**4.3 Saponins and terpenes**

Saponins consist of a broad family of structurally similar substances with steroid or triterpenoid glycone (Sapogenin) containing more than a fraction of oligosaccharides. The saponins and their derivatives are reported in several edible legumes. Several *in vivo* studies documented legume saponins' health benefits, including antioxidant, antidiabetic, hepatoprotective, hypocholesterolemic, anti-cancer, antitumor, antiviral [75]. Treatment of Akebia saponin D (100 μM) to oleic acidinduced BRL cells reduced lipid accumulation, increased BNip3 levels and mitophagy [76]. Hou et al. (2020) demonstrated oral administration of ginsenoside-Rg1 (30 mg/kg/day) reduced SREBP-1c expression, lipid accumulation and alleviated liver inflammation in NAFLD rats [52]. Sea cucumber-derived saponins echinoside A (EA) ameliorated orotic acid induced-NAFLD mainly by inhibiting lipogenesis genes. Andrographolide, a diterpene lactone present in *Andrographis paniculata* treatment to choline-deficient amino acid-defined mice, prevented liver inflammation, reduced macrophage infiltration and inflammation activation [53, 77].

Wang et al. observed glycyrrhizic acid (a natural triterpene glycoside) administration to MCD diet-fed mice significantly inhibited hepatic stellate cell activation and collagen deposition [54]. In a similar study, glycyrrhizic acid suppressed lipid accumulation and reduced the levels of SREBP-1c, FAS, SCD-1 in HFD fed mice [78]. Glycyrrhetinic acid, a bioactive triterpenoid from licorice, reduced the inflammation and fat content in the mouse liver and inhibited AKR1B10 activity [79].

### **4.4 Phytosterols**

Phytosterols or plant sterols are cholesterol-like molecules which perform vital structural functions in plants. Phytosterols are best known for their cholesterollowering effects and recent investigations highlighted their anti-fibrotic developments in key NAFLD models [80]. Plant sterol and stanol ester supplementation significantly reduced plasma lipids and prevented HFD induced inflammation in experimental animals [81]. In a high-fat Western-style diet-induced mice study, stigmasterol and β-sitosterol markedly reduced the liver TGs, cholesterol, intestinal bile acid levels and alleviated NAFLD [55]. Intragastric administration of phytosterol esters for 12 weeks to HFD fed rats reduced liver size, lipid content and improved intestinal flora [82]. In combination with EPA and DHA, phytosterol

esters significantly reduced the levels of TGs, cholesterol, LDL cholesterol and decreased the pro-inflammatory cytokines in NAFLD subjects [83]. β-sitosterol supplementation for 12 weeks mitigated high-fructose diet-induced macrovesicular steatosis and progression of steatohepatitis [84].

## **4.5 Carotenoids**

Carotenoids are a family of poly-isoprenoid structured and fat-soluble pigments that occur naturally in plants and microbes. The primary sources of carotenoids in the human diet are yellow, orange and red-colored fruits and vegetables. In the last few decades, carotenoids have been the main focus of research mainly due to their potent antioxidant, anti-inflammatory and anticancer properties [85]. Besides, carotenoids were also reported for their anti-fibrotic effect in several experimental NAFLD models [86]. Several clinical studies revealed circulatory carotenoid levels to NAFLD risk [56, 87]. β-carotene is reported for strong antioxidant potential and a vast number of *in vitro* and *in vivo* studies revealed the hepatoprotective and anti-fibrotic effect of β-carotene [88–90]. Lycopene, a non-provitamin A carotenoid, mainly exhibited hepatoprotective effect through scavenging ROS. Supplementation of lycopene significantly lowered steatosis and obesity-induced inflammation in NAFLD animals [91–94]. The xanthophyll carotenoid, astaxanthin is reported for various biological effects such as free radical scavenging, ocular protective, hepatoprotective, anti-aging, anti-diabetic, anti-inflammatory, anticancer, etc. [95–97]. In experimental liver fibrosis models, astaxanthin offered hepatoprotection by reducing liver pro-inflammatory cytokines, attenuating insulin resistance, downregulating key signaling pathways [98–102]. Other carotenoids like α-carotene, lutein and zeaxanthin also exerted hepatoprotection in experimental NAFLD models [103–105].

#### **4.6 Functional foods from plant/animal origin/carbohydrates**

Oats (*Avena sativa*) rich in β-glucan, a polysaccharide responsible for its functional properties and other active compounds such as antioxidants, vitamins, minerals and phenolic compounds and dietary fibers. A study conducted in Sprague–Dawley rats found that a diet rich in oats increased liver LDLR, reduced liver TGs and cholesterol, thereby preventing NAFLD development to liver cirrhosis [106]. Flaxseed (*Linum usitatissimum*) is a highly nutritional functional food due to active components such as polyunsaturated fatty acid (PUFA), α-linolenic acid, proteins, lignans, soluble and insoluble dietary fibers, antioxidants and phytoestrogens. In a clinical trial conducted with 50 subjects, supplementation of flaxseed diet significantly reduced the body weight, liver enzymes, insulin resistance, hepatic fibrosis and steatosis. A significant difference was observed between control and flaxseed groups in ALT, AST, GGT, fibrosis score and steatosis score, etc. [107].

Choline is an essential nutrient present in eggs, liver, soy wheat and vegetables. Choline is either produced in the body or is absorbed from a diet rich in phospholipids such as phosphatidylcholine. Phosphatidylcholine is a significant component of cell membranes and present in egg yolk and soy. Choline and betaine supplementation effectively alleviated NAFL in dairy cattle, PEMT- deficient mice by increasing AMPK, reducing mRNA levels of DGAT2 and lipid accumulation, decreased expression of genes such as acyl-CoA synthase-1 and -4, mitochondrial glycerol phosphate acyltransferase, etc. [108]. Studies conducted in humans showed that betaine supplementation reduced serum concentrations of ALT and AST and lowered hepatic steatosis [109, 110]. In Balb/c mice, administration of betaine increased serum ALT, decreased hepatic and visceral mass accumulation by reducing glucose

production through inhibiting gluconeogenesis and promoting the use of glucose in glycogen production leading to improved serum glucose levels. Thereby, betaine reversed insulin resistance by promoting IRS1 phosphorylation and enhanced downstream pathways of gluconeogenesis and glycogen synthesis and effectively alleviated NAFLD [111].
