Comprehensive chemical profiling of Bassia indica Wight. aerial parts extract using UPLC-ESI–MS/MS, and its antiparasitic activity in Trichinella spiralis infected mice: in silico supported in vivo study

Characterization of the triterpenoidal saponins and the phenolics

LC–ESI–MS/MS was conducted as the best choice for the identification of compounds in complex extracts with co-eluting peaks. LC/MS analysis was performed in -ve mode which produced a prominent deprotonated molecular ion [M–H]– with weaker fragment ions. Whereas, the most abundant fragment ions corresponded to cleavage of the glycosidic bond accompanied by transfer of a hydrogen atom from the leaving sugar were resulted from + ve mode [38].

A total of 19 compounds (Fig. 1a, b) were identified based on the comparison of their pseudo-molecular ions and mass fragmentation patterns with the mass spectral data from the literature and compounds libraries.

The characterized compounds include 13 triterpenoidal saponins (Fig. 1a) of the oleanane-type base skeleton and were identified as; oleanolic acid 3-O-6´-O-methyl-β-D-glucuronopyranoside (1), chikusetsusaponin-IVa (2) and its methyl ester (3), chikusetsusaponin IV (4) and its methyl ester (5), momordin-Ic (6) and its methyl ester (7), betavulgaroside-I (8), -II (9) -IV (10), -X (11), licorice-saponin-C2 (12) and -J2 (13). In addition, 6 more phenolics (Fig. 1b) were identified as syringaresinol (14), 3,4-di-O-caffeoylquinic acid (15), 3-O-caffeoyl-4-O-dihydrocaffeoylquinic acid (16), 3,4-di-O-caffeoylquinic acid butyl ester (17), 3,5-di-O-galloyl-4-O-digalloylquinic acid (18) and quercetin 3-O-(6´´-feruloyl)-sophoroside (19). 

As appeared presented in (Table 1), compounds (14, 6, 811, 13, 1519) produced at the same retention time both the protonated [M + H]+ and sodiated [M + Na]+ adduct in the + ve mode along with the deprotonated ion [M–H]– in the –ve mode, which made it easy to confirm the molecular masses of these compounds, in addition, all compounds recorded an acceptable deviation (≤ 1 ppm) between their experimental and accurate masses. Moreover, compounds (5) and (7) are the methylated derivatives of (4) and (6) respectively, compound (13) is the hydroxy-methylene derivative of compound (12). Briefly, CID of compounds (1–13) showed the presence of fragment ions around m/z 439 and 425 corresponding to the aglycone moiety oleanolic acid (OA) with the loss of OH and/or OCH3 respectively, other fragment ions were generated upon the loss of GlcUAp at m/z 193, Glc at m/z 179, Xylp at m/z 149. Furthermore, compounds (1517) produced the main characteristic fragment ions including loss of one or two caffeoyl moieties at m/z 179 characteristic for chlorogenic acid derivatives. To the best or our knowledge, compounds (13, 5, 811) were reported from K. indica [39], compounds (4, 6, 7) were reported from K. scoparia [40] and compounds (14 and 19) were isolated before from B. indica [15] and B. muricata [41, 42] respectively. In addition, compounds (12, 13, 18) and (1517) are reported in our study from B. indica for the first time [42, 43].

Table 1 LC–MS/MS of all identified compounds from B. indica aerial parts n-BuOH fracIn vitro Anthelmintic activity

The developmental stages of T. spiralis (adult, migratory and encysted) occur in the same host, hence it has been used as an interesting experimental model to assess the effectiveness of many anthelminthic agents [44].

SEM findings of adult T. spiralis

For infected control group cultured in an incubation medium only, the cuticle of the adult worm teguments retained the normal structure in the form of ridges, transverse creases and annulations, along with the appearance of openings of the hypodermal gland. Whereas, in B. indica BuOH frac. treated-groups, the adult worms showed shrinking, sloughing of some areas of the cuticle, loss of the normal annulations of the cuticle and marked destruction of the adult worm cuticle, besides, the albendazole-group showed destruction of the adult worms and sloughing with loss of annulations (Fig. 2).

Fig. 2figure 2

Scanning electron microscope findings of the cultured T. spiralis adult worm. A normal control group showing adult worm with intact annulated cuticle and openings of hypodermal glands, B infected control group showing normal adult worm cuticle with tapering end, C normal control group showing intact adult worm cuticle with normal longitudinal striation (green arrows), D and E B. indica-groups showing shrunked T. spiralis adult, sloughing of some areas of the cuticle (yellow arrow) and loss of the normal annulations in the cuticle, F B. indica-group showing marked destruction of the adult worm cuticle, sloughing of some areas of the cuticle (red arrows), G albendazole-group showing destruction of the adult worms and sloughing (yellow arrows) with loss of annulations

SEM findings of T. spiralis larval stage

The infected control-group revealed coiled posterior with intact cuticular folds, transverse striations and shallow longitudinal grooves. Whilst, B. indica BuOH frac. group showed shrunken in T. spiralis larva, destructed cuticle with large blebs and loss of the normal annulations in the cuticle, aside from, albendazole group revealed large blebs with loss of the normal cuticular folds and striations (Fig. 3).

Fig. 3figure 3

Scanning electron microscope findings of the cultured T. spiralis larvae. A infected control group showing normal comma shaped larva with intact cuticle and coiled posterior end. B and C normal control group showing larva with intact cuticular folds, transverse striations and shallow longitudinal grooves, D, E, and F B. indica BuOH frac groups showing T. spiralis larva with destructed shrunked cuticle, loss of its normal annulations and larval cuticle showing large blebs (blue and red arrows), G and H albendazole group showing large blebs (green arrow), and loss of the normal cuticular folds and striations

The shape of the parasite, nutrition and protection are dependent on cuticle integrity, which is an essential part of Trichinella’s body wall, along with the hypodermis and the somatic musculature it is necessary for osmoregulation [45]. Based on Thompson and Geary, who reported the principal mechanism of drug entry into the helminths to be a transcuticular passive diffusion, so anti-anthelmintics targeted the destruction of the worm’s surface [46, 47]. Which faced by the parasite’s blebbing as an attempt to replace damaged surface membrane in response to drug action. Therefore, the tegumental changes can be considered as a good indicator for the possible anthelmintic activity of a drug [48]. In our study, the electron microscopy scans showed severe destruction of the adult worm and larvae, marked cuticle swelling, areas with vesicles, blebs and loss of annulations in both B. indica BuOH frac. and albendazole treated-groups, while it retained its normal morphology when incubated in the culture medium only.

Anthelmintic drugs (BzC) can disrupt the parasite metabolism or destruct its cuticle/cytoskeleton which is finally lead to paralysis and eventual death [49]. Hence, the efficient antiparasitic activity of B. indica BuOH frac., could be mainly attributed to its chemical constituents (1–19) of saponins [13,14,15] and phenolics [19]. Briefly, the biological potential of saponins are basically due to their ability to cause changes in cell permeability via their specific interaction with the cell membrane [50]. Cavalcanti Gomes et al., hypothesized that saponins may be able to cause larval death via their ability to interfere with enzymatic pathways involved in larval development [51]. However, large concentrations of saponins are potentially toxic. Anyhow, in moderate concentrations, they can reduce the parasitic burden [52]. Moreover, Ekeanyanwu and Etienjirhevwe proved that phenolics can interfere with the energy generated by helminth parasites through the uncoupling of specific reductase-mediated reactions [53]. In addition, the high content of flavonoids, phenolic acids and tannins are correlated with high antioxidant property, which is in turn exhibited anthelmintic potential by interposing with energy generation in helminth parasites, uncoupling oxidative phosphorylation, and binding to free proteins in the gastrointestinal tract of the host animal and/or glycoprotein on the cuticle of the parasite and leading to death [54].

In vivo results

To validate the potential in vitro outcomes, an in vivo study was conducted to assist the anti-parasitic activity of B. indica BuOH frac. compared with albendazole, during the (a)-intestinal phase (3–5 days p.i.), (b)-muscular phase (30–32 days p.i.) and (c)-intestinal then muscular phases (3–5 days p.i. and 30–32 days p.i.) separately (Fig. 4a, b).

Fig. 4figure 4

Photograph Represents T. spiralis. a Adult in the intestinal fluid on day 5 post infection (X 10). b Larvae in the diaphragm of mice on day 35 post infection (X 40)

Adult worm count in the small intestine

As appeared in (Table 2), prophylactic treatment of the infected mice with B. indica BuOH frac. resulted in a significant reduction (P < 0.05) in the mean adult worm count (51 ± 8.4) with efficacy of 42.7%, compared to the control infected untreated group (G-III) with (89 ± 8.5). Moreover, a significant decrease in the mean number of adult worms (46.5 ± 3.5), with a drug efficacy of 47.8% was obtained in B. indica BuOH frac., treated group (G-Va) with (P < 0.01) in comparison to the control infected untreated group.

Table 2 The counts of T. spiralis adult worm and encysted larvae per gram muscleEncysted larvae count in muscles

Prophylactic treatment of the infected mice by B. indica BuOH frac. significantly reduced (P < 0.001) the mean larval count per gram muscle (1525 ± 330.4) with an efficacy of 80.7% compared to the control infected untreated group (7920 ± 973.1). A significant decrease in the mean larval count per gram muscle was detected in the treated groups (P < 0.001) compared to the control infected untreated group. Moreover, better larvae eradications were found in groups that received two doses than groups received a single dose. Furthermore, the best reduction of the mean larval count (1600 ± 556.8) in the tested extract was found in G-Vc with efficacy of (79.8%). A significant reduction of the mean larval count (2166.7 ± 351.2) was observed in G-Vb with an efficacy of 72.7% for the single-dose regimen (Table 2).

In the present study, single-drug treatment significantly decreased the count of T. spiralis, but better larvae eradications were found in groups that received the drugs in two doses regimen (during intestinal and muscular phases). In the same context, Lu and co-workers demonstrated the in vivo anthelmintic activity of the MeOH extract of K. scoparia against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus) [55]. Moreover, Javed and colleagues reported the antifungal activity of n-BuOH frac. of the MeOH leaf extract of K. indica against Macrophomina phaseolina [22]. In addition, El-Wakil and co-workers evaluated the anti-trichinellosis activity of Annona muricata leaves ext. and stated that larval eradication was better with biphasic treatment over intestinal phase only [56].

Histopathological resultsSmall intestine changes

Histopathological examination of sections from the small intestine of the control negative (G-I) revealed a preserved villous pattern (Fig. 5a). Whereas, other sections of the infected control (G-III) showed dense intervillous inflammatory cellular infiltration consisting of mononuclear cellular infiltrate in the form of lymphocytes and plasma cells, there were broadening and atrophy of the intestinal villi with crypt hyperplasia with fragments of the adult worms were detected within the intestinal lumen (Fig. 5b). Sections examined from (G-IV) received B. indica BuOH frac. for seven days before infection, showed some Trichinella cyst with mostly preserved villous (Fig. 5c). However, sections examined from the treated group showed an obvious reduction in the intensity of the inflammatory cellular infiltration, with remarkable improvement of the other histopathological changes of the intestine, with a returning of the normal villous pattern (G-Va) with mostly preserved villous pattern (Fig. 5d). Furthermore, sections in the intestine of albendazole treated mice (G-VIa) showed the absence of Trichinella cysts and preserved villous pattern (Fig. 5e).

Fig. 5figure 5

Sections in the intestine showing;  A Preserved villous pattern_ G-I. B Some Trichinella cysts and distorted villous pattern_G-III. C Some Trichinella cysts with mostly preserved villous pattern_G-IV. D Mostly preserved villous pattern_G-Va. E Absence of Trichinella cysts and preserved villous pattern_G-VIa. a, c, d, e; (H&E stain, X200) and b; (H&E stain, X400)

Skeletal muscle changes

Histopathological examination of muscular sections from the infected control group (G-III) revealed the presence of a massive number of encysted T. spiralis larvae, diffusely in the sarcoplasm of the muscles and several chronic inflammatory cells in the form of lymphocytes, plasma cells, histiocytes infiltrating muscle bundles and surrounding the encysted larvae (Fig. 6b) compared to the control negative (Fig. 6a). Furthermore, the muscles from the prophylaxis group (G-IV) showed a focally degenerated trichina capsule and pericapsular histio-lymphocytic inflammatory cellular infiltration (Fig. 6c). Concerning the histopathological examination of muscular sections from mice groups that received the B. indica BuOH frac. for a single dose in the muscular phase only (G-Vb), there were fewer trichina capsules with focally degenerated capsule and dense pericapsular plasma-lymphocytic inflammatory cellular infiltration (Fig. 6d). In addition, muscular sections from groups that received the B. indica BuOH frac. for two doses showed marked improvement of the histopathological finding compared to the infected control (Fig. 6e). Furthermore, muscular sections of albendazole treated mice showed focally degenerated capsule with mild to dense pericapsular plasma-lymphocytic inflammatory cellular infiltration (Fig. 6f, g).

Fig. 6figure 6

Histopathological muscular sections showing; A Non-infected and non-treated (control negative)_G-I. B Intact capsules (yellow arrow) and pericapsular plasma-lymphocytic inflammatory cellular infiltration (green arrow)_G-III. C Degenerated trichina capsule and larva and dense pericapsular plasma-lymphocytic inflammatory cellular infiltration (green arrow)_G-IV. D Degenerated capsule (black arrow) and focal pericapsular plasma-lymphocytic inflammatory cellular infiltration (green arrow)_G-Vb. E Markedly degenerated capsule (black arrow) and larva with invasion by many macrophages (green arrow)_G-Vc. F Focally degenerated capsule (black arrow) and mild pericapsular plasma-lymphocytic inflammatory cellular infiltration (green arrow)_G-VIb. G Degenerated trichina capsule (black arrow) and dense pericapsular plasma-lymphocytic inflammatory cellular infiltration (green arrow)_G-VIc. a, b, d, f: (H&E stain X200) and c, e, g: (H&E stain X400)

Hence, histopathological examination of sections from the small intestine of the infected control group showed dense intervillous inflammatory cellular infiltration, with broadening and atrophy of the intestinal villi with crypt hyperplasia. Moreover, fragments of the adult worms were detected within the intestinal lumen. Muscular sections from the infected control group revealed the presence of a massive number of encysted T. spiralis larvae diffusely present in muscle sarcoplasm and a number of chronic inflammatory cells. In addition, the reduction of these destructive and inflammatory changes was evident in the treated groups. Groups with the combination therapy exhibited the best improvement in restoring the normal architecture, the presence of the least number of trichina capsule with degenerated capsule and focal pericapsular plasma-lymphocytic inflammatory cellular infiltration [57].

Immunostaining for TNF-α (a-proinflammatory cytokines)

Section in skeletal muscle of the infected untreated control positive (G-III) showed trichina capsules with focal capsular degeneration and degenerated contents, surrounded by some mono- and polymorphnuclear inflammatory cells exhibiting moderate expression of TNF-α (Fig. 7a). While, section in skeletal muscle of B. indica prophylaxis (G-IV) showed trichina capsules surrounded by large number of mononuclear inflammatory cells exhibiting marked expression of TNF-α (Fig. 7b). However, section in skeletal muscle of (G-Vb) received B. indica BuOH frac. in muscular phase only showed trichina capsules surrounded by large number of mono and polymorphnuclear inflammatory cells exhibiting marked expression of TNF-α (Fig. 7c). Moreover, in (G-Vc) that received the drug in both intestinal and muscular phases, showed trichina capsules surrounded by large number of mono and polymorph nuclear inflammatory cells, and exhibited only focal expression of TNF-α (Fig. 7d). Besides, a moderate to mild expression of TNF-α was detected in (G-VIb and -VIc), (Fig. 7e & 7f) respectively. Hence, it can be concluded that B. indica BuOH frac. lowered the parasite burden in the intestine and muscle tissues, and alleviating muscular inflammatory reaction, which is in a good agreement with the evidences indicated that inflammatory mediators are involved in the muscular pathogenesis of T. spiralis infection and accelerate the development and progression of myositis associated with this phase [58].

Fig. 7figure 7

Sections in skeletal muscle showing T. spiralis trichina capsules (IHC, TNF-α, DAB, X400). A With focal capsular degeneration and degenerated contents, surrounded by some mono and polymorphnuclear inflammatory cells exhibiting moderate expression of TNF-α (arrow)_G-III control positive (infected untreated). B Surrounded by large number of mononuclear inflammatory cells exhibiting marked expression of TNFα (arrow)_G-IV (prophylactic). C Surrounded by large number of mono and polymorphnuclear inflammatory cells exhibiting marked expression of TNF-α (arrow)_G-Vb. D Surrounded by large number of mono and polymorphnuclear inflammatory cells exhibiting only focal expression of TNF-α (arrow)_G-Vc. E Surrounded by moderate number of mono and polymorphnuclear inflammatory cells exhibiting high percentage cellular expression of TNF-α (arrow)_G-VIb. F Surrounded by small number of mono and polymorphnuclear inflammatory cells exhibiting mild expression of TNF-α (arrow)_G-VIc

Monokines, such as IL-1, -6, -8, TNF-α and G-CSF, are immune molecules that are secreted mainly by monocyte and macrophages, and play a vital role in mediating and regulating immune and inflammatory reactions as reflected by proinflammatory factor, which perform its biological function in a pleiotropic way on multiple cell types and play a pivotal role in the pathogenesis of chronic inflammatory diseases [59]. Our results proved the anti-inflammatory activity of that B. indica BuOH frac. in T. spiralis infection via depressing the expression of TNF-α in the muscle tissues. However, the experimental results clearly demonstrated that both B. indica BuOH frac. and albendazole were effective against intestinal adult worms and muscle larvae. In addition, TNF-α was controlled at low levels due to the bioactivity of B. indica BuOH frac., which indicated its powerful anti-inflammatory function in T. spiralis disease. These results are in consistent with Xu and co-workers who reported that Vaccaria n-BuOH extract lowered the production of proinflammatory cytokines as TNF-α and the infection burden of T. spiralis in vivo [60]. Therefore, B. indica BuOH frac. may achieved anti-inflammatory effect by relieving the inflammatory response of the infected mice and eventually achieved the nematicidal effect of T. spiralis. Notably, although albendazole plays a more effective role in reducing the burden of T. spiralis larvae, but due to its limitation in clinical use, B. indica BuOH frac. may be an ideal option to treat human trichinellosis. The resulted promising anti parasitic potential of B. indica BuOH frac. constituents arouse our interest to go further for an in silico molecular docking investigation of its identified compounds (1–19) as novel anti T. spiralis.

Molecular docking results

Recently, drug discovery development expressed in the computational studies which involve the use of algorithms and programs, have led to a regenerated interest for predictions of therapeutic interventions in biological processes. Molecular docking approach, which foretells binding interactions between target receptors and ligands at the binding pocket active site, it represents an intrinsic technique that virtually checking several thousands of ligands against target receptors and identify their potential inhibitors with high accuracy and speed [61].

MOE (2019,0102) was conducted to perform a detailed in silico study of all identified compounds (1–19) from B. indica BuOH frac. with respect to selected protein targets (Table 3). Briefly, as reported that albendazole targets selectively β-tubulin monomer of the parasite and inhibits its microtubule polymerization [9, 10]. So, (PDB ID: 1OJ0) was the one of choice for this goal, all the docked compounds (1–19) fit into the binding site with different binding affinities compared to the co-crystalized ligand (ABZ) with RMSD < 2 Å. Out of this list, only compounds (14–17) revealed closest binding energies to ABZ, while the others revealed a relatively high (+ ve) unfavorable binding energies, which could be attributed to their high molecular weights (418–954 Da) compared to that of ABZ (265 Da) and its active pocket. However, their binding energies may be improved via multipose binding in the docking process [62].

Table 3 Docking scores and receptors amino acids involved in the interactions with the ligand compounds

Nevertheless, the binding affinities reflect the potential inhibition of B. indica BuOH frac. constituents (1–19) against microtubule polymerization. However, the ligand size plays a vital role in docking process i.e., larger ligands preserve a considerable docking challenge due to their higher flexibility, beside they are more susceptible to form higher number of interactions with the protein receptor. Thus, the binding energy of the resulted complexes may not be considered as a determinant property to clarify their activity against the parasite, and we can relate the inhibition of microtubule polymerization of the parasite to the interactions of all compounds (1–19) with the 3 key interacting residues amino acids (Val236, Ser165 and Gln134) of the active pocket [63].

Molecular docking of ligand molecules (1–19) with the protein receptor proved that all molecules bonded with one or other amino acids in the active pocket (Table 3), out of all frontrunner compounds (1–19), six compounds (4, 5, 8, 11, 15, 19) exhibited two amino acid interactions, three compounds (12, 17, 18) were found to have exploited the same binding pocket and interacted with the same 3 key amino acid residues within the receptor site (Fig. 8a), and only compound (17) revealed the most favorable and lowest binding energy among others. It was observed that all the frontrunner compounds exhibited one or more interactions with the key amino acids at distances much better than the co-crystallized ligand itself. In addition, Aguayo-Ortiz and co-workers, identified that (Phe167, Glu198 and Phe200) are the most important amino acids associated with the resistance mutations of helminths [64]. Moreover, Robinson and colleagues, affirmed that (Tyr50, Gln134, Thr165 and Met257) are the amino acids identified as BzC resistance mutations [65]. Hence, it can be documented from (Table 3) that compounds (1, 3–8, 11, 14–16, 18, 19) out of the others interacted with one or more of amino acids associated with the resistance mutations, which strengthen their potential anti-anthelmintic.

Fig. 8figure 8

a Docking poses of the 3D structures of selected compounds with the active pocket residues (1OJ0). b Docking poses of the 3D structures of selected compounds with the active pocket residues (2AZ5). c Docking poses of the 3D structures of selected compounds with the active pocket residues (Ts-CF1). d Docking poses of the 3D structures of selected compounds with the active pocket residues (Ts-CRT)

As proved from the in vivo study that the activity of B. indica BuOH frac. may be attributed to its potential anti-inflammatory via depressing the expression of TNF-α in the muscle tissues, this encourage our group to explore the in-silico binding affinities of all identified compounds (1–19) from the BuOH frac. with tumor necrosis factor alpha (TNF-α). Zia and co-workers, asserted that Tyr119 is a crucial for TNF-α inhibition along with Leu57, Tyr59, Ser60, Gln61 and Tyr151 [66]. Molecular docking results of the ligand compounds (1–19) yielded complexes with binding energies ranged from (–9.97 to –6.98 kcal/mol) far less than that of ABZ (–5.74 kcal/mol), and even superior with less bonding distances < 4 Å. Out of the frontrunner list, ABZ and compounds (2, 6, 11–14, 16, 18) interacted with the pivotal amino acid Tyr119, in addition, compounds (2, 5, 8) represented the most energetical favorable ligands with outstanding binding energies (> –9.5 kcal/mol) and thus are speculated to have better binding affinities (Fig. 8b).

Tracking the life cycle of T. spiralis revealed that cathepsin F specific (Ts-CF1) is a cysteine protease existed in cuticle and stichosome, and it is an essential for their life stages, this is favoring its potential use as a drug target against T. spiralis infection [6]. Moreover, Shao and fellows, proved that T. spiralis lean to escape the host innate and adaptive immune attack via some sophisticated mechanisms [7], one of which is the secretion of calreticulin protein (Ts-CRT) on the surface of T. spiralis at different stages of adult and muscle larvae worms, which binds, interferes and overlaps with the host immune system to facilitate their survival, so targeting this protein receptor may help against their infection [67].

To unveil the effect of all identified compounds (1–19) against T. spiralis infection, a homology modeling protocol was performed to prepare the 3D structures of both Ts-CF1 and Ts-CRT, and were averred via their overall quality factors to be within the range of a high-quality model. The cysteine protease conserved active site of Cys173, His309 and Asn333 which were identified and revealed in the propeptide of Ts-CF1 [6]. While, in silico docking studies predicted that C1q binding sites in Ts-CRT are mainly located between Lys163 in the N-domain and Asn286 in the P-domain, with an active pocket composed of Lys163, Asn164, Glu187, Asp193, Lys194, Glu195, Tyr197, Arg199, Asp202, Trp223, Glu278, Trp279, Ala280, Glu282, Gln283 and Asn286 [

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