23 ± 6.90 C	104.12 ± 8.61 C	86.47 ± 4.01 BC
(G5) Antioxidant 5 mg/kg	149.49 ± 7.04 C	85.31 ± 3.50 CD	74.86 ± 3.83 C
Total	183.77 ± 10.84	111.37 ± 9.61	90.26 ± 4.17
LSD	27.142	21.850	11.311
P-Value	<.001	<.001	<.001

 

Thus, Chlorpromazine causes a dose-related decrease in bile secretion and changes the fluidity of the hepatocyte and canalicular membranes, which in turn affects the function integrity of these sites and is well known to cause hepatotoxicity in humans (Derby et al., 1993).

Ninety percent of drug-induced hepatotoxicity is caused by hepatocellular damage, which is linked to abnormally high serum alanine aminotransferase (ALT) and little or no rise in alkaline phosphatase (ALP); a high serum bilirubin level that is present in cases of severe hepatocellular damage which is a sign of a bad prognosis (Telles-Correia et al., 2017). It can be prevented by regular monitoring with liver function tests and in case of the early detection of liver injury, the drug should be stopped, and symptomatic management should start (Velayudham and Farrell, 2003; Morgan et al., 2019).

The current investigation revealed an increase in serum as AST, ALT, and ALP activities in rats treated with CPZ alone compared to control group. This results in leakage of specific intracellular enzymes such as AST, ALT, and ALP to the plasm boosting their activities there. Similar findings have been published in a number of researches (Abdel-Wahhab et al., 2017; Al-Fatlawi et al., 2019; Dejanovic et al., 2014). Additionally noted was an increase in ALT, AST, and ALP levels.

Table 2 shows that the ethanol extract of S. costus is reducing the toxic effect of chlorpromazine since the plasma levels of AST, ALT, and ALP in prophylactic groups of rats by S. costus were much lower than those of the CPZ group. Previous investigations have revealed that an extract of Indian costus decreased the activity of liver enzymes (Hegazy et al., 2020; Toussoni et al., 2020) and this may be attributed to phytochemical compounds such as flavonoids and chlorogenic acid that serve as antioxidant material that help to inhibit free radicals that caused lipid peroxidation and prevents chlorpromazine poisoning. These results correspond with (Ansari et al., 2018; Kadhem, 2019).

Whereas reactive oxygen species (ROS) are produced at a higher rate in the organ, which is well known to cause oxidative stress (Abdel-Wahhab et al., 2016). and this contributes in inhibiting the defensive system. Concerning this same issue, Jorgačević et al. (2014) found that although neutrophils and Kupffer cells are significant sources of ROS, the main sources of ROS within hepatocytes are iron overload, cyclooxygenase, lipoxygenase, cytochrome P450 2E1, mitochondria, and nicotinamide adenine dinucleotide phosphate oxidase. Consequently, the reduction of hepatocyte antioxidant ability adds to the ROS-induced liver damage.

 

Histological Examination

The histopathological changes of the rat’s liver tissue revealed the normal architecture in the G1 or control group (normal structure of the cords of the hepatocytes, the central vein and the blood sinusoids) (Figure 1) with many disorders lesions in the G2 or CPZ group (in a dose of 2 mg/kg body weight/day of chlorpromazine only). This is histologically represented by hepatocyte suffering from acute cell swelling, where most hepatocyte were in necrosis and inflammatory cells infiltration was observed. Also, blood vessels congestion and perivascular inflammatory cells infiltration are manifested in the field of view X400, (Figures 2, 3, and 4). In the portal tract, dilation was in the portal vein with perivascular edema, bile ducts epithelial hyperplasia, and hemosiderin deposition also peri central area of Hydropic Degeneration were observed in the field of X100 (Figures 5, 6, and 7).

 

 

As previously reported, the liver section of CPZ-treated rat had lipid droplets, necrotic cytoplasm, and pyknotic or apoptotic nuclei, as well as congestion and fibrous thickening of the portal tube with extended bile canaliculi (Abdel-Wahhab et al., 2017). These results mirrored those in the liver published by Yang et al. (2015). Additionally, this result is consistent with other studies that show the contribution of oxidative stress in the pathogenesis of cholestasis (Parola et al., 1996; Ohta et al.,1999) and can be explained as a consequence of generation of CPZ cation radicals and metabolic activation of CPZ to quinonimine derivatives (Tolar, 2004).

 

 

 

 

Recent investigation has shown that pathological alterations of varied severity in liver tissue occur when CPZ is administered at different doses (Bailo et al., 2023).

In this work, the ethanolic costus extract at a dosage of 5 mg/kg effectively corrected the changes in liver tissue structure caused by CPZ, therefore providing a stronger hepatoprotective effect against CPZ-induced liver damage (Figure 8). By removing too many free radicals, where a great number of earlier research have demonstrated that costus can donate electrons to reactive radical cells and transform them into more stable, non-reactive species (Shediwah et al., 2019; Benedetto et al., 2019; El-Kholie et al., 2022; AL-Zayadi et al., 2023).

 

Ayaz (2017) report that costus extract is rich in bioactive compounds including; dihydrocostus lactone, costonolide, cinnarobicrin, monoterpenes, sesquiterpenoids, flavonoids, lignans, triterpene steroids, and glycosides that are produced as antioxidants. As such, costus extract can be regarded as a suppressor of the negative effects of CPZ in rats and as an inhibitor of the generation of radicals that cause liver damage.

CONCLUSIONS AND RECOMMENDATIONS

The published results confirm that CPZ leads to changes in Weight and responsible for remarkable functional and structural abnormalities in the liver. The pathological changes involve the hepatocytes, and the hepatic connective tissues as well as induced inflammation of the hepatic tissues. Also, treatment with costus root extract significantly reduces CPZ-induced hepatotoxicity histologically in hepatic rats. Therefore, this encourages the use of Indian costus in future studies as therapeutic alternatives.

ACKNOWLEDGEMENTS

We thank the College of Education for womens at Anbar University for approving the work in the Life Sciences Laboratories

NOVELTY STATEMENT

The study demonstrates that ethanolic extract of costus root offers effective hepatic protection against the adverse effects of chlorpromazine, contributing to improved functional and histological liver parameters in rats.

AUTHOR’S CONTRIBUTIONS

Nbaa Mutea Abid AL-Alh and Nagam Khudhair contributed to the conceptualization, methodology, investigation, writing of the original draft, and visualization. Nuha Hatem Khalaf and Ahmed Khalid reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.

Financial Support and Sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.

REFERENCEs

Abd El-Rahman GI, Behairy Elseddawy ANM, Batiha GE, Hozzein WN, Khodeer MDM, Abd-Elhakim MYM (2020). Saussurea lappa Ethanolic Extract Attenuates Triamcinolone Acetonide-Induced Pulmonary and Splenic Tissue Damage in Rats via Modulation of Oxidative Stress, Inflammation, and Apoptosis. Antioxidants, 9: 396. https://doi.org/10.3390/antiox9050396

Abdel-Wahhab MA, Eid NI, Ahmed LA, Ragab GM, El-Nekeety AA, El-Hakim MM, Hassan NS (2017). Inulin nanoparticles and silymarin counteract chlorpromazine-induced injury in the liver and kidney of rats. J. Appl. Pharm. Sci., 7(08): 104-115.

Abdel-Wahhab MA, Salman AS, Ibrahim MIM, El-Kady AA, Abdel-Aziem SH, Hassan NS, Waly AI (2016). Curcumin nanoparticles loaded hydrogels protects against aflatoxin B1-induced genotoxicity in rat liver. Food Chem. Toxicol., 94: 159-71. https://doi.org/10.1016/j.fct.2016.06.005

Aithal GP, Watkins PB, Andrade RJ, Larrey D, Molokhia M, Takikawa H, Hunt CM, Wilke RA, Avigan M, Kaplowitz N, Bjornsson E, Daly AK (2011). Case definition and phenotype standardization in druginduced liver injury. Clin. Pharmacol. Ther., 89: 806-815. https://doi.org/10.1038/clpt.2011.58

Ak M, Sezlev D, Sutcigil L, Akarsu S, Ozgen F, Yanik T (2013). The investigation of leptin and hypothalamic neuropeptides role in first attack psychotic male patients: olanzapine monotherapy. Psychoneuroendocrinology, 38(3): 341-347. https://doi.org/10.1016/j.psyneuen.2012.06.012

Al-Fatlawi Ather AY, Al-Fatlawi Anees AY (2020). Tocopheronic acid attenuates chlorpromazine induced liver injury in rats, targeting oxidative stress and same biochemical markers. Int. J. Pharm. Res., 12(4): 2001-2011. https://doi.org/10.31838/ijpr/2020.12.04.285

Allison DB, Mentore JL, Heo M (1999). Antipsychotic-induced weight gain: a comprehensive research synthesis. Am. J. Psychiatry, 156(11): 1686-1696. https://doi.org/10.1176/ajp.156.11.1686

Al-Mushhadani TM, Arteen HE, Jumaa HJ (2020). Effect of Plant Seeds Peganum harmala L-Evaporation on Liver and Heart Enzymes Activity and Total Protein in Male White Rats (Rattus norvegicus) Treated with Chlorpromazine. Rafidain J. Sci., 29(1): 68-79. https://doi.org/10.33899/rjs.2020.164482

AL-Zayadi ZA, SHanan HK, Akool AK (2023). Evaluation of the Anticancer Effect of Saussurea costus Root Extract Against Induced Hepatic and Renal Cancer in White Mice: A Histopathological Study. Adv. Anim. Vet. Sci.,11(7): 1065-1076. https://doi.org/10.17582/journal.aavs/2023/11.7.1065.1076

Ansari S, Siddiqui MA, Maaz M (2018). Hepatocurative effect of Saussurea lappa CB Clarke and Artemisia absinthium, Linn in chronic hepatitis B. J. Young Pharm., 10(3):354. https://doi.org/10.5530/jyp.2018.10.78

Amara U, Mashwani ZR, Khan A, Laraib S, Wali R, Sarwar U (2017). Conservation Status and Therapeutic Potential of Saussurea lappa: An Overview. Am. J. Plant Sci., 8: 602-614. https://doi.org/10.4236/ajps.2017.83041

Ayaz NO (2017). Modulating impacts of coustus Sassura lappa extract against oxidative stress and genotoxicity induced by deltamethrin toxicity in rat kidneys. Int. J. Pharm. Res. Allied Sci., 6(2).

Bailo OV, Rykalo NA (2023). Morphological changes in the liver of rats after administration of chlorpromazine, depending on the dose and duration of administration. Rep. Morphol., 29(1): 67-76. https://doi.org/10.31393/morphology-journal-2023-29(1)-10

Belfied A, Goldberg DM (1971). Alkaline phosphatase colorimetric method. J. of Enzyme, (12): 561. https://doi.org/10.1159/000459586

Benedetto C, D’Auria M, Mecca M, Prasad P, Singh P, Singh S, Milella L (2019). Chemical and biological evaluation of essential oil from Saussurea costus (Falc.) Lipsch. from Garhwal Himalaya collected at different harvesting periods. Nat. Prod. Res., 33(16): 2355-2358. https://doi.org/10.1080/14786419.2018.1440219

Boyd-Kimball D, Gonczy K, Lewis B, Mason T, Siliko N, Wolfe J (2019). Clas sics in Chemical Neuroscience: Chlorpromazine. ACS Chem. Neurosci., 10(1): 79-88. https://doi.org/10.1021/acschemneuro.8b00258

De Hert M, Detraux J, van Winkel R, Yu W, Correll CU (2011). Metabolic and cardiovascular adverse effects associated with antipsychotic drugs. Nat. Rev. Endocrinol.,8(2): 114-126. https://doi.org/10.1038/nrendo.2011.156

Dejanovic B, Stevanovic I, Ninkovic M, Stojanovic I, Vukovic Dejanovic V (2014). Protective effect of agmatine in acute chlorpromazine hepatotoxicity in rats. Acta. Vet. Brno., 83: 305-312. https://doi.org/10.2754/avb201483040305

Derby LE, Gutthann SP, Jick H, Dean AD (1993). Liver disorders in patients receiving chlorpromazine or isoniazid. Pharmacotherapy. 13:353-358. https://doi.org/10.1002/j.1875-9114.1993.tb02742.x

Drury RAB, Wallington EA (1980). Preparation and fixation of tissues. In: Drury RAB, Wallington EA, editors. Carleton ’s histological technique. 5 Oxford: Oxford University Press, 41-5.

El-Kholie E, El Sheikh N, Hassan M (2022). Potential Anti-obesity Effects of Costus (Saussurea costus) and Bitter Melon (Citrullus colocynthis) in Rats Fed on High Fat Diet. J. Home Econ., 32 (4): 127-140.

Enseleit F, Sudano I, Periat D, Winnik S, Wolfrum M, Flammer AJ (2012). Effects of Pycnogenol on endothelial function in patients with stable coronary artery disease: a double-blind, randomized, placebo-controlled, cross-over study. Eur. Heart J., 33: 1589- 97. https://doi.org/10.1093/eurheartj/ehr482

Fleischhacker WW, Heikkinen ME, Olie JP (2010). Effects of adjunctive treatment with aripiprazole on body weight and clinical efficacy in schizophrenia patients treated with clozapine: a randomized, double-blind, placebo-controlled trial. Int. J. Neuropsychopharmacol.,13(8): 1115-1125. https://doi.org/10.1017/S1461145710000490

Gandhi A, Guo T, Shah P, Moorthy B, Ghose R (2013). Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice. Toxicol. Appl. Pharmacol., 266: 430-8. https://doi.org/10.1016/j.taap.2012.11.030

Goncalves P, Araujo JR, Martel F (2015). Antipsychotics-induced metabolic alterations: focus on adipose tissue and molecular mechanisms. Eur. Neuropsychopharmacol., 25(1):1-16. https://doi.org/10.1016/j.euroneuro.2014.11.008

Hafkenscheid JC (1979). Determination of GOT. Clin. Chem.; 25: 155. https://doi.org/10.1093/clinchem/25.1.55

Hayta S, Tasar N, Cakilcioglu U, Gedik O (2014). Morphological, karyological features and pollen morphology of endemic Ebenus haussknechtii Bornm. Ex hub.-Mor. From Turkey: A traditional medicinal herb. J. Herb. Medi., 4(3):141-146. https://doi.org/10.1016/j.hermed.2014.04.006

Hegazy MM, Abonama OM, Mohammad AS, Abouelnour ES, Badr MM, Elhalfawy IA (2020). The role of Indian Costus against toxicity of thermally oxidized Palm oil in albino rats. Egypt J. Forensic Sci. Appli. Toxicol., 20(3): 23-40. https://doi.org/10.21608/ejfsat.2020.18984.1107

Henary RJ (1974). Clinical Chemist: Principals and Techniques. 2nd, Edition, Hagerstoun (MD), Harcer, ROW, P. 882.

Jorgačević B, Mladenović D, Ninković M, Prokić V, Stanković MN, Aleksić V, Cerović I, Vukićević RJ, Vučević D, Stanković M, Radosavljević T (2014). Dynamics of oxidative/nitrosative stress in mice with methionine-choline-deficient diet-induced nonalcoholic fatty liver disease. Hum. Exp. Toxicol., 33: 701-709. https://doi.org/10.1177/0960327113506723

Kadhem MAJ (2019). Protective of Ethanolic Extract Of Saussurea Lappa Against Paracetamol-Induced Hepatic And Renal Damage In Male Rabbits. Asian J. Pharm. Clin. Res., 12(8): 68-73. https://doi.org/10.22159/ajpcr.2019.v12i18.34218

Keefe EB, Blankenship NM, Scharchmid BF (1980). Alteration of rat liver plasma membrane fluidity and ATPase activity by chlorpromazine hydrochloride and its metabolites. Gastroenterology, 79: 222-231. https://doi.org/10.1016/0016-5085(80)90134-1

Kumar JV, Pratap CG (2023). Assessment Of The Anti-Obesity Efficacy Of Ethanolic Extracts Of Saussurea lappa (Costus) Roots In Cafeteria Diet Fed Obese Rats. Eur. Chem. Bull., 12(Special Issue 5), 1390 - 1402.

Lu ML, Wang TN, Lin TY (2015). Differential effects of olanzapine and clozapine on plasma levels of adipocytokines and total ghrelin. Prog. Neuropsychopharmacol. Biol. Psychiatry, 58: 47-50. https://doi.org/10.1016/j.pnpbp.2014.12.001

Messelmani T, Morisseau L, Sakai Y, Legallais C, Le Goff A, Leclerc E (2022). Liver organ-on-chip models for toxicity studies and risk assessment. Lab Chip., 22(13):2423-50. https://doi.org/10.1039/D2LC00307D

Morgan K, Martucci N, Kozlowska A, Gamal W, Brzeszczyński F, Treskes P, Samuel K, Hayes P, Nelson L, Bagnaninchi P, Brzeszczynska J, Plevris J (2019). Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line. Biomed. Pharmacother., 111:1408-1416. [PubMed] https://doi.org/10.1016/j.biopha.2019.01.020

Naseer S, Iqbal J, Naseer A, Kanwal S, Hussain I, Tan Y (2022). Deciphering chemical profiling, pharmacological responses and potential bioactive constituents of Saussurea lappaDecne.extracts through in vitro approaches.Saudi J. BiolSci., 29(3): 1355- 66. https://doi.org/10.1016/j.sjbs.2022.01.040

Ohta Y, Kongo M, Sasaki E (1999). Harada Change in hepatic antioxidant defence system with liver injury development in rats with single α-naphthyl isothiocyanate intoxication .Toxicology, 39:265-275. https://doi.org/10.1016/S0300-483X(99)00131-6

Parola M.Leonardnzzi G, Albano E, Poli G, Diazani MU (1996). On the role of lipid peroxidation in the pathogenesis of liver damage induced by long standing cholestasis. Free Radic Biol. Med., 20: 351-359 https://doi.org/10.1016/0891-5849(96)02055-2

Reuben A, Koch DG, Lee WM (2010). Drug-induced acute liver failure: results of a US multicenter, prospective study. Hepatology, 52: 2065-2076. https://doi.org/10.1002/hep.23937

Saleem TS, Lokanath N, Prasanthi AM, Madhavi M, Mallika G, Vishnu MN (2013). Aqueous extract of Saussurea lappa root ameliorate oxidative myocardial injury induced by isoproterenol in rats. Adv. Pharm. Technol. Res., 4(2): 94-100. https://doi.org/10.4103/2231-4040.111525

Samuels AM, Carey MC (1978). Effect of chlorpromazine hydrochloride and its metabolites on Mg+2 and Na+/K+ ATPase activities of canalicular-enriched rat liver plasma membrane . Gastroenterology,47:1183-1190. https://doi.org/10.1016/0016-5085(78)90690-X

Sciences SSPS (2012). Statistical Software for Windows Version 16.0 Microsoft. Chicago, IL, USA: SPSS Statistical Packages for the Social Science,

Seeman MV (2021). History of the dopamine hypothesis of antipsychotic action. World J. Psychiatry,11(7): 355-364. [PMC free article] [PubMed] https://doi.org/10.5498/wjp.v11.i7.355

Shediwah FMH, Naji KM, Gumaih HS, Alhadi FA, Al-Hammami AL, D’Souza MR (2019). Antioxidant and antihyperlipidemic activity of Costus speciosus against atherogenic diet-induced hyperlipidemia in rabbits. J. Integr. Med., 17(3): 181-191. https://doi.org/10.1016/j.joim.2019.02.002

Singh R, Chahal KK, Singla N (2017). Chemical composition and pharmacological activities of Saussurea lappa: A review. J. Pharmacogn. Phytochem., 6 (4): 1298-1308.

Telles-Correia D, Barbosa A, Cortez-Pinto H, Campos C, Rocha NBF, Machado S (2017). Psychotropic drugs and liver disease: A critical review of pharmacokinetics and liver toxicity. World J. Gastrointest. Pharmacol. Ther., 8(1): 26-38. https://doi.org/10.4292/wjgpt.v8.i1.26

Tolar MS (2004). Oxidative stress plays an important role in the pathogenesis of drug - induced retinopathy. Exp. Biol. Med.,229: 607-615 https://doi.org/10.1177/153537020422900704

Toussoni DE, El-Atrsh A, Mansour M, Assem A (2020). Costus root aqueous extract modulates rat liver toxicity, DNA damage, injury, proliferation alterations induced by plant growth regulator Ethephon. Braz. J. Pharm. Sci., 56: e18500 : 1-10. https://doi.org/10.1590/s2175-97902019000318500

Uzbekova D, Makarova V, Khvoynitskaya L, Mirgorodskaya L (2002). Protective effects of beehive products and lactulose on chlorpromazine-induced liver damage in rats. Journal of Hepatology.;36:165. https://doi.org/10.1016/S0168-8278(02)80597-9

Velayudham LS, Farrell GC (2003). Drug-induced cholestasis. Expert Opin Drug Saf., 2(3): 287-304. [PubMed] https://doi.org/10.1517/eods.2.3.287.21377

Yang Q, Yang F, Tang X, Ding L, Xu Y, Xiong Y, Wang Z, Yang L (2015). Chlorpromazine-induced perturbations of bile acids and free fatty acids in cholestatic liver injury prevented by the Chinese herbal compound Yin-Chen-Hao-Tang. BMC Complement Altern. Med., 15:122. https://doi.org/10.1186/s12906-015-0627-2

Zahara K, Tabassum S, Sabir S, Arshad M, Qureshi R, Shoaib AM (2014). A review of therapeutic potential of Saussurea lappa-An endangered plant from Himalaya Asian Pac. J. Trop. Med., 7(1): 60-69. https://doi.org/10.1016/S1995-7645(14)60204-2

Zhang X, Ouyang J, Thung S (2013). Histopathologic Manifestations of Drug-induced Hepatotoxicity. Clinics Review Articles: Drug Hepatotoxicity. Philadelphia: Elsevier, 547-559. https://doi.org/10.1016/j.cld.2013.07.004