Ameliorative Effects of Thunbergia erecta L. Leaves Against the Initiation of Hepatocarcinogenesis Induced by Diethylnitrosamine in the Rat Model

Thunbergia erecta L. contains cytotoxic and liver-protective compounds. Thunbergia erecta L. leaves were macerated in 70% aqueous ethanol, then fractionated with ethyl acetate (9.3 g) and butanol (12.7 g), and attenuated Den-induced liver cancer in a Wistar rat experimental model. Ethyl acetate and butanol fractions were chromatographed using column chromatography and solid-phase extraction (SPE); Vicenin-II (1), kaempferol (2), biochanin A, sissotrin 7-O-β-glucopyranoside (3), gentianose (4), acacetin 7-O-β-glucopyranoside (5), apigenin 7-O-β-glucopyranoside (6), and rosmarinic acid (7) were extracted, and their structures were determined using NMR spectroscopy and ESI-mass spectrometry. Sixty rats were divided into six groups (ten each): control group, Den group, doxorubicin/Den-treated group, butanol fraction/Den-treated group, and isolated acacetin 7-O-β-glucopyranoside/Den-treated group. The liver enzymes and proinflammatory biomarkers were used to estimate the liver function. In addition, liver tissues were collected for analysis of oxidative stress markers, gene expression, and histopathology. There is a significant increase in the levels of liver enzymes, AFP, and TNF-ἁ. This was conveyed by a significant increase of IL-1 and caspase-3, elevation of MDA and reduction of GSH, and suppression of Bcl2 and elevation of Bax expression. All parameters in butanol, ethyl acetate fractions, and isolated acacetin 7-O-β-glucopyranoside (major constituents) of T. erecta L. were significantly improved to values close to those of the control group. Supplementary Information The online version contains supplementary material available at 10.1007/s12010-022-04292-x.


Introduction
Plant-derived natural compounds have played a vital role in human life.Many scientific types of research have revealed that medicinal plants may be used as natural remedies and are now being used as an alternative for medications [1.].Thunbergia erecta L. is vigorous, a woody shrub which belongs to Acanthaceae family.This shrub has small, ovate leaves with entire margins borne opposite on thin, brown stems [2.].The purple flowers have a yellow throat and may appear singly or in small clusters.This plant produces rounded seed capsules that end in a beak.Thunbergia genus has ornamental value and is native to tropical regions of Africa and South Asia [3.].Thunbergia species have a number of bioactive chemicals with established pharmacological effects include alkaloids, naphthalene, coumaroyl malic acid, benzyl β-glucopyranoside, delphinidin, apigenin, and phenolic compounds such as tannin, feruloyl malic acid, and rosmarinic acid [4. , 5. ].Traditionally, Thunbergia species leaves, stems, and roots was used as anti-inflammatory and antipyretics agents [6.].It has been also reported to possess antibacterial activities against gram positive as well as gram-negative bacteria such as Escherichia coli, Klebsiella pneumonia, and Streptococcus pyogenes [7.].Thunbergia species also exhibit antinociceptive and antitumor [8.], cytotoxic, and antioxidant activities [9. , 10. ].Regarding the previous reports and our phytochemical analysis, it has been revealed that T. erecta L. contains several bioactive compounds which have ameliorative effects against hepatocellular carcinoma.Liver cancer is one of the world's most prevalent diseases, especially in Asia and Africa [11.].Hepatocellular carcinoma (HCC), the fourth most common cause of cancer mortality, accounts for 90% of all liver cancer [12. , 13. ].Diethylnitrosamine (Den) is a well-known hepatocarcinogenic agent found in cigarette smoke, water, cured and fried foods, cheese from cheddars, chemicals from the farming industry, cosmetics, and pharmaceutical products [14. , 15. ].Despite continuous advances in managing chronic liver diseases, tumor detection, and treatment, the prognosis of HCC remains lower in comparison to other tumors [16.].The incidence of high mortality and associated side effects following chemotherapy and/or radiotherapy increase the demand for alternative medicines for cancer treatment.Not surprisingly, many potent anticancer compounds have been isolated from plants, e.g., doxorubicin, taxol, etoposide, cisplatin, vinblastine, vincristine, and topotecan [17.].This has led to the current interest in alternative medicine that will aid in therapy and help to avoid its unjustifiable use.Liver enzymes AST (aspartate transaminase), ALT (alanine transaminase), and ALP (alkaline phosphatase protein) are indicative of diagnosis and severity of the liver injury [18.] and when hepatocytes were injured or destructed the pro-inflammatory factors like TNF-α, IL-1β, IL-6, and ROS (reactive oxygen species) are released [19.], MDA (malonaldehyde) is an indicator of lipid peroxidation [20.].Caspases are a family of protease enzymes playing essential roles in programmed cell death [21.]; α-fetoprotein (AFP) is an important tumor marker with the best sensitivity and specificity for hepatocellular carcinoma (HCC) diagnosis and monitoring of its therapy [22.].The members of the Bcl-2 family involving of both proapoptotic and antiapoptotic proteins contribute to the regulation of programmed cell death in hepatocytes [23. , 24. ].Numerous of the genetic amendments of fibrosis initiate an imbalance in the proapoptotic and anti-apoptotic proteins of the Bcl-2 family [25.].The anti-apoptotic proteins, including Bcl-2, is known to hinder mitochondrial apoptotic pathway by delaying the release and oligomerization of proapoptotic proteins, and is downregulated in fibrotic cell [26.].On the other hand, proapoptotic members of the Bcl-2 family, such as Bax, activate mitochondrial apoptosis by enabling pore development and cytochrome c release from the inner mitochondrial membrane with consequent initiation of caspases resulting in cell death.These proapoptotic members are over expressed in fibrosis [27.].An amendment in Bax/Bcl-2 ratio plays a critical role in determining whether a cell should switch towards proliferation or apoptosis [28.].Den was therefore used in this study to construct a liver cancer model in Wistar rats.The potential bioactive chemical components of T. erecta L. leaf extract and their anti-cancer effects were then studied.

Plant Material
T. erecta L. fresh leaves were harvested in May 2018 from the Attia Shoala flower plantation in Kaliobeya, Egypt.Mrs. Therese Labib, botanical specialist, Department of Flora and Taxonomy, El-Orman Garden, Giza, Egypt, generously identified the plant.A voucher specimen of the plant (Reg.No. T7) was deposited at the Herbarium of the Pharmacognosy and Medicinal Plants Department at Al-Azhar University's Faculty of Pharmacy in Cairo, Egypt.

Extraction, Fractionation, and Purification
Air-dried leaves powder (1.4 kg) of T. erecta L. macerated using 70% aqueous ethanol (5 × 3 L) at 25 ± 2 °C.The extract was filtered, and the filtrate was evaporated using a rotary evaporator (Buchi Co., Switzerland) at 70 °C.The total leaf extract (155 g), 0.1 g of leaf extract used for quantitative estimation of total flavonoid and phenolic compounds, and the remaining extract fractionated at 25 ± 2 °C ethyl acetate (3 × 1 L) and butanol (5 × 1 L), yielding 9.3 g, and 12.7 g respectively.The ethyl acetate fraction was applied on a successive silica gel column (100 g, 70 × 4 cm) using CH 2 Cl 2 :CH3OH to obtain compounds 1, 2, and 3 while the butanol fraction was subjected to polyamide S6 CC (100 g, 80 × 5 cm) eluted with H2O/EtOH mixtures up to EtOH.By using PC, UV light, and spray reagents, similar fractions were collected to obtain nine collective fractions (I-IX), which were purified by applying on successive silica gel columns using EtOH and CH 2 CL 2 .Then, the subfractions were purified using sephadex LH-20/ethanol, which obtained compounds 4, 5, 6, and 7.

Total Phenolic and Flavonoid Assessment
Total phenolic content was evaluated using the Folin-Ciocalteau assay technique [23.], and total flavonoids were quantified using [29.].

Animals
Adult male Wister albino rats weighing between 100 and 120 g were obtained from the National Organization for Drug Control and Research (NODCAR), Cairo, Egypt, were used.Rats were housed in standard polypropylene cages in the institute's animal house facility at 25 °C with 50-60% relative humidity, 12-h light-dark cycles, and free access to food and water.Care and use of the animals were conducted under the supervision of the Animal Ethics Committee of the National Organization for Drug Control and Research (NODCAR) with an approved ethical number (NODCAR/II/13/2020).The study was also conducted according to the National Regulations on Animal Welfare and the Institutional Animal Ethical Committee (IAEC).

Experimental Protocol
Fifty rats after 2 weeks of acclimatization were randomly divided into five groups of ten each, as follows: control group: ten rats received saline intraperitoneally.Forty animals received diethylnitrosamine (Den) at a dose of 55 mg/kg body weight twice a week [30.] then classified into 4 groups as follows: Den-treated group, doxorubicin/Den-treated group: Dox was administered intraperitoneally for 24 h at a dose of 10 mg/kg body weight, twice a week; butanol fraction/Dentreated group: at a dose of 50 mg/kg body weight by oral gavage of butanol fraction intragastrical every day for 4 weeks; ethyl acetate fraction/Den-treated group: at a dose of 50 mg/kg ethyl acetate body weight by oral gavage intragastrical every day for 4 weeks; acacetin 7-O-glucoside/ Den-treated group: at a dose of 0.44 mg/kg, by oral gavage [31.].The total treatment period during the whole experimental study was 6 weeks.All treatments were administered orally.
At the end of the experiment, the rats were euthenized by decaption.Blood and liver samples were collected.The Sera were separated using centrifugation and stored at − 80 °C until analysis.The livers were washed in ice-cold isotonic saline and blotted dry between two filter papers.The portions of liver tissue samples were homogenized in ice-cold water.1.15% KCl was added to make a (10% w/v) homogenate with a Glass-Col motor-driven homogenizer (USA), and the homogenate was used for the determination of malondialdehyde (MDA).The other portion of the liver tissue was homogenized in ice-cold water.Five percent sulfosalicylic acid to make a (10% w/v) homogenate for the estimation of reduced glutathione (GSH).The sections of the liver were kept in 10% neutral buffer formaldehyde for histological examination.

Determination of Liver Toxicity Indices
The serum levels of ALT and AST were determined by a colorimetric method using a Biodiagnostics kit (AL 1031, AS 1061), while the activity of ALP was performed according to the method of Belfield and Goldberg using the Reactive Gpl kit ref. EZ002LQ-SP.

Determination of Oxidative Stress Markers in Liver Tissue
The supernatant of homogenized liver samples was used for the determination of GSH and MDA levels using bio-diagnostics kits (CAT NO.GS 2511 and MD 2529, respectively).

Gene Expression Determination of Bax&Bcl2 in Liver Tissue by RT-PCR
Gene expression RT-PCR approximately 30 mg of the liver tissue was stored in an RNA lysis solution at 80 °C until genetic processing.Assessment of Bax and Bcl2 gene expression was done by real-time quantitative reverse transcription PCR (RT-PCR): total RNA was extracted from frozen samples using TRIzol® reagent (Invitrogen, Sigma-Aldrich, St. Louis, MO, USA) according to a standard protocol.The isolated total RNA was converted into complementary DNA (cDNA) using SMARTScribeTM Reverse Transcriptase (Clontech Laboratories, Inc., a Takara Bio Company).RT-PCR was performed using a Real-Time PCR v 7.9 System (DTlite, DNA Technology, LLC, 125Zh Varshavskoe highway, bld.6, Moscow, Russia, 117587) and SYBR® Green PCR Master Mix (QIAGEN) in a final volume of 25 L with the following thermal cycling conditions: 95 °C for 15 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s.The sequences of PCR primer pairs used for each gene are shown in Table 1.The ABI Prism sequence detection system software was used to analyze the data, and the PE Biosystems (Foster City, CA) v17 Sequence Detection Software was used to quantify it.The relative expression of the studied genes was calculated using the comparative threshold cycle method.All values were normalized to the GAPDH gene as an invariant endogeneous control (reference gene).

Histological Examination
Liver tissue embedded in paraffin blocks was sectioned at 5-μm thickness, de-paraffinized, rehydrated, and stained with H&E stain to assess histopathological changes and Picro-Sirius red stain for detection of collagen fibers, and blindly examined for the extent of liver damage under an Axio research microscope (Zeiss) with an attached digital camera (MRC5, Zeiss).Measurement of tissue fibrosis by morphometric analysis using the Image-J software program on Windows 10.

Statistical Analysis
Statistical analysis was achieved using Graph Pad Prism.Software Inc., Program, version 5.0.The data were presented as mean ± SE, and the level of significance was set at p ˂ 0.05.Multiple comparisons were done using one-way ANOVA followed by the Dunnett test as a multiple comparison test.

Quantitative Estimation of Total Phenolic and Flavonoid Content
The phenolic and flavonoid content of T. erecta L. leaf extract were 78.6 ± 5.15 (gallic acid equivalent) μg/g and 79.0 ± 6.30 (rutin equivalent) μg/g, respectively, as shown in Table 2.

Isolated Compounds Identification
Compound 1 Yellowish white amorphous powder (

Liver Function
Significant increase in liver enzymes (ALT, AST, and ALP) (p ≤ 0.05) was observed in the Den group when compared to control group.In contrast, Dox / Den-treated animal caused decrease in their levels when compared to Den-treated rat.Both fractions of T. erecta L. supplementation resulted in a moderate reduction in the activity of ALT, AST, and ALP, while ethyl acetate fraction showed a significant reduction in ALT as compared to the Den-treated group.Acacetin glycoside was able to restore liver enzyme activity as shown in Table 3. Amendment in the level of pro-inflammatory cytokines and inflammatory mediators, specifically, caspase-3, TNF-α, and IL1β, is enlightened in Table 4. Den intoxication markedly enhanced the pro-inflammatory cytokine and inflammatory mediators when compared to normal control.The current study revealed that both fractions of T. erecta L. attenuated the poisonous effect of Den by reducing the activity of enhanced inflammatory mediators.Acacetin glycoside has the most amelioration effect.

Oxidative Stress Markers (MDA, Reduced Glutathione GSH)
Our data shows that DEN administration was found to diminish the hepatic antioxidants GSH but markedly increase in the MDA level when compared to the control group.However, both fractions of T. erecta L. caused a significant amelioration in the GSH content and MDA level.
Acacetin glycoside supplementation has the same effect of ethyl acetate fraction (Table 5).

Alpha-Fetoprotein (AFP)
Activity of the serum alpha-fetoprotein (AFP) level was significantly increased in Den-treated group when compared to the normal group (Table 6).Significant decline in the serum AFP level was observed in the treatment groups when compared with the DEN group.

On Bax and Bcl2 RT-PCR Gene Expression
The diethylnitrosamine-treated group revealed significant increase in expression of Bax (apoptotic gene) but significant decrease in Bcl2 (anti-apoptotic gene) gene when compared to the control group, while there were significant differences between all treated groups, but the treatment with T. erecta L. Ethyl acetate fraction was more effective in decreasing the expression of Bax gene than other treatments.And, the most effective fraction was acacetin 7-O-β-glucopyranoside in increasing the expression of Bcl2 gene (Table 7).

Histopathological Examination
Histopathological examination of liver sections from different groups showed the highest score of liver fibrosis in the control-positive group with portal tract deposition of collagen fibers (group 2) [Table 8 and Fig. 2 (2-6)], with high significant difference in comparison with the negative control group (group 1) [Fig. 2 (2)].All treated groups (3-6) [Fig. 2 (4-6)] showed significantly higher fibrosis score in the form of portal tract deposition of collagen, compared to the negative control group (1) and significantly lower fibrosis scores compared to the positive control group 2. Group 6 showed the least mean percentage of fibrotic area.
Compound 5 from its chromatographic properties may be flavone in nature.

Biological Study
The study aims to evaluate the antioxidant, anti-inflammatory, anti-apoptotic, and antifibrogenic effect of T. erecta L. ethyl acetate, butanol fractions, and the major isolated metabolite; acacetin 7-O-glucopyranoside against diethylnitrosamine induced liver toxicity.The polyphenolic compounds and flavonoids are identified in T. erecta L. and the presence of these biomolecules in erecta leaves makes them a potential bioantioxidant source.A chronic hepatocellular injury is the main cause of liver fibrosis, a serious condition with considerable morbidity and mortality.Patient mortality is increasing with liver fibrosis because it manifests a variety of pathological conditions, sometimes leading to liver cancer [42.].Hepatocellular damage is caused by oxidative stress, chronic inflammation, and cellular proliferation caused by nitrosamines, which are associated with liver fibrosis [43.].Furthermore, Den changes the expression of enzyme markers in the serum [44.] and in tissues affected by hepatocyte damage [45.].In this study, the researchers noted that hepatic cellular injuries attributable to a number of pathological conditions such as hepatitis caused the elevation of these enzymes.According to previous studies, Den-induced liver injury is associated with increased activity of these enzymes [46.].Reactive oxygen species (ROS), generated from oxidative stress, are responsible for contributing to fibrosis, a significant factor in chronic disease [42.].Hepatocellular fibrosis induced by Den results in significant increases in inflammatory markers such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) [45.].Accordingly, macrophages and neutrophils constituting the liver fibrosis pathogenic cells are thought to release pro-inflammatory cytokines (IL-1β and TNF-α) upon activation [41.].Caspases 3 are implicated in the mediation of nuclear apoptosis, including chromatin condensation and DNA fragmentation, as well as bleeding in cells [47.].The present study showed that Den has been exhibiting an increase in the expression of the proapoptotic gene Bax, while Bcl-2 expression has declined [27. ]; as a result, the ratio of Bax/Bcl-2 surged dramatically.These results demonstrate that Den induces intrinsic apoptosis during hepatocellular fibrosis as well as facilitates the removal of transformed cells through targeting the Bcl-2 family members [24. , 25. ].In our study, it was demonstrated that T. erecta L. bioactive constituents initiate cell proliferation and survival possible by overexpressing Bcl2this is attributed to phenolic acids and flavonoids, which have anti-apoptotic properties as has been described previously [48.].Due to the presence of hydroxyl groups inside their aromatic rings, phenolic phytoconstituents have strong antioxidant properties [49.].Acacetin 7-O-glucopyranoside is the most active ingredient isolated from this plant and it provides powerful healing properties.Our current study showed that Den treatment resulted in reduced GSH levels and higher MDA levels in liver tissue of rats diethylnitrosamine causes fibrotic lesions to form in the liver during oxidative damage [44.].The antioxidant effect of T. erecta L. arises from its antioxidants content as quercetin-3-O-xyloside [48.], kaempferol [50.], and acacetin7-O-glucoside [51.] which protect the cells from oxidative damage.Many signaling pathways are blocked by flavonoids, resulting in reduced proliferation, angiogenesis, and metastasis [52.].A marked reduction in inflammatory cytokine biomarkers (interleukin 1β & TNF-α) was seen in the treated groups (EE, EB, & acacetin 7-O-glucoside).As a result of this inhibition, hepatic fibrosis and hepatocellular toxicity associated with Den may have been reduced.In conjunction with these findings, recent studies have shown that herbal bioactive molecules can be effective in preventing liver fibrosis [53. , 54. ].Histopathological evaluation of H&E and Picro-Sirius red staining showed that from normal control: polyhedral hepatocytes and nuclei were round, prominent nucleoli, arranged around the central vein in a radial pattern with no vacuolated cytoplasm, dispersed chromatin and separated by normal sinusoids, histopathological examination of H&E for routine morphological evaluation, and Picro-Sirius red staining for fibrosis scoring and estimation showed that normal control: conventional hepatic lobular architecture in the form of polyhedral hepatocytes with nuclei were round, arranged around the central vein in a radial pattern with no vacuolated cytoplasm, separated by normal sinusoids.Den treatment induced histopathological damage and increased collagen deposition.With disordered lobular structure, congestion of the centrilobular vein, appearance of foci of steatosis, proliferation of Kupffer cells, dilatation of sinusoids, and infiltration of inflammatory cells.In addition, fragmented chromatin was prevalent in the hepatocytes of intoxicated group by diethylenediamine.While doxorubicin inhibits liver fibrosis induced by Den, and treatment with EE, EB, and acacetin7-O-glucoside markedly alleviated and achieved marked improvements in reversing this pathological injury the Den induced in the rats' livers ( reduction in fibrosis scoring).Furthermore, chromatin fragmentation was much less or absent in the Den-treated DoX.EE, EB, and acacetin 7-O-glucoside rats.This histopathological analysis is in accordance with the results of the biochemical diagnostic indicators.This could be due to the observed ability of extracts to recover endogenous antioxidant mechanisms, resulting in the free radicals being scavenged to allow hepatocyte regeneration.This antioxidant effect breaks the vicious cycle of continuous inflammation fibrosis resulting from the continuous activation of KCs, causing a release of pro-inflammatory factors and aiding fibrosis.

Figure 1 and
Supplementary materials: Fig 1S-14 S, showed all isolated compounds.

Fig. 2 ( 1 )
Fig. 2 (1) Sections in control no infected mice with normal hepatic lobular architecture ((H&E stain and Picro-Sirius red stains, × 100).(2) Section in control non-treated liver tissue showing normal hepatic architecture with no excess deposition of fibrous tissue (H&E stain and Picro-Sirius red stains, × 100).(3) Section in liver of group (2) showing deposition of fibrous tissue, mostly in the portal tract (H&E stain and Picro-Sirius red stains, × 200).(4) Sections in liver of group(3 and 4)  showing deposition of variable amounts of fibrous tissue in hepatic parenchyma and the portal tract (H&E stain and Picro-Sirius red stains, × 200).(5) Liver section in group(5) showing nearly preserved hepatic lobular architecture, with no evidence of fibrosis or nodular transformation (H&E stain and Picro-Sirius red × 100).(6) Liver section in group 6 showing nearly preserved hepatic lobular architecture, with no evidence of fibrosis or nodular transformation (H&E stain, Picro-Sirius red X100).No other significant histopathological changes were detected within different groups concerning hepatocytic degeneration or lobular inflammation & 4S)[34.].Compound 3 showed in aromatic region an AX spin coupling system of two ortho doublets, 2H/each, characteristic of 1,4 di-substituted B-ring.The relative downfield location of the X-type doublet (H3′/5′) at δ H 7.09 (̴ ∆ + 0.2 ppm) was indicative for 4′-O-methoxy, which was assigned as singlet at 3.85.The isoflavone was also supported by the characteristic location of H-2 resonance at about δ H 8.52 ppm.As well as the downfield shift of H-8 proton (≈ ∆ + 0.25 ppm) together with the presence of a β-anomeric proton signal at 4.91 (H1′′), were two evidence of the attachment of β-glucopyranoside moiety on OH-7 as O-glycoside.Negative ESI/MS showed a molecular ion peak at m/z 445.19 [M-H] − corresponding to mol.weight 446 assigned for molecular formula C 22 H 22 O 10 , together with other fragment ion at 281[M-hexose-2H] − assigned for a glycone[35.] to confirm the structure as sissotrin (Fig.1& 5-6S).

Table 6
Effect of T. erecta leaves fractions (ethyl acetate, butanol) & acacetin 7-O-glucoside on each of the following: Bax and Bcl2 gene expression Data are represented as mean ± SEM (n = 6).*p <0.05 vs. normal control, #p <0.05 vs Den, *#p <0.05 vs Den + Dox.Statistical analysis was done using one-way ANOVA followed by the Dunnett test as multiple comparison test.ALT,

Table 7
Effect of T. erecta leaves fractions (ethyl acetate, butanol) & acacetin 7-O-glucoside on each of the following: tumor necrosis factor AFP IU/I Data are represented as mean ± SEM (n = 6).*p <0.05 vs. normal control, #p <0.05 vs Den, *#p <0.05 vs Den +Dox.Statistical analysis was done using one-way ANOVA followed by the Dunnett test as multiple comparison test.(*significant from -ve control &# significant from +ve control)

Table 8
Effect of T. erecta leaves fractions (ethyl acetate, butanol) & acacetin 7-O-glucoside on each of the following: histopathological examination Histological assessment of percentage of tissue fibrosis in liver sections in the four examined treated groups (Den., diethylnitrosamine; dox,