Hepatotoxicity and Potential Effects of Phytochemicals

HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Editor Sinan İNCE Lyon 2025 HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Editor Sinan İNCE Lyon 2025 Hepatotoxicity and Potential Effects of Phytochemicals Editor • Prof. Dr. Sinan İNCE • Orcid: 0000-0002-1915-9797 Cover Design • Motion Graphics Book Layout • Motion Graphics First Published • October 2025, Lyon e-ISBN: 978-2-38236-924-1 DOI: 10.5281/zenodo.17382131 copyright © 2025 by Livre de Lyon All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission from the Publisher. The author or authors of the relevant section are responsible for any copyright infringement that may occur due to the images and graphics used in the book. The editor or publisher does not assume responsibility in this regard. Publisher • Livre de Lyon Address • 37 rue marietton, 69009, Lyon France website • http://www.livredelyon.com e-mail • [email protected] i PREFACE The liver is a pivotal organ responsible for sustaining metabolic homeostasis, detoxification, and biotransformation processes. Owing to its central role in these functions, it is also among the organs most susceptible to toxic insults. A wide spectrum of xenobiotics—including pharmaceutical agents, industrial chemicals, environmental pollutants, and dietary constituents—can compromise hepatic physiology and result in hepatotoxicity. Beyond its clinical implications for individual health, hepatotoxicity constitutes a major concern with respect to food safety, public health, and socioeconomic stability. In light of the increasing awareness of the adverse and often unpredictable side effects of synthetic drugs, scientific attention has progressively shifted toward natural compounds as safer alternatives. Within this framework, phytochemicals—bioactive molecules of botanical origin—have attracted particular interest as potential hepatoprotective agents. Polyphenols, flavonoids, alkaloids, terpenoids, and related phytoconstituents have been reported to exert protective effects through antioxidant, anti-inflammatory, and cytoprotective mechanisms, thereby supporting hepatic resilience against toxic challenges. This book aims to elucidate the mechanistic aspects of hepatotoxicity while critically evaluating the protective and therapeutic potential of phytochemicals. By integrating insights from modern toxicology with evidence derived from traditional medicinal knowledge, this work aspires to contribute to the establishment of scientifically validated phytotherapeutic approaches. It is anticipated that the outcomes will provide a meaningful foundation for future research and inform innovative strategies for safeguarding liver health. Editor Prof. Dr. Sinan İNCE iii CONTENTS PREFACE i CHAPTER I. LIVER ANATOMY AND HISTOLOGY 1 Hasan Hüseyin DEMİREL CHAPTER II. LIVER INJURY 11 Selcan CESUR CHAPTER III. THYMOQUINONE 19 Emine SEVER CHAPTER IV. BERBERINE 29 Berrin YALINBAŞ KAYA CHAPTER V. APIGENIN 39 Sinan İNCE CHAPTER VI. QUERCETIN 53 Fahriye KAN & İsmail KÜÇÜKKURT CHAPTER VII. THYMOL AND CARVACROL 65 Funda KARABAĞ CHAPTER VIII. 6-GINGEROL – ZINGERON 75 Yusuf ABUL CHAPTER IX. SILYMARIN 87 Ali TÜREYEN CHAPTER X. RESVERATROL 95 İsmail KÜÇÜKKURT & Fahriye KAN CHAPTER XI. CAFFEIC ACID 105 Ayşenur SEVİNÇ CHAPTER XII. EPIGALLOCATECHIN-3-GALLATE 115 Fahriye ZEMHERİ-NAVRUZ CHAPTER XIII. HESPERIDIN 127 Ulaş ACARÖZ Ömer ÇAKMAK Damla ARSLAN-ACARÖZ CHAPTER XIV. KAEMPFEROL 149 Mehmet BAŞEĞMEZ iv   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS CHAPTER XV. NARINGENIN 161 Ömer ÇAKMAK & Damla ARSLAN-ACARÖZ & Ulaş ACARÖZ CHAPTER XVI. SCHISANDRA DERIVATIVES 181 Duygu İSKENDER MAZMAN CHAPTER XVII. SYRINGIC ACID 195 Duygu YILMAZ CHAPTER XVIII. OLEANOLIC ACID 207 Muhammed Fatih DOĞAN & Nurcan DOĞAN CHAPTER XIX PROCYANIDINS 217 Sevilay Süreyya ERMİŞ CHAPTER XX. ANTHOCYANINS 233 Muhammet KOCABAŞ LIVER ANATOMY AND HISTOLOGY   7 2.2. Intrahepatic Bile Ducts The intrahepatic bile ducts have thin walls lined by a single layer of squamous epithelial cells. Their origin is from small capillary-like channels without defined walls located between the hepatic cell cords, known as canaliculi biliferi. After emerging from the cell cords, the canaliculi biliferi develop an independent wall, and several of them merge to form the ductuli biliferi. The ductuli biliferi then converge to form the ductus interlobularis, which is situated within the spatia interlobulares (Kiernan spaces). Subsequent fusion of the ductus interlobularis gives rise to larger bile ducts. These ducts unite on the visceral surface of the liver into two main channels called the ductus hepaticus dexter and ductus hepaticus sinister, which collect bile from the right and left lobes of the liver, respectively (21). 3. Liver Histology Hepatocytes are the parenchymal cells that form the cellular plates within the liver lobule and constitute approximately 80% of the total liver cells. The liver is encased by Glisson’s capsule, a fibrous connective tissue layer covering its external surface. Fine connective tissue fibers emerging from this capsule partition the liver into lobes and lobules, while blood vessels, nerves, and bile ducts course within this connective tissue framework. The fundamental structural and functional unit of the liver is the lobule, which is classified into three types: the classical lobule, the portal lobule, and the liver acinus. The classical lobule has a hexagonal shape, bounded by portal areas at its periphery and featuring a thinwalled central vein at its center. A delicate connective tissue layer surrounds the central vein. Hepatocytes are arranged radially in anastomosing cords extending outward from the central vein, with sinusoidal capillaries located between these cords. In humans, the connective tissue surrounding the classical lobules is thin and sparse, which sometimes complicates the delineation of lobular boundaries. Portal areas situated at the corners of the lobule contain branches of the portal vein and hepatic artery, the bile duct (forming the portal triad), lymphatic vessels, and nerve fibers (22). The portal vein extensively branches to form small portal venules within the portal areas. These venules further branch around the lobule to create distributing veins. Fine incoming venules arising from the distributing veins open into the sinusoidal vessels. The sinusoidal vessels progress radially and converge to form the central or centrilobular vein located at the center of the 8   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS lobule. The central vein collects blood from the sinusoids along the lobule, increasing in diameter as it progresses, eventually exiting the lobule and joining larger lobular veins. These lobular veins merge to form the hepatic veins, which drain into the inferior vena cava. The hepatic arteries also branch extensively to form interlobular arterioles; some of these arterioles supply the portal structures, while others open directly into 4. Conclusion In summary, the liver’s complex anatomical structures, extensive vascular networks, and intricate histological organization collectively underpin its vital functions in metabolism, detoxification, immune regulation, and homeostasis. A thorough understanding of these features not only enriches our knowledge of human physiology but also provides critical insights for surgical practice, clinical interventions, and ongoing biomedical research (24). References: 1.Banerjee A, Hariharan D. History of liver surgery. Clin Liver Dis. 2024;23(1):e0237. doi:10.1097/CLD.0000000000000237 2.Tebala GD, Avenia S, Cirocchi R, Delvecchio A, Desiderio J, et al. Turning points in the practice of liver surgery: A historical review. Ann Hepatobiliary Pancreat Surg. 2024;28(3):271-282. doi:10.14701/ahbps.24-039 3.Cavalcanti de A Martins A, Martins C. History of liver anatomy: Mesopotamian liver clay models. HPB (Oxford). 2013;15(4):322-323. doi:10.1111/j.1477-2574.2012.00555.x 4.Akın Saygın D, Aydın Kabakçı AD, Yılmaz MT. Adım Adım Anatomi. İstanbul: İstanbul Tıp Kitabevleri; 2023:410-413. 5.Arıncı K, Elhan A. Anatomi. 7. Baskı. 1. Cilt. Ankara: Güneş Tıp Kitabevleri; 2020:266. 6.Gökmen FG. Sistematik Anatomi. 2. Baskı. İzmir: Nobel Tıp Kitabevleri; 2022. 7.Schraegle AE, Millard ND, King BG. Human anatomy and physiology. Am J Nurs. 1951;51:40. Pearson Education; London. 8.Skandalakis JE. Clinically Oriented Anatomy. Vol 282. JAMA. Wolters Kluwer India Pvt Ltd; 1999:1485. 9.Akın Saygın D, Aydın Kabakçı AD, Yılmaz MT. Adım Adım Anatomi. İstanbul: İstanbul Tıp Kitabevleri; 2023. s. 410-413. LIVER ANATOMY AND HISTOLOGY   9 10.Gövsa Gökmen F. Sistematik Anatomi. 2. Baskı. İzmir: Nobel Tıp Kitabevleri; 2023:333-533. 11.Anatomi OHO. Ankara: Nobel Tıp Kitabevleri; 2005. 12.Arıncı K, Elhan A. Anatomi 1. cilt. Güneş Kitabevi, Ankara. 388, 1995. 13.Lev-Toaff AS, Friedman AC, Cohen LM, Radecki PD, Caroline DF. Hepatic infarcts: New observations by CT and sonography. Am J Roentgenol. 1987;149(1):87-90. 14.Mathew RP, Venkatesh SK. Liver vascular anatomy: a refresher. Abdom Radiol (NY). 2018;43(8):1886-1895. 15.Charnsangavej C, Clary B, Fong Y, Grothey A, Pawlik TM, Choti MA. Selection of patients for resection of hepatic colorectal metastases: Expert consensus statement. Ann Surg Oncol. 2006;13(10):1261-1268. 16.Balcerzak A, Tubbs RS, Waśniewska-Włodarczyk A, Rapacka E, Olewnik Ł. Classification of the superior mesenteric artery. Clin Anat. 2022;35(4):501-511. 17.Washke J, Böckers TM, Paulsen F. Sobotta Anatomi Konu Anlatımı. 2016. 18.Gray’s Anatomy. 18th ed. Services P, editor. 2006:251-259. 19.Mortele KJ, Ros PR. Anatomic variants of the biliary tree: MR cholangiographic findings and clinical applications. AJR Am J Roentgenol. 2001;177(2):389-394. 20. Kozan B. Biliyer sistemin anatomik varyasyonlarında MR kolanjiyopankreatografi bulguları [Uzmanlık Tezi]. İstanbul: Fatih Sultan Mehmet Eğitim ve Araştırma Hastanesi Radyoloji Anabilim Dalı; 2006. 21.Grays H. Gray’s Anatomy. The Anatomical Basis of Clinical Practice, 40.Th Churcill Livingstone: Elseiver Science, 2008: 27-40,1163-1180 22.Gökçe H, Soylu NK, Vardı N, Yakıncı C. Histoloji ve Patoloji. 2. Baskı. İzmir: Nobel Tıp Kitabevleri; 2023:426-427. 23.Junqueira LC, Carneiro J. Temel Histoloji. Çevirenler: Solakoğlu S, Aytekin Y. İstanbul: Nobel Tıp Kitabevleri; 2009:328. 24. Kalra A. Physiology, Liver. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. 11 CHAPTER II LIVER INJURY Selcan CESUR (MD, Specialist) Department of Gastroenterology, Ministry of Health Eskisehir City Hospital, E-mail: [email protected], Orcid: 0000-0002-1504-7069 1. Introduction The liver is a vital organ with unique regenerative capacity and a central role in drug metabolism and detoxification. It is highly susceptible to toxic insults because it serves as the first organ exposed to orally ingested compounds via the portal circulation(1). Hepatotoxicity can be defined as liver damage caused by chemical substances, including pharmaceutical drugs, environmental agents, alcohol, and herbal products. Clinically, it may present as asymptomatic biochemical abnormalities, acute hepatitis, cholestatic injury, or even acute liver failure requiring transplantation(2). The rising global use of herbal remedies and dietary supplements has introduced new dimensions to hepatotoxicity. Phytochemicals—naturally occurring bioactive compounds derived from plants—are often consumed for their perceived health benefits. Classes of phytochemicals such as polyphenols, alkaloids, terpenoids, and flavonoids have been shown to modulate key cellular pathways involved in oxidative stress, inflammation, and apoptosis(3,4). Despite their therapeutic potential, not all phytochemicals are benign. Several herbal preparations have been linked to clinically significant liver injury, highlighting the need to balance potential benefits with toxicological risks(5,6). Understanding the underlying molecular mechanisms of hepatotoxicity and hepatoprotection is therefore critical for the safe clinical use of phytochemicals. 12   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 1. Pathophysiology of Liver Injury Liver injury develops through the interaction of multiple cellular and molecular mechanisms. Upon exposure to toxic agents, hepatocytes undergo endoplasmic reticulum stress and mitochondrial dysfunction. The accumulation of reactive oxygen species (ROS) depletes intracellular glutathione reserves, induces lipid peroxidation and protein damage, and triggers cell death pathways (6,7,19,20). Liver injury is generally categorized into three major mechanisms: 1. Hepatocellular injury: Caused by direct toxicity or ROS-mediated damage, typically associated with elevated ALT and AST levels. 2. Cholestatic injury: Involves impairment of bile canaliculi and bilirubin transport, reflected by increased ALP and GGT. 3. Mixed-type injury: A combination of hepatocellular and cholestatic patterns. Additionally, inflammatory cytokines such as TNF-α, IL-6, and IL-1β amplify hepatocellular damage. Kupffer cell activation leads to further release of pro-inflammatory mediators, contributing to systemic inflammation and disruption of hepatic microcirculation (7,21,22). 2. Mechanisms of Hepatotoxicity 2.1. Xenobiotic metabolism and bioactivation The liver’s capacity for xenobiotic metabolism is mediated by Phase I and Phase II enzymatic pathways. Cytochrome P450 (CYP) enzymes play a particularly important role in oxidative biotransformation(7). While these reactions often detoxify harmful compounds, they can also generate reactive intermediates capable of damaging hepatocytes. These reactive metabolites may covalently bind to cellular macromolecules (DNA, proteins, lipids), disrupt homeostasis, and initiate necrosis or apoptosis (Figure 1). 2.2. Oxidative stress and mitochondrial dysfunction A central feature of hepatocellular injury is oxidative stress, resulting from the excessive production of reactive oxygen species (ROS) and an insufficient antioxidant response(8). ROS generation leads to lipid peroxidation of cellular membranes, protein oxidation, and mitochondrial impairment. Mitochondrial LIVER INJURY   13 permeability transition pores open, leading to loss of membrane potential, decreased ATP synthesis, and release of pro-apoptotic factors such as cytochrome c, triggering cell death cascades (9). 2.3. Inflammatory responses and immune-mediated injury Kupffer cells, the resident macrophages of the liver, play a crucial role in amplifying hepatic injury(10). Upon activation, they release proinflammatory cytokines including TNF-α, IL-1β, and IL-6, which promote further hepatocyte damage and recruit neutrophils. This immune-mediated amplification can convert mild injury into severe necroinflammation. 2.4. Bile acid dysregulation and cholestasis Certain toxic insults target bile acid transporters such as the bile salt export pump (BSEP), leading to impaired bile flow and cholestatic hepatotoxicity(11). Accumulation of bile acids induces endoplasmic reticulum stress, mitochondrial dysfunction, and hepatocyte death. 2.5. Idiosyncratic reactions Unlike dose-dependent toxicities (e.g., acetaminophen overdose), idiosyncratic drugor herb-induced liver injury is unpredictable and may be immune-mediated or metabolic(12). It remains a significant challenge for clinicians due to its low incidence but severe outcomes. Figure 1. Mechanisms of Hepatotoxicity (18) 14   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 3. Phytochemicals: Classification and Biological Actions Phytochemicals encompass a diverse group of compounds with varying structural and pharmacological properties (Figure 2) • Polyphenols: These compounds, including resveratrol, curcumin, and quercetin, are potent antioxidants that modulate Nrf2, NF-κB, and MAPK signaling pathways(10). They scavenge ROS, enhance phase II detoxifying enzymes, and suppress proinflammatory gene expression. • Alkaloids: Berberine and sanguinarine exhibit anti-inflammatory and metabolic regulatory effects but can be toxic at high concentrations(11). • Terpenoids: Silymarin from Silybum marianum demonstrates membrane-stabilizing, antioxidant, and anti-fibrotic effects, making it one of the most widely used herbal hepatoprotective agents(12). • Flavonoids: Quercetin, catechins, and related molecules are known to enhance antioxidant defense systems and modulate inflammatory mediators(13). These compounds interact with cellular pathways involved in oxidative stress, inflammation, apoptosis, and mitochondrial stability. Figure 2. Phytochemicals, Classification and Biological Actions (18) LIVER INJURY   15 4. Hepatoprotective Effects of Phytochemicals 4.1. Antioxidant and cytoprotective mechanisms Many phytochemicals exert hepatoprotective effects by reducing oxidative stress and enhancing the cellular antioxidant defense system. Curcumin activates Nrf2 signaling, promoting the transcription of antioxidant response element (ARE)-regulated genes, including HO-1 and NQO1(14) Similarly, quercetin increases the activity of SOD, CAT, and GPx, counteracting ROS-induced injury(13). 4.2. Anti-inflammatory actions Polyphenols and alkaloids can suppress NF-κB activation, reducing the transcription of proinflammatory cytokines such as TNF-α and IL-6(10). This contributes to reduced Kupffer cell activation, attenuated neutrophil infiltration, and minimized hepatocellular damage. 4.3. Membrane stabilization and anti-fibrotic properties Silymarin stabilizes hepatocyte membranes, limits toxin penetration, and inhibits collagen deposition, slowing the progression of hepatic fibrosis(12) Experimental studies show significant reductions in transaminase levels and histopathological improvement in toxin-induced liver injury models. 4.4. Modulation of gut-liver axis Emerging evidence indicates that phytochemicals can influence gut microbiota composition, which in turn affects liver metabolism and inflammation(13,14,15) Polyphenols improve intestinal barrier function and reduce endotoxin translocation, indirectly protecting the liver. 5. Hepatotoxic Potential of Phytochemicals Despite their beneficial effects, phytochemicals may exert hepatotoxicity under certain conditions: • Dose-dependent toxicity: High doses of berberine or green tea extracts have been linked to elevated transaminases and clinical hepatitis(5,15). • Idiosyncratic herb-induced liver injury: Some individuals may develop immune-mediated liver injury after exposure to specific herbal preparations, even at therapeutic doses(16). 16   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS • Pyrrolizidine alkaloid toxicity: These compounds, found in several herbal teas, can cause hepatic sinusoidal obstruction syndrome, a severe and often irreversible condition(5,17,18). • Herb–drug interactions: Phytochemicals may modulate CYP450 activity, altering the metabolism of co-administered drugs and increasing hepatotoxic risk(7,18,19) The perception that “natural equals safe” is misleading. Standardization, pharmacovigilance, and clinical monitoring are essential to ensure patient safety. 6. Clinical Implications and Regulatory Challenges The increasing use of herbal medicines necessitates robust safety assessments. Currently, many herbal products are marketed as dietary supplements with limited regulatory oversight. This lack of standardization contributes to variability in phytochemical content and potential contamination with hepatotoxic compounds(16,17) Clinicians should consider herbal supplement use in patients presenting with unexplained liver injury. Detailed pharmacokinetic and toxicological studies are needed to define therapeutic windows and identify risk factors for hepatotoxicity. Integration of mechanistic biomarkers and genomic susceptibility profiling may also help predict adverse reactions in the future. 7. Clinical Presentation and Consequences of Liver Injury The clinical spectrum of hepatotoxicity ranges from asymptomatic biochemical abnormalities to fulminant liver failure. • Early stage: Fatigue, anorexia, right upper quadrant pain, and elevated ALT/AST. • Advanced stage: Jaundice, coagulopathy, hepatic encephalopathy, ascites, and systemic inflammatory response. • Chronic progression: Fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Diagnosis relies on a combination of laboratory markers, imaging, liver biopsy, and causality assessment tools such as the RUCAM score (23,24,25). These methods help differentiate between drug-induced liver injury (DILI) and other liver diseases. THYMOQUINONE   23 The study demonstrated that THQ prevented TGF-β induced HSC activation and ethanol-induced liver injury. Hepatic stellate cells (HSCs) are the primary producers of ECM; however, once activated, they increase the production of various ECM proteins such as α-SMA, MMPs, TIMPs, collagen, and TGF-β, thereby contributing to the progression of fibrosis. The findings revealed that TGF-β suppressed the phosphorylation of AMPK and LKB1, whereas THQ enhanced AMPK phosphorylation, thereby inhibiting HSC activation. These results were further confirmed in ethanol-fed mice. Cholestasis occurs as a result of impaired bile flow due to obstruction of the bile ducts or liver damage. It is also one of the major causes of liver cell injury, fibrosis, cirrhosis, and fatal liver failure (9). According to Kong et al. (21), administering THQ as a pretreatment at doses of 25 and 50 mg/kg improved the liver’s antioxidant capacity while reducing oxidative stress-mediated injury in cholestatic rats. According to the findings, THQ administration decreased hydroxyproline (HP) and MDA levels in liver tissue, while increasing SOD and GPx activities. Histopathological analyses further revealed that necrosis and fibrotic scores were lower in THQ-treated groups. In parallel, Oğuz et al. (22) demonstrated that THQ (50 mg/kg, orally) alleviated oxidative stress and liver injury in bile duct-ligated rats. In this study, THQ increased antioxidant enzyme (SOD and GPx) activities while reducing HP and MDA levels. Histological evaluations revealed that THQ attenuated fibrosis, bile duct hyperplasia, and portal hepatitis. It was also reported to decrease the immunostaining positivity of proliferating cell nuclear antigen and α-SMA. These results indicate that THQ may protect liver function in cholestasis and exert hepatoprotective effects by strengthening the antioxidant defense and suppressing oxidative damage. In a study investigating the effects of THQ on hepatocarcinogenesis models, Sayed-Ahmed et al. (23) demonstrated that THQ (4 mg/kg/day) prevented diethylnitrosamine (DENA)-induced hepatocarcinogenesis in rats by reducing oxidant status and enhancing the antioxidant activity. In addition, THQ improved histopathological alterations in liver tissues and exerted beneficial effects on various biochemical parameters (total bilirubin, ALT, ALP, MDA, NOx, GST, GSH, GPx, and CAT). Another study reported that THQ increased p21 expression while suppressing cyclin D1 and CDK2 levels, thereby leading to cell cycle arrest in HCC cells. At the same time, it triggered apoptosis by up-regulating Bax protein and downregulating Bcl-2 expression. In experiments conducted on nude mice, tumor development was inhibited through increased p21 levels and decreased Bcl-2 and NICD1 expression (24). According to the 24   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS findings of Raghunandhakumar et al. (25), treatment with THQ (20 mg/kg) suppressed NDEA-induced hepatocarcinogenesis, largely by regulating the G1/S phase transition and thereby exhibiting anti-proliferative activity. THQ administration reduced tumor proliferation, decreased liver injury markers, and prevented nodule formation. In addition, Xu et al. (26) reported that THQ suppressed cell proliferation in human cholangiocarcinoma (CCA) cell lines (TFK-1 and HuCCT1) by inducing cell cycle arrest and apoptosis and also inhibited angiogenesis and tumor growth in in vivo experiments. Overall, THQ suppresses tumor progression by modulating key biological processes involved in liver cancer development, including immune regulation, apoptosis, cell proliferation, autophagy, angiogenesis, invasion, and metastasis. Molecules such as p53, NF-κB, cyclin B1, cyclin D1, ROS, VEGF, and XIAP, which play pivotal roles in these processes, are among the major biomarkers targeted by THQ. It has been suggested that THQ may protect the liver against hepatotoxicity induced by various agents, similar to certain natural and chemical drugs (27). Studies have reported that THQ exerts protective effects against liver injury induced by cisplatin (28), tamoxifen (29), methotrexate (30), cyclophosphamide (31), cyclosporine A (32), acetaminophen (33), aflatoxin B1 (34), CCl4 (18), and pesticides (1,3) . 5. Conclusion Growing attention has been directed toward natural agents in the prevention and therapy of liver diseases. Among them, THQ has emerged as an important candidate with therapeutic potential for hepatic as well as extrahepatic disorders. Evidence from cell-based and animal studies suggests that THQ exerts beneficial effects in conditions such as hepatic fibrosis, cholestasis, liver cancer, and drug-related hepatotoxicity, largely through its antioxidant and anti-inflammatory mechanisms. 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Anti-fibrotic effect of thymoquinone on hepatic stellate cells. Phytomedicine. 2014;21(3):254-260. doi:10.1016/J.PHYMED.2013.09.014 21. Kong L, Li G, Yang P, Res ZX. Protective effect of thymoquinone on cholestatic rats with liver injury. Genet Mol Res. 2015;14(4):12247-12253. https://m.jstshuichan.com/sites/default/files/articles/year2015/vol14-4/pdf/ gmr5925.pdf 22. Oguz S, Kanter M, Erboga M, Erenoglu C. Protective effects of thymoquinone against cholestatic oxidative stress and hepatic damage after THYMOQUINONE   27 biliary obstruction in rats. J Mol Histol. 2012;43(2):151-159. doi:10.1007/ S10735-011-9390-Y/FIGURES/7 23. Sayed-Ahmed MM, Aleisa AM, Al-Rejaie SS, et al. Thymoquinone Attenuates Diethylnitrosamine Induction of Hepatic Carcinogenesis Through Antioxidant Signaling. Oxid Med Cell Longev. 2010;3(4):254-261. doi:10.4161/ OXIM.3.4.12714 24. Ke X, Zhao Y, Lu X, et al. TQ inhibits hepatocellular carcinoma growth in vitro and in vivo via repression of Notch signaling. Oncotarget. 2015;6(32):32610. doi:10.18632/ONCOTARGET.5362 25. Raghunandhakumar S, Paramasivam A, Senthilraja S, et al. Thymoquinone inhibits cell proliferation through regulation of G1/S phase cell cycle transition in N-nitrosodiethylamine-induced experimental rat hepatocellular carcinoma. Toxicol Lett. 2013;223(1):60-72. doi:10.1016/J. TOXLET.2013.08.018 26. Xu D, Ma Y, Zhao B, et al. Thymoquinone induces G2/M arrest, inactivates PI3K/Akt and nuclear factor-κB pathways in human cholangiocarcinomas both in vitro and in vivo. Oncol Rep. 2014;31(5):20632070. doi:10.3892/OR.2014.3059/DOWNLOAD 27. Khader M, Eckl PM. Thymoquinone: an emerging natural drug with a wide range of medical applications. Iran J Basic Med Sci. 2014;17(12):950. https://pmc.ncbi.nlm.nih.gov/articles/PMC4387230/ 28. Al-Malki AL, Sayed AAR. Thymoquinone attenuates cisplatininduced hepatotoxicity via nuclear factor kappaβ. BMC Complement Altern Med. 2014;14(1):1-8. doi:10.1186/1472-6882-14-282/FIGURES/4 29. Suddek GM. Protective role of thymoquinone against liver damage induced by tamoxifen in female rats. https://doi.org/101139/cjpp-2014-0148. 2014;92(8):640-644. doi:10.1139/CJPP-2014-0148 30. El-Sheikh AAK, Morsy MA, Abdalla AM, Hamouda AH, Alhaider IA. Mechanisms of Thymoquinone Hepatorenal Protection in MethotrexateInduced Toxicity in Rats. Mediators Inflamm. 2015;2015(1):859383. doi:10.1155/2015/859383 31. Laskar AA, Khan MA, Rahmani AH, Fatima S, Younus H. Thymoquinone, an active constituent of Nigella sativa seeds, binds with bilirubin and protects mice from hyperbilirubinemia and cyclophosphamide-induced hepatotoxicity. Biochimie. 2016;127:205-213. doi:10.1016/J.BIOCHI.2016.05.020 32. Farag MM, Ahmed GO, Shehata RR, Kazem AH. Thymoquinone improves the kidney and liver changes induced by chronic cyclosporine A 28   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS treatment and acute renal ischaemia/reperfusion in rats. Journal of Pharmacy and Pharmacology. 2015;67(5):731-739. doi:10.1111/JPHP.12363 33. Aycan IÖ, Tüfek A, Tokgöz O, et al. Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats. International Journal of Surgery. 2014;12(3):213-218. doi:10.1016/J.IJSU.2013.12.013 34. Nili-Ahmadabadi A, Tavakoli F, Hasanzadeh GR, Rahimi HR, Sabzevari O. Protective effect of pretreatment with thymoquinone against Aflatoxin B1 induced liver toxicity in mice. Daru. 2011;19(4):282. https://pmc. ncbi.nlm.nih.gov/articles/PMC3304388/ 29 CHAPTER IV BERBERINE Berrin YALINBAŞ KAYA (Assoc. Prof. Dr.) Department of Gastroenterology, Ministry of Health Eskisehir City Hospital, E-mail: [email protected], Orcid: 0000-0002-1414-4115 1. Introduction Berberine is an isoquinoline alkaloid widely distributed in plants, belonging to the protoberberine group and existing as a quaternary ammonium salt. It has the molecular formula C₂₀H₁₈NO₄⁺ (berberine ion), and is particularly abundant in Berberis vulgaris, Berberis aristata, Coptis chinensis, and Hydrastis canadensis (1–3). Its pharmacological properties include antioxidant, anti-inflammatory, antitumor, antidiabetic, and hypolipidemic effects (4,5). In the liver, berberine is primarily metabolized by cytochrome P450 enzymes (CYP2D6, CYP1A2, CYP3A4), generating four main metabolites— demethylberberine, berberrubine, jatrorrhizine, and palmatine—through oxidative demethylation (6,7). However, due to intestinal P-glycoprotein (P-gp)- mediated efflux mechanisms, the oral bioavailability of berberine is extremely low (8,9). In contrast, high tissue concentrations have been detected in the liver, spleen, kidneys, and lungs, suggesting that some of its pharmacological actions may be related to tissue accumulation (10). In recent years, interest has grown in the pharmacokinetic characteristics, hepatoprotective effects, and potential hepatotoxicity mechanisms of berberine (11–13). This review discusses the pharmacokinetic profile, hepatoprotective mechanisms, potential hepatotoxicity, and future clinical applications of berberine based on the current literature. 30   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Figure 1. Chemical structure of berberine (C₂₀H₁₈NO₄⁺, berberine ion; berberine chloride (commercial salt): C₂₀H₁₈ClNO₄·xH₂O). 2. Hepatoprotective Mechanisms of Berberine The hepatoprotective effects of berberine involve multiple mechanisms: 2.1. Antioxidant Activity Berberine reduces free radicals and increases glutathione (GSH) levels, thereby mitigating oxidative stress. It inhibits lipid peroxidation, protecting hepatocyte membranes from damage. Activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway upregulates antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). 2.2. Anti-inflammatory Activity Arachidonic acid pathways (COX and LOX) play roles in the production of inflammatory mediators. Liver injury is often associated with inflammatory responses. Berberine suppresses these pathways, lowering levels of inflammatory lipid mediators. Moreover, by inhibiting the nuclear factor kappa B (NF-κB) signaling pathway, berberine suppresses pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, reducing inflammatory cell infiltration and hepatocyte apoptosis (9,14). 2.3. Regulation of Lipid Metabolism Among its most extensively studied mechanisms in NAFLD, berberine activates AMPK, inhibiting fatty acid synthesis (ACC, FAS) while enhancing β-oxidation (PPARα). This prevents hepatic triglyceride and free fatty acid BERBERINE   31 accumulation, reversing steatosis. Furthermore, by upregulating hepatic LDL receptor expression, berberine decreases plasma cholesterol levels (5–7). 2.4. Reduction of Fibrosis and Cirrhosis Chronic liver injury may progress to fibrosis and cirrhosis. Berberine suppresses hepatic stellate cell activation and collagen synthesis, thereby attenuating fibrosis. It modulates profibrogenic pathways such as TGF-β/Smad and increases the activity of matrix metalloproteinases (MMPs). Experimental models (e.g., CCl₄, TAA) demonstrate regression of fibrotic nodules. Hepatotoxic anticancer drugs such as methotrexate, doxorubicin, and cyclophosphamide often cause hepatitis, steatohepatitis, or fibrosis. In animal studies, berberine treatment significantly reduced these histopathological changes (11,12). Chronic liver damage can progress to fibrosis and subsequent cirrhosis. Berberine holds the potential to slow liver fibrosis by reducing hepatic stellate cell activation and collagen synthesis. It is reported to modulate profibrogenic pathways like the TGF−β/Smad signaling pathway and increase the activity of matrix metalloproteinases (MMPs). Fibrotic nodules regress significantly in experimental models (CCl4 , TAA, etc.). Since many anticancer agents (like methotrexate, doxorubicin, and cyclophosphamide) are hepatotoxic (causing hepatitis, steatohepatitis, liver cell necrosis, liver fibrosis, or cirrhosis), finding ways to limit this hepatotoxicity is crucial. Accordingly, BBR treatment has been shown in animal studies to significantly reduce liver histopathological changes, including fibrosis, caused by anticancer drugs (11,12). 2.5. Interaction with Gut Microbiota Berberine is converted by intestinal bacteria into dihydroberberine (dhBBR), a more bioavailable form (14). 2.6. Modulation of Microbiota By increasing the production of short-chain fatty acids (particularly butyrate), berberine exerts hepatoprotective effects through the gut–liver axis (14). 2.7. Regulation of Bile Acid Homeostasis In conjunction with gut microbiota, berberine regulates bile acid metabolism, reducing the risk of cholestasis (15). 32   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 3. Potential Hepatotoxic Effects: Dose and Mechanistic Considerations Although generally regarded as safe, certain studies indicate that berberine may exhibit hepatotoxic potential at high doses or with prolonged use. Such effects have typically been observed in animal models at doses exceeding 100 mg/kg. 3.1. Dose Dependence Berberine has a narrow therapeutic window, and elevated doses may increase liver enzymes (ALT, AST). In diabetic rats, chronic administration of >50–150 mg/kg for 16 weeks induced hepatic injury, whereas such effects were absent in healthy rats (9,13). These variations may reflect interindividual differences in metabolism and clearance. 3.2. Mitochondrial Dysfunction In vitro evidence suggests that high concentrations of berberine can inhibit mitochondrial respiratory chain function, leading to oxidative stress and hepatocyte injury. At low to moderate doses, however, berberine stabilizes mitochondrial membranes and activates the Nrf2/HO-1 pathway, enhancing antioxidant enzyme expression (SOD, CAT, GSH-Px) and reducing ROS levels (9,10). 3.3. Drug–Drug Interactions Berberine may inhibit cytochrome P450 enzymes, particularly CYP3A4 and CYP2D6. This can increase plasma concentrations of co-administered drugs metabolized by these enzymes (e.g., statins, cyclosporine, anticoagulants), raising toxicity risk (17). 3.4. Disturbance in Bile Acid Homeostasis Inhibition of transporters such as BSEP and MRP2 may result in cholestasis (18). 4. Pharmacokinetic Properties of Berberine Although berberine exerts beneficial effects across various diseases, its poor oral absorption and low bioavailability limit clinical use. Intravenous administration can raise plasma levels, but clinical trials report adverse events such as hypotension and respiratory depression (22). 39 CHAPTER V APIGENIN Sinan İNCE (Prof. Dr.) Afyon Kocatepe University, Faculty of Veterinary, Department of Pharmacology and Toxicology, E-mail: [email protected], Orcid: 0000-0002-1915-9797 1. Introduction Plant species rich in pharmaceutically active constituents are considered complementary or alternative agents in modern therapeutic approaches (1). Naturally derived compounds obtained from these plants possess therapeutic potential in the management of various pathophysiological conditions. In particular, an increasing body of scientific evidence supports the efficacy of plant-based therapeutic strategies in complex diseases such as cancer (2) and metabolic dysfunctions (3,4). Recent experimental and clinical studies have demonstrated that plant extracts exhibit a wide range of biological activities. These extracts have been shown to possess hepatoprotective effects that safeguard liver tissue from damage, as well as anti-inflammatory, free radical-scavenging, and tumor cell proliferation-inhibiting properties (2,5–7). Furthermore, the relatively low toxicity levels of these natural products enhance their safety profile for clinical applications (8). Flavonoids have held a prominent position among plant-derived bioactive compounds for over a century and have been extensively investigated in various clinical and experimental studies (9). These compounds, which exhibit a broad structural diversity in plants, belong to the group of secondary metabolites containing a phenyl-substituted chroman core and are classified as natural products (4). Based on their chemical structures, flavonoids are categorized into six main classes: flavones, flavonols, isoflavones, anthocyanins, flavan-3-ols, and flavanones (1). Flavonoids have been reported to exhibit diverse pharmacological 40   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS activities, including immunomodulatory, antioxidant, anti-inflammatory, and anti-cancer effects, particularly by preventing tumor cell proliferation (10–13). Moreover, their antimicrobial, anti-genotoxic, anti-allergic, neuroprotective, and cardioprotective activities have also been demonstrated in various studies (14–16). 2. Flavonoids and Flavones: Structural and Pharmacological Perspectives Plant-derived phenolic compounds are classified into four main groups based on their chemical structures and constituent profiles: flavonoids, phenolic acids, stilbenes, and lignans. The primary criteria for this classification include the number of phenolic rings, the nature of the linkages between these rings, and the overall organization of the molecular skeleton (17). Among these groups, flavonoids, in particular, have attracted significant scientific interest due to their occurrence as naturally synthesized low-molecular-weight polyphenolic secondary metabolites in plants (18). Biosynthetically, flavonoids are produced via the phenylpropanoid pathway, playing roles both in the plant’s physiological defense systems and in exerting a wide range of biological effects on human health. Chemically, flavonoids are 2-phenyl-benzo-γ-pyran derivatives characterized by a C6–C3–C6 skeleton and a chromone structure. Their molecular architecture comprises two aromatic benzene rings (A and B) and an attached heterocyclic pyran ring (C). This three-ring system plays a critical role in determining both the biological activity and the metabolic transformations of flavonoids. In terms of carbon atom positioning, a specific numbering system is used: carbons in the A and C rings are numbered sequentially with standard numerals, while those in the B ring are denoted with superscript numbers (19). This structural framework serves as an important reference for defining flavonoid subclasses and analyzing functional groups. Based on molecular variations, flavonoids are further classified into subclasses such as flavones, flavanones, flavonols, and polymethoxyflavones. These subclasses differ according to the degree of hydroxylation, methylation patterns, glycosidic linkages, and conjugation structures, each possessing a distinct biological activity profile (20). Moreover, this classification is of considerable importance for understanding the pharmacokinetic properties, bioavailability, and interactions of flavonoids with cellular targets. In both plant physiology and pharmaceutical sciences, flavonoids represent a structurally diverse and functionally versatile group of compounds. APIGENIN   41 3. Apigenin (Apig): A Bioactive Flavone 3.1. The Structure and Chemical Characteristics of Apigenin Apigenin is commonly found in nature in glycosidic conjugated forms, such as apigenin-7-O-glucoside, as well as in various acylated derivatives (21). This flavonoid is synthesized as a secondary metabolite in many plant species and occurs in both monomeric and dimeric forms in nature. The dimeric form of apigenin, known as biapigenin, is particularly isolated from the buds and flowers of Hypericum perforatum (St. John’s Wort), a plant recognized for its neuroprotective properties (9). In addition, members of the Artemisia family, including the genera Tanacetum, Achillea, Artemisia, and Matricaria, have been reported to be rich sources of apigenin (22). Numerous fruits, vegetables, and medicinal plants contain high levels of apigenin, making it readily obtainable from natural dietary sources (23). Notably, plant-derived foods such as parsley, artichoke, onion, chamomile, and orange are considered significant sources for apigenin production (4,24). Furthermore, products such as grapefruit, corn, rice, tea, and wheat germ also contain high concentrations of apigenin and its derivatives (9). In certain plants, apigenin occurs in O-glycosidic or C-glycosidic forms, allowing its purification from sources such as celery, grapes, apples, and vervain (8). In this context, parsley and celery are among the richest natural sources of apigenin. Overall, a variety of food groups—including fruits, vegetables, aromatic and medicinal plants, nuts, honey, and herbal teas—can be identified as the primary natural sources of apigenin (21). Apigenin occurs in nature in both monomeric and dimeric forms, with its dimeric form referred to as “biapigenin.” From a physicochemical perspective, this flavonoid compound exhibits very low solubility in water, approximately 1.35 µg/mL, while demonstrating high solubility in organic solvents such as dimethyl sulfoxide (DMSO) and dilute potassium hydroxide (KOH) (24). Its solubility in hot alcohol is moderate. Apigenin has a melting point ranging between 345 and 350 °C, presents as yellow crystalline solids in its physical form, and possesses relatively low stability. Therefore, storage at −20 °C is recommended for prolonged preservation (25). With a molecular formula of C₁₅H₁₀O₅ and a molecular weight of 270.24 g/mol, apigenin is chemically defined as 4′,5,7-trihydroxyflavone and classified as a natural aglycone within the flavone subclass (9). This structure indicates that apigenin serves as the core unit of numerous plant-derived glycosides. In nature, it predominantly occurs as 7-O-glycosides or as C-glycosides at the 6or 8-positions, and upon oral 42   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS administration, these glycosidic forms can be metabolized into free apigenin within the gastrointestinal tract (26). Moreover, apigenin has been reported to exhibit lower toxicity compared to related flavonoid derivatives, supporting its safe intake through daily dietary consumption (24). 3.2. The Pharmacokinetic Profile of Apigenin The circulating concentration and tissue distribution of apigenin are closely associated with its conversion into larger conjugated compounds. In particular, glucuronidation products formed in the intestinal mucosa are re-secreted into the intestinal lumen, thereby reducing the systemic absorption of apigenin (27). Furthermore, apigenin undergoes conjugation reactions such as methylation, sulfation, and glucuronidation, which significantly influence its biological activity (17). The enzyme uridine 5′-diphosphoglucuronosyltransferase (UDP-glucuronosyltransferase), located in intestinal epithelial cells, serves as the primary catalyst responsible for apigenin glucuronidation (28). In addition, apigenin is metabolized more rapidly in the intestine than in the liver (9). In a study conducted on rat liver, apigenin was reported to be metabolized in vivo and ex vivo via Phase I metabolic reactions into monohydroxy derivatives, including luteolin, scutellarein, and isoscutellarein. These metabolites subsequently undergo various conjugation reactions as part of Phase II metabolism, the second stage of biotransformation in the body. Specifically, the Phase I metabolites scutellarein and isoscutellarein are conjugated into three distinct mono-glucuronide and one mono-sulfated derivatives, whereas luteolin, a major Phase I product of apigenin, is converted into four different monoglucuronides, two sulfated compounds, and a methylated product, diosmetin. Additionally, the unmetabolized apigenin molecule was also found to be converted into two mono-glucuronide and one mono-sulfated conjugated forms (29). Comparative analyses of metabolite excretion revealed that in immature male and female rats, the apigenin mono-glucuronide conjugate was excreted at higher rates (10.0–31.6%) compared to the mono-sulfate conjugate (2.0–3.6%). A similar trend was observed in mature female rats; however, in mature male rats, the excretion of the mono-glucuronide conjugate was lower (4.9%) relative to the mono-sulfate conjugate (13.9%). These differences are believed to arise from sexand maturity-dependent variations in metabolic enzyme activities (30). These findings highlight the complex nature of apigenin’s metabolic APIGENIN   43 transformations and suggest that its pharmacokinetic properties may vary according to biological sex and age. 3.3. Pharmacological Properties Apigenin, a natural compound belonging to the flavonoid family, has recently attracted considerable scientific attention due to its low toxicity profile and selective effects on cancer cells (9). Numerous preclinical and clinical studies have supported its therapeutic and protective effects not only in cancer treatment but also in various chronic and inflammatory diseases. The antioxidant, anti-inflammatory, anticarcinogenic, and immunomodulatory activities of apigenin demonstrate its potential to target multiple stages of pathophysiological processes, thereby inhibiting disease progression (9). Moreover, compared to other flavonoids, apigenin exhibits fewer adverse effects, contributing to a favorable safety profile even with long-term use. Owing to these characteristics, apigenin is considered a promising molecule in the development of pharmacological agents. Apigenin has become a focal point of scientific research due to its low toxicity profile and its selective biological effects on cancer cells compared to other flavonoids (9). This compound has been supported by experimental and clinical studies demonstrating both protective and therapeutic effects against various diseases (9). Notably, in lipopolysaccharide (LPS)-stimulated astrocytes and RAW 264.7 macrophage cells—key players in inflammatory responses—apigenin has been shown to significantly reduce nitric oxide (NO) production by suppressing the expression of inducible nitric oxide synthase (iNOS) (31). In the inflammatory process, not only enzymes such as iNOS and cyclooxygenase-2 (COX-2) but also a variety of cytokines—including interleukin (IL)-1β, IL-6, interferon-gamma (IFN-γ), IL-4, IL-5, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 alpha (MCP-1α), and macrophage inflammatory protein-1 alpha (MIP-1α)—play critical regulatory roles. Apigenin modulates the inflammatory process by suppressing the gene expression of these cytokines through the modulation of various protein kinases involved in signaling pathways, such as protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and mitogen-activated protein kinase (MAPK) (31). These mechanisms enable apigenin to attenuate the inflammatory response, thereby slowing the progression of chronic diseases and preventing tissue damage. Thus, apigenin demonstrates pharmacological potential not only 44   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS through its antioxidant properties but also via regulatory effects on cellular signaling pathways. 3.4. Hepatoprotective Effects of Apigenin Apigenin, as a natural flavonoid compound, exhibits multiple biological effects that protect and support liver health. In particular, in carbon tetrachloride (CCl₄)-induced toxicity models frequently used to simulate liver injury, apigenin has been demonstrated to reduce oxidative stress in hepatocytes and enhance antioxidant defense mechanisms. Apigenin not only inhibits free radical generation but also increases the activity of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), thereby preventing oxidative damage at the cellular level (5). Through these mechanisms, apigenin contributes to the preservation of liver function by preventing liver injury characterized by elevated liver enzymes (32). Moreover, apigenin plays a significant role in the inflammatory processes underlying chronic liver diseases. By inhibiting the nuclear factor kappa B (NFκB) signaling pathway, which is critical for inflammation regulation, apigenin reduces the production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), thereby attenuating inflammatory damage in liver tissue (7). The benefits of apigenin have also been observed in liver fibrosis, which develops as a consequence of chronic inflammation. Apigenin suppresses the transforming growth factor-beta 1 (TGF-β1) signaling pathway and inhibits the activation of hepatic stellate cells, resulting in the reduction of fibrotic tissue formation (33). Due to these properties, apigenin is considered a promising natural compound for the prevention or retardation of liver fibrosis progression. Additionally, apigenin exhibits significant anticarcinogenic effects against liver cancer, particularly hepatocellular carcinoma. Apigenin induces apoptosis and inhibits cell proliferation by promoting the activation of tumor suppressor genes such as p53 in cancer cells (34). Furthermore, apigenin reduces the invasion and metastatic potential of tumor cells, thereby contributing to the control of cancer progression. With these multifaceted activities, apigenin is regarded as an important molecule with both protective and therapeutic potential in the treatment of liver diseases. Yang et al. (35) were the first to demonstrate that apigenin (100 and 200 mg/kg) exerts notable protective effects against acetaminophen-induced acute liver injury in mice. These effects were attributed to enhanced glutathione APIGENIN   45 reductase (GR) activity, leading to elevated hepatic GSH levels. In line with these findings, Rašković et al. (36) reported that apigenin (10 mg/kg) mitigated acetaminophen-induced liver damage in rats by suppressing lipid peroxidation and strengthening enzymatic antioxidant defenses. Subsequent studies by Zhao et al. (37) and Zhang et al. (38) further corroborated the hepatoprotective properties of apigenin in both in vivo and in vitro models. Specifically, Zhao et al. (37) showed that apigenin promotes autophagy through regulation of the SIRT1-p53 axis, thereby reducing inflammation and oxidative stress. Meanwhile, Zhang et al. (38) demonstrated that apigenin alleviates acetaminophen-induced liver injury by activating the AMPK/GSK-3β signaling cascade, enhancing CPT1A activity, and stimulating the Nrf2-mediated antioxidant response. Evidence indicates that pretreatment with apigenin (20 mg/kg) markedly enhances hepatic antioxidant defenses (including GSH, CAT, GPx, and SOD) in mice exposed to methotrexate-induced hepatotoxicity, while simultaneously lowering serum markers of liver damage (AST, ALT, ALP) and pro-inflammatory cytokines (TNF-α, IL-1β) (39). Similarly, Sahindokuyucu-Kocasari et al. (40) reported that apigenin administration (3 mg/kg) mitigated methotrexate-induced liver injury by restoring oxidative balance (MDA, SOD, CAT, GSH-Px, GSH) and suppressing the expression of apoptotic (Caspase-3) and inflammatory mediators (CRP, G-CSF, iNOS). Collectively, these findings suggest that apigenin protects against methotrexate-induced hepatotoxicity primarily through its antioxidant and anti-inflammatory activities. Al-Amarat et al. (41) reported that pretreatment with apigenin (20 and 40 mg/kg) markedly alleviated cyclophosphamide-induced hepatic damage in rats. This protective effect was evidenced by decreased serum levels of ALT, AST, ALP, and LDH, as well as suppression of ROS, LPO, NF-κB, pro-inflammatory cytokines (TNF-α, IL-6, iNOS), and apoptotic markers (Bax, Caspase-3). The underlying mechanism appears to involve activation of the Nrf2/HO-1 signaling cascade and enhancement of the antioxidant defense system. Wang et al. (42, 43) were the first to reveal, using both in vivo and in vitro models, that apigenin exerts protective effects against alcohol-induced hepatic injury. In animal studies, apigenin (150 and 300 mg/kg) lessened liver damage by enhancing acetaldehyde degradation, improving PPARα-driven lipid metabolic pathways, and attenuating CYP2E1-mediated oxidative stress (42). In cell culture experiments, apigenin (6, 12, and 24 µM) downregulated NF-κB and upregulated IκB-α expression, thereby suppressing inflammatory cytokine production and reducing alcohol-induced hepatocellular inflammation (43). 46   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Collectively, these results suggest that apigenin may counteract alcohol-related liver injury by functioning as a GR activator and CYP2E1 inhibitor, offering a mechanistic basis for potential therapeutic applications in ALD. According to Fehaid et al. (44), apigenin (20 mg/kg) attenuated lead induced oxidative stress by enhancing Nrf2/HO-1 pathway activity and stimulating the expression of downstream antioxidant enzymes, which collectively diminished pro-oxidant generation. Furthermore, apigenin downregulated pro-inflammatory cytokine levels and significantly inhibited lead-induced apoptosis in both hepatocytes and renal cells. Ali et al. (45) reported that apigenin exerts a protective effect against N-nitrosodiethylamine-induced liver toxicity in rats. Treatment with apigenin (10, 20, and 40 mg/kg) produced a dose-dependent decline in serum ALT, AST, ALP, and LDH activities, along with marked decreases in lipid peroxidation and protein carbonyl levels. Moreover, comet assay results indicated that apigenin administration significantly lessened DNA damage in hepatocytes, circulating lymphocytes, and bone marrow cells. In a murine model of LPS-induced hepatic injury, apigenin (100 and 200 mg/kg) showed marked hepatoprotective activity by suppressing NF-κB and MAPK signaling pathways, boosting enzymatic and non-enzymatic antioxidant defenses, and markedly decreasing oxidative stress, neutrophil infiltration, and overall inflammatory response (46). Tsalkidou et al. (47) and Tsaroucha et al. (48) demonstrated that apigenin confers notable protection in hepatic ischemia/reperfusion (I/R) injury. Administration of apigenin (15 mg/kg) modulated the expression of Bcl-2 and Bax, thereby lowering the activity of pro-apoptotic factors and reducing ICAM-1 levels, which in turn limited inflammatory mediator release and ameliorated I/R-induced hepatic damage (48). Furthermore, apigenin treatment significantly downregulated Fas gene expression in hepatocytes during reperfusion (47). Since the Fas gene encodes the Fas receptor, a key initiator of apoptosis (49), these findings suggest that apigenin’s hepatoprotective effects may be partly mediated through suppression of the Fas/FasL apoptotic signaling pathway. 4. Conclusion In recent years, there has been a significant increase in interest toward natural products, particularly plant-derived agents. This trend is primarily driven by the growing acceptance of using natural compounds therapeutically in the treatment of numerous diseases that adversely affect quality of life. Numerous studies in APIGENIN   47 the literature have demonstrated that plant extracts exhibit a broad spectrum of biological and pharmacological effects. For instance, certain phytochemicals such as epicatechin, quercetin, myricetin, puerarin, kaempferol, naringin, hesperidin, proanthocyanidins, and genistein are known to possess regulatory effects on hyperglycemia. Research focused on flavonoids has also revealed that compounds in this class exhibit various biochemical and therapeutic properties. However, studies specifically investigating apigenin, a member of the flavonoid group, remain limited. In this context, the present review has addressed apigenin and its potential effects on several diseases in detail. As a result, apigenin, increasingly studied due to its beneficial health effects, may hold a significant position in the future as a bioactive phytochemical and is considered to have a critical role in the prevention and treatment of the rising global health challenges. Furthermore, apigenin stands out as a multifunctional bioactive compound exhibiting hepatoprotective, antioxidant, anti-inflammatory, antifibrotic, and anticancer effects that support liver health. Nevertheless, further studies are required to translate these effects into clinical applications. Current preclinical data indicate that apigenin is a promising natural agent for the prevention and treatment of liver diseases. References 1. Vazhappilly CG, Amararathna M, Cyril AC, et al. Current methodologies to refine bioavailability, delivery, and therapeutic efficacy of plant flavonoids in cancer treatment. J Nutr Biochem. 2021;94:108623. doi:10.1016/j. jnutbio.2021.108623 2. Şirin N, Elmas L, Seçme M, Dodurga Y. Investigation of possible effects of apigenin, sorafenib and combined applications on apoptosis and cell cycle in hepatocellular cancer cells. Gene. 2020;737:144428. doi:10.1016/j. gene.2020.144428 3. Panda S, Kar A. Apigenin (4’,5,7-trihydroxyflavone) regulates hyperglycaemia, thyroid dysfunction and lipid peroxidation in alloxan-induced diabetic mice. J Pharm Pharmacol. 2007;59(11):1543-1548. doi:10.1211/ jpp.59.11.0012 4. Kilit AC, Aydemir D. Apiin’in sitotoksik etkisi. Bilim Armonisi. 2021;4(2):64-70. 5. Singh JPV, Selvendiran K, Banu SM, Padmavathi R, Sakthisekaran D. Protective role of apigenin on the status of lipid peroxidation and antioxidant 48   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS defense against hepatocarcinogenesis in Wistar albino rats. Phytomedicine. 2004;11(4):309-314. doi:10.1078/0944711041495184 6. Wang J, Liu YT, Xiao L, Zhu L, Wang Q, Yan T. Anti-inflammatory effects of apigenin in lipopolysaccharide-induced inflammatory in acute lung injury by suppressing COX-2 and NF-kB pathway. Inflammation. 2014;37(6):2085-2090. doi:10.1007/s10753-014-9960-9 7. Yue S, Xue N, Li H, Huang B, Chen Z, Wang X. Hepatoprotective effect of apigenin against liver injury via the non-canonical NF-κB pathway in vivo and in vitro. Inflammation. 2020;43:1634-1648. doi:10.1007/s10753-02001222-6 8. Bi CC, Han WW, Yu JR, Zhang HF, Xing GY, Liu Z. Insights into the pharmacological and therapeutic effects of apigenin in liver injuries and diseases. Heliyon. 2023;9(5):e15609. doi:10.1016/j.heliyon.2023.e15609 9. Ali F, Rahul, Naz F, Jyoti S, Siddique YH. Health functionality of apigenin: A review. Int J Food Prop. 2017;20(6):1197-1238. doi:10.1080/10942 912.2016.1207188 10. Huang J, Wang S, Zhu M, Chen J, Zhu X. Effects of genistein, apigenin, quercetin, rutin and astilbin on serum uric acid levels and xanthine oxidase activities in normal and hyperuricemic mice. Food Chem Toxicol. 2011;49(9):1943-1947. doi:10.1016/j.fct.2011.06.054 11. Banerjee K, Mandal M. Oxidative stress triggered by naturally occurring flavone apigenin results in senescence and chemotherapeutic effect in human colorectal cancer cells. Redox Biol. 2015;5:153-162. doi:10.1016/j. redox.2015.05.005 12. Ghițu A, Schwiebs A, Radeke HH, et al. A comprehensive assessment of apigenin as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. Nutrients. 2019;11(4):858. doi:10.3390/ nu11040858 13. Costa ACDF, de Sousa LM, dos Santos Alves JM, et al. Antiinflammatory and hepatoprotective effects of quercetin in an experimental model of rheumatoid arthritis. Inflammation. 2021;44(5):2033-2043. doi:10.1007/ s10753-021-01427-5 14. Zhao L, Wang JL, Liu R, Li XX, Li JF, Zhang L. Neuroprotective, anti-amyloidogenic and neurotrophic effects of apigenin in an Alzheimer’s disease mouse model. Molecules. 2013;18(8):9949-9965. doi:10.3390/ molecules18089949 QUERCETIN   55 Figure 1: Structural Formula of Quercetin The amount of quercetin consumed in the diet varies depending on eating habits and the type of food consumed. Some literature suggests that daily quercetin intake in a Western diet ranges from 5–40 mg/day, with an average of approximately 15 mg/day. A study in China reported an average of 20.9 mg/day, while in Japan, the average was approximately 15–16 mg/ day. However, while no studies have directly measured quercetin levels in the Mediterranean diet, where fruit and vegetable consumption is high, it has been emphasized that these levels may be higher than in other regions. (8,9,10) Quercetin’s natural form is mostly glycoside-bound, and this form undergoes hydrolysis in the intestines to form an aglycone structure. This conversion is important for the compound’s absorption and biological activity. Furthermore, the amount of quercetin can vary depending on the food’s growing conditions, storage time, and cooking method. For example, cooking onions has been reported to significantly reduce quercetin content. (8) 3. Bioavailability and Metabolism Despite its potent biological activity, quercetin is a compound with low bioavailability. Absorption occurs primarily in the small intestine. Quercetin glycosides ingested with food are hydrolyzed to the free aglycone form in the intestinal mucosa or by microbial enzymes. Following absorption, quercetin undergoes metabolism in both the intestinal epithelium and the liver. 56   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Its bioavailability is influenced by diet and accompanying nutrients, with fatty meals especially enhancing solubility and uptake. Furthermore, the intestinal microbiota plays an important role in quercetin metabolism; quercetin can be broken down into phenolic acids by microbial enzymes, and these metabolites can exhibit biological activity. (8) After quercetin is metabolized in the body, it is eliminated primarily through urine, bile, and feces. Studies in humans have reported that the plasma half-life of quercetin ranges from approximately 11 to 28 hours. This long plasma half-life suggests that it may accumulate after regular and continuous consumption. A significant portion of orally ingested quercetin is metabolized in the liver and intestinal mucosa and excreted in the urine. Some is excreted into the intestines via bile, where it enters the enterohepatic cycle, and some is reabsorbed. Another route of elimination is breakdown by microbial enzymes in the gastrointestinal tract and excreted in the feces. (11) In the human diet, quercetin is predominantly present in the form of glycosides. Since this form is not readily absorbed, it undergoes enzymatic hydrolysis in the digestive tract, where it is converted into its aglycone form. After being digested and absorbed, quercetin undergoes metabolic processing in several organs, including the liver, kidneys, and both the small and large intestines. Due to its biological activities, quercetin has been studied as both a dietary supplement and a phytochemical agent for managing various liver conditions, including inflammatory, fibrotic, and metabolic disorders. These encompass hepatitis, sudden liver failure, alcoholic liver disease, and fatty liver not related to alcohol. These therapeutic effects are largely attributed to quercetin’s potent antioxidant activity. Quercetin’s effects on the liver are not limited to its antioxidant properties; it also plays an active role in protecting liver tissue through its anti-inflammatory, antifibrotic, and antiapoptotic effects. (12) These protective effects of quercetin are schematized in Figure 1. 4. Quercetin’s Hepatoprotective Effect Mechanisms 4.1. Antioxidant and Detoxification Effects In the body, free radicals are naturally produced as by-products of cellular metabolism, and under normal physiological conditions, their potentially harmful effects are neutralized by antioxidant systems. When the balance between free radical production and antioxidant defense shifts toward an excess of radicals, oxidative stress arises. This imbalance can damage cellular structures QUERCETIN   57 and functions, accelerate aging, and contribute to the development of various diseases, including cardiovascular disorders, diabetes, and liver injury. Growing data indicate that oxidative stress is a key factor in triggering and driving the progression of hepatic diseases. Accordingly, antioxidants are widely used in the management of oxidative liver damage, and consistent intake of antioxidant compounds is regarded as a preventive measure to reduce the risk or delay the onset of liver disorders. Moreover, the liver itself is central to the metabolism of many compounds that can promote the generation of free radicals. (13,14,15) Figure 2: Hepatoprotective effect of quercetin Quercetin is widely acknowledged for its potent antioxidant capacity, which has attracted considerable attention in studies investigating its protective effects on the liver and other organs. In vitro experiments have confirmed that quercetin exhibits significant antioxidant and cytoprotective effects in human liver cell lines, highlighting its potential in maintaining hepatic health. Complementary in vivo studies have demonstrated that quercetin alleviates carbon tetrachloride (CCl4)–induced liver toxicity in mice, achieving dose-dependent reductions in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and triglycerides. In addition to mitigating biochemical markers of liver damage, quercetin also suppresses malondialdehyde (MDA) formation, a key indicator of lipid peroxidation, while enhancing hepatic glutathione (GSH) content and the activity of endogenous antioxidant enzymes. Together, these findings suggest that quercetin represents a promising natural compound for counteracting oxidative liver injury and supporting overall hepatic function. (16) 58   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS In a related investigation, Wang et al. (17) evaluated the impact of quercetin on ovarian aging in rats at doses of 12.5, 25, and 50 mg/kg. Using Western blot analysis, they assessed the expression of genes associated with oxidative stress, including catalase and glutathione synthetase, in ovarian cells. Their results indicated that quercetin had minimal effect on ovarian morphology, hormone secretion, or the estrous cycle. Interestingly, even at low concentrations, quercetin upregulated genes involved in oxidative stress defense, suggesting that it may strengthen the ovarian antioxidant system and enhance cellular resilience against oxidative damage. These findings underscore quercetin’s potential to modulate redox balance in both hepatic and ovarian tissues, reflecting its broader significance as a bioactive phytochemical with multifaceted protective properties. Zhang et al. (18) investigated the in vivo therapeutic detoxification effects of quercetin against acute liver injury caused by carbon tetrachloride (CCl₄), which is toxic to the liver. In the study, quercetin administered orally within 30 minutes after CCl₄ toxicity resulted in a significant decrease in serum ALT/AST activity. Quercetin also reduced MDA levels, an indicator of lipid peroxidation in the liver, and offset the CCl₄-induced increase in GSH levels, contributing to the alleviation of oxidative stress. These findings strongly support the notion that quercetin may function as a detoxifying agent against CCl₄-induced acute hepatotoxicity, and that this effect is mediated by activating cellular antioxidant defense systems. One of the liver’s primary functions is to transform ingested drugs, toxins, and endogenous compounds into water-soluble compounds, rendering them less toxic. This detoxification process occurs in two stages. Phase I involves oxidation, reduction, and hydrolysis reactions. Phase II consists of the addition of water-soluble groups to compounds modified in Phase I. Cytochrome P450 (CYP) enzymes play the most significant role in Phase I. Isoenzymes such as CYP1A2, CYP2C9, CYP2C19, and CYP3A4 are particularly active in the first metabolic steps of many drugs and xenobiotics. (19,20) A study conducted by Rastogi et al. (21) investigated the regulatory effects of quercetin on these enzymes. In vitro analyses using human liver microsomes revealed that quercetin significantly inhibited these CYP isoenzymes. These findings suggest that quercetin may slow the degradation rate of some drugs or toxic compounds by interfering with Phase I metabolism, potentially leading to drugdrug interactions. Therefore, quercetin’s effects on liver detoxification are not limited to its antioxidant capacity; it also stands out as an important bioactive QUERCETIN   59 substance that can influence pharmacokinetic processes via CYP enzyme systems. 4.2. Anti-inflammatory Effects Quercetin is generally found in glycoside form in natural plant sources, particularly in foods such as fruits, vegetables, tea, and wine. Quercetin ingested in this glycoside form is converted to quercetin by microorganisms in the intestines. This converted quercetin has been observed to have greater antiinflammatory activity in vivo compared to directly ingested quercetin. In other words, quercetin released from its glycoside form in the intestines exhibits better antioxidant and anti-inflammatory properties. The anti-inflammatory activity of quercetin is partly attributed to its ability to inhibit NF-κB signaling, thereby lowering pro-inflammatory cytokine production and mitigating inflammatory processes. (22) In a study conducted by Nouri et al. (23), they evaluated the protective effects of quercetin on diclofenac-induced hepatotoxicity in terms of reducing the inflammatory response. Research has shown that diclofenac administration resulted in a pronounced inflammatory response in the liver, characterized by a significant increase in the levels of proinflammatory cytokines (e.g., TNF-α and IL-1β). Furthermore, quercetin administration significantly suppressed the expression of these inflammatory mediators, and a reduction in histopathologically observed inflammatory infiltration in liver tissue was also reported. Research has shown that liver damage from various sources can lead to the synthesis and release of proinflammatory cytokines, including TNF-α and IL-1β. Quercetin, combined with its antioxidant capacity, has been shown to suppress the inflammatory response and reduce inflammatory damage triggered by oxidative stress. Based on these findings, quercetin is considered an effective agent not only in reducing oxidative stress but also in regulating inflammatory processes. The study provides important data supporting the pharmacological potential of quercetin and suggests that this flavonoid may be a protective strategy against inflammatory damage induced by hepatotoxic agents such as diclofenac. Flavonols are bioactive compounds known to exert strong anti-inflammatory effects in both cellular and animal models of inflammation. In one study, the anti-inflammatory potential of quercetin, a naturally occurring flavonol, was assessed in lipopolysaccharide-stimulated macrophages. The results indicated that quercetin inhibited nitric oxide production, reduced interleukin-6 (IL-6) expression, and suppressed NF-κB activation. Furthermore, histopathological analyses showed a marked decrease in inflammation and IgE levels following 60   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS quercetin treatment. These findings suggest that quercetin possesses promising therapeutic potential for the prevention and management of inflammatory conditions. (24) 4.3. Antiapoptotic Effects Efforts to develop alternative approaches with fewer side effects continue to overcome the limitations of current therapies for liver cancer, a prevalent disease worldwide. Among natural strategies, quercetin stands out due to its strong antioxidant properties, which enable it to modulate key processes involved in hepatocellular carcinoma, including inflammation, fibrosis, and apoptosis. Quercetin has been shown to upregulate pro-apoptotic proteins such as Bax and caspase-3, thereby promoting programmed cell death, while downregulating proteins like Bcl-2 that support cell survival and proliferation. Extensive studies on quercetin’s anticancer potential highlight its promise as a preventive agent against cancer development. (25) In another study, Wang et al. (26) investigated the potential role of quercetin, a natural flavonoid, in enhancing the antitumor efficacy of doxorubicin, a chemotherapeutic agent commonly used in liver cancer treatment, and in reducing drug-induced liver cytotoxicity. The study demonstrated a synergistic effect of the combination of quercetin and doxorubicin on hepatocarcinoma cell lines using both in vitro and in vivo experimental models. This combination significantly enhanced the cytotoxic effect of doxorubicin, suppressed cell proliferation, and increased apoptosis rates. In contrast, quercetin was found to have a protective effect on normal liver cells, reducing cytotoxicity and mitigating cellular damage. Molecular analyses showed that doxorubicin administered with quercetin increased the activation of caspase-9 and caspase-3 in the mitochondrial apoptotic pathway, reduced mitochondrial membrane potential, and triggered the release of cytochrome c. Quercetin also decreased the levels of the anti-apoptotic Bcl-xl protein, while increasing the translocation of the pro-apoptotic Bax protein to mitochondria and the activation of the tumor suppressor p53. Similarly, the in vivo portion of the study, conducted with an animal model, revealed that quercetin used together with doxorubicin significantly inhibited tumor growth and reduced doxorubicin-induced liver damage. These findings suggest that quercetin can be considered an adjuvant agent that enhances chemotherapeutic efficacy and has the potential to alleviate chemotherapyinduced hepatotoxicity. QUERCETIN   61 Quercetin and other flavonoids possess the potential to act as potent antioxidants, and this view has been supported by studies. This antioxidant property of plants is crucial in alleviating liver fibrosis and maintaining a balanced apoptosis process. The strong chemical properties of the benzene ring in its structure, along with the hydrophilic-hydrophobic balance created by its hydroxyl groups and sugar bonds, contribute to the improvement of cell apoptosis pathways and liver fibrosis. (27) Indeed, A comprehensive study by Wang et al. (28) demonstrated that the ameliorative effect of quercetin on liver fibrosis is closely related to the aforementioned factors, highlighting the importance of quercetin’s therapeutic potential. Quercetin administration (5–15 mg/kg) dose-dependently suppressed NF-κB activation by inhibiting the degradation of IκBα and simultaneously limited the activity of this pathway by reducing p38 MAPK phosphorylation. It also contributed to the reduction of Bax expression, the elevation of Bcl-2 levels, and thus the inhibition of caspase-3 activity. These findings suggest that quercetin suppresses the progression of liver fibrosis in rats. The results suggest that quercetin exerts its anti-fibrotic effects by limiting inflammation by targeting the NF-κB/IκBα and p38 MAPK pathways and by inhibiting apoptosis by regulating the Bcl-2/ Bax balance. 5. Conclusion Quercetin is recognized for its strong ability to neutralize free radicals, thereby influencing critical cellular pathways related to oxidative stress, inflammation, and programmed cell death. Through these mechanisms, it contributes to hepatoprotection and the maintenance of liver function. In nature, quercetin generally occurs as glycoside derivatives in various fruits and vegetables. Following ingestion, these glycosides undergo hydrolysis in the intestine to release the active aglycone form. Once absorbed, quercetin is further metabolized within enterocytes and hepatocytes, shaping its bioavailability and biological activity. Liver disease progression is largely driven by oxidative damage, inflammatory mechanisms, and apoptosis-related processes. Quercetin exerts its protective effect on the liver by affecting these three fundamental processes through different mechanisms. It exerts its antioxidant effects by suppressing lipid peroxidation and increasing the activity of antioxidant enzymes (MDA, GSH, SOD, and CAT). In the event of hepatotoxicity, it reduces oxidative stress by lowering ALT and AST 62   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS levels. Its anti-inflammatory effect reduces the inflammatory response in liver tissue by inhibiting the NF-κB pathway, suppressing proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, and reducing COX-2/iNOS expression. It has been shown to significantly suppress inflammation in models of hepatotoxicity induced by agents such as diclofenac and LPS. 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Rajput JD, Bagul SD, Bendre RS (2017) Synthesis, biological activities and molecular docking simulation of hydrazone scaffolds of carvacrol, thymol and eugenol. Res Chem Inter 43: 6601-6616. 33. Harwood SH, Moldenke AF, Berry RE (1990) Toxicity of peppermint monoterpenes to the variegated cutworm (Lepidoptera: Noctuidae). J EcoEntomol 83: 1761-1767. 34. Nieto G (2017) Biological activities of three essential oils of the Lamiaceae family. Med 4: 63. 35. Ryan MF, Byrne O (1988) Plant-insect coevolution and inhibition of acetylcholinesterase. Journal Chemical Ecology 14: 1965-197. 36. Jukic M, Politeo O, Maksimovic M, Milos M (2007) In vitroacetylcholinesterase inhibitory properties of thymol, carvacrol and theirderivatives thymoquinone and thymohydroquinone. Phyto Res 21:259-261. THYMOL AND CARVACROL   73 37. 19. Cole LM, Casida JE (1992) GABA-gated chloride channel: Binding sitefor 4′-ethynyl-4-n-[2, 3-3H2] propylbicycloorthobenzoate ([3H] EBOB) in vertebrate brain and insect head. Pest Biochem Physiol 44: 1-8. 38. Suntres ZE, Coccimiglio J, Alipour M (2015) The bioactivity and toxicological actions of carvacrol. Critic Rev Food Sci Nutr 55: 304-318. 39. Hagan EC, Hansen WH, Fitzhugh OG, Jenner PM, Jones WI (1967) Foodflavourings and compounds of related structure. II. Subacute and chronic toxicity. Food Cosmetics Toxicol 5: 141-157. 75 CHAPTER VIII 6-GINGEROL – ZINGERON Yusuf ABUL (MD) Department of Gastroenterology, Ministry of Health Eskisehir City Hospital, E-mail: [email protected], Orcid: 0009-0006-4099-5614 1. Introduction 6-gingerol is found in fresh ginger, which is commonly encountered in everyday life. Ginger is a spice and phytochemical widely used in Southeast Asian countries (Figure 1). Although ginger has not been approved for medical treatment of any specific disease, it is commonly used without a prescription in folk medicine for conditions such as gastrointestinal problems, nausea, vomiting, dyspeptic complaints, osteoarthritis, joint pain, anxiety, and agitation (1). Figure 1: Appearance of ginger 2. Chemical properties It is a substance that activates heat receptors on our tongue after being consumed by the body (1) (Figure 2). When ginger is cooked, 6-gingerol 76   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS transforms into zingerone, which has a sweet profile in its solid form. If ginger is subjected to drying or heating, gingerol undergoes another reaction, forming shogaol, a sharper compound than gingerol. (2) This is why dried ginger is more pungent. Ginger also contains 8-gingerol, 10-gingerol, and 12-gingerol. (3) All of these compounds are collectively referred to as gingerol. Figure 2. Chemical formula of 6-Gingerol. Ginger is composed of nearly half starch by weight and serves as a carbohydrate reservoir. Animal studies have shown that ginger extracts can increase body secretions such as saliva, stomach acid, and bile. It is also believed that ginger may be effective in treating conditions such as anxiety, depression, psychological issues, inflammation, and pain due to its serotonin and cyclooxygenase inhibition. In a clinical study, ginger was found to be effective against nausea and vomiting during pregnancy. (4) The normal dose of ginger depends on various factors such as the product’s purity and concentration, with some components being in raw powder form while others are semi-purified. The daily dose is equivalent to 1000 mg of dried powdered extract. It is typically used in concentrations ranging from one to three doses per day. Ginger is generally considered a safe compound, and no conclusive evidence has been found to suggest it causes liver damage. The most commonly observed side effects are mild heartburn, dyspeptic complaints, diarrhea, or constipation. In many studies, side effects were not significantly different from those of a placebo. Ginger has been evaluated for pregnancyrelated nausea and has not been associated with fetal toxicity or teratogenicity. (4) Although some hypersensitivity reactions have been reported, these were not severe. No cases of anaphylaxis or Stevens-Johnson syndrome related to ginger use have been observed. 3. Physiological potential of gingerol It is stated that gingerol has anti-inflammatory, antioxidant, neuroprotective (5), and gastroprotective effects. Many studies have been conducted in this 6-GİNGEROL – ZİNGERON   77 regard. (6) In some studies, it has been observed that gingerol increases the success rate in glycemic regulation. (7) Additionally, in another study, the effect of gingerol on cardiovascular protection in diabetic rats was investigated. (8) In these rats, which had better glycemic regulation, it was observed that the cardiovascular prognosis was also better. Although many studies (9-13) have been conducted on the effect of gingerol on various malignant tumors, these studies have mostly focused on animal experiments, and we can say that human studies are still not at a sufficient level. Although many studies have been conducted on gingerol, only a few of them have been clinical trials. The variability of these phytochemicals is high, and the efficacy observed in research is limited. (14, 15) Most herbal products are under serious control by local health authorities, such as the FDA, and due to various restrictions, the experimental studies on herbal products do not adequately translate into clinical practice. Therefore, this situation diminishes the value of herbal product research. (14, 15) In regions like Asia and Africa, where herbal products are more commonly used, the lack of financial support for research has hindered clinical investigations of products like gingerol in terms of both safety and efficacy. (14) Most research on gingerol has been conducted on mice or human tissue cultures, and these studies may be considered as part of the control, prevention, and treatment of various diseases. In a study where a type of ginger grown in Africa was found to contain higher levels of gingerol compared to other ginger varieties, it was observed that this ginger showed better results in terms of its antifungal effects. (16) In a meta-analysis evaluating the effects of herbal products in patients with prostate cancer, two separate studies conducted in mouse models demonstrated that gingerol increases apoptosis in cancer cells by acting on the mitochondrial membrane (9). Similarly, other anti-cancer studies have reported that gingerol exerts enhancing effects on the degradation of proteins involved in the G1 phase of the cell cycle (9,11,12,13). In a study on skin cancer in mice, gingerol was found to prevent the malignant transformation of normal cells and to induce apoptosis in tumor cells (17,18). Gingerol suppresses cell proliferation by inhibiting the translation of cyclin proteins involved in replication during the G1 and G2 phases of the cell cycle (19). In addition, by inhibiting the anti-apoptotic Bcl-2 protein in mitochondria, gingerol promotes apoptosis (Figure 3). 78   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Figure 3. Established cellular pathways targeted by 6-gingerol, leading to apoptosis in a cancer cell (17). Abbreviations: CDK: Cyclin-dependent kinase; PI3K: Phosphoinositide 3-kinase; p-Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; AMPK: 5′ adenosine monophosphateactivated protein kinase; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2 In a study conducted on a cell culture prepared from human breast cancer cells, a 16% reduction in cell viability was observed at a concentration of 10 μM gingerol (10). In this study, gingerol was found to target three distinct proteins involved in the induction of metastasis in breast cancer, while also inhibiting the infiltration and growth of malignant cells (10). Another beneficial effect of gingerol has been observed in patients treated with cisplatin. Since high doses of cisplatin may cause renal failure, this adverse effect represents a limiting factor in its clinical use. In a study conducted in rats, the administration of gingerol was found to prevent cisplatin-induced renal failure (20). Gingerol has been shown to increase glutathione production in a dosedependent manner, suggesting that higher doses enhance its efficacy. Through the antioxidant activity of glutathione, gingerol is thought to improve glycemic control in diabetic patients (21). In a study conducted on severely obese diabetic mice, gingerol was observed to enhance glucose uptake into cells even in the absence of exogenous insulin, leading to beneficial effects on fasting glycemia and glucose tolerance (7). The reduction in lipoproteins and the improvement in glucose tolerance were attributed to increased glutathione production (21). Furthermore, the anti-inflammatory effects of gingerol were found to contribute 6-GİNGEROL – ZİNGERON   79 to glycemic regulation, thereby reducing the risk of cardiac arrhythmia, a common complication in diabetic patients (8). Gingerol also possesses neuroprotective properties (22). In one study, gingerol demonstrated antioxidant activity in the hippocampus of mice and in human neuroblastoma cells, which was found to reduce the risk of Alzheimer’s disease (22). Although numerous studies have reported the antioxidant activity of gingerol and its low risk of toxicity, some evidence also suggests potential genotoxic effects. In a study conducted on human hepatoma cells, very high doses of gingerol were shown to cause DNA damage and chromosomal abnormalities (23). With increasing concentrations, gingerol may exhibit prooxidant effects. In another study, intraperitoneal injection of gingerol in rats was found to induce hypothermia, whereas no hypothermic effect was observed in rats receiving excessive amounts of gingerol orally (24). In an animal study investigating the effects of gingerol on hepatic ischemiareperfusion injury, gingerol was found to reduce liver ischemia-reperfusion damage. This finding supports its antioxidant properties, which have been confirmed in numerous studies (25). 4. Zingeron and its biological effects Zingerone is a solid form of ginger with low solubility in water (Figure 3). It is not present in fresh ginger but is produced through cooking or drying. Zingerone is a potent antioxidant and is responsible for the antidiarrheal effects of ginger (26). In a study conducted in rats, the role of zingerone in cisplatininduced liver injury was investigated (27). The results showed that in rats treated with zingerone, there was a significant reduction in elevated liver enzyme levels associated with cisplatin administration. Similar to gingerol, zingerone is considered a phytochemical compound with beneficial effects on the liver and can be used safely. Figure 3. Chemical formula of Zingerone ( 4-(4-Hydroxy-3-methoxyphenyl)butan-2-one. 80   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 5. Conclusion Extensive research has demonstrated that ginger and its biologically active constituents exert beneficial effects in the management of various health conditions. When consumed at appropriate doses, ginger is generally well tolerated and does not cause significant adverse effects. Nevertheless, excessive intake may induce hypoglycemia in individuals with diabetes and may slightly increase the risk of bleeding in patients receiving anticoagulant therapy.” Gingerol and zingerone, the two principal bioactive phenolic compounds of Zingiber officinale (ginger), have been widely studied for their pharmacological and therapeutic potential across multiple disease models. Accumulating preclinical evidence highlights their pleiotropic biological actions, ranging from antioxidant and anti-inflammatory properties to anticancer and organ-protective effects. Gingerol has been shown to exert anticarcinogenic activity through several molecular mechanisms. Studies in prostate and skin cancer models demonstrate that gingerol interacts with the mitochondrial membrane, promoting apoptosis via inhibition of the anti-apoptotic protein Bcl-2 and activation of Bax-mediated pathways. In addition, gingerol interferes with the cell cycle by suppressing cyclin protein translation in the G1 and G2 phases, thereby reducing malignant proliferation. Investigations on breast cancer cells revealed that gingerol at a concentration of 10 μM reduced cell viability by approximately 16%, while concurrently downregulating three metastasis-inducing proteins and preventing tumor cell invasion and growth. Collectively, these findings suggest that gingerol targets multiple oncogenic pathways, positioning it as a promising adjuvant candidate for cancer therapy. Beyond its anticancer role, gingerol displays notable chemoprotective effects. In rodent models, gingerol supplementation mitigated cisplatin-induced nephrotoxicity and hepatotoxicity, conditions that often limit cisplatin’s clinical utility. The protective effect is thought to be mediated via upregulation of glutathione synthesis, enhancement of cellular antioxidant defenses, and suppression of oxidative stress–related pathways. Furthermore, gingerol exhibited antidiabetic properties in obese diabetic mice by enhancing glucose uptake into cells independently of exogenous insulin, improving fasting glycemia and glucose tolerance, and reducing circulating lipoprotein levels. These metabolic effects were attributed to glutathione-dependent redox regulation, in combination with gingerol’s anti-inflammatory actions, which also conferred protection against diabetes-associated cardiac arrhythmias. 87 CHAPTER IX SILYMARIN Ali TÜREYEN (Assoc. Prof. Dr.) Department of Gastroenterology, Ministry of Health Eskisehir City Hospital, E-mail: alitur[email protected], Orcid: 0000-0001-8991-720X 1. Introduction The protective effects of herbal medicines on various organs have been demonstrated in numerous scientific studies. The liver, the largest internal organ of the body, plays a central role in vital functions such as metabolism, detoxification, digestion, and immune regulation. Liver diseases adversely affect these functions and can have serious consequences on overall health. For this reason, interest in phytotherapy for the protection of liver health and the treatment of liver diseases has markedly increased in recent years. In this context, silymarin has attracted attention for centuries due to its diverse biological effects. Research has shown that silymarin is one of the most extensively studied herbal agents in the treatment of liver diseases. Its most remarkable property lies in its pronounced protective and therapeutic effects on liver tissue (1,2). 2. Chemical Structure, Properties, and Derivatives Silymarin is a plant-derived compound obtained from the seeds of Silybum marianum (milk thistle), a member of the Asteraceae family. Chemically, silymarin is composed of a mixture of flavonolignans, including silibinin, isosilibinin, silychristin, silydianin, and dehydrosilibinin isomers. The main active constituent of silymarin is silibinin, which itself consists of two diastereomers: silibinin A and silibinin B. Silibinin accounts for approximately 50–70% of the total chemical composition of silymarin (3,4). The chemical structure of silymarin is shown in Figure 1. 88   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Figure 1. Chemical structure of silymarin 3. Synthesis, Bioavailability, and Metabolism of Silymarin After oral administration, the absorption of silymarin is relatively low, and studies have shown that in rats and humans, it reaches peak plasma concentration 4 to 6 hours after intake. After 24 hours, 2–3% has been detected in bile. In the liver, it is conjugated with sulfate and glucuronic acid and excreted through the bile. To a much lesser extent, it is also eliminated via the urine (2,4). The halflife of silymarin is 6–8 hours. In adult humans, it is administered three times daily at a dose of 100–300 mg/kg. Acute toxicity studies have shown that it is well-tolerated (5). Due to the compound’s low solubility and poor oral bioavailability, nanoemulsion formulations have been developed to enhance its therapeutic effects. Since silymarin is not soluble in water, it cannot be consumed as herbal tea. In the market, it is commercially available in capsule and tablet forms under brand names such as Livergol, Silipide, and Legalon (2,6). 4. Pharmacological Effects of Silymarin Silymarin’s potential benefits in the treatment of liver diseases remain a subject of debate. It is a plant extract that has been repeatedly investigated in numerous studies for its positive effects on liver health (4). It has been reported that silymarin, through its antioxidant activity, exerts anti-inflammatory, antifibrotic, antitoxic, antiviral, anticarcinogenic, pro-apoptotic, and antiandrogenic effects (Figure 2). Its antioxidant activity is considered predominant, mainly through increasing intracellular glutathione content, suppressing lipid peroxidation, scavenging free radicals, and reducing reactive oxygen species (3,7,8). SILYMARIN   89 Figure 2. Major pharmacological effects of silymarin mediated by its antioxidant activity By stabilizing hepatocyte membranes, it prevents toxins from entering the cells. It exerts an antifibrotic effect by inhibiting the transforming growth factor-beta (TGF-β) signaling pathway. In addition, it reduces inflammation by inhibiting TNF-alpha and 5-lipoxygenase (9). Long-term use has been shown to reduce inflammatory markers. Although its exact mechanism of action is not yet fully understood, it is thought to support liver health through its antioxidant and anti-inflammatory properties. In liver cells, it has also been reported to enhance the activity of the reticuloendothelial system and stimulate ribosomal RNA, thereby increasing protein synthesis (10,11). Through this effect, silymarin has been reported in clinical studies to exert beneficial effects on the liver’s major function of protein synthesis, particularly in patients with cirrhosis and hepatitis (3,12). Silymarin has a well-established safety profile. Apart from headache, gastrointestinal disturbances, and rare allergic skin rashes, no significant adverse effects have been reported, even at high doses (2,13). The use of natural silymarin complexes combined with phosphatidylcholine has also been investigated to enhance its efficacy. This combination may improve the solubility and bioavailability of silymarin. In particular, when combined with phosphatidylcholine, silymarin contains its primary active flavonolignan, silibinin, which is thought to exert protective effects against liver inflammation and damage (1,14). Beyond liver diseases, studies in mice have shown that silymarin prevents cisplatin-induced nephrotoxicity and improves outcomes in 90   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS cold ischemic nephropathy. As an anticarcinogenic agent, it has been reported to protect human melanoma cells against ultraviolet radiation and to exert preventive effects on photocarcinogenesis. Furthermore, numerous studies have demonstrated its use as an anticarcinogenic compound in neurotoxicity, depression, in vitro fertilization, as well as in lung, prostate, and ovarian cancers (3,7,15). 5. Hepatoprotective Properties of Silymarin It is used as a hepatoprotective agent to support conventional therapies and dietary interventions in cases of cirrhosis, alcoholic and non-alcoholic fatty liver disease, hepatitis, and mushroom poisoning (6). Recent studies have shown that higher doses of silymarin (420–700 mg/ day) and long-term use may reduce liver enzyme levels; however, a clear effect on liver histology (fibrosis, steatosis) has not been demonstrated. In particular, there is no evidence that silymarin reduces viral load or provides histological improvement in viral hepatitis B and C (16,17). In mice, administration of silymarin immediately before Amanita mushroom poisoning has been shown to completely prevent hepatotoxicity, while administration within the first 24 hours was found to reverse liver damage. Studies have also reported that silymarin reduces liver injury induced by ethanol, carbon tetrachloride, acetaminophen, radiation, iron overdose, phenylhydrazine, and cold ischemia in mice (11,18). Successful treatment with silymarin has been reported in patients with drug-induced liver injury. Liver regeneration refers to the liver’s ability to regrow when it is damaged or partially resected. This process allows the liver to restore its functionality (19,20). For maximum benefit, it is recommended that silymarin therapy be initiated as early as possible in patients with significant liver diseases such as fatty liver disease and acute liver failure, where the regenerative potential of the liver is still high and oxidative stress, causing cytotoxicity, can be alleviated. Meta-analyses of studies conducted in cirrhotic patients have found that silymarin treatment is associated with a significant reduction in liver-related mortality. Based on the current evidence, we conclude that silymarin may play a therapeutic role in the management of alcoholic liver cirrhosis (16,21). Silymarin’s antioxidant activity, stimulation of protein synthesis in the liver, and promotion of cell regeneration suggest that it may reduce the progression of various cancer types. Among its active components, Silybum marianum has been shown to be effective in the regeneration of damaged liver tissues. In SILYMARIN   91 particular, it has been observed to facilitate the rapid restoration of lost tissue in cases such as liver resection. Moreover, studies have reported that silibinin prevents fibrosis by reducing the proliferation of hepatic stellate cells (22,23). In addition, silymarin has been shown to improve glycemic parameters in patients with diabetes and alcoholic cirrhosis (4,24). It has also been reported that silymarin inhibits hepatic cytochrome P450 enzymes and contributes to an increase in HDL cholesterol while reducing total cholesterol levels (25,26). 6. Conclusion Silymarin frequently shows beneficial effects on liver enzymes; however, its histological benefits remain unclear, although it is generally well tolerated. Newly established dosages, improved quality, and standardization of silymarin are expected to provide the long-awaited evidence regarding its efficacy in the treatment of liver diseases. The future of silymarin research appears promising, and it remains a plant open to investigation for its therapeutic potential in various other diseases. To further confirm its safety and efficacy, high-quality, randomized, placebo-controlled clinical trials may be required. References 1. Flora K, Hahn M, Rosen H, Benner K. Milk thistle (Silybum marianum) for the therapy of liver disease. Am J Gastroenterol. 1998;93(2):139-43. 2. Loguercio C, Festi D. Silybin and the liver: From basic research to clinical practice. World J Gastroenterol. 2011;17(18):2288-301. 3. Mohammadi S, Ashtary-Larky D, Asbaghi O, et al. Effects of silymarin supplementation on liver and kidney functions: A systematic review and doseresponse meta-analysis. Phytother Res. 2024;38(5):2572-93. 4. Ding T, Tian S, Zhang Z, et al. Determination of active component in silymarin by RP-LC and LC/MS. J. Pharmacol. Biomed. Anal 2001; 26: 155-61. 5. Kren V, Walterova D. Silybin and Silymarin-new effects and applications. Biomed Papers 2005;149: 29-41. 6. Kang JS, Jeon YJ, Park SK, Yang KH, Kim HM. Protection against lipopolysaccharide-induced sepsis and inhibition of interleukin-1 and prostaglandin E2 synthesis by silymarin. Biochem Pharmacol 2004; 67: 17581. 7. Fraschini F, Demartini G, Esposti D. Pharmacology of silymarin. Clinical Drug Investigation 2002; 22: 51-65. 92   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 8. Ahmad N, Fazal H, Abbasi BH, Anwar S, Basir A. DPPH free radical scavenging activity and phenotypic difference in hepatoprotective plant (Silybum marianum L.) Toxicol Ind Health 2013; 29: 460-7. 9. Matveev AV, Koniaeva EI, Kurchenko VP, Shchekatikhina AS. Hepatoprotective properties of silymarin. Eksp Klin Gastroenterol 2011; 2: 130-5. 10. Gupta OP, Sing S, Bani S, et al. Anti-inflammatory and antiarthritic activities of silymarin acting through inhibition of 5-lipoxygenase. Phytomedicine 2000; 7: 21-4. 11. Song Z, Deaciuc I, Song M, Lee DY, Liu Y, Ji X, et al. Silymarin protects against acute ethanol-induced hepatotoxicity in mice. Alcohol Clin Exp Res. 2006;30:407–413. 12. Dehmlow C, Erhard J, de Groot H. Inhibition of Kupffer cell functions as an explanation for the hepatoprotective properties of silibinin. Hepatology. 1996;23:749–754. 13. Perumpail BJ, Li AA, Iqbal U, Sallam S, Shah ND, Kwong W, et al. Potential therapeutic benefits of herbs and supplements in patients with NAFLD. Diseases. 2018;6:80. 14. Valenzuela A, Garrido A. Biochemical bases of the pharmacological action of the flavonoid silymarin and of its structural isomer silibinin. Biol Res. 1994;27:105–112. 15. Javed S, Kohli K, Ali M. Reassessing bioavailability of silymarin. Altern Med Rev. 2011;16:239–249. 16. Doehmer J, Tewes B, Klein KU, Gritzko K, Muschick H, Mengs U. Assessment of drug-drug interaction for silymarin. Toxicol In Vitro. 2008;22:610–617. 17. Serviddio G, Bellanti F, Stanca E, Lunetti P, Blonda M, Tamborra R, et al. Silybin exerts antioxidant effects and induces mitochondrial biogenesis in liver of rat with secondary biliary cirrhosis. Free Radic Biol Med. 2014;73:117– 126. 18. Cacciapuoti F, Scognamiglio A, Palumbo R, Forte R, Cacciapuoti F. Silymarin in non alcoholic fatty liver disease. World J Hepatol. 2013;5:109–113. 19. Milosević N, Milanović M, Abenavoli L, Milić N. Phytotherapy and NAFLD--from goals and challenges to clinical practice. Rev Recent Clin Trials. 2014;9:195–203. 20. de Avelar CR, Pereira EM, de Farias Costa PR, de Jesus RP, de Oliveira LPM. Effect of silymarin on biochemical indicators in patients with SILYMARIN   93 liver disease: Systematic review with meta-analysis. World J Gastroenterol. 2017;23:5004–5017. 21. Javed S, Ahsan W, Kohli K. Pharmacological influences of natural products as bioenhancers of silymarin against carbon tetrachloride-induced hepatotoxicity in rats. Clin Phytosci. 2018;4:18. 22. Gu HR, Park SC, Choi SJ, Lee JC, Kim YC, Han CJ, et al. Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells. Clin Mol Hepatol. 2015;21:49–59. 23. Abrol S, Trehan A, Katare OP. Comparative study of different silymarin formulations: formulation, characterisation and in vitro/in vivo evaluation. Curr Drug Deliv. 2005;2:45–51. 24. Mayer KE, Mayer RP, Lee SS. Silymarin treatment of viral hepatitis: a systematic review. J Viral Hepatitis. 2005;12:559–567. 25. Shaker E, Mahmoud H, Mnaa S. Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food Chem Toxicol. 2010;48:803–6. 26. Stickel F, Schuppan D. Herbal medicine in the treatment of liver diseases. Digest Liver Dis. 2007;39:293–304. 95 CHAPTER X RESVERATROL İsmail KÜÇÜKKURT1 & Fahriye KAN2 1(Prof. Dr.), Afyon Kocatepe University Faculty of Veterinary Medicine, Department of Biochemistry E-mail: [email protected] ORCİD: 0000-0003-0198-629X 2(Dr.), Afyon Kocatepe University Faculty of Veterinary Medicine, Department of Biochemistry E-mail: [email protected] ORCİD: 0000-0002-6366-8396 1. Introduction The liver plays a central role in maintaining the body’s vital functions. As an organ with critical functions such as metabolism, detoxification, energy balance, and bile production, it can be damaged by numerous factors, including drugs, industrial and environmental toxins, alcohol consumption, and viral infections. Chronic hepatotoxicity caused by these factors can lead to serious consequences such as tissue fibrosis, cirrhosis, and, in more advanced cases, liver failure. Therefore, it is important to investigate protective agents against liver damage and elucidate their mechanisms. Naturally derived polyphenols are attracting attention in this field and may offer significant benefits against liver damage through their cytoprotective effects. One of the most prominent compounds in this group is resveratrol, which has been shown to provide significant protection for liver health in both animal and cell culture studies through its properties, including reducing oxidative stress, suppressing inflammatory cytokine expression, regulating apoptosis, and improving mitochondrial function. (1,2,3) Studies on resveratrol began with epidemiological data from the so-called “French paradox.” These data revealed that in the French population, where 96   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS red wine consumption is high, cardiovascular disease rates were relatively low despite a diet rich in saturated fat. This suggests that the polyphenolic content of red wine, when compared with similar amounts of other beverages, may have a protective effect on cardiovascular health. The biological effects of resveratrol, a key polyphenol in red wine, have garnered attention. (4) Studies to date have reported that resveratrol exhibits beneficial biological activities associated with many diseases, including inflammation, cardiovascular health, cancer, and aging. For example, it has regulatory effects on inflammatory processes in rheumatoid arthritis and inflammatory bowel diseases. (5,6) In a study conducted on healthy volunteers, it was concluded that resveratrol, administered in doses of 0.5–5.0 g for 29 days, is a potential substance that may reduce or delay the risk of cancer development. (7) In animal studies, resveratrol administration to middle-aged mice fed a standard diet was reported to provide improvements in functional aging parameters such as bone density, muscle coordination, balance, and cataract development, but did not prolong lifespan; however, resveratrol was found to prolong lifespan in obesity-prone mice fed a high-calorie diet (8,9). Resveratrol, a polyphenolic phytoalexin, has the potential to protect against the harmful effects of oxidative stress and inflammatory processes. This section examines the hepatoprotective effects that resveratrol may have on liver tissue through its antioxidant, anti-inflammatory, and antiapoptotic properties. 2. Resveratrol: Structure and Sources Resveratrol is a polyphenolic compound belonging to the stilbenoid class (Figure 1). Stilbenoids are phenol-based plant metabolites found widely in nature and associated with human health benefits against conditions such as cancer, inflammation, neurodegenerative diseases, and heart disease. Red grapes, especially their skins and seeds, are one of the richest sources of resveratrol. Their high concentration in the skin protects against fungal infections. Red wine, especially the skins, contains high amounts of resveratrol. (10) Forest fruits such as blueberries, raspberries, and blackberries also contain resveratrol. Although in lower amounts than grapes, they are valuable for their antioxidant properties. Cocoa and peanuts have also been found to contain resveratrol, albeit in lower amounts. Japanese knotgrass (Polygonum cuspidatum) is the most concentrated source of resveratrol used in herbal supplements. (11) RESVERATROL   103 11. Walle T, Hsieh F, DeLegge MH, Oatis JE Jr, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos. 2004;32(12):1377-1382. 12. Seeram NP, Kulkarni VV, Padhye S. Sources and chemistry of resveratrol. In: Resveratrol in Health and Disease. CRC Press; 2005:16-32. 13. Walle T. Bioavailability of resveratrol. Ann N Y Acad Sci. 2011;1215(1):9-15. 14. Li F, Han Y, Wu X, et al. Gut microbiota-derived resveratrol metabolites, dihydroresveratrol and lunularin, significantly contribute to the biological activities of resveratrol. Front Nutr. 2022; 9:912591. 15. Woting A, Clavel T, Loh G, Blaut M. Bacterial transformation of dietary lignans in gnotobiotic rats. FEMS Microbiol Ecol. 2010;72(3):507-515. 16. Spaleniak W, Cuendet M. Resveratrol as a circadian clock modulator: mechanisms of action and therapeutic applications. Mol Biol Rep. 2023;50(7):6159-6170. 17. Salehi B, Mishra AP, Nigam M, et al. Resveratrol: a double-edged sword in health benefits. Biomedicines. 2018;6(3):91. 18. Smoliga JM, Blanchard O. Enhancing the delivery of resveratrol in humans: if low bioavailability is the problem, what is the solution? Molecules. 2014;19(11):17154-17172. 19. Björnsson ES. Hepatotoxicity by drugs: the most common implicated agents. Int J Mol Sci. 2016;17(2):224. 20. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci. 2002;65(2):166-176. 21. Kayes T, Ho V. Amanita phalloides-associated liver failure: molecular mechanisms and management. Int J Mol Sci. 2024;25(23):13028. 22. Kobayashi A, Suzuki Y, Sugai S. Specificity of transaminase activities in the prediction of drug-induced hepatotoxicity. J Toxicol Sci. 2020;45(9):515-537. 23. Algefare AI, Alfwuaires M, Famurewa AC, Elsawy H, Sedky A. Geraniol prevents CCl₄-induced hepatotoxicity via suppression of hepatic oxidative stress, pro-inflammation and apoptosis in rats. Toxicol Rep. 2024; 12:128-134. 24. Hartwig A, Arand M, MAK Commission. Vinyl chloride. MAK Collect Occup Health Saf. 2023;8(2): Doc038. 25. Sebai H, Sani M, Yacoubi MT, et al. Resveratrol, a red wine polyphenol, attenuates lipopolysaccharide-induced oxidative stress in rat liver. Ecotoxicol Environ Saf. 2010;73(5):1078-1083. 104   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 26. Zhou R, et al. Protective effects of resveratrol on liver injury induced by toxic agents: a review. Food Chem Toxicol. 2019. 27. Palsamy P, Subramanian S. Resveratrol protects diabetic kidney by attenuating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines. J Nutr Biochem. 2010. 28. Yu W, et al. Resveratrol, a therapeutic agent for non-alcoholic fatty liver disease. Mol Nutr Food Res. 2019. 29. Faghihzadeh F, Adibi P, Hekmatdoost A. The effects of resveratrol supplementation on cardiovascular risk factors in patients with non-alcoholic fatty liver disease: a randomised, double-blind, placebo-controlled study. Br J Nutr. 2015;114(5):796-803. 30. Theodotou M, Fokianos K, Moniatis D, et al. Effect of resveratrol on non-alcoholic fatty liver disease. Exp Ther Med. 2019;18(1):559-565. 31. Bishayee A, Darvesh AS, Politis T, McGory R. Resveratrol and liver disease: from bench to bedside and community. Liver Int. 2010;30(8):11031114. 32. Meng X, Zhou J, Zhao CN, Gan RY, Li HB. Health benefits and molecular mechanisms of resveratrol: a narrative review. Foods. 2020;9(3):340. 33. Lee HJ, Kang MG, Cha HY, Kim YM, Lim Y, Yang SJ. Effects of piceatannol and resveratrol on sirtuins and hepatic inflammation in high-fat dietfed mice. J Med Food. 2019;22(8):833-840. 34. Chupradit S, Bokov D, Zamanian MY, Heidari M, Hakimizadeh E. Hepatoprotective and therapeutic effects of resveratrol: a focus on anti‐inflammatory and antioxidative activities. Fundam Clin Pharmacol. 2022;36(3):468-485. 35. Elgizawy HA, Ali AA, Hussein MA. Resveratrol: isolation, and its nanostructured lipid carriers, inhibits cell proliferation, induces cell apoptosis in certain human cell lines carcinoma and exerts protective effect against paraquatinduced hepatotoxicity. J Med Food. 2021;24(1):89-100. 105 CHAPTER XI CAFFEIC ACID Ayşenur SEVİNÇ (MD), Department of Gastroenterology, Ministry of Health Eskişehir City Hospital, E-mail: [email protected], ORCID: 0009-0007-1382-469X 1. Introduction Hepatotoxicity or drug-induced liver injury (DILI) is characterized by liver function test abnormalities and exclusion of causes other than drug, environmental or herbal preparation exposure. DILI can be classified by its clinical presentation (hepatocellular, cholestatic, mixed), hepatotociticy mechanism or histopathologic findings in liver biopsy (1). True epidemiologic data is scarce so determining real incidence of hepatotoxicity is challenging. Studies on aetiology of acute liver failure (ALF) shows that drugs are the most common cause of ALF in US , Europe and Japan (2). In the United States and Europe reaction against medical preparations are the cause of DILI most of the time, but in Asia herbal preparations are the most common causes (3). DILI can be classified as intrinsic (direct) vs. idiosyncratic. Direct DILI is foreseeable, it occurs in dose dose-dependent fashion and it has short onset (hours to days). On the contrary, idiosyncratic DILI is generally not dose dependant and it occurs only in small number of exposed patients thus makes it unforeseeable (2). In both idiosyncratic and intrinsic DILI, drug exposure exposes liver to unstable molecules (e.g. reactive oxygen species) that covalently attach to proteins, activate free radical damage, induce transmission of signal pathways and produce organelle stress, prevent bile acid transport and either result in cell death or start adaptive reaction which inhibits these actions (e.g. antioxidant protection, mitochondria or endoplasmic reticulum misfolded protein reactions, mitochondria regeneration) so that damage does not end in apoptosis (4). 106   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS For neutralizing the destructive actions of oxidative stress, cells utilize couple of pathways. Whether produced in vivo or taken from external sources, antioxidants, has pivotal role in eliminating reactive oxygen species (ROS), reducing the stress caused by oxidation. Most plant derived chemicals acts as antioxidants and are crucial to eliminate oxydative stress. Plant derived chemicals depending on their roles in plant metabolism, are classified as primary and secondary metabolites. Some of primary metabolites like carbohydrates, amino acids, proteins, lipids, purines, and pyrimidines of nucleic acids, are crucial for vegetative survival. Secondary metabolites serve as the rest of chemical compunds produced from byproducts from metabolic pathways producing primary metabolites. Even though not having direct effect on vitality of plants, secondary metabolites augment the capability of plant to grow by enabling the communication and adaptabilty to their surrondings (5). Chemical structures of secondary plant metabolites are used for classification as; phenolics, alkaloids, saponins, terpenes, lipids and carbohydrates (6). 2. Chemical Aspects of Caffeic Acid Phenolic metabolites produced by most of the plants and part of a complex group of organic chemicals. The phenolic compound can be described in two subgroups as, simple phenolic and polyphenolic compounds (Figure 1). Figure 1. Chemical classification of phenol compounds CAFFEIC ACID   107 Cinnamic acid derivatives (phenylpropanoids) are nine carbon molecules. One member of this class is caffeic acid (3,4dihydroxycinnamic) (7). In most plant tissues caffeic acid (3,4dihydroxycinnamic) can be found. In many products that consumed Daily like coffee beverages, blueberries and apples as well as propolis; this phytochemical can be found. It has known benefits as carcinogenic inhibitor, antioxidant and antibacterial in vitro, and it can asist in preventing atherosclerosis and other caridovascular diseases (7). Caffeic acid is a hydroxycinnamic acid, which has a pheylpropanoid (C6C3) structure with a 3,4 dihyroxlated aromatic ring attached to a carboxylic acid by a transthylene wire is a part of he phenolic acid family. Endogenous shikimate pathwayi in plants that is responsible for the making of aromatic amino acids form glucose results in synthesis of this molecule (Figure 2). Figure 2. Chemical structure of caffeic acid The reaction that produces caffeic acid begins with shikimic acid and after that first step is phosphorylation by the enzyme shikimatokinase, second step is conjugation of a molecule of phosphoenolpyruvate by 5-enolpyruvylshikimate3phosphate (EPSP) synthase then by action of chorismate synthetase, concluding at one of the most important intermediary metabolites of this pathway, chorismic acid Prephenic acid is produced by enzyme chorismate mutase (a precursor of L-phenylalanine) from chorismic acid. Production of L-phenylalanine is carried out by two molecules. In the deamination process, as a coenzyme pyridoxal phosphate (PLP) and as an electron exchanger nicotinamide adenine dinucleotide (NAD). Cinnamic acid is formed by deamination of L-phenylalanine with the help of enzyme phenylalanine ammonia lyase (PAL). Cinnamic acid then transformed into p-coumaric acid with the help of cinnamate-4-hydroxylase (C4H) then at last to caffeic acid with the help of the enzyme 4-coumarate 3-hydroxylase (C3H) (Figure 3) (8). 108   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Figure 3. The synthesis of caffeic acid Large volume of plant material is needed for extraction of caffeic acid from plants with solvents like methanol and ethyl acetate at high temperatures, because output is very low. Alternatively organic synthesis is used to produce the compound. Nevertheless, issue with production of byproducts has brougt to mind the posibility of synthesis of secondary metabolites like caffeic acid by synthesis with the help of microbes. So, in microorganisms like Echerichia coli strains, modifications in genetic code is made, which made synthesis of 3-hydroxylase hydroxyphenylacetate (4HPA3H) and tyrosine ammonia lyase (TAL) possible. These enzymes act on L-tyrosine producing p-coumaric acid and L-dopa. Generation of caffeic acid is achieved by action of these two enzymes on intermediate molecules (8). 3.Pharmacokinetics of Caffeic Acid Free and esterified forms of caffeic acid can be found in phenolic acid in large volumes. It can be found as 75 to 100% of hydroxycinnamic acid in fruits. Nevertheless, caffeic acid is present in foodstuff on it’s ester form, so it’s absorbtion is challenging. Caffeic acid needs to be broken down in intestinal microflora to be absorbed, because cells in human body (mucosa of intestines, liver and stomach) and bodily fluids (plasma, gastric juice, duodenal fluid) don’t contain esterase enzymes needed for hydrolysing the chlorogenic acid to produce caffeic acid. Ingestion of caffeic acid in bound form begins with it’s arrival in the stomach, then small part of it is absorbed. In the colon, esterases made by microflora cleave the ester part of the caffeic acid, then it’s free form is absorbed by intestinal mucosa (most 95%). CAFFEIC ACID   109 Monocarboxylic acid transporters (MCT) assists in absorbtion of caffeic acid from cell membrane into intestinal cells. After 1 hour from ingestion maximum blood level of caffeic acid is seen. Repeated dosing every 2 hours is needed for high blood levels, because plasma levels are decreased rapidly. With the help of sulfotransferase enzymes, UDP-glucotransferases and catecholo-methyltransferases, caffeic acid goes through three enzymatic conjugation processes just after absorbtion; methylation, sulphation, and glucuronidation. These enzymatic reactions make caffeic acid more hypophilic, by which means decreasing it’s toxicity and expediting it’s elimination. The elimination of caffeic acid (5.927%) happens mainly by urine (8). 4. Caffeic Acid in the Foodstuff Various foods, beverages, nuts, herbs, vegetables, fuits and oils contain caffeic acid. There is no sufficient data on what dosage of caffeic acid should be consumed but clinical data shows that from almost zero to 1 gram in humans is acceptable for consumption (Table 1-8) (9). Table 1. Fruits and Fruit Products Plum, prune 1.11 mg/100 g FW Date, dried 2.52 mg/100 g FW American cranberry 2.31 mg/100 g FW Black chokeberry 141.14 mg/100 g FW Cloudberry 1.00 mg/100 g FW Lingonberry, raw 6.34 mg/100 g FW Grapefruit 2.00e-03 mg/100 g FW Peach, peeled 0.63 mg/100 g FW Date, fresh 3.37 mg/100 g FW Apple (Dessert), whole, raw 0.33 mg/100 g FW Pear, peeled 0.14 mg/100 g FW Table 2. Vegetables Cauliflower, raw 1.00e-02 mg/100 g FW Eggplant (Purple), whole, raw 0.38 mg/100 g FW Olive (Black), raw 2.10 mg/100 g FW Olive (Green), raw 1.33 mg/100 g FW Tomato, whole, raw 0.45 mg/100 g FW Carrot, raw 0.02 mg/100 g FW Potato, raw 1.62 mg/100 g FW 110   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Table 3. Alcoholic beverages Beer (Alcohol free) 0.01 mg/100 ml Beer (Dark) 0.03 mg/100 ml Beer (Regular) 0.03 mg/100 ml Wine (Red) 1.88 mg/100 ml Wine (Rosé) 0.33 mg/100 ml Wine (White) 0.24 mg/100 ml Table 4. Non alcoholic beverages Coffee beverage (Filter) 0.03 mg/100 ml Grape (Green), pure juice 0.16 mg/100 ml Plum, prune, juice from concentrate 5.10 mg/100 ml Apple (Cider), juice from concentrate 0.24 mg/100 ml Apple (Cider), pure juice 0.34 mg/100 ml Apple (Dessert), juice from concentrate 0.15 mg/100 ml Apple (Dessert), pure juice 0.68 mg/100 ml Pear, pure juice 0.74 mg/100 ml Pomegranate, pure juice 0.07 mg/100 ml Table 5. Oils Olive, oil, extra virgin 0.02 mg/100 g FW Olive, oil, virgin 0.02 mg/100 g FW Soy, oil 8.00e-04 mg/100 g FW Table 6. Seeds and Nuts Walnut, dehulled 0.24 mg/100 g FW Sunflower seed, meal 8.17 mg/100 g FW Table 7. Cereals and cereal products Bread, rye, whole grain flour 0.77 mg/100 g FW Common wheat, whole grain flour 0.04 mg/100 g FW Maize, refined flour 0.04 mg/100 g FW Oat, refined flour 0.04 mg/100 g FW Oat, whole grain flour 0.16 mg/100 g FW Rice, parboiled 0.34 mg/100 g FW Rice, whole grain 0.05 mg/100 g FW Rye, whole grain flour 0.20 mg/100 g FW CAFFEIC ACID   111 Table 8. Seasonings (herbs, spices) Common sage, dried 26.40 mg/100 g FW Common sage, fresh 7.42 mg/100 g FW Common thyme, dried 21.28 mg/100 g FW Common thyme, fresh 11.70 mg/100 g FW Italian oregano, fresh 10.40 mg/100 g FW Marjoram, dried 1.90 mg/100 g FW Oregano, dried (wild marjoram) 10.70 mg/100 g FW Rosemary, dried 9.67 mg/100 g FW Rosemary, fresh 2.08 mg/100 g FW Spearmint, dried 25.00 mg/100 g FW Welsh onion, fresh 0.02 mg/100 g FW Vinegar 0.28 mg/100 ml Caraway 16.40 mg/100 g FW Ceylan cinnamon 24.20 mg/100 g FW Cumin 16.60 mg/100 g FW Ginger, dried 15.50 mg/100 g FW Nutmeg 16.30 mg/100 g FW Star anise 20.20 mg/100 g FW 5. Caffeic Acid Phenethyl Ester (CAPE) and Hepatotoxicity There is a natural biologically active molecule called Caffeic acid phenethyl ester (CAPE) occuring in plants widely. It’s main source is honey beehives and it’s made by extraction. 2-phenylethyl (2E)-3-(3,4dihydroxyphenyl) acrylate is chemical name of CAPE. Alternative naming of CAPE is phenylethyl caffeate or phenethyl caffeate. Molecular formula is C17H16O4 (Figure 4). Other way of synthesizing this polyphenolic ester is reaction of caffeic acid with phenethyl alcohols. This polyphenol has crucial role in many biological activities and has hydroxyl groups within the catechol ring. Figure 4. Caffeic acid phenylethyl ester molecule structure 112   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS Infections, cancer, oxidative stress, diabetes, neurodegeneration, inflammation, and anxiety are some of the conditions that CAPE has positive effect on (10). In traditional medicine CAPE is used for hepatoprotective purposes for long time. Inhibition of both lipoxygenase activity and lipid peroxidation is thought to be the mechanism of its anti-oxidant, antiproliferative, cytoprotective, and anti-inflammatory effects. Transcription factor inhibition of nuclear factorkappa B (NF-κB) by CAPE is linked to inhibition of phorbol ester-induced H2O2 production and tumor production (11). Chronic liver disease is characterized by aggregation of extracellular matrix proteins which is seen in liver fibrosis resulting form chronic damage to the liver. Hepatocyte damage can occur by oxidative stress with mechanisms of protein alkylation and lipid peroxidation. Activation of primary fibrogenic cell type producing collagen type I in the liver, known as hepatic stellate cells (HSCs), is triggered by oxidative stress related mediators. In the mechanism of liver fibrosis oxidative stress plays a pivotal role. Encoding of antioxidant proteins and phase 2 detoxifying enzymes, which is involved in drug metabolism, detoxification and antioxidant defenses regulated by NF-E2related factor 2 (Nrf2) which is a key transcription factor for regulation of induction. Nrf1 and Nrf2 proteins were identified as part of the NAD(P)H quinine oxidoreductase 1 antioxidant response element (ARE) DNA/protein complex, in nuclear protein extracts from normal and activated HSCs. The concurrent induction of the antioxidant response element (ARE)-mediated cytoprotective pathway and augmented proliferative signaling may confer resistance to electrophile-induced cytotoxicity in hepatic stellate cells (HSCs) during the progression of liver fibrosis. Collectively, these findings support the hypothesis that CAPE exerts superior inhibitory effects on liver fibrosis and oxidative stress through the activation of Nrf2 expression, compared to vitamin E (a well-established antioxidant agent) (11). 6. Conclusion With the rise of the longevity and wellness sectors in recent years, antioxidants, and particularly plant-derived polyphenols, have become increasingly popular. Instead of taking medications, the importance of therapeutic dietary modification has become increasingly prominent. Therefore, EPIGALLOCATECHIN-3-GALLATE   119 enhancer accessibility; as a result, transcriptional control over nodes such as the cyclin–CDK machinery and the BAX/BCL2 balance shifts, yielding phenotypes ranging from growth arrest to apoptosis or continued proliferation (2). 9. Clinical potential and future directions Within cancer biology, EGCG is framed—particularly in reproductivesystem cancers and broader reviews—as a multi-target agent with a comparatively favorable toxicity profile (3,24). When integrated with NAFLD/hepatotoxicity data and inflammation control, clinical translation will hinge on defining patient-specific dose windows, managing drug–drug interactions, and careful candidate selection (1). On the production side, mapping galloylation-related genes such as the serine carboxypeptidase-like (SCPL) family and optimizing agronomic/processing strategies help preserve raw-material quality and EGCG content, bridging pharmaceutical input to clinical output (6). Strengthening the gut microbiota–tryptophan–aryl hydrocarbon receptor (AhR) axis offers barrier benefits against environmental toxins, suggesting system-level gains along the gut–liver axis (25). In parallel, system-level targeting of miRNA networks can broaden vascular and metabolic benefits, supporting integration of extrahepatic advantages into clinical design (25). 10. Conclusion Taken together, current evidence indicates that EGCG establishes a multi-target protective architecture in the liver: activation of Nrf2/Keap1 and suppression of NF-κB lower oxidative stress and inflammation, thereby braking steatosis, fibrogenesis, and tumorigenic progression in NAFLD across multiple mechanistic layers (including NRF2, AMPK, SIRT1, TLR4/MYD88, TGF-β/SMAD, and PI3K/Akt/FoxO1) (30). In experimental models, EGCG reduces hepatic lipid accumulation, corrects insulin-signaling defects, and eases oxidative burden to ameliorate steatohepatitis, with efficacy sensitive to dosing regimens (30,31,32,33). In APAP injury, EGCG limits CYP-mediated NAPQI formation, lowers oxidative stress, and supports GSH homeostasis— effects replicated across multiple preclinical studies (12,34). In alcohol-related liver injury, its ability to augment fatty-acid oxidation and suppress steatosis counteracts ethanol-driven metabolic derangements (8). At the epigenetic level, the classical finding that EGCG inhibits DNMT and reactivates methylation-silenced genes suggests top-down reprogramming of hepatic stress responses (15). In fibrosis, miR-221–mediated osteopontin (OPN) 120   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS degradation points to direct interception of pro-fibrogenic signaling (26). Along the gut–liver axis, EGCG’s modulation of microbiota–bile acid interactions and the AhR pathway may improve lipid/cholesterol metabolism and injury-recovery gene programs (32,25); in the liver, a rise in PPARα-centered fatty-acid oxidation likely contributes to this metabolic correction (8), while miRNA/circRNA remodeling offers a transcriptomic basis for the system-level effects (25,22). Overall, these antioxidant–anti-inflammatory–epigenetic actions substantiate clinical potential; however, rigorous human studies are needed to define dose ranges, optimize formulation/stability, and clarify pharmacokinetics (3,24,1,12,4,6). Practically, EGCG’s benefits appear strongest in well-defined targets (NAFLD, alcohol-related injury, APAP toxicity), and a rational path forward is to pair individualized dosing and safety monitoring with strategies that enhance stability and bioavailability (12,8,1,3,4). When considered collectively, current evidence indicates that EGCG can concurrently modulate multiple hepatic targets spanning metabolism, inflammation, and fibrosis; in experimental NAFLD models, increases in hepatic IDE levels and activity have been associated with enhanced insulin clearance, accompanied by dose-dependent improvements in steatosis and metabolic abnormalities (10–40 mg/kg, i.p.) (35). At the clinical level, interventions using green tea enriched in EGCG or purified EGCG have been reported to yield signals of improvement in hepatic steatosis and liver enzymes, and a recent randomized controlled trial has provided additional indications of clinical feasibility (36,37). Mechanistically, attenuation of FGF21 resistance and strengthening of the FGFR/ AMPK pathway have been reported to reduce oxidative stress and hepatocyte injury, thereby providing a molecular basis for metabolic improvement (38). In a cholestatic fibrosis model (bile duct ligation), suppression of collagen deposition and α-SMA activation has been demonstrated, consistent with histological and functional recovery (39). Under stress-induced liver injury, preservation of hepatic functional indices and immune responses has also been reported (40). 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Cholestatic effect of epigallocatechin gallate in rats is accompanied by Cyp7a1 up-regulation and reduced bile formation. Toxicol Appl Pharmacol. 2013;269(2):187-194. doi:10.1016/j.taap.2012.12.019. 127 CHAPTER XIII HESPERIDIN Ulaş ACARÖZ1,2, Ömer ÇAKMAK3, Damla ARSLAN-ACARÖZ4 1(Assoc. Prof. Dr.) Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03200 Afyonkarahisar, Türkiye 2Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Kyrgyz-Turkish Manas University, KG-720038 Bishkek, Kyrgyzstan E-mail: ulasacar[email protected] ORCID: 0000-0002-1533-4519 3(Assist. Prof. Dr.)Department of Gastronomy and Culinary Arts, Faculty of Arts and Social Sciences, Istanbul Esenyurt University, 34513 Istanbul, Türkiye E-mail: omer[email protected] ORCID: 0000-0001-7658-1284 4(Assoc. Prof. Dr.) Department of Biochemistry, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03200 Afyonkarahisar, Türkiye E-mail: [email protected] ORCID: 0000-0001-9230-6725 128   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 1. Introduction Flavonoids constitute a large and structurally diverse group of secondary metabolites that are synthesized extensively throughout the plant kingdom. These polyphenolic compounds are widely recognized for their fundamental roles in plant physiology, where they contribute to processes such as cellular growth, differentiation, defense against pathogens, protection from ultraviolet radiation, and regulation of signaling pathways involved in pollination and symbiotic interactions. Beyond their ecological importance, flavonoids also serve as key bioactive molecules with significant implications for human nutrition and health (1). Since prehistoric times, plants rich in flavonoids have been used medicinally, and their remarkable therapeutic promise has fueled studies spanning extraction and characterization through animal models and human clinical trials (2). Within this family, flavanones constitute a comparatively less abundant subclass that occurs both as glycosides and as aglycones. Hesperidin is a naturally occurring bioflavonoid abundantly found in various plant sources. From a chemical perspective, it belongs to the flavanone glycosides, consisting of the aglycone hesperetin conjugated with the disaccharide rutinose, which is formed by the combination of rhamnose and glucose units (3). Hesperidin was originally identified in 1828 by the French chemist Lebreton, who extracted it from the peel of citrus fruits. The compound’s name originates from hesperidium, a botanical term referring to citrus species characterized by a thick outer rind, including oranges, lemons, limes, and mandarins (4). Hesperetin is a naturally occurring flavanone that is biosynthesized in a wide range of plant species through the flavonoid metabolic pathway. It is especially concentrated in a variety of edible fruits and vegetables, most notably in citrus species such as oranges, lemons, and grapefruits, as well as in tomatoes, cherries, bergamot, and figs. These dietary sources contribute significantly to the human intake of hesperetin and related polyphenolic compounds, which are recognized for their diverse biological and pharmacological properties (5). Extensive research has demonstrated that hesperidin exerts a wide spectrum of biological effects, including antioxidant, anti-inflammatory, anti-adipogenic, antiallergic, anticancer, antiviral, insulin-sensitizing, lipid-lowering, neuroprotective, and vascular-protective properties (6). Liver injury can arise from alcohol consumption, heavymetal toxicity, viral hepatitis, biliary obstruction, or malnutrition. Chronic damage promotes HESPERIDIN   135 5.3. Anticancer Effects Cancer continues to represent one of the most serious and complex global health burdens, affecting millions of individuals each year and accounting for a substantial proportion of premature deaths worldwide (52). Its development is influenced by a wide array of environmental and genetic determinants, including lifestyle-related factors such as tobacco consumption, unhealthy dietary habits, chronic exposure to ionizing radiation, and certain infectious agents. At the molecular level, oxidative stress and the resultant generation of reactive oxygen species play a pivotal role in DNA damage, mutation accumulation, and disruption of cellular signaling pathways that collectively contribute to malignant transformation and tumor progression (53). Accordingly, antioxidants have been explored as adjuncts or alternatives in cancer therapy (54). Owing to their comparatively modest sideeffect profiles, chemosensitizing potential, and ability to act on multiple cellular signaling pathways, natural phytochemicals can complement or, in some contexts, outperform conventional strategies. Flavonoids—including hesperidin—exhibit strong antioxidant activity, and in human cancer cell models hesperidin demonstrates antiproliferative, antitumor, and broader anticancer properties (55). Magura et al. conducted an in-depth investigation into the anticancer potential of the flavonoids hesperidin and luteolin using the MCF-7 human breast cancer cell line as an in vitro model. Their experimental findings revealed that both compounds significantly reduced the viability of cancer cells in a manner dependent on both treatment duration and concentration. Furthermore, hesperidin and luteolin were shown to trigger programmed cell death (apoptosis) through the activation of both extrinsic (death receptor-mediated) and intrinsic (mitochondria-mediated) apoptotic pathways. Molecular analyses demonstrated that treatment with these flavonoids downregulated the expression of the anti-apoptotic protein Bcl-2 while simultaneously upregulating the proapoptotic protein Bax, thereby shifting the cellular balance toward apoptosis. In addition to these protein-level effects, the compounds modulated several microRNAs associated with cancer regulation, including a marked increase in miR-16 and miR-34a expression and a significant suppression of miR-21 levels. Collectively, these data suggest that hesperidin, in conjunction with luteolin, exerts potent antiproliferative and pro-apoptotic effects on breast cancer cells and holds considerable promise as a prospective therapeutic or adjuvant agent in the management of various cancer types (56). 136   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS 5.4. Neuroprotective Effects Neurodegenerative disorders—including Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS)—represent a heterogeneous group of chronic, progressive neurological conditions marked by the gradual dysfunction and irreversible loss of neurons in specific regions of the central nervous system. These diseases are often multifactorial in origin, involving complex interactions among genetic predisposition, oxidative stress, mitochondrial impairment, and neuroinflammatory processes that together drive neuronal damage and death. Clinically, they manifest through a wide range of symptoms such as memory impairment, cognitive decline, motor deficits, emotional instability, and behavioral alterations, leading to a profound reduction in quality of life. The incidence of these disorders increases sharply with aging, making them a major health and socioeconomic concern in the elderly population worldwide (57, 58). Hesperidin has shown protective effects in several PD models, plausibly through antioxidant actions and dopamineenhancing mechanisms (59). In an ADrelevant study, Wang et al. tested hesperidin in APPswe/PS1dE transgenic mice, a model characterized by amyloidβ (Aβ)–driven pathology (60). Daily treatment (100 mg/kg) attenuated memory and learning deficits, improved motor function, and increased the activities of mitochondrial complexes I– IV along with key antioxidant defenses. The hesperidin group also showed a marked rise in GSK3β phosphorylation. Collectively, these results suggest that hesperidin may enhance cognitive performance, bolster endogenous antioxidant systems, and mitigate mitochondrial dysfunction associated with AD (60). 5.5. Cardioprotective Effects Cardiovascular diseases (CVDs) represent one of the leading causes of early mortality and long-term disability worldwide, contributing significantly to the global economic burden through increasing healthcare expenditures (61). The majority of CVDs originate from atherosclerosis, the pathological process underlying peripheral artery disease, coronary and cerebrovascular disorders, myocardial infarction, arrhythmias, venous thromboembolism, and stroke (62). With the growing recognition of the therapeutic potential of plantderived compounds, the use of botanical and herbal interventions has become more widespread. Numerous phytochemicals—such as flavonoids, polyphenols, carotenoids, and isoflavones—have been shown to possess cardioprotective activities (63). HESPERIDIN   137 Rezaee et al. investigated the cardioprotective potential of hesperidin using an experimental model of carbon monoxide (CO)-induced cardiac injury. Their study demonstrated that administration of hesperidin markedly reduced myocardial necrosis and structural tissue damage caused by CO exposure. At the molecular level, treatment with hesperidin led to an upregulation of the prosurvival Akt protein and a concurrent decrease in the pro-apoptotic BAX/ BCL-2 ratio, indicating a shift toward enhanced cellular survival signaling. Furthermore, hesperidin ameliorated CO-induced electrocardiographic (ECG) abnormalities, reflecting improvements in cardiac electrical function. Collectively, these findings suggest that hesperidin confers significant protection against CO-related cardiotoxicity, likely through the modulation of apoptotic and survival pathways within cardiac tissues (64). In a complementary experimental study utilizing a mouse model of atherosclerosis, administration of hesperidin was shown to exert multiple beneficial metabolic and vascular effects. Treatment significantly limited body weight gain and mitigated hyperlipidemia, while concurrently improving insulin responsiveness and overall glucose homeostasis, reflecting a restoration of insulin sensitivity. Histopathological and biochemical analyses further revealed that hesperidin effectively reduced hepatic lipid accumulation (steatosis), inhibited the formation of macrophage-derived foam cells within arterial walls, and markedly decreased the size of atherosclerotic plaques. On a molecular level, these improvements were associated with the downregulation of key lipogenic enzymes involved in acetyl-CoA metabolism and fatty acid synthesis pathways, including fatty acid synthase. Taken together, these observations point to a multifaceted mechanism in which hesperidin alleviates the progression of atherosclerosis, likely through its anti-inflammatory, antioxidant, and lipid-modulatory actions (65). 5.6. Effects on Wound Healing Wound repair is a complex, multistage biological process culminating in full reepithelialization. In the United States alone, more than 6.5 million people are affected by chronic, nonhealing wounds, with annual costs exceeding $25 billion. An ideal dressing should maintain a moist milieu, protect against infection, and accelerate repair—criteria that hydrogels meet particularly well, explaining their widespread clinical use (66). Within this context, hesperidin has shown promising prohealing potential. Li et al. (2018) evaluated hesperidin in diabetic rats and reported reduced blood glucose, along with significant increases in woundclosure percentage and 138   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS serum insulin (67). In a related model of diabetic foot ulcers in rats, hesperidin improved insulin concentrations, reduced wound area, and lowered blood glucose and glycated hemoglobin (HbA1c) (68). Across studies, hesperidin was also associated with favorable modulation of redox and inflammatory markers— commonly assessed via SOD, GSH, MDA, and NO—consistent with support for the healing microenvironment (67, 68). 5.7. AntiAging Activity Aging is a complex biological process influenced by multiple factors and can be accelerated by exposure to various chemical and physical stressors. The buildup of oxidized and glycated proteins contributes to persistent inflammation and the development of age-associated diseases. One promising preventive approach focuses on inhibiting protein glycation, where several natural compounds—such as hesperidin—have demonstrated protective potential (69). In a 2023 study, Novotná et al. examined hesperidin and related flavonoids and observed modulation of hyaluronidase, elastase, and collagenase activities, together with significant reductions in MMP1 and MMP2 expression—enzymes implicated in extracellularmatrix degradation (70). Earlier work by Sun et al. (2012) used a K6001 yeast replicativelifespan assay to screen commercial flavonoids as dietary antiaging candidates; hesperidin (Citrus spp.) extended lifespan at 5 and 10 mg doses compared with controls (p < 0.01 for both) (71). 5.8. Antimicrobial and Antiviral Effects Hesperidin has been reported to exert antiviral actions by inhibiting replication of specific viral genomes and altering viral gene expression (72). It shows activity against human rotavirus (73) and has been identified—based on preclinical evidence and insilico analyses—as a potentially interesting antiCOVID19 candidate (74). Beyond antiviral effects, in vivo and in vitro studies suggest antiparasitic activity (75). Interestingly, hesperidin has been identified as a potential contraceptive candidate due to its ability to inhibit sperm hyaluronidase—an enzyme essential for the penetration of the oocyte. Modified forms of the molecule, particularly its sulfonated and phosphorylated derivatives, have demonstrated even greater inhibitory potency. Furthermore, hesperidin exhibits antimicrobial activity against several sexually transmitted pathogens, such as Neisseria gonorrhoeae, Chlamydia trachomatis, HSV-2, and HIV, while showing no detectable cytotoxicity toward host cells or the normal vaginal Lactobacillus flora (76). HESPERIDIN   139 Other reported actions include modulation of ghrelin secretion through serotoninreceptor antagonism, with implications across multiple pathophysiological settings, and clinical benefit in a randomized, controlled trial for hemorrhoids (77). Preclinical literature also describes antiepileptic, antidepressant, and memoryenhancing properties (78). Finally, hesperidin can complement and potentiate the functions of other natural compounds—such as vitamin C, caffeine, naringenin, and additional flavonoids—highlighting its potential in combination strategies (79). 6. Hesperidin’s Role in Hepatotoxicity Liver disorders are a major cause of morbidity and mortality worldwide. They often stem from unhealthy lifestyle factors—excess adiposity, alcohol use, and illicit drugs—and can culminate in acute liver failure (ALF), an abrupt loss of hepatic function. In ALF, systemic complications such as renal failure, cardiovascular collapse, and cerebral edema are among the principal causes of death (80). Longstanding clinical experience and experimental work indicate that medicinal plants can ameliorate hepatic conditions; notably, several botanically derived actives—including hesperidin—have shown benefits in diseases that impair the liver (81). Methotrexateinduced injury. In adult male Sprague–Dawley rats, hesperidin mitigated methotrexate (MTX)induced hepatotoxicity. Treatment lowered malondialdehyde (MDA), elevated catalase (CAT) and glutathione (GSH), and attenuated histopathological changes, reflecting reduced hepatic oxidative stress. Relative to MTX alone, coadministration of hesperidin significantly upregulated Nrf2 and HO1, while suppressing NFκB and TNFα, thereby dampening MTXdriven hepatic inflammation. By decreasing proinflammatory and proapoptotic mediators and bolstering antioxidant capacity, hesperidin exerted a clear protective effect; pretreatment also prevented lipid disturbances, shifts in antioxidant status, and aberrant enzyme activities (82). In a model of cisplatin-induced hepatic injury, hesperidin demonstrated notable protective effects. Administration of cisplatin (CIS) led to elevated levels of total and indirect bilirubin, glucose, cholesterol, and triglycerides, along with reductions in total serum protein and globulin concentrations. CIStreated rats also showed increased hepatic MDA, depletion of antioxidant enzymes, and distinct histological damage within connective tissues. Pretreatment with hesperidin (200 mg/kg) markedly ameliorated these biochemical and structural disturbances, indicating its hepatoprotective 140   HEPATOTOXICITY AND POTENTIAL EFFECTS OF PHYTOCHEMICALS potential mediated through anti-inflammatory, antioxidant, and anti-apoptotic mechanisms (83). 7. Conclusion Hesperidin is among the most compelling and promising bioflavonoids owing to its wideranging pharmacological activities. Citrus fruits and their juices are consumed globally and provide an easily accessible dietary source; hesperidincontaining supplements—alone or combined with other citrus bioflavonoids—are likewise commercially available. Across diverse conditions, including neurological and psychiatric disorders and cardiovascular disease, hesperidin is increasingly studied for its role in modulating inflammatory processes, with particular interest in liver disease. Despite these prospects, important gaps remain. The optimal human dose, regimen, and treatment duration have not been firmly established. Moreover, hesperidin’s poor aqueous solubility and limited bioavailability constrain its therapeutic potential, suggesting that watersoluble forms and other formulation strategies could enhance clinical benefit. While antiinflammatory and lipidlowering actions make hesperidin a promising candidate for both prevention and treatment of hepatic disorders, more rigorous investigation is warranted. 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