Ailanthus excelsa leaf extract: Chemical characterization, antischistosomal activity, and in silico study of isolated phenolic compounds as promising thioredoxin glutathione reductase inhibitors

Hepatic schistosomiasis is the most well-known chronic condition, with a variety of clinical symptoms. In the current study, the metabolite profile of the Ailanthus excelsa leaf butanol extract was investigated using chromatographic isolation and Liquid chromatography electrospray ionization /mass spectrometry (LC-ESI-MS/MS) analysis. Also, its schistosomicidal effect was examined in vivo regarding disease progression in a comparative experimental study to praziquantel (PZQ). The parasitological parameters (total worm burden, tissue egg load, and oogram pattern) of the infected and the treated mice were counted. A histopathological examination of the liver granuloma took place as well. The extract (at a dose of 500 mg/kg) caused a significant worm reduction of 41.30% and also a significant reduction in intestinal egg load (6.36 ± 1.12 vs. 17.82 ± 2,024.6). Data also showed a reduction in immature eggs in the extract group (33.00 ± 2.45 vs. 57.4 ± 3.89) when compared to the infected untreated group. Five phenolic compounds were isolated and identified as gallic acid (1), methyl gallate (2), quercitrin (3), isoquercitrin (4), and kaempferin (5). Furthermore, molecular docking was utilized for the first time to evaluate the efficacy of the isolated compounds (1–5) in disrupting the essential worm enzyme thioredoxin glutathione reductase. Compounds 3–5 showed high docking scores ranging from −7.054 to −11.370 kcal/mol that were comparable to that of PZQ (−6.407 kcal/mol). Moreover, LC-ESI-MS/MS profiling led to the identification of 33 secondary metabolites. These compounds were classified as phenolic acids, flavonoids, iridoids, stilbenoids, chalcones, tannins, and coumarins. The findings suggest that the A. excelsa leaf extract could be used as a naturally occurring antischistosomal agent and this emphasizes the significance of phenolic components.


INTRODUCTION
Schistosomiasis is endemic in 75 countries in Africa, Asia, South America, and the Middle East, rendering it one of the most important neglected tropical illnesses (Gray et al., 2011;Hotez and Kamath, 2009). In the control of schistosomiasis, the use of safe and effective drugs will remain the main control tool until a successful vaccine is produced. Praziquantel (PZQ) is one of the most important and widely effective antibilharzial drugs against humans' four main pathogenic schistosomes (Gönnert and Andrews, 1977), and it has proven effective in large-scale therapies. Population-level schistosomiasis control with PZQ has various drawbacks. PZQ susceptibility has been recently implemented in schistosomes by laboratory selection (Fallon and Doenhoff, 1994). Reduced cure rates and failure of treatment after PZQ were reported in Senegalese, Kenyan, and Egyptian patients (Ismail et al., 1994). However, Schistosoma developed resistance to PZQ; in endemic regions, PZQ-resistant parasites had been established. Therefore, it is essential to develop new therapeutics for the treatment of schistosomiasis (Doenhoff et al., 2008). Natural products have risen to power in recent years as potential sources of novel schistosomiasis medications (El-Sayed et al., 2011). This article attempts to update the antischistosomal natural products and/or derived compounds. Ailanthus excelsa (A. excelsa) (Roxb.) belongs to the Simaroubaceae family and is a deciduous tree commonly known as the "tree of heaven." The plant is widely distributed throughout Asia and North Australia, and it is indigenous to China (Adamik and Brauns, 1957;Khushbu et al., 2011). Traditionally, various parts of A. excelsa are used to treat a variety of health problems, such as wounds and skin eruptions, fevers, bronchitis, asthma, diarrhea, and dysentery (Asolkar et al., 1992;British Pharmacopoeia, 1988). Ailanthus excelsa has also been used as a source of medication. For example, in Chinese medicine, the bark is used to treat diarrhea and dysentery, particularly when there is blood in the stool (Dash and Padhy, 2006). In Asian and Australian medicine, the bark has also been used for worms, excess vaginal discharge, malaria, and asthma (Chevellier, 1996;Kirtikar and Basu, 1995). In Africa (Sharma and Guna-Vijnana, 1996), the plant is useful in the treatment of gonorrhea, epilepsy, tapeworm infection, and high blood pressure (Sharma and Guna-Vijnana, 1996). Ailanthus excelsa contains a wide range of phytochemicals, including quassinoids, alkaloids, flavonoids, terpenoids, and proteins (Joshi et al., 2003;Kapoor et al., 1971;Loizzo et al., 2007;Nag and Matai, 1994;Ogura et al., 1977;Said et al., 2010;Sherman et al., 1980). Moreover, β-sitosterol and vitexin were isolated from the leaf part (Kapoor et al., 1971). From a biological activity point of view, the plant possesses a wide spectrum of biological activities, such as analgesic (Khushbu et al., 2011), antimicrobial (Ghumare et al., 2014Manikandan et al., 2015), antidepressant (Chauhan et al., 2011), anticancer (Ogura et al., 1977;Said et al., 2012), antifungal (Joshi et al., 2003;Ratha et al., 2013), antidermatophytic (Pandith, 2012, antiviral (Rashed et al., 2013), antioxidant (Said et al., 2010), antiproliferative (Said et al., 2010, hepatoprotective (Hukkeri et al., 2002), antiasthmatic (Kumar et al., 2010), antidiabetic (Cabrera et al., 2008, and antibacterial (Shrimali et al., 2001). The root bark has also been shown to have significant cytotoxic and anticancer properties (Ogura et al., 1977). For schistosome survival inside the mammalian host, the parasite selenoprotein thioredoxin glutathione reductase (TGR) is required. Several inhibitors of Schistosoma mansoni TGR have been discovered through high-throughput screening programs (Alger and Williams, 2002). A flavoprotein is homodimeric where a glutaredoxin (Grx) domain is linked to a normal thioredoxin reductase (TR) domain in each subunit. In Schistosoma, the schistosomal redox homeostasis is dependent entirely on TGR, which guides nicotinamide adenine dinucleotide phosphate to reduce equivalents to thioredoxin and glutathione (Alger and Williams, 2002). TGR is the only enzyme that can reduce both thioredoxin and glutathione disulfide, implying that the parasite's redox mechanism is heavily reliant on it. Due to the relevance of biological redox systems and the variations in redox metabolism between S. mansoni and its host (Kuntz et al., 2007), TGR could be an essential parasite protein and therapeutic target. The polyphenolic components of the n-butanol extract of A. excelsa leaves were characterized and their antischistosomal activity in vivo was investigated in the current study. Additionally, isolated chemicals were molecularly docked as strong TGR inhibitors.

Plant materials
In May 2019, leaves of A. excelsa (Roxb.) were collected from the Zoo Garden in Giza Governorate, Egypt. Dr. Therese Labib, Consultant at Orman Botanical Garden in Giza, Egypt, and the National Gene Bank, graciously recognized the plant. The Herbarium of the Medicinal Chemistry Department, Theodor Bilharz Research Institute, Giza, Egypt, received a voucher specimen (No. A.e/L/2019).

Extraction, fractionation, and chromatographic isolation
Dry powdered leaves of A. excelsa (2 kg) were extracted with ethanol at room temperature. A rotavapor was used to concentrate the crude extract under reduced pressure to afford 420 g (a yield of 21%). The ethanolic extract was defatted using petroleum ether (60°C-80°C) (400 g). The residue (375 g) was dissolved in distilled water and then consecutively extracted with CH 2 Cl 2 , ethyl acetate, and n-butanol to afford 65, 38, 74, and 105 g, respectively, for CH 2 Cl 2 , ethyl acetate, n-butanol, and aqueous extracts (Mohammed et al., 2019). The n-butanol extract (55 g) was subjected to polyamide column chromatography and eluted with H 2 O/ethanol via a gradient mix elution system with gradually decreasing polarity up to 100% ethanol. One hundred ninety-eight individual fractions were obtained and were combined according to their chemical profiles on paper chromatography (PC) and/ or thin-layer chromatography. The main fractions were further purified by successive Sephadex LH-20 column chromatography using different mobile phases: BIW (4:1:5 v/v/v upper layer), 20% ethanol, and 100% ethanol to afford five pure, isolated compounds.

Animals
Forty laboratory male Swiss albino mice (CD-1) aged 6-7 weeks with a weight range of 18-20 g were obtained from the Schistosome Biological Supply Centre (SBSC), Theodor Bilharz Research Institute, Giza, Egypt. They were then housed in an appropriate environment at 20°C-22°C with a 12 hours light/dark cycle and a humidity range of 50%-60%. During acclimatization and experimental periods, mice were provided with food and water ad libitum.

Infection of animals
The cercarial suspension (0.1 ml) was gently mixed, stained with a picric acid solution, and counted. Infection of mice with S. mansoni cercariae was conducted using the body immersion method and through exposure to 60-10 cercariae/ mouse (Liang et al., 1987).

Compounds assessed in vivo
The A. excelsa leaf extract and PZQ were evaluated for their antischistosomal activity in vivo. PZQ was obtained as tablets (Distocide, Egyptian International Company, EIPICO, Egypt). Both were freshly suspended in 2% Cremophor EL before use.

Experimental design
The A. excelsa leaf extract and PZQ were orally administered for five consecutive days in the seventh week after infection.
Group 1: S. mansoni-infected mice (infected control). Group 2: Infected mice were administered PZQ at a dose of 200 mg/kg. Group 3: Infected mice were administered A. excelsa extract at a dose of 200 mg/kg. Group 4: Infected mice were administered A. excelsa extract at a dose of 500 mg/kg.

Antischistosomal activity
Mice were sacrificed and tamed; after that, the worm burden was estimated and sexed to estimate worm reduction proportion (Duvall and De Witt, 1967). Liver or intestinal tissues were examined to count the number of eggs per gram (Kamel et al., 1977). During various stages, the percentage of egg production was calculated, and then three parts of the intestine were studied to identify and count the eggs in various stages of maturation, accompanied by calculating the average numbers for each stage (Pellegrino et al., 1962).

Histopathological investigations
Histopathological investigations were performed according to the reported procedures with some modifications (Ibrahim et al., 2022).

Molecular docking study
The Molecular Operating Environment (MOE-DOCK/2014.09) was used for molecular modeling. All compounds were sketched using the ChemDraw program and subjected to an energy minimization process to be introduced into MOE, where the conformational search was performed. Their 3D conformers were then docked into the active site of the TGR enzyme [protein data bank (PDB) ID: 2V6O]. During the docking process, London dG was kept for ranking, and Generalized Born Volume Integral/ Weighted Surface Area dG was utilized for scoring the generated poses. Water molecules were deleted. With default parameters, the docking process was used. London dG ranked the top 30 poses, and they were stored.

Statistical analysis
All data were entered and analyzed in the Statistical Package for the Social Sciences (SPSS) software (IBM SPSS Statistics for Windows, Version 23, IBM Corp., Armonk, NY).

Chromatographic isolation and chemical profiling of the n-butanol extract using Liquid chromatography electrospray ionization /mass spectrometry (LC-ESI-MS/MS) analysis
Five phenolic metabolites were isolated from n-butanol as a polyphenolic-rich extract using successive column chromatography. Spectroscopic approaches such as 1 H-and carbon-13 magnetic resonance ( 13 C-NMR), as well as chemical tests, were utilized to determine their chemical structures ( Fig. 1, 4S-11S). The chemical metabolites were tentatively identified using LC-ESI-MS/MS analysis in the negative ion mode based on their MS fragmentation patterns, molecular weights, and previous data (Table 1 and Fig. 1S, 12S-15S). The examined extract included 33 secondary metabolites in total; the identified secondary metabolites were classified as phenolic acids, organic acids, flavonoids (aglycones and glycosides), iridoids, stilbenes, chalcones, tannins, and coumarins (Supplementary Materials: 2S).

Worm burden and distribution
Administering PZQ at an oral dose of 200 mg/kg to S. mansoni-infected mice for five consecutive days was effective in reducing total worm counts (93.5% worm reduction), while A. excelsa extract administration at doses of 200 and 500 mg/kg reduced total worm counts (33.2% and 41.3% worm reduction, respectively). The difference was statistically significant at p < 0.01 (Table 2).

Tissue egg load
In comparison to PQZ (200 mg/kg body weight), mice given A. excelsa at doses of 200-500 mg/kg body weight showed a greater reduction in mean total tissue (hepatic and intestinal). The difference between the infected and untreated control mice (10,938.41911.32 and 17,815.002024.66) was statistically significant at p < 0.01 (Table 3).

Percentage of egg developmental stages (oogram pattern)
PZQ at 200 mg/kg was given to S. mansoni-infected mice for five consecutive days, and this revealed the disappearance of all immature stages of ovary development with an increase in the dead ova. The difference was significant between the infected and untreated mice at p < 0.01 (Table 3). Regarding the total immature eggs, a significant decrease in the percentage was observed upon using the A. excelsa extract, and an increase in the percentage of dead and mature eggs was observed when compared with the infected group.

Hepatic granuloma volume
Histopathological investigation of liver segments from various examined groups showed a meaningful reduction in the number of egg granulomas in the PZQ-treated group in relation to all other treated and control groups (p < 0.01). However, no significant difference in granuloma counts was found between the control and treated groups at either of the doses assessed (200 and 500). As regards the granuloma diameter, there was a significant reduction in granuloma size in the PZQ-and A. excelsa-treated groups (500 mg/kg) compared to the control group (4,222.366.52 m). There was no statistically significant difference in granuloma diameter between the control and A. excelsa-treated groups (200 mg/kg). In addition, most of the egg granulomas of diverse groups were fibrocellular, with a mild increase in the number of cellular granulomas in the control group compared to the other treated groups. Worm granulomas could be detected only in the PZQtreated group. Considering the measurement of liver fibrosis in tissue sections, it was found that the PZQ-treated group showed a significant reduction in fibrosis [measured as area/ low-power field (LPF)] compared to the control group. The A. excelsa-treated group (500) showed a less significant reduction in fibrosis than the control group relative to the PZQ-treated group. No considerable variation in fibrosis was noticed between the control and the A. excelsa-treated group (200). Studying the cellular pattern of inflammatory cells showed a predominance of neutrophils over mononuclear inflammatory cells (Table 4 and Figures 2  and 3). Herbal medicines are of immense importance as a vital source of bioactive compounds, which are the basic nucleus of the pharmaceutical industry (Phillipson, 1994). Moreover, the efficacy of antischistosomiasis drugs is inferred by the reduced egg and worm burden index in the treated mice (Andrews, 1985). Consequently, the reduction rate of eggs in the mice treated with PZQ is a strong indicator of the drug's efficacy. Furthermore, our study of the A. excelsa extract recorded a reduction in egg count after treatment. The reduced egg load in the treated mice may be due to several factors, including reduced worm burden, decreased productivity of the female already present, and/or obliteration of a few eggs caused by the host's tissue reaction produced by the host's Ailanthus excelsa (500 mg/kg) 0.00 ± 0.00* 3.60 ± 0.60** 10.80 ± 0.92** 41.30 PZQ and A. excelsa leaf extract were administered orally 7 weeks after S. mansoni infection.
Results are presented as mean ± standard error of mean (SEM). **Significant difference from infected control at p < 0.01. *Significant difference from infected control at p < 0.05. tissue reaction. In the current work, data revealed the reduction in tissue egg load in the mice treated with the A. excelsa extract was coupled with a significant increase in the percentage of mature and dead eggs for A. excelsa compared with the infected untreated mice. These results agree with those of Pellizgo et al. (1962), who stated that the absence of immature eggs in the oogram pattern is a significant indicator of drug efficacy. The antioxidants β-carotene and N-acetylcysteine increased the reduction in the total number of worms and tissue egg loads, while increasing the percentage of dead ova and decreasing the percentage of mature ova phases (Ebeid et al., 2007;Seif el-Din et al., 2006). Histopathological examination of the liver sections in the different studied mice groups showed an enormous number of egg granulomas of large sizes and irregular outlines, with a high percentage of newly formed cellular granulomas. This was in accordance with the results of El-Nahal et al. (1998). Otherwise, the PZQ-treated group exhibited a considerable decrease in both egg granulomas' number and diameter, with more regular outlines and higher percentages of fibrocellular granulomas. Many large worm granulomas were detected in the liver sections of this group due to the shift of dead worms from the portal circulation to the liver (El-Lakkany et al., 2012). No significant reduction in granuloma count was detected  between the control and the treated groups at both examined doses (200 and 500 mg/kg). This observation pointed to the weak effect of the A. excelsa extract in decreasing the number of living worms or their oviposition in both examined doses. As regards the granuloma diameter, there was a significant reduction in granuloma size in the PZQ-and A. excelsa-treated groups (500 mg/kg) relative to the control group. No considerable variation in granuloma diameter was detected between the control and A. excelsa-treated group (200 mg/kg). Conversely, a considerable decrease in the mean granuloma diameter was detected at the high dose of the A. excelsa extract compared to the control-infected group. This could be attributed to an anti-inflammatory effect of the tested extract, especially at higher doses, resulting in a decrease in the inflammatory pool of each granuloma with a consequent decrease in size. This study sheds light on the in vivo antischistosomal potential of the A. excelsa extract.

Molecular docking study
Schistosoma mansoni's reliance on TGR to protect itself from expected oxidative stress makes it an attractive therapeutic target (Sharma et al., 2009). The skeleton of TGR from S. mansoni  was found as a fusion of two domains: Grx (1-106) and TR (107-598) (Angelucci et al., 2010;Huang et al., 2015). Based on the abovementioned findings, we decided to perform a molecular docking study of the five isolated compounds inside the active site of TGR comparing the results with the reference drug PZQ. Herein, a molecular docking study was done to predict and score the poses of the ligand-protein binding. The docking studies were performed using MOE 2014.0901. The TGR crystal structure was downloaded from a protein data bank (PDB:2V6O). The results are shown in Figures 4-7 and Table 1S and Figures 2S and 3S.
PZQ revealed a docking score of −6.407 kcal/mol showing two hydrogen bonds with Cys159 and Thr442. The least binding scores were observed for compounds gallic acid and methyl gallate displaying binding energies of −3.278 and −4.718, respectively. Kaempferol-3-O-rhamnoside was able to fit inside the pocket by forming three hydrogen bonds with Asp433, Cys159, and Thr442, besides one arene-H interaction with Tyr296, revealing a binding score of −8.758 kcal/mol. Also quercitrin was bounded via four hydrogen bonds with the Asp433, Lys162, Thr442, and Ser276 amino acids, whereas isoquercitrin among all compounds  displayed seven H-bonds with the most important amino acids inside the active site with an energy score of −11.370 kcal/mol.

CONCLUSION
The A. excelsa leaf extract has a high concentration of polyphenolic metabolites, which have been tentatively identified by LC-ESI-MS/MS and characterized as antischistosomal. The current investigation found a significant reduction in the number of worms and eggs after treatment with the plant extract. Therefore, the plant is considered a strong candidate as a promising source of natural antischistosomiasis agents.

CONFLICT OF INTEREST
No potential conflicts of interest were reported by the authors.

FUNDING
There is no funding to report.

ETHICAL APPROVAL
All the experiments on animals were conducted according to the internationally valid guidelines after the approval of the Institutional Theodor Bilharz Research Institute-Research Ethical Committee (TBRI-REC). The serial number of the protocol is PT (606).

AUTHORS' CONTRIBUTIONS
HSM and MAG conceived and designed the experiments; searched for information; performed extraction, fractionation, chromatographic isolation, structural elucidation, and LC-ESI-MS/MS interpretation; drafted the original paper; and revised the last version. SW evaluated in vivo antischistosomal activity. TA performed the histopathological examination. HA performed the statistical analysis. RS performed the molecular docking experiment. All authors contributed to manuscript revision and read and approved the submitted version.

DATA AVAILABILITY
All the data obtained during the study are presented in this manuscript. Any further inquiries for additional information are available upon request from the corresponding author.

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