Phytochemical profiling and anti-fibrotic activities of Plumbago indica L. and Plumbago auriculata Lam. in thioacetamide-induced liver fibrosis in rats

This study aimed at investigating the chemical composition and the hepatoprotective activities of Plumbago indica L. and P. auriculata Lam. LC–MS/MS analyses for the hydroalcoholic extracts of the aerial parts of the two Plumbago species allowed the tentative identification of thirty and twenty-five compounds from P. indica and P. auriculata, respectively. The biochemical and histopathological alterations associated with thioacetamide (TAA)-induced liver fibrosis in rats were evaluated in vivo where rats received the two extracts at three different dose levels (100, 200 and 400 mg/kg p.o, daily) for 15 consecutive days with induction of hepatotoxicity by TAA (200 mg/kg/day, i.p.) at 14th and 15th days. Results of the present study showed a significant restoration in liver function biomarkers viz. alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl transferase and total bilirubin. The liver homogenates exhibited increased levels of antioxidant biomarkers: reduced glutathione (GSH) and catalase (CAT), accompanied with decline in malondialdehyde (MDA). Furthermore, treated groups exhibited a significant suppression in liver inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6), and fibrotic biomarker: alpha smooth muscle relaxant. Histopathological examination of the liver showed normality of hepatocytes. Noteworthy, P. indica extract showed better hepatoprotective activity than P. auriculata, particularly at 200 mg/kg. To sum up, all these results indicated the hepatoprotective properties of both extracts, as well as their antifibrotic effect was evidenced by reduction in hepatic collagen deposition. However, additional experiments are required to isolate their individual secondary metabolites, assess the toxicity of the extracts and explore the involved mechanism of action.


In silico investigation.
To get an insight about the mechanism by which the tested extracts ameliorated the deleterious effects of TAA, we docked the most abundant compounds into the active site of TNF-α and IL-6, two molecular targets involved in liver fibrosis. The crystal structures of TNF-α (PDB id: 2AZ5) and IL-6 PDB id: 1ALU) were downloaded from protein data bank (www. pdb. org). Molecular operating environment (MOE), 2013.08; Chemical Computing Group Inc., Montreal, QC, Canada, H3A 2R7, 2016 was utilized to perform the docking analysis according to the previously described method 11 . In vivo investigation. Acute toxicity study. Acute oral toxicity was conducted according to the OECD 423 guidelines 12 . Wistar albino rats (n = 5) were randomly selected and fasted for 4 h with free access to water. Hydroalcoholic extracts of both Plumbago species (suspended in 0.5% NaCMC) were daily administered orally at doses 250, 500 and 1000 mg/kg for each plant extract. The animals were monitored for behavioral alterations, toxic symptoms or mortality. Both plant extracts didn't show any toxic signs, behavior changes or mortality at the tested doses.
Experimental animals and design. Fifty-four adult male albino rats weighing 200-220 g were utilized in this study. They were purchased from the animal house of Faculty of Science, Cairo University. The animals were housed in separated in steel mesh cages and kept under standard conditions (ventilation, temperature (25 ± 2 °C), humidity (60-70%) and light/dark condition (12/12 h)) and fed on a standard rat pellet diet and fresh, clean drinking water. The animals were acclimatized for a period of 15 days prior to the beginning of study. Rats were randomly allocated into nine groups (6 rats each). All groups excluding group I received TAA (El-Gomhouria Company for drug and chemicals, Egypt, 200 mg/kg; i.p.) on the 14th and 15th days of the experiment. Group I; rats served as control negative group received saline on the 14th and 15th days of the experiment and vehicle (0.5% NaCMC, Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) daily throughout the experiment. www.nature.com/scientificreports/ Group II served as control positive received two doses of thioacetamide (TAA; 200 mg/kg; i.p.) at 24 h intervals on the 14th and 15th days and also received vehicle (0.5% NaCMC) daily throughout the experiment 13 14 . These dose levels were also used in order to evaluate the dose response curve for each plant. Drugs and extracts used in this experiment were suspended in 0.5% of NaCMC. Each group except the first and second groups received their corresponding treatments for fifteen days. At the end of experimental period, blood sample were withdrawn, the animals were sacrificed, and liver tissues were isolated.
Blood samples collection. The blood samples were withdrawn from the retro orbital vein of each rat, under mild anesthesia with phenobarbital. Blood samples were left to coagulate and centrifuged at 3000 rpm for 15 min. The sera were used to determine the activities of serum transaminases, alanine transaminase (ALT) and aspartate transaminase (AST) 15 , the level of gamma glutamyltransferace (GGT) 16 and total bilirubin 17 using the Biodiagnostic kits, Egypt.
Liver tissue homogenate. Liver specimens were rinsed with cold PBS in cold pH 7.2 and homogenated by Teflon homogenizer and then centrifuges at 3500 rpm for 15 min at −4 °C. Supernatants were utilized to determine reduced glutathione (GSH) 18 and catalase (CAT) activities 19 and malondialdehyde (MDA) 20 levels as oxidative biomarkers, as well as tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) as inflammatory biomarkers 21 and alpha smooth muscle relaxant (α-SMA) 22 as fibrotic biomarker using appropriate commercial kits.
Histological and histochemical assessment studies. The Livers were dissected immediately after death and the specimens were fixed in neutral-buffered formalin saline (10%, at least 72 h). All the specimens were washed in tap water for 30 min and dehydrated in ascending grades of alcohol, cleared in xylene, and embedded in paraffin. Serial sections of 4-5 µm thick were cut and stained with haematoxylin and eosin for histopathological investigation 23 .
Statistical analysis. The significant differences between the means of the groups (mean ± standard error of mean) were analyzed with one-way analysis of variance (ANOVA) followed by the Tukey's post hoc multiple comparisons test. Results were considered as statistically significant when p < 0.05. The statistical analysis and figures were generated using GraphPad Prism 8.4.3.

Results
Phytochemical analyses. Profiling of the phenolic metabolites in the hydroalcoholic extracts of P. indica and P. auriculata aerial parts were performed by LC-MS/MS. In total, thirty and twenty-five secondary metabolites were detected in P. indica and P. auriculata, respectively. Twenty-one compounds were common in both extracts. The compounds were identified and tentatively annotated based on their retention times, molecular weight, and fragmentation pattern as well as comparison with reported data found in literature and online data base (Mass Bank) ( www.nature.com/scientificreports/  Table 2. Effect of pretreatment with hydroalcoholic extracts of the aerial parts of P. indica or P. auriculata on serum hepatic functions biomarkers in TAA-induced liver toxicity in rats. Results were expressed as Mean ± S.D., n = 5. SLM: Silymarin, * ,@ Significantly different from normal control and TAA groups at p > 0.05, respectively.  (Fig. 1).

Parameters ALT (U/L) AST(U/L) GGT(U/L) Bilirubin (umol/L)
Effects of pretreatment with hydroalcoholic extracts of the aerial parts of P. indica or P. auriculata on the hepatic inflammatory biomarkers in TAA-induced injury in rats. Initially, we virtually explored the interactions of the individual secondary metabolite from both extracts with the active site of both hepatic inflammatory biomark- www.nature.com/scientificreports/ ers (TNF-α and IL-6). The docked compounds furnished considerable binding energies and several hydrogen bonding and hydrophobic and ionic interactions, Table 3 and Fig. 2. These results were further confirmed in vivo where injection of TAA significantly elevated liver TNF-α and IL-6 by 1.33 and 3.01 fold, respectively compared to normal group values. Doses of the hydroalcoholic extract of P. indica aerial parts (100, 200 and 400 mg/kg) significantly decreased the raised TNF-α levels by about 8.43%, 19.04% and 11.93%, respectively, as well as IL-6 levels by about 24.44%, 60.54% and 37.40%, respectively compared to TAA control group. The rats pretreated with the hydroalcoholic extracts of P. auriculata aerial parts at doses of 100, 200 and 400 mg/kg showed remarkable reductions in the elevated TNF-α by about 5.92%, 15.57% and 9.12%, respectively and IL-6 by about 17.21%, 53.25% and 35.01%, respectively, compared to TAA control groups. Both extracts showed an obvious reduction in TNF-α and IL-6 levels. Moreover, pretreatment with silymarin showed a reduction by about 23.78% and 65.55% for TNF-α and IL-6 levels, respectively (Fig. 3).
Effect of pretreatment with hydroalcoholic extracts of the aerial parts of P. indica or P. auriculata on hepatic α-SMA in TAA-induced liver injury in rats. Induction of liver dysfunction in rats using TAA significantly provoked the hepatic α-SMA by 6.04 fold as compared to the normal group (Fig. 4). Both plant extracts revealed a noticeable protection against the elevation of α-SMA by about 33.13%, 69.61% and 51.45% for 100, 200 and 400 mg/ kg of P. indica extract, respectively and 33.79%, 63.44%, and 44.95% for 100, 200 and 400 mg/kg of P. auriculata extract, respectively compared to TAA group. In spite of this observable protection accompanied with both plant extracts against fibrosis which is comparable by the protection showed by silymarin, there is still different in α-SMA values compared with control group indicating an antifibrotic effect.
Histopathological examination of liver tissue. In regards to the histopathological examination, there was no histopathological alteration and the normal histological structure of the central vein and surrounding hepatocytes in the parenchyma were detected in normal control rats (Fig. 5A) while, examination of the livers of TAA rats revealed swelling besides vacuolar degeneration observed in hepatocytes (Fig. 5B) and associated with focal mononuclear leucocytes inflammatory cells aggregation in the hepatic parenchyma (Fig. 5C). The hepatic parenchyma showed also focal necrosis, as well as congestion in the portal and central veins (Fig. 5D-F). Collagen fibers deposition was noticed around central vein and portal tract besides pseudolobulation of hepatocytes with fibroblasts was also observed (Fig. 5G, H). Normal hepatic architecture was observed at silymarin treated group (Fig. 5I). Rats protected by P. indica (100 mg/kg) showed fibrosis in the portal area of liver and few inflammatory cells with many apoptotic cells (Fig. 5J) and parenchymal cells with marked apoptosis was also observed (Fig. 5K). Much better improvement was detected in P. indica group (200 mg/kg), as there was a normal hepatic architecture observed (Fig. 5L) while P. indica (400 mg/kg) treated group showed also normal parenchymatous architecture of the liver but there is a little cellular infiltration, inflammatory cell and vascular congestion were noted (Fig. 5M, N). Some livers of P. auriculata administrated rats at the dose of 100 mg/kg showed fine strands of fibrous tissue extending from portal areas toward the parenchyma (Fig. 5O) and the hepatic parenchymal cells are near to the normal appearance with mild vacuolation (Fig. 5P). Liver sections of rats treated by P. auriculata (200 mg/kg) signifying that the portal area and surrounding hepatocytes in the parenchyma was histological normal (Fig. 5Q), but the central veins and sinusoid were dilated (Fig. 5R) while preserved liver architecture against damage was observed but small foci of inflammatory cells were noted in some fields at the rats protected by P. auriculata (400 mg/kg) (Fig. 5S, T).

Discussion
In the current study, phytochemical analyses by LC-MS/MS were performed in order to investigate the bioactive compounds present in of P. indica and P. auriculata. LC-MS/MS analysis revealed the presence of numerous compounds that was tentatively identified for the first time in both plants such as genistein 8-C-glucoside, 3-hydroxy-3' ,4' ,5'-trimethoxyflavone, 6-ethoxy-3(4'-hydroxyphenyl)-4-methylcoumarin and luteolin. The findings of LC-MS/MS analyses also in parallel with previous reports that revealed the presence of plumbagin in both plants 6 .
The current study also investigated the protective role of P. indica and P. auriculata hydroalcoholic extracts on liver injury induced by TAA-administration in male albino Wistar rats. To the best of our knowledge, this is the first in-vivo assessment for the anti-inflammatory and anti-fibrotic activities of both plants against TAA-induced fibrosis in rats. TAA is a well-established model for induction of hepatic fibrosis by producing free radicals during its metabolism resulting in oxidative stress mediated acute hepatic inflammation and destruction of hepatocytes in the liver [24][25][26] . Thioacetamide metabolites covalently bind to the liver macromolecules causing dramatic elevation of serum levels of ALT, AST, GGT, and bilirubin, a decrease in antioxidant capacity as proved by depletion of GSH and CAT levels and an increase in lipid peroxidation as confirmed by elevation of MDA levels and by increasing ROS production. ROS are shown to enhance pro-inflammatory cytokines production (TNF-α and interleukin-6 (IL-6)). There are also solid links between inflammation, oxidative stress, fibrogenesis and angiogenesis 27 . Liver fibrosis induced by TAA is characterized by several amendments occurred at hepatic ECM 28 .
The outcomes of the present investigation showed that pretreatment of TAA-induced hepatic fibrotic rats with P. indica extract at 200 mg/kg exhibited a pronounced protection on the levels of serum hepatic enzymes, antioxidant, anti-inflammatory and anti-fibrotic marker: α-SMA, against the toxic and fibrotic changes induced by TAA. The current study also revealed that the hydroalcoholic extract of P. indica aerial parts has a potency similar to the root alcoholic extract of the same plant that was assessed previously against the same hepatotoxic agent, TAA 29 . Moreover, administration of both extracts ahead of TAA reduced the elevated enzyme levels in serum plasma resulting from the stabilization of these biomarkers, clearly showing a preventive effect of the plants on TAA intoxication. Reduced levels of GSH have been associated with TAA induced hepatitis and are closely www.nature.com/scientificreports/ www.nature.com/scientificreports/ correlated to the lipid peroxidation and disturbance of calcium ions induced by toxic agents 30,31 . In extractstreated groups, there was an observed boost in tissue GSH content showing that both extracts tend to reverse the tissue depletion of GSH in hepatic tissues. The hydroalcoholic extracts of both plants cause a significant increase in hepatic CAT activities and, thus, diminishes the oxidative injury in the liver due to the free radical establishment by the action of TAA. Lipid peroxidation is considered as one of the important characteristics of oxidative stress 32 . Rats treated with TAA showed significantly elevated levels of lipid peroxidation, which is characterized by increase in the levels of MDA resulted in the failure of the antioxidant defense mechanism 33 . There was a decrease in the levels of MDA in plants-treated rats previously to their intoxication with TAA, showing that both extracts may exert a preventive action on hepatic tissue. In case of silymarin, this compound also exerted antioxidant activities indicated by prevention of GSH and CAT depletion besides maintain MDA at normal levels. Consistent with these data, Eldhose et al. 29 was previously reported that silymarin have a clear antioxidant activity at 100 mg/kg against TAAinduced liver injury. Furthermore, a chronic subjection of rats to TAA resulted in an increase in hepatic TNFα and IL-6 levels. This is in parallel with previous studies 26,34 . Silymarin also revealed anti-inflammatory potential where it decreased hepatic TNF-α and IL-6 contents which is in line with a previous study 35 .
The anti-inflammatory action of both plants also observed as a reduction in hepatic TNF-α and IL-6 levels compared to TAA group. Here, induction of liver fibrosis via TAA increased liver α-SMA that comes in agreement with several previous studies 36,37 . α-SMA is an actin isoform and a specific marker for smooth muscle cell differentiation 38 . Silymarin, on the other, demonstrated anti-fibrotic activities in rats evidenced by significant www.nature.com/scientificreports/ reduction in hepatic α-SMA. Similar results were reported where daily administration of silymarin resulted in modulation of fibrosis by reducing the hepatic α-SMA contents 39,40 .
Numerous studies have highlighted the hepatoprotective and antioxidants effects of several Plumbago species 41,42 . Rajasekaran and Periasamy 43 studied the protective potential of ethanolic root extract of P. indica on paracetamol-induced liver damage at a dose of 200 and 400 mg/kg of the extract. Eldhose et al. 29 has also described the curative effect of P. indica roots using its methanolic extracts at different doses, 100 and 200 mg/ kg bw, on TAA-induced liver damage in Wistar albino rats. Moreover, P. auriculata was proven to have an antioxidant capacity verified by several assessments such as oxygen radical absorbance capacity (ORAC), the thiobarbituric acid reactive substances (TBARS), ferric reducing antioxidant power (FRAP), and the nitroblue tetrazolium (NBT) 44 . The results of current study suggested that both Plumbago species furnished marked  . Effects of pretreatment with hydroalcoholic extracts of the aerial parts of P. indica or P. auriculata on the hepatic α-SMA in TAA-induced liver injury in rats. Animals were given TAA (200 mg/kg/day; i.p.) on 14th and 15th days. Other regimens were given orally and daily for 15 days. Results were expressed as Mean ± SD, n = 5. SLM: Silymarin, * ,@ Significantly different from normal control and TAA groups at p < 0.05, respectively. www.nature.com/scientificreports/ hepatoprotective properties owing to their antioxidant, antiinflammatory and antifibrotic effects against TAA induced liver toxicity.

Conclusion
The result of the current work highlighted that pretreatment of P. indica or P. auriculata hydroalcoholic extracts have significant hepatoprotective effects on TAA-induced liver oxidative stress and liver fibrosis in rats through their robust antioxidant and anti-inflammatory potentials. Both doses of P. indica and P. auriculata at 200 mg/kg showed promising protection against TAA-induce liver fibrosis with superior hepatoprotective activity accounted for P. indica. The bioactive compounds present at both plants, identified by LC-MS/MS, could be responsible for these activities. However, further clinical studies are needed in order to validate the usage of these extracts as hepatoprotective supplements, to ameliorated fibrosis and thus could be used as antifibrotic agents.
Institutional review board statement. The design of the experiments and the protocol conforms the guidelines of the National Institute of Health (NIH) and National Research Center-Medical Research Ethics Committee for the use of animal subjects and approved by ethical research committee of Faculty of Pharmacy, Cairo University (Approval Numbered N-14052020). The study was carried out in compliance with ARRIVE guidelines. www.nature.com/scientificreports/

Data availability
All data generated and analyzed during this study are included in this published article.