Elucidation of Natural Components of Gardenia thunbergia Thunb. Leaves: Effect of Methanol Extract and Rutin on Non-Alcoholic Fatty Liver Disease

The rising prevalence of non-alcoholic fatty liver disease NAFLD has strained the healthcare system. Natural products could solve this problem, so the current study focused on the impact of G. thunbergia Thunb. against this ailment. LC–ESI–MS/MS revealed the phytochemical profile of the methanol extract from Gardenia thunbergia leaves (GME). Forty-eight compounds were tentatively identified, and stigmasterol, fucosterol, ursolic acid, and rutin were isolated. The separation of the last three compounds from this plant had not before been achieved. The anti-NAFLD effect of the methanol extract of the leaves of G. thunbergia, and its major metabolite, rutin, was assessed in mice against high-fructose diet (HFD)-induced obesity. Male mice were allocated into nine groups: (1) saline (control), (2) 30% fructose (diseased group), (3) HFD, and 10 mg/kg of simvastatin. Groups 4–6 were administered HFD and rutin 50, 75, and 100 mg/kg. Groups (7–9) were administered HFD and methanol extract of leaves 100, 200, and 300 mg/kg. Methanol extract of G. thunbergia leaves at 200 mg/kg, and rutin at 75 mg/kg significantly reduced HFD-induced increments in mice weight and hepatic damage indicators (AST and ALT), steatosis, and hypertrophy. The levels of total cholesterol, LDL–C, and triglycerides in the blood decreased. In addition, the expressions of CYP2E1, JNK1, and iNOS in the diseased mice were downregulated. This study found that GME and rutin could ameliorate NAFLD in HFD-fed mice, with results comparable to simvastatin, validating G. thunbergia’s hepatoprotective effects.


Introduction
The active ingredients of herbal medicine and natural products have recently received increased attention as prospective therapeutic agents for the treating, and preventing of NAFLD, owing to their outstanding levels of effectiveness and minimal risks of adverse effects [1]. Gardenia thunbergia Thunb. (family Rubiaceae), Star Gardenia, is a shrub or small tree that thrives in Egypt and many African and Asian nations [2]. The flavonoids, terpenoids, iridoids, and steroids found in the Gardenia family have many therapeutic advantages, including anti-inflammatory, analgesic, and antioxidant properties, which may be attributed to flavonoids [3,4]. In the present study, LC-ESI-MS/MS was utilized to reveal, tentatively, the phytochemicals of the leaves of the plant. Recently, products from natural sources have become a substantial basis of pharmaceuticals, and they are actively being researched as potential therapeutic candidates [5]. They have a lot of benefits, including being risk-free, effective, biocompatible, and affordable [6]. Natural products, as a result, can be a successful method for preventing the emergence of various disorders, including NAFLD.
NAFLD (non-alcoholic fatty liver disease) is a type of liver ailment that negatively impacts people's lives. The global prevalence of NAFLD has risen to 40% [7]. NAFLD

Identification of Compounds' Structures Isolated from G. thunbergia Methanol Extract of Leaves
Four compounds isolated from G. thunbergia methanol extract of leaves were fully elucidated by their 1 H-and 13 C-NMR, APT, HMBC, HSQC, and EI-MS, ESI-MS spectra. Based on their spectra, compounds 1-4 were identified as Fucosterol, Stigmasterol, Ursolic acid, and Rutin. Their structures are drawn in Figure 4.

Identification of Compounds' Structures Isolated from G. thunbergia methanol extract of leaves
Four compounds isolated from G. thunbergia methanol extract of leaves were fully elucidated by their 1 H-and 13 C-NMR, APT, HMBC, HSQC, and EI-MS, ESI-MS spectra. Based on their spectra, compounds 1-4 were identified as Fucosterol, Stigmasterol, Ursolic acid, and Rutin. Their structures are drawn in Figure 4.

Effect of Rutin and Leaves Methanol Extract on Liver Index and Liver Function Tests of Mice
The presented data (Table 2) of the diseased group exhibited a remarkable increase in aspartate aminotransferase, alanine transaminase, and liver index, related to the control group (p < 0.05). Simvastatin significantly decreased the liver index and liver enzymes (p < 0.05) according to the diseased group. Rutin and methanolic extract of leaves displayed a remarkable decrease in ALT and AST serum levels at dose levels of rutin, 50 and 75 mg/kg, and methanol extract, 100 and 200 mg/kg (p < 0.05), related to the diseased group, while large doses of rutin, 100 mg/kg, and methanolic extract, 300 mg/kg, did not show significant changes according to the diseased group. The results revealed that rutin at 75 mg/kg might significantly decrease the liver index, ALT, and AST levels (p < 0.05) when related to the mice group administered simvastatin (10 mg/kg). Moreover, the mice group that received methanol extract at 200 mg/kg displayed a remarkable reduction in liver index related to the mice group managed with simvastatin. Data displayed as mean ± SD, n = 6 per group. NC: normal control, DG: diseased group. Statistics were calculated by one-way ANOVA followed by an LSD comparison test. a : significant according to the normal control group, b : significant according to the diseased group, c : significant according to the simvastatin group. Significance was assessed at p < 0.05. Table 3 demonstrates that the diseased group had high serum levels of total cholesterol, triglycerides, and low-density lipoprotein-cholesterol, while there was a noticeable decrease in the high-density lipoprotein-cholesterol of mice in the normal group (p < 0.05). The group administered simvastatin (10 mg/kg) exhibited a notable decrease in serum triglycerides, total cholesterol, and LDL-C and a notable increase in HDL -C in the diseased group (p < 0.05). Both rutin and methanolic extract at different doses exhibited considerable improvement in total cholesterol levels, compared to the diseased group. However, rutin at 75 mg/kg and GME at 200 mg/kg showed a reduction in triglyceride level in the diseased group (p < 0.05). As observed in Table 3, rutin at the dose of (50 and 100 mg/kg) and GME at (100 and 200 mg/kg) showed a notable reduction in LDL-C serum levels in the diseased group (p < 0.05). On the other hand, HDL-C significantly improved only at a dose of 75 mg/kg of rutin and also; improved at doses of 200 and 300 mg/kg of GME, compared to the diseased group (p < 0.05). The present data revealed that rutin at the dose of 75 mg/kg significantly improved dyslipidemia caused by the consumption of a high fructose diet, compared to the diseased group. In comparison, rutin at 50 mg/kg did not show noteworthy changes in the lipid profile of mice when compared to the diseased group. Our data demonstrated a substantial improvement of NAFLD combined dyslipidemia at 200 mg/kg of GME. On the other hand, the dose of 100 mg/kg of this extract did not show notable changes in the diseased group.

Effect of Rutin and Methanolic Extract on Oxidative Stress-Related Gene Expression
The diseased group of mice showed a significant rise in the relative messenger ribonucleic acid expression of iNOS and CYP2E1 in the liver (p < 0.05) by qRT-PCR, compared to the normal control group ( Figure 5A,B). It was observed that different doses of rutin and leaves methanolic extract exhibited a significant decrease in relative mRNA expression of both iNOS and CYP2E1 (p < 0.05), compared to the diseased group ( Figure 5A,B). Interestingly, the mice group that was administered rutin at the dose of 75 mg/kg exhibited the most significant downregulation of CYP2E1 relative gene expression (decreased by 86%), relative to the diseased group. Administration of GME at 200 mg/kg and 300 mg/kg exhibited valuable downregulation in CYP2E1 relative gene expression (82% and 71%, respectively), relative to the diseased mice ( Figure 5A,B). Figure 5C displays a notable rise in the relative mRNA expression of hepatic JNK1 of mice in the diseased group (p < 0.05) relative to the normal control group. It was also observed that different doses of rutin and total methanolic leaves extract exhibited a significant decrease in relative mRNA expression of JNK1 (p < 0.05) compared to the diseased group ( Figure 5C). Furthermore, the present work displayed that the mice group taking rutin at 75 mg/kg, and the mice group receiving total G. thunbergia methanol extract of leaves at a dose of 200 mg/kg, exhibited the most significant downregulation of JNK1 relative gene expression (decrease by 93% and 80%, respectively) compared to the diseased mice ( Figure 5C). . Data displayed as mean ± SD, n = 6 per group. Statistically, a significant difference resulted from a one-way ANOVA followed by Tukey's multiple comparison test. Data was significant at p-value < 0.05. *: significant according to the normal control group. #: significant according to the diseased group. GME: G. thunbergia methanol extract of leaves. Control: normal control group. Diseased: diseased group. Figure 5C displays a notable rise in the relative mRNA expression of hepatic JNK1 of mice in the diseased group (p < 0.05) relative to the normal control group. It was also observed that different doses of rutin and total methanolic leaves extract exhibited a significant decrease in relative mRNA expression of JNK1 (p < 0.05) compared to the diseased group ( Figure 5C). Furthermore, the present work displayed that the mice group taking rutin at 75 mg/kg, and the mice group receiving total G. thunbergia methanol extract of leaves at a dose of 200 mg/kg, exhibited the most significant downregulation of JNK1 relative gene expression (decrease by 93% and 80%, respectively) compared to the diseased mice ( Figure 5C).

Histopathology of Liver in Different Mice Groups
The liver tissues of mice in the normal group displayed a regular-sized central vein surrounded by normal hepatocytes ( Figure 6A). On the contrary, the liver tissues of mice in the diseased group showed marked inflammation around the central vein and hepatocytes with marked steatosis; also, some cells exhibited necrosis ( Figure 6B). Liver sections of mice that received simvastatin (10 mg/kg) revealed mild inflammatory cellular infiltrate surrounded by hepatocytes with mild steatosis ( Figure 6C). Liver tissues of the mice group that were administered rutin at 75 mg/kg displayed a portal tract surrounded by minimal inflammatory cellular infiltrate surrounded by hepatocytes with minimal steatosis and degeneration ( Figure 7B), relative to the marked inflammatory cellular infiltrate surrounded by hepatocytes with moderate steatosis observed in the tissues from mice in the group administered rutin at 50 mg/kg ( Figure 7A). Moreover, the liver tissues from mice in the group that received rutin at 100 mg/kg displayed a portal tract surrounded by marked inflammatory cellular infiltrate surrounded by hepatocytes with moderate steatosis ( Figure 7C).
The results also exhibited that the liver tissues of the mice administered GME at 200 mg/kg showed normal hepatocytes with no inflammation or steatosis ( Figure 8B), when compared to the marked inflammatory cellular infiltrate surrounded by hepatocytes with mild steatosis observed in the liver tissues of mice that received GME at 100 mg/kg ( Figure  8A). A mildly dilated congested central vein accompanied by mild steatosis was observed in the liver tissues of mice that received total leaves extract at 300 mg/kg ( Figure 8C). . Data displayed as mean ± SD, n = 6 per group. Statistically, a significant difference resulted from a one-way ANOVA followed by Tukey's multiple comparison test. Data was significant at p-value < 0.05. *: significant according to the normal control group. #: significant according to the diseased group. GME: G. thunbergia methanol extract of leaves. Control: normal control group. Diseased: diseased group.

Histopathology of Liver in Different Mice Groups
The liver tissues of mice in the normal group displayed a regular-sized central vein surrounded by normal hepatocytes ( Figure 6A). On the contrary, the liver tissues of mice in the diseased group showed marked inflammation around the central vein and hepatocytes with marked steatosis; also, some cells exhibited necrosis ( Figure 6B). Liver sections of mice that received simvastatin (10 mg/kg) revealed mild inflammatory cellular infiltrate surrounded by hepatocytes with mild steatosis ( Figure 6C). Liver tissues of the mice group that were administered rutin at 75 mg/kg displayed a portal tract surrounded by minimal inflammatory cellular infiltrate surrounded by hepatocytes with minimal steatosis and degeneration ( Figure 7B), relative to the marked inflammatory cellular infiltrate surrounded by hepatocytes with moderate steatosis observed in the tissues from mice in the group administered rutin at 50 mg/kg ( Figure 7A). Moreover, the liver tissues from mice in the group that received rutin at 100 mg/kg displayed a portal tract surrounded by marked inflammatory cellular infiltrate surrounded by hepatocytes with moderate steatosis ( Figure 7C).    The results also exhibited that the liver tissues of the mice administered GME at 200 mg/kg showed normal hepatocytes with no inflammation or steatosis ( Figure 8B), when compared to the marked inflammatory cellular infiltrate surrounded by hepatocytes with mild steatosis observed in the liver tissues of mice that received GME at 100 mg/kg ( Figure 8A). A mildly dilated congested central vein accompanied by mild steatosis was observed in the liver tissues of mice that received total leaves extract at 300 mg/kg ( Figure 8C).
Molecules 2023, 28, x FOR PEER REVIEW 12 of 21 Figure 8. Representative photomicrograph of a liver section of mice groups that received different doses of total G. thunbergia methanol extract of leaves (H&E, ×200) as follows: (A); mice group received GME at 100 mg/kg: section of the liver displayed portal tract surrounded by marked inflammatory cellular infiltrate (red arrows) surrounded by hepatocytes with mild steatosis (blue arrow). (B); mice group received GME at 200 mg/kg: A section of the liver showed a portal tract (red arrow) surrounded by average-sized cords of hepatocytes (blue arrows) with no inflammation or steatosis. (C); mice group received GME at 300 mg/kg; showed dilated congested central vein tract (red arrow) with no inflammation and hepatocytes with mild steatosis.

Discussion
The spread of non-alcoholic fatty liver disease is associated with increasing cases of obesity, which loads the global health care system. Due to the extraordinary consumption of high-density foods, such as high-fat or high-sugar intake diets, organ damage raises the white adipose tissue, leading to health-threatening inflammatory alterations in humans, accompanied by metabolic and hormonal changes [5].
Nutraceuticals are active phytochemical components found in plants, such as flavonoids. They are employed as a combination therapy in up to 82 percent of affected persons with fatty liver, cancer, and other disorders. Flavonoids and other phenolic compounds Figure 8. Representative photomicrograph of a liver section of mice groups that received different doses of total G. thunbergia methanol extract of leaves (H&E, ×200) as follows: (A); mice group received GME at 100 mg/kg: section of the liver displayed portal tract surrounded by marked inflammatory cellular infiltrate (red arrows) surrounded by hepatocytes with mild steatosis (blue arrow). (B); mice group received GME at 200 mg/kg: A section of the liver showed a portal tract (red arrow) surrounded by average-sized cords of hepatocytes (blue arrows) with no inflammation or steatosis. (C); mice group received GME at 300 mg/kg; showed dilated congested central vein tract (red arrow) with no inflammation and hepatocytes with mild steatosis.

Discussion
The spread of non-alcoholic fatty liver disease is associated with increasing cases of obesity, which loads the global health care system. Due to the extraordinary consumption of high-density foods, such as high-fat or high-sugar intake diets, organ damage raises the white adipose tissue, leading to health-threatening inflammatory alterations in humans, accompanied by metabolic and hormonal changes [5].
Nutraceuticals are active phytochemical components found in plants, such as flavonoids. They are employed as a combination therapy in up to 82 percent of affected persons with fatty liver, cancer, and other disorders. Flavonoids and other phenolic compounds are essential active components among other active components [47]. The LC-ESI-MS/MS analysis of GME explored the tentative recognition of 48 known compounds contributing to various health benefits. Antioxidant properties are one of the most critical possible health benefits of phenolics (chlorogenic acid) and flavonoids, such as (rutin, hesperetin, naringenin, luteolin, kaempferol glycosides, and methoxylated flavones) [48,49].
The isolation of stigmasterol, fucosterol, ursolic acid, and rutin was performed from the unsaponifiable part of the petroleum ether fraction and n-butanol fraction of leaves extracts. Their structures were elucidated via spectroscopic analysis of these compounds and by comparison with the literature.
Compound (2) Figure S8). The 1 H-NMR spectrum, displayed in the Supplementary Materials, Figure S9, showed singlet signals at δ 0.67 (3H, s) and 1.00 (3H, s), which were assigned to the presence of two methyl groups at 18 and 19 positions; three methyl doublets resonating at δ 0.81 (3H, d, J = 6.5 Hz), 0.83 (3H, d, J = 6 Hz) 0.92 (3H, d, J = 6.5 Hz) were assigned to carbons 26, 27 and 21, respectively; and a methyl triplet at δ 0.88 (3H) at C-29. It also displayed signals resonating at δ 5.00 (2H, m), 5.14 (2H, m), and 5.35 (1H, m), suggesting the existence of three protons, according with those of a trisubstituted and a disubstituted olefinic bond at C-22, C-23, and C-6 respectively. The proton ascribed to the H-3 of a sterol moiety appeared as a multiplet signal at δ 3.51; this characteristic signal was assigned to an oxygenated methine proton. The 13 C-NMR spectral data (demonstrated in the Supplementary Materials, Figure S10) reinforced the presence of 29 carbon signals, including six methyls and nine methylenes. The chemical shift at δ (C-3) 71.80 was due to oxymethine carbon, and signals at δ C 140.73 and δ C 121.72 were due to a double bond between C-5 and C-6, respectively. Carbon signals at δ C 138.32 (C-22) and δ C 129.24 (C-23) were ascribed to carbon in a trans-conformation of a triterpenoid nucleus. The structure was elucidated as stigmasterol, by analyzing the spectroscopic data in the literature [42,50].
The EI-MS spectrum of compound (3)  which is common in an ursane-type triterpenes skeleton [50]. A down-field signal at δ 2.97 (1H, m) represented H-3, which was assigned to the methine proton on the carbon, which bore the hydroxyl group (its proton signal was displayed at δ 4.30), and an olefinic proton signal resonating at δ 5.09 (1H, s) ppm was assigned to H-12 of a triterpene. Finally, a doublet signal resonated at δ 2.46 (1H, d, J = 11.3 Hz) and was assigned to C-18. The 13 C-NMR spectrum indicated that compound (3) had a total of 30 carbon atoms and showed the presence of signals characteristic to the triterpene group of α-amyrine: δ C 77.44, 125.10, 138.70, and 178.91 ppm. The signal resonating at δ 77.44 ppm was ascribed to C-3 (CH-O-) of a triterpenoid and the signals at δ 125.10 and 138.70 ppm at position 12 and 13, respectively, were for the double bond of ursane type of triterpenes [28]. The signal resonating at δ C 178.91 ppm was attributed to a carbon of a carbonyl group at position 28. It was identified as ursolic acid, according to the reported data in the literature [43]. Different spectra are provided in the Supplementary Materials, Figures S11-S13.
The ESI-MS spectrum of compound (4) 18.21 (rha-6), were attributed to the presence of rhamnose moiety. The up-field signal resonating at δ 18.21 ppm of (rha-6) was noticed with signals for the dihydroxy groups at 3', 4' of ring B. Signals which were ascribed at δ 101.63 and 101.21 ppm, were attributed to acetal carbon of (C-glc-1) and (C-rha-1). A recognized down-field signal showed at δ 177.81 ppm for C-4. The spectrum also showed different down-field signals resonating at δ 164.65, 157.06, 161.66, and 156.88 ppm for C-7, 9, 5, and 2, respectively. Correlation between carbons and protons displayed the protoncarbon coupling with nearest carbon skeletons and was confirmed by the Heteronuclear Multiple Bond Correlation spectrum, as shown in the Supplementary Materials, Figure S18. It was identified according to the reported data in the literature [44][45][46]. Different spectra were found in the Supplementary Materials, Figures S15-S17.
In the present work, induction of NAFLD, mediated by administration of HFD in drinking water, was confirmed by the significant increase in the liver index and elevated serum levels of AST, ALT, TG, TC, LDL-C, and the low concentration of HDL-C. Histopathological examination of mice from the disease control group showed marked inflammation around the central vein and hepatocytes with marked steatosis, confirming the induction of NAFLD, as previously described by Zhang et al. [51]. Furthermore, the significantly increased levels of triglyceride and total cholesterol in mice of the disease control group indicated fat deposition in the liver tissue.
Both rutin and methanol extract of leaves of Gardenia thunbergia showed notable improvement in the lipid profile of treated mice groups, compared to the diseased group. Interestingly, rutin at the dose of 75 mg/kg and GME at 200 mg/kg were the doses that displayed a significant decrease in triglyceride levels, compared to the diseased group. Rutin at (50 and 100 mg/kg) and GME at (100 and 200 mg/kg) showed a notable reduction in LDL-C serum levels, compared to the diseased group. HDL-C improved only at a dose of 75 mg/kg of rutin and 200 and 300 mg/kg of GME, compared to the diseased group.
These results indicated that administration of GME or pure rutin exhibited a decrease in fat accumulation in hepatocytes, resulting in the decreased inflammation and steatosis observed in liver sections of mice from the treated groups.
The reducing effect of GME on AST and ALT levels indicated that it might reduce the adverse effects of a high fructose diet on the liver and lessen hepatic injury, which agreed with Li et al. [49], who demonstrated that flavonoids had a positive effect on liver structure and function.
The inflammatory response plays an essential role in developing hepatic steatosis [52]. Accumulation of fatty acids could be handled by cytochrome (CYP2E1), a member of the oxidoreductase cytochrome family [53][54][55]. However, increased CYP2E1 expression contributes considerably to oxidative stress in NAFLD livers according to various studies, including human and mouse models of NAFLD [56][57][58]. In addition, CYP2E1 triggers hydroxylation of fatty acids, resulting in the accumulation of cytotoxic lipid intermediates and ROS and activating of cell death by activating the ERK1/2 pathway [56].
It was found that upregulation of CYP2E1 played a potential role in the activation of the death signaling c-Jun N-terminal kinases (cJNK) pathway [59][60][61]. Activation of cJNK would, consequently, phosphorylate and activate other proteins, such as Bax and Bcl-2, which would, finally, trigger mitochondrial apoptotic pathways and subsequent development and progression of NAFLD. Increased steatosis and fatty acid β-oxidation would stimulate reactive oxygen species and oxidative stress, leading to increased cJNK activity, and, subsequently, increased insulin resistance, provoking hepatocyte death [59][60][61].
These results indicated that flavonoids of G. thunbergia, especially rutin, could hinder the development and progression of NAFLD through the downregulation of CYP2E1, which, subsequently, inhibits the c-JNK apoptotic pathway. Histopathological examination of liver tissues of the mice groups administrated rutin (at the dose of 75 mg/kg) or GME (at the dose of 200 mg/kg) revealed the absence of inflammation and steatosis, which was considered as further evidence for the potential role of G. thunbergia in inhibiting NAFLD development. Our results were consistent with other studies that reported that TNF-α, FFAs, and ROS activated JNK and promoted initiation and progression of insulin resistance and the development of NAFLD [62,63]. GME and rutin also reduced iNOS expression levels. Free-radical scavenging activity of GME polyphenolics could decrease ROS and prevent inflammatory reactions. These results were consistent with Li et al. [49], who reported that the downregulation of iNOS inhibited the development of insulin resistance in mice fed HFD.
Further assessment of liver tissues of mice given GME (200 mg/kg) displayed a portal tract surrounded by average-sized cords of hepatocytes with no inflammation or steatosis. Mice treated with rutin (75 mg/kg) showed a portal tract surrounded by minimal inflammatory cellular infiltrate and hepatocytes with minimal steatosis and degeneration. GME (300 mg/kg) showed a dilated congested central vein tract (red arrow) with no inflammation and hepatocytes with mild steatosis. Mice treated with rutin (100 mg/kg) revealed marked inflammatory cellular infiltrate surrounded by hepatocytes with mild steatosis and a congested central vein tract, compared to normal hepatocytes, and the absence of steatosis or inflammation was observed in mice given rutin at the dose of (75 mg/kg) or GME (200 mg/kg).
Administration of G. thunbergia extract of leaves at a dose of (200 mg/kg) and rutin (75 mg/kg) could prevent the development of NAFLD in HFD-fed mice through decreasing the oxidative stress mediated by β-oxidation of fatty acids, and decreasing inflammatory steatosis development through downregulation of the CYP2E1-cJNK apoptotic signaling pathway.

Plant Material
Gardenia thunbergia Thunb. Leaves were obtained from the shrubs growing in Al Sadat City, Egypt, in March, 2018. Professor Dr. Ibrahim Abd El Rahim Mashaly, Professor of Plant Ecology and Flora, Department of Botany, Faculty of Science, Mansoura University, certified the plant's legitimacy. The leaves were air-dried and then ground before use for this study. The voucher specimen (PG00415-M) was kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Tanta University.

Extraction and Isolation Procedure
Gardenia thunbergia leaves were dried at room temperature and ground to obtain 1.5 kg dry powder. They were extracted by cold maceration with methanol (3 × 3 L, 48 h each) till exhaustion and then evaporated using a rotary evaporator under reduced pressure at 40 • C to provide a dry leaves methanol extract of (220 g) yield. A part of the methanol extract (175 g) was resuspended in methanol and successively partitioned with petroleum ether 60-80, methylene chloride, ethyl acetate, and n-butanol. The yield of each fraction was petroleum ether (45 g), methylene chloride (40.1 g), ethyl acetate (4.6 g), and n-butanol (55.6), represented in the Supplementary Materials, Figure S1.
The petroleum ether fraction obtained from the methanolic extract of the leaves was used to investigate the saponifiable and unsaponifiable matter. Two grams of petroleum ether extract of the methanol extract of the leaves was saponified by heating for 6 h using a boiling water bath with a reflux condenser, accompanied by 30 mL alcoholic KOH (10%). To eliminate the majority of the alcohol, the saponified liquid was distilled, then the aqueous liquid was diluted with 20 mL water before being extracted with ether till exhaustion. The combined ethereal extracts were washed with distilled water, dehydrated over anhydrous Na 2 SO 4 , and distilled under a vacuum to yield the unsaponifiable matter (USM) of G. thunbergia methanol extract of leaves, represented in the Supplementary Materials, Figure S2. The unsaponifiable matter of the petroleum ether fraction (1 g) was applied to a silica gel column (40 g, ϕ two × 30 cm, fraction collected 30 mL), using the wet method. The mobile phase was composed of gradient mixtures with increasing polarities of petroleum ether-CH 2 Cl 2, then methylene chloride: methanol mixtures of increasing polarities. The petroleum ether-CH 2 Cl 2 (98:2) afforded compound (1) (24 mg). The petroleum ether-CH 2 Cl 2 (30:70) eluate (100 mg) was applied to another CC, using silica gel (6 g, ϕ one × 6 cm, fraction collected 10 mL), using a mobile phase of increasing polarity (gradient elution) of n-Hexane-EtOAc. The n-Hexane-EtOAc (10:90) afforded compound (2) (33 mg). The first column afforded eluate (82 mg) by methylene chloride-methanol (98:2) was applied to another CC using silica gel (6 g, ϕ 1 × 6 cm, fraction collected 10 mL). Elution was done successively with a mixture of increasing polarities of CH 2 Cl 2 -MeOH to give compound (3) (18 mg) at (93:7), displayed in Figure  S4 of the Supplementary Materials.
The n-butanol fraction (55.6 g) was mixed with deionized water and the suspension was passed through a Diaion HP-20 column [60 × 3 cm, 80 g]. The mobile phase was composed of deionized water (1 L), followed by MeOH-H 2 O (50:50) and 100% MeOH. Next, the n-butanol fraction, eluted with 100% MeOH (5.5 g), was applied to a column of silica gel (200 g, ϕ 4.5 × 40 cm, fractions of 50 mL each) using the dry method and eluted, consecutively, with a mixture of increasing polarities of CH 2 Cl 2 -MeOH containing 5% water. A yellow matter was obtained from the CH 2 Cl 2 -MeOH containing 5% water (70:30) eluate (435 mg). This matter was applied to Sephadex LH-20 for purification to obtain a compound (4) (412 mg), represented in the Supplementary Materials, Figure S14. in serum samples [69] were performed using colorimetric kits purchased from Spectrumdiagnostics ® , Cairo, Egypt.

Determination of Lipid Profile in the Serum
At the end of the experiment (8 weeks), mice were kept for overnight fasting. Then, mice of all groups were anesthetized and sacrificed by cervical dislocation, and aliquots of mice serum samples were collected and stored at −20 • C for biochemical analysis. Kits for determining serum biomarkers of lipid profile, adopting manufacturer protocol [70,71], were purchased from Bio-diagnostic ® , Cairo, Egypt.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) for Liver Tissue
The liver tissue samples of mice were homogenized to determine iNOS, CYP2E1, and c-JNK1 relative gene expression. A total RNA extraction kit (Bio E-Technology, Xi'an, China) was used to extract total RNA from the liver tissue of mice; purity and concentration of mRNA were estimated. The extracted mRNA was used to form cDNA. Primer sequences were mentioned in the Supplementary Materials. Each sample was analyzed by the qRT-PCR, normalized to the reference gene level (GAPDH) level, and expressed as a relative copy number (RCN). Threshold cycle (Ct) values were calculated, and the transcript levels were calculated by using 2 −∆Ct formula [68].

Histopathological Examination of Liver Tissue
Liver tissue was cut into five µm sections, which were kept in paraffin, hematoxylin, and eosin were used in staining. A professional histopathologist examined these sections with a light microscope (Leica DM 500, Switzerland). The liver sections were investigated for inflammatory cell infiltration, necrosis, and the areas scored steatosis [72].

Statistical Analysis
Statistical analysis was performed with the statistical package for social science (SPSS) software version 20. One-Way ANOVA test was done for one-independent variables. The least significant difference (LSD) test is used in the analysis of variance for multiple comparisons in studying liver index, liver function tests, and the lipid profile of mice. Data displayed as mean ± SD, n = 6 per group. Statistically, a significant difference was determined in the qRT-PCR experiment by a one-way analysis of variance followed by Tukey's multiple comparison test. Data was significant at p-value < 0.05.

Conclusions
The findings of this study indicated that G. thunbergia methanol extract of leaves and rutin isolated from this plant had a distinguishable effect in reducing lipid serum levels. Fucosterol, ursolic acid, rutin, and stigmasterol were isolated from the leaves. The first three actives were separated for the first time from this species. Different compounds (48) from several phytochemical classes were tentatively identified using LC-ESI-MS/MS to explore the chemical composition of this plant. The presence of the major metabolites of rutin, isoquercitrin, delphinidin rhamnoglucoside, kaempferol glycosides, quinic acid, 4 ,5,7trihydroxyflavonol, chlorogenic acid, hesperetin, acacetin, daphnetin, 3,4,5 -trihydroxy-3glucopyranosylstilbene, and 3,5,7-trihydroxy-4 -methoxyflavone with anti-inflammatory, and antioxidant properties, could explain the therapeutic effects of G. thunbergia methanol extract of leaves in the non-alcoholic fatty liver disease (NAFLD). G. thunbergia extract and rutin could hinder the development of NAFLD by reducing oxidative stress and triggering the downregulation of CYP2E1 and c-JNK cell death pathway. Therefore, G. thunbergia extract and rutin could be potential therapeutic options for treating NAFLD. However, further clinical studies are required to assess potentially toxic and effective doses.