Tentatively Identified (UPLC/T-TOF–MS/MS) Compounds in the Extract of Saussurea costus Roots Exhibit In Vivo Hepatoprotection via Modulation of HNF-1α, Sirtuin-1, C/ebpα, miRNA-34a and miRNA-223

Saussurea costus is a plant traditionally used for the treatment of several ailments. Our study accomplished the UPLC/T-TOF–MS/MS analysis of a methanol extract of Saussurea costus roots (MESC), in addition to lipoidal matter determination and assessment of its in vivo hepatoprotective activity. In this study, we were able to identify the major metabolites in MESC rather than the previously known isolated compounds, improving our knowledge of its chemical constituents. The flavones apigenin, acacetin, baicalein, luteolin, and diosmetin, and the flavonol aglycones quercetin, kaempferol, isorhamnetin, gossypetin, and myricetin and/or their glycosides and glucuronic derivatives were the major identified compounds. The hepatoprotective activity of MESC was evaluated by measuring catalase activity using UV spectrophotometry, inflammatory cytokines and apoptotic markers using ELISA techniques, and genetic markers using PCR. Paracetamol toxicity caused a significant increase in plasma caspase 2, cytokeratin 18 (CK18), liver tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), miRNA-34a, and miRNA-223, as well as a significant decrease in liver catalase (CAT) activity and in the levels of liver nuclear factor 1α (HNF-1α), sirtuin-1, and C/ebpα. Oral pretreatment with MESC (200 mg/kg) showed a significant decrease in caspase 2, CK18, TNF-α, IL-6 and a significant increase in liver CAT activity. MESC decreased the levels of liver miRNA-34a and miRNA-223 and induced HNF-1α, sirtuin-1, and C/ebpα gene expression. The histological examination showed a significant normalization in rats pretreated with MESC. Our findings showed that Saussurea costus may exert a potent hepatoprotective activity through the modulation of the expression of cellular cytokines, miRNA-34a, and miRNA-223.


Introduction
The use of medicinal plants for treating various ailments has been continuously explored and developed as an adjuvant to synthetic medicine; these plants also offer a broad range of bioactive secondary metabolites. Saussurea costus (syn. Saussurea lappa, Asteraceae) is considered one of the most important traditional Chinese medicinal plants. It is a rich source of various bioactive phytoconstituents, and its genus comprises about 300 species [1]. S. costus is generally known as costus root and Kust or Qist Hindi (Arabia). S. costus is traditionally used for treating asthma, gastric ulcer, inflammation, and liver diseases [2]. Many authors reported that its bitter roots have anxiolytic [3], anti-inflammatory, and antirheumatic activities [4]. S. costus was proved to be a potent inducer of apoptosis because of the presence of costunolide [5]. An aqueous extract of S. costus was found to

UPLC/T-TOF-MS/MS
To obtain full coverage of the metabolomics of MESC, ESI-MS/MS was performed in negative and positive modes. The total ion chromatograms (TIC) of MESC are presented in Figure 1. The flavonoid aglycones Oand C-glycosides were identified; flavones and flavonol and their glycosides or glucuronic derivatives were the major components, reflecting the plant diverse biological activities [20]. Flavanones, isoflavones, and anthocyanidin glycosides or their derivatives, in addition to organic and phenolic acids and coumarins derivatives, were detected ( Table 2, Figures 2 and 3).

Amino, Organic, and Hydroxybenzoic Acid Derivatives
The common neutral loss of 44 Daltons was observed in MS2 fragmentations of phenolic and organic acids due to the loss of CO 2 as in succinic and malic acids (at m/z 73.0277 and 89.0259, respectively) [30]. The identified amino acids were oxoproline, dimethylglycine, and 2-aminoadipic acid.

In Vivo Hepatoprotective Activity
In the present study, MESC was evaluated for its hepatoprotective activity in paracetamolinduced liver toxicity. Plasma caspase 2 and cytokeratin 18 (CK18) were elevated in the group Gp II in comparison with the control group. Hofer et al. reported that caspase-cleaved cytokeratin 18 (CK18-Asp396) values were increased significantly in Gp II [36]. Caspase-2 is an activator caspase involved in a number of apoptotic pathways, remarkably, in response to intracellular stress factors (e.g., DNA damage, ER stress). Caspase-2 is found in injured hepatocytes, and its activity has indeed been found to be strongly raised in non-alcoholic steatohepatitis patients and mouse models [37]. CK18 is the most widely available intermediate filament protein in the liver and helps caspase substrates during hepatic cells apoptosis. High CK18 levels have been found in hepatocellular carcinoma, viral liver infections, alcoholic liver disease, NAFLD, and cholestatic liver disease [38].
Toxicity by paracetamol (1 g/kg) led to a significant elevation (p < 0.01) in plasma caspase 2 and CK18 levels, corresponding to 346.39% and 139.31% increases, respectively, as compared to the levels in Gp I, considering the values in the control group as 100% (Table 3). Pretreatment with silymarin significantly decreased plasma caspase 2 and CK18 levels by 42.36% and 29.38%, respectively compared to the levels in Gp II. Pretreatment with MESC significantly decreased the level of plasma caspase 2 and CK18 by 54.93% and 23.17%, respectively compared to those in Gp II. MESC and silymarin pretreatment reduced the levels of caspase 2 and CK18 compared with those in Gp II. The depletion of caspase 2 and CK18 was demonstrated to enhance apoptosis and decrease cell viability by affecting caspase-2 activity in hepatocellular carcinoma cells [39]. Table 3. Effect of MESC and silymarin on plasma caspase 2, cytokeratin 18 (CK18), and liver TNF-α, interleukin 6 (IL-6), and catalase (CAT) in experimental rats. Toxicity by paracetamol led to a significant increase in liver TNF-α and IL-6 levels by 294.70% and 217.35%, respectively, as well as a significant decrease (p < 0.01) in liver CAT level by 50.37%, compared to the levels in Gp I (Table 3). Pretreatment with silymarin significantly decreased liver TNF-α and IL-6 levels by 51.69% and 54.12%, respectively, as well as induced a significant elevation in liver CAT level by 151.59%, compared to the levels in Gp II. Pretreatment with MESC significantly decreased the levels of liver TNF-α and IL-6 by 65.22% and 52.06%, respectively, as well as significantly increased liver CAT level by 164.73% compared to the levels in Gp II.

Gps
Paracetamol-induced liver toxicity and cell inflammation led to infiltration of inflammatory cells and to inflammatory cytokine upregulation (TNF-, IL-1, and IL-6), resulting in inflammation [40,41]. MESC and silymarin pretreatment suppressed the elevation of serum TNF-α and IL-6, reducing their inflammatory effect and inducing CAT activity in the liver of Gp II rats. Therefore, the hepatoprotective effect of MESC was related to its anti-inflammatory activity.
A significant decrease of the expression of HNF-1α, sirtuin-1, and C/ebpα genes by 55.10%, 61.22%, and 77%, respectively, as well as a significant increase of the expression of liver miRNA-34a and miRNA-223 genes by 447.27% and 344.79%, respectively, in Gp II rats as compared with Gp I rats (p < 0.05) were observed (Figures 4 and 5). Silymarin administration showed a significant increase (p < 0.05) in C/ebpα, HNF-1α, and sirtuin-1 gene expression and a significant decrease (p < 0.05) of liver miRNA-34a and miRNA-223 genes expression compared to the levels in Gp II. MESC administration showed a significant increase (p < 0.05) in the expression of liver C/ebpα, HNF-1α, and sirtuin-1 genes by 404.35%, 179.55%, and 223.64%, respectively, as well as a significant decrease (p < 0.05) of liver miRNA-34a and miRNA-223 genes expression by 46.27% and 40.49%, respectively, compared with the levels in Gp II. Paracetamol overdose could suppress C/ebpα gene expression in hepatocytes in vivo, which was accompanied by a significant accumulation of cytokines. MESC and silymarin protected rats against paracetamol-induced hepatotoxicity. Oxidative stress due to C/ebpα gene expression depletion and uncontrolled ROS is known to be the primary pathogenic mechanism of paracetamol-induced liver toxicity, as well as the main inhibitor of hepatocytes protective factors. Our metabolomics results revealed that MESC is rich in flavonoid compounds that exerted pronounced antioxidative effects against paracetamol-induced depletion of C/ebpα gene expression in the hepatocytes of Gp II rats [42].  HNFs were discovered to be liver-enriched transcription factors with various functions in the transcription of liver-specific genes [43]. Although HNF-1 affects many organs, hepatic responses are the most noticeable due to its high liver concentration and local production. [44]. Inactivation of HNF-1 has been linked to a variety of tumor-promoting pathways [44]. In our study, HNF-1α gene expression was significantly decreased compared to the control levels after paracetamol administration. No data are available about the direct effect of paracetamol administration on the fold changes in HNF-1α gene expression. Our study suggests that the inactivation of HNF-1α gene expression may be due to the elevation of TNF-α in paracetamol-treated rats. A study by Bao et al. implicated TNF-α-induced inhibition of HNF-1α in the promotion of HCC disease progress [45]. MESC and silymarin pretreatment suppressed TNF-α production, which might lead to the induction of HNF-1α expression. Flavonoids have the potential of exerting positive health benefits and increase the expression of HNF-1α [46,47].
Sirtuin-1 has a role in many biological processes such as energy metabolism, apoptosis, and inflammation. Our results showed a significant decrease in liver sirtuin-1 gene expression in paracetamol-treated rats, in agreement with Wojnarová et al. [48]. MESC and silymarin pretreatment activated the expression of liver sirtuin-1 in Gp II rats. Many studies reported the activation of liver sirtuin-1 gene expression after treatment [48,49].
Our data showed an increase in hepatic miRNA-34a and miRNA-223 gene expression in Gp II rats. miRNA-34a was shown to be increased in hepatic fibrosis, HCV infection, alcoholic liver disease, and in the presence of cardiotoxicity [50,51]. Inhibiting miRNA-34a suppressed lipid accumulation and improved the degree of steatosis. Additionally, silencing miRNA-34a led to an increase in the expression of sirtuin-1, regulating the activity of AMP kinase [52]. miRNA-223 is elevated in the serum of patients with hepatitis B and HCC, implying that miRNA-223 could be used as a new biomarker for liver injury. [53]. MESC pretreatment induced the downregulation of hepatic miRNA-34a and miRNA-223 gene expression in Gp II rats. The above results indicated that miRNA-34a and miRNA-223 are components of very important mechanism underlying the treatment of liver toxicity by MESC.
Liver sections of the control group (Gp I) demonstrated normal hepatocytes with no fibrosis or inflammation (Figure 6a), while liver tissue of Gp II showed degenerated hepatocytes (hydropic degeneration, yellow arrows in Figure 6b) with fibrosis ( Figure 6b). The hepatocytes of Gp III rats were normal, without fibrosis or inflammation after silymarin pretreatment (Figure 6c). Additionally, the hepatocytes showed recovery without hydropic degeneration in MESC-pretreated rats (Gp IV) as compared with paracetamol-treated rats (Figure 6d). According to these histological studies, MESC and silymarin have a hepatoprotective effect against paracetamol-induced liver toxicity.
Our LC-MS/MS results showed that flavonoids were the major components of S. costus; which supports the hepatoprotective activity of this plant [54]. Coumarins as well possess potent antioxidant activity and are therefore regarded as potent hepatoprotective compounds [55]. Tejaswi et al. proved the anti-inflammatory activity of S. lappa root extract in rats [54]; moreover, ALT, AST, total protein and albumin levels showed an improvement after the extract administration in rabbits [56,57]. This study showed for the first time an improvement in all measured genetic, apoptotic markers and inflammatory cytokines after S. costus root extract pretreatment.

Plant Materials and Extraction Process
Saussurea costus (Falc.) Lipsch. roots were purchased from a local herbalist (Giza, Egypt, 2020). Identification of the plant was performed by the Agriculture Research Center, Cairo, Egypt. In Soxhlet, powdered roots (1 kg) were extracted with methanol (4 × 500 mL, 1 h). The extract was left to cool, filtered, and then evaporated (Rotavapor ® , BÜCHI, Flawil, Switzerland) [58]. The obtained dried extract was used for biological and chemical investigations.

Gas Chromatography-Mass Spectrometry Analysis (GC-MS) of the Lipoidal Matter
Powdered roots (100 g) were macerated in n-hexane (2 × 100 mL), filtered, and evaporated. The crude oil was investigated for its composition in a GC-MS system (Agilent Technologies, Santa Clara, CA, USA) equipped with a mass spectrometer detector (5977A) at the National Research Centre, Cairo, Egypt. The GC was equipped with an HP-5MS column.
3.5. In Vivo Hepatoprotective Activity 3.5.1. Experimental Design Adult male albino rats (150 ± 10 g, 40) were purchased from the National Cancer Institute, Cairo University, Giza, Egypt. Rats were provided water and standard diet ad libitum, observed daily, and kept in polypropylene cages under normal environmental conditions (22 ± 2 • C). The prophylactic potential of MESC against paracetamol hepatotoxicity was evaluated. Ethical approval was obtained by the ethic committee of the faculty of applied medical science (20210802, 2 August 2021). Animals and the treatment schedule (4 weeks) were as follows: Gp I (normal control) and Gp II (positive control); rats were orally given Tween 80 (3 mL, 1%) in saline daily; Gp III rats were orally treated with silymarin (50 mg/kg, in 1% tween 80), daily [62]. Gp IV rats were orally treated with MESC (200 mg/kg, in 1% tween 80) daily [63]. On day 27, the animals of all groups fasted for 18 h. On day 28, all animals in Gp II, III, and IV received paracetamol (1 g/kg) [64]. On day 29, blood samples from a retro-orbital vein were collected in heparin-containing tubes and centrifuged (1000 rpm, 20 min). Plasma was collected, and caspase 2 and CK18 were evaluated using the ELISA technique (BMG Labtech Spectrostar Nano, Ortenberg, Germany). The Caspase-2 ELISA kit is based on the competitive enzyme immunoassay technique, which employs a monoclonal anti-Caspase-2 antibody and a Caspase-2-HRP conjugate. In a microplate reader, the intensity of color is spectrophotometrically measured at 450 nm. The CK18 kit employs the Double Antibody Sandwich Technique, which relies on the antigen characteristics with more than two valances and can recognize the coated antibody and the detection antibody at the same time. The liver tissue was homogenized and centrifuged in phosphate buffer (3 mL, pH 7.2; 3000× g, 10 min). The clear supernatant was tested for CAT using a Cayman Chemical Company kit (An Arbor, MI, USA). The UV spectrophotometric method for measuring catalase activity is centered on monitoring the change in 240 nm absorbance at high concentrations of hydrogen peroxide (30 mM). TNF and IL-6 levels in the liver were measured using a microplate reader at 450 nm (Thermo Electric Corp., Shanghai, China). An anti-IL-6 or an anti-TNF-α monoclonal antibody and a biotin-conjugated monoclonal anti-IL-6 or anti-TNF-α antibody that binds to IL-6 or TNF-α captured by the first antibody were used in an indirect sandwich enzyme-linked immunosorbent assay to evaluate IL-6 and TNF-α. Streptavidin-HRP was added to the wells, followed by a substrate solution reacting with HRP. After stopping the reaction with acid, the absorbance was measured.

Real-Time PCR
The TRIzol method was used to isolate total RNA from the rats' liver (Life Technologies Corp., Grand Island, NY, USA). RNA (1 µg) in reaction buffer was mixed with dithiothreitol (10 nmol/L), oligo (dT) primer (25 pg), 0.5 mmol/L of each deoxyribonucleoside triphosphate (dNTP), and 200 units superscript II Rnase H-Reverse Transcriptase. The reactions were kept at 42 • C for 2 min, 42 • C for 50 min, 70 • C for 15 min, and then chilled to 4 • C. The PCR reaction mixture consisted of PCR buffer, MgCl 2 , (1.5 mM), 0.2 mM of each dNTP, 0.4 µM of specific primers (Table 4) for hepatocyte nuclear factor 1α (HNF-1α), sirtuin-1, CCAAT-enhancer-binding proteins (C/ebpα), miRNA-34a, and miRNA-223. The PCR reaction mixtures were incubated at 94 • C for 3 min, then continued for each molecule to be analyzed with the respective number of cycles at 94 • C for 45 s and at their corresponding annealing temperatures for 30 s, followed by 1 min 30 s at 72 • C. Following that, a 10 min extension step at 72 • C was performed. β-Actin mRNA was used as a housekeeping gene to normalize the CYP mRNA content [65]. Table 4. Primers used in real-time PCR.

Histological Assessment
The liver pieces were fixed in formaldehyde solution (10%) and then were examined for histopathological changes.

Statistical Analysis
Statistical analyses were carried out using GraphPad Prism (version 5.01, San Diego, CA, USA). ANOVA with posttest Tukey's multiple comparisons were performed. Values (n = 10) are presented as mean ± standard deviation for ELISA measurements and PCR analyses of gene expression (p < 0.05 was considered statistically significant).

Conclusions
Herein for the first time, we were able to identify the chemical profile of Saussurea costus root through LC-MS analysis. The tentatively identified phytoconstituents (49) were characterized as belonging to different chemical classes, flavonoids being the major constituents besides organic acids, coumarins, and anthocyanidin. S. costus has a potent hepatoprotective activity through the modulation of cellular cytokines release and miRNA-34a and miRNA-223 expression. S. costus reduced the levels of caspase 2 and CK18, TNF-α, and IL-6 and increased catalase activity. Furthermore, it increased HNF-1α, sirtuin-1, and C/ebpα expression and decreased miRNA-34a and miRNA-223 gene expression. S. costus minimized some negative symptoms and pathological changes. We hope that this study will attract attention towards this plant myriad chemical constituents and its great potential in health care.