Polyphenolic Profile of Herniaria hemistemon Aerial Parts Extract and Assessment of Its Anti-Cryptosporidiosis in a Murine Model: In Silico Supported In Vivo Study

Herniaria hemistemon J.Gay is widely used in folk medicine to treat hernia. The present study aimed to annotate the phytoconstituents of H. hemistemon aerial-part extract and investigate its in vivo anticryptosporidial activity. The chemical characterization was achieved via the LC–ESI–MS/MS technique resulting in the annotation of 37 phytocompounds comprising flavonoids and phenolic acids. Regarding the anticryptosporidial activity, fifty dexamethasone-immunosuppressed mice were separated into five groups: GI, un-infected (normal control); GII, infected but not treated (model); GIII, infected and received NTZ, the reference drug; GIV, infected and received H. hemistemon extract (100 mg/kg); and GV, infected and received H. hemistemon extract (200 mg/kg). When GIII, GIV, and GV were compared to GII, parasitological analyses displayed highly significant differences in the mean numbers of Cryptosporidium parvum oocysts in the stool between the different groups. GV demonstrated the highest efficacy of 79%. Histopathological analyses displayed improvement in the small intestine and liver pathology in the treated groups (GIII, IV, and V) related to the model (GII), with GV showing the highest efficacy. Moreover, the docking-based study tentatively highlighted the potential of benzoic acid derivatives as lactate dehydrogenase inhibitors. The docked compounds showed the same binding interactions as oxamic acid, where they established H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195. To sum up, H. hemistemon is a promising natural therapeutic agent for cryptosporidiosis.


Introduction
Cryptosporidium spp. are intracellular protozoan of the phylum Apicomplexa that can invade the microvillous area of epithelial cells in the digestive tract of humans and other mammals [1]. In immunocompromised patients, cryptosporidiosis can produce major complications such as severe diarrhea, dehydration, electrolyte imbalance, and hepatic and respiratory disorders [2]. Although the nature of cryptosporidiosis is widely understood, the therapeutic options are still restricted. Supportive therapy and specific treatments by Nitazoxanide (NTZ), the only Food and Drug Administration (FDA)-approved medicine in the United States, are still the only options for these serious infections. Nitazoxanide has an impact on the parasite by preventing its metabolism-necessary anaerobic energy, which makes its clearance easier [3]. Unfortunately, NTZ's impact on children suffering from malnutrition is limited, and it has no effect on immunocompromised hosts [4].
Despite its prevalent utility and numerous traditional uses, there are no adequate reports regarding the effects of H. hemistemon against cryptosporidiosis in a murine model. Therefore, this study aimed to annotate the chemical constituents of H. hemistemon aerialpart extract and to explore its activity against cryptosporidiosis in a murine model and possible modes of action via a molecular docking study.

Plant Material, Extraction, and Fractionation
Aerial parts (1.2 Kg) of Herniaria hemistemon J.Gay were collected after permission from and in compliance with relevant international guidelines and legislation of Matrouh governorate, Egypt in April 2020. The identification and authentication of the plant were performed by specialists at El-Orman Botanical Garden, Giza, Egypt. A voucher specimen was preserved at the Medicinal Chemistry Department, TBRI under the accession code (H.h.ap.2020). The dry, powdered plant materials were extracted with 85% methanol (4 × 2 L) at room temperature. The obtained extract was concentrated under reduced pressure using Rotavapor (Buchi R-300) at 40 • C. The total extract was then defatted using petroleum ether to afford 103 g (the extraction yield was 8.5%).

Phytochemical Analysis, Total Phenolic (TPC) and Total Flavonoid (TFC) Contents and Antioxidant Properties
The chemical components of H. hemistemon aerial-part extract were tentatively identified using high-performance liquid chromatography-mass spectrometry (HPLC-PDA-MS/MS). The LC system was SHIMADZU LC MS 8050 (Shimadzu, Japan, USA) coupled with a triple quadruple spectrometer with an ESI source. The separation was performed via a C18 reversed-phase column (Zorbax Eclipse XDB-C18, rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, USA). A gradient of water and acetonitrile (ACN) (0.1% formic acid each) was applied from 5% to 30% ACN over 45 and increased to 60 over the last 15 min with a flow rate of 1 mL/min. The samples were automatically injected using autosamplerSIL-40C xs. The instrument was controlled by LC solution software (Shimadzu, Japan). The MS operated in the negative mode. TPC, TFC, DPPH (2,2-diphenyl-1-picrylhydrazyl) and total antioxidant capacity (TAC) were assayed as previously described [18].

Animals
The experimental work was permitted by the ethical committee of Theodor Bilharz Research Institute (TBRI-REC) in accordance with internationally valid guidelines (protocol serial number: PT: 612). TBRI-REC operates in a manner consistent with the National Institute of Health (NIH) guide for the care and use of laboratory animals (Eighth edition) and adhered to the ARRIVE guidelines. Theodor Bilharz Biological Center provided laboratory male-bred, CDI-strain white Albino mice, which were about 4-6 weeks old and weighed 20-25 g. The experiments were conducted in the TBRI's Animal House, in a well-ventilated plastic cage in conditioned rooms (24 ± 2 • C) with clean wood-chip bedding that were distant from direct sunlight, keeping a clean environment.

Immunosuppression and Induction of Infection
A total of 50 laboratory-bred white albino male mice were orally given 0.25 µg/g/day dexamethasone sodium phosphate (Dexazone) by gavage through an esophageal tube to suppress their immune systems. Dexazone was applied daily for two weeks before oral inoculation of Cryptosporidium oocysts, and it was given once per week for the entire study duration for each group [19].
The mice were separated into five groups. GI: immunosuppressed and uninfected (negative control). Mice (GII-GV) were gavaged and orally infected using an esophageal tube (day 0) with C. parvum oocysts (concentrated from the feces of naturally infected neonatal calves and genetically identified as C. parvum [8]). The infection dose was approximately 1000 oocysts of C. parvum dissolved in 200 µL PBS for each mouse. To ensure infection establishment, fecal pellets were collected and studied after one week of mice infection (7th day post-infection (PI)).
At the 7th day PI, drugs were administered via oral gavage: -GII: Immunosuppressed and infected (model); -GIII: Immunosuppressed, infected, and received NTZ (Nanazoxid, 100 mg/5 mL suspension, Medizen Pharmaceutical industries for Utopia Pharmaceuticals) at 100 mg/kg every day for 5 successive days [20]. The doses were derived by extrapolating therapeutic human doses to animal doses [21]; -GIV: Immunosuppressed, infected, and received H. hemistemon extract at a dose of 100 mg/kg every day for 5 days; -GV: Immunosuppressed, infected, and received H. hemistemon extract at a dose of 200 mg/kg every day for 5 days.

Parasitological Examination
Fecal pellet collection was performed on the 12th day PI (the time of the end of therapeutic doses), stained using modified Zheil Nelsen stain (MZN), and examined with the x100 oil-immersion lens according to the reported procedures [22]. The parasite number was detected per gram of feces [23]. The efficacy percentages of NTZ and H. hemistemon were determined according to the following equation: efficacy (%) = mean value of infected non-treated group-mean value of infected treated group × 100/mean value of infected non-treated group [24].

Histopathological Examination
All mice were sacrificed 12 days after infection under light anesthesia by using isoflurane inhalation (Forane ® , Baxter, UK). The jejunum, ileum, and sections from all segments of the liver tissue were collected and placed in a 10% buffered formalin solution for fixation, embedded in paraffin wax blocks, sectioned, and stained in the TBRI pathology lab with hematoxylin and eosin (H&E) to detect the pathological abnormalities [25].

Docking-Based Virtual Screening
The annotated compounds of the H. hemistemon aerial-part extract were virtually screened for their ability to inhibit lactate dehydrogenase (PDB codes: 4ND1) [26] using AutoDock Vina software [27]. Detailed methods are provided in the Supplementary File.

Statistical Analysis
Data analysis was performed using Microsoft Excel 2016 and the statistical package for social science 'IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, NY, USA)'. Continuous, normally distributed variables were represented as mean ± SE with a confidence interval of 95%. The ANOVA test was used to compare the means of normally distributed variables between groups. p-values ≤ 0.05 were considered statistically significant.

Chemical Profiling
The LC-ESI-MS/MS analysis of H. hemistemon aerial-part extract furnished 37 secondary metabolites belonging to organic and phenolic acids, coumarins, flavonoids, and their glycosides and sulfate derivatives ( Figure 1 and Table 1 (Table 1). Another signal demonstrated an [M-H]m/z at 395 and daughter ions at 151, 255, 300 and 315; it was tentatively identified as isorhamnetin sulfate (Table 1).

Docking-Based Virtual Screening
The annotated compounds of the H. hemistemon aerial-part extract were virtually screened for their ability to inhibit lactate dehydrogenase (PDB codes: 4ND1) [26] using AutoDock Vina software [27]. Detailed methods are provided in the supplementary file.

Statistical Analysis
Data analysis was performed using Microsoft Excel 2016 and the statistical package for social science 'IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, NY, USA)'. Continuous, normally distributed variables were represented as mean ± SE with a confidence interval of 95%. The ANOVA test was used to compare the means of normally distributed variables between groups. p-values ≤ 0.05 were considered statistically significant.

Chemical Profiling
The LC-ESI-MS/MS analysis of H. hemistemon aerial-part extract furnished 37 secondary metabolites belonging to organic and phenolic acids, coumarins, flavonoids, and their glycosides and sulfate derivatives ( Figure 1 and Table 1 (Table 1). Another signal demonstrated an [M-H] -m/z at 395 and daughter ions at 151, 255, 300 and 315; it was tentatively identified as isorhamnetin sulfate (Table 1).

Total Polyphenols, Total Flavonoids and Antioxidant Properties
The H. hemistemon aerial-part extract displayed considerable total phenolic (TPC) and total flavonoid (TFC) contents when assayed using the Folin-Ciocalteu and aluminum chloride methods ( Table 2). As expected, it also furnished promising antioxidant potential in two assays: DPPH and TAC (Table 2). These results might be attributed to the presence of a series of polyphenolics with antioxidant properties, among them kaempferol, quercetin, isorhamnetin and myricetin, along with their glycosides and sulfate derivatives, as well as several phenolic acids (Table 1).

Parasitological Examination
C. parvum was not found in the uninfected mice (GI, the negative control group). On the other hand, all infected mice began excreting C. parvum oocysts, which were confirmed after seven days post-infection (PI). At the end of the experiment (12 days PI), the model (GII, infected, untreated) developed the highest oocyst intensity with a mean score of 90.4. In contrast, all treated groups showed lower oocyst intensities compared to the model (GII), with GV showing the best efficacy with an inhibition of 79% (Table 3).  Table 1.   Table 1.

Effects of H. hemistemon Aerial-Part Extract on the Small Intestine
Following C. parvum oocyst induction, several deleterious pathological changes in the small intestine were observed. These included a marked broadening of the villi with a decreased ratio of villous height to crypt length, a dense infiltration of mononuclear inflammatory cells inside the villous core, villous tip-region degeneration, and high mucin production. Cryptosporidium oocysts were also present along the villi brush border and in the intestinal lumen as oval-to-rounded bodies. NTZ (the reference drug) and the extract, at the two dose levels, attenuated the aforementioned effects of the extract, and at 200 mg/kg, retained a nearly normal villous pattern (Figure 3).

Effects of H. hemistemon Aerial-Part Extract on the Liver
Similar to the small intestine, the induction of C. parvum oocysts induced caused pathological changes to the liver tissue. These included hepatocyte degeneration and focal infiltration with mononuclear cells. The reference drug (NTZ) and the extract, at 100 mg/kg, moderately improved the above-mentioned effects. Interestingly, the extract, at 200 mg/kg, retained the hepatic architecture ( Figure 4).

Docking-Based Virtual Screening
Molecular modeling has become integral in biomedical research, minimizing lab work and facilitating the discovery of the most probable molecular targets and/or signaling pathways [28]. To putatively determine the mode of action of the H. hemistemon aerial-part extract, we subjected the structures of all annotated compounds to molecular inverse docking experiments using the idTarget platform (http://idtarget.rcas.sinica.edu.tw accessed on 1 October 2022) [29]. We found lactate dehydrogenase (PDB ID: 4ND1) as a probable target for gallic acid, hydroxybenzoic acid, and methyl gallate with affinity scores of −9.35, −8.89, and −8.73, respectively. As a validation step, the whole annotated structures were then re-docked into the enzyme's active site using AutoDock Vina [30]. From the docked structures, quinic acid, hydroxybenzoic acid, gallic acid, and methyl gallate relatively better scores compared co-crystalized inhibitor (oxamic acid, Table 4). They also showed several interactions such as the co-crystalized inhibitor oxamic acid, including H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195 ( Figure 5). This docking-based study tentatively highlighted the potential of benzoic acid derivatives as C. parvum-derived lactate dehydrogenase inhibitors.
Following C. parvum oocyst induction, several deleterious pathological changes in the small intestine were observed. These included a marked broadening of the villi with a decreased ratio of villous height to crypt length, a dense infiltration of mononuclear inflammatory cells inside the villous core, villous tip-region degeneration, and high mucin production. Cryptosporidium oocysts were also present along the villi brush border and in the intestinal lumen as oval-to-rounded bodies. NTZ (the reference drug) and the extract, at the two dose levels, attenuated the aforementioned effects of the extract, and at 200 mg/kg, retained a nearly normal villous pattern (Figure 3).

Effects of H. Hemistemon Aerial-Part Extract on the Liver
Similar to the small intestine, the induction of C. parvum oocysts induced caused pathological changes to the liver tissue. These included hepatocyte degeneration and focal infiltration with mononuclear cells. The reference drug (NTZ) and the extract, at 100 mg/kg, moderately improved the above-mentioned effects. Interestingly, the extract, at 200 mg/kg, retained the hepatic architecture ( Figure 4).

Docking-Based Virtual Screening
Molecular modeling has become integral in biomedical research, minimizing lab work and facilitating the discovery of the most probable molecular targets and/or signaling pathways [28]. To putatively determine the mode of action of the H. hemistemon aerialpart extract, we subjected the structures of all annotated compounds to molecular inverse docking experiments using the idTarget platform (http://idtarget.rcas.sinica.edu.tw accessed on 1 October 2022) [29]. We found lactate dehydrogenase (PDB ID: 4ND1) as a probable target for gallic acid, hydroxybenzoic acid, and methyl gallate with affinity scores of −9.35, −8.89, and −8.73, respectively. As a validation step, the whole annotated structures were then re-docked into the enzyme's active site using AutoDock Vina [30]. From the docked structures, quinic acid, hydroxybenzoic acid, gallic acid, and methyl gallate relatively better scores compared co-crystalized inhibitor (oxamic acid, Table 4). They also showed several interactions such as the co-crystalized inhibitor oxamic acid, including H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195 ( Figure  5). This docking-based study tentatively highlighted the potential of benzoic acid derivatives as C. parvum-derived lactate dehydrogenase inhibitors.

Discussion
Cryptosporidiosis represents a serious health issue that can cause life-threatening diarrhea in immunocompromised people [2]. The lack of effective cryptosporidiosis therapies and vaccinations had led to the quest for an effective and safe anticryptosporidiosis therapy, particularly for immunocompromised hosts [4]. H. hemistemon has been proven to have antifungal effects against Aspergillus niger and Candida albicans, and antibacterial properties against both Gram-positive bacteria such as Enterococcus faecalis, Staphylococcus aureus, and Bacillus subtilis and Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa [16].
In this study, Herniaria hemistemon (H. hemistemon), which belongs to the family Caryophyllaceae, was tested against cryptosporidiosis in immunocompromised mice. The administration of H. hemistemon (GIV (100 mg/kg) and GV (200 mg/kg)) following challenged infection resulted in marked reductions in Cryptosporidium oocyst shedding (reduction of 61% and 79%, respectively) related to the infected control group. Interestingly, the H. hemistemon extract, at a dose of 200 mg/kg, displayed better activities than the reference drug, Nitazoxanide (reduction of 79% versus 67% in favor of the extract).
Additionally, a histopathological examination of the small intestine of GII (immunosuppressed and infected) revealed a deleterious effect on the structure of the intestinal mucosa compared to GI (immunosuppressed and not infected). Villous shortening and atrophy were observed, as well as a reduction in the ratio of villous height to mucosal ulceration, goblet cell depletion, crypt length, and infiltration of the lamina propria with inflammatory cells, primarily eosinophils and lymphocytes, as well as a diffuse loss of the brush border microvillous surface area. This was in line with several earlier studies [8,9]. Interestingly, these deleterious changes were inversed in GV (immunosuppressed, infected, and treated with H. hemistemon extract at a dose of 200 mg/kg). Noteworthily, H. hemistemon attenuated the adverse effects of cryptosporidiosis on the liver, where it retained a normal structure at the high dose of the extract.
The H. hemistemon aerial-part extract exhibited promising antioxidant activities, as well. The demonstrated activities (anticryptosporidiosis and antioxidant) could be explained by the presence of the 37 phytocompounds, detected in the extract, with reported antiparasitic and antioxidant properties. This was also supported by the docking study, which suggested that benzoic acid derivatives in the H. hemistemon extract (i.e., gallic acid, hydroxybenzoic acid, and methyl gallate) could act as C. parvum lactate dehydrogenase inhibitors and may serve as promising starting skeletons for the further development of more potent anti-C. parvum agents. Additionally, previous reports revealed that some identified compounds in the H. hemistemon extract showed potent antiparasitic effects against various types of parasites. For instance, rutin was evaluated in vitro for its ability to inhibit the replication of C. parvum [31]. Moreover, caffeoylquinic acids exhibited anthelmintic effects against Entamoeba histolytica [32]. Peña-Espinoza and his co-workers reported the antiparasitic effects of hydroxycinnamic acids, quercetin, and kaempferol derivatives against gastrointestinal parasites [33]. Furthermore, it was reported that polyphenolic compounds could be responsible for the anticryptosporidial activity of plant extracts through numerous modes of action, including interfering with essential parasite enzymes [34].
In general, the positive therapeutic effects of Herniaria in this study are consistent with previous reports that documented the medicinal use of the genus Herniaria as antispasmodic, antihypertensive, lithophytic, and diuretic agents. They demonstrated antioxidant and antimicrobial activities, as well [14]. Additionally, similar results were observed from several polyphenolic-rich extracts. These included pomegranate peels (red and white), Egyptian propolis, Verbena Officinalis, Ficus carica, and Olea europaea extracts; they significantly diminished the C. parvum oocyst count in infected mice with comparable activities to the reference drug, NTZ, and displayed a potential improvement in the shape and structure of the villi of ileal sections, as well [8,9,35].

Conclusions
The current work annotated 37 secondary metabolites from the H. hemistemon aerialpart extract via LC-ESI-MS/MS. It also suggested H. hemistemon as a promising anticryptosporidiosis agent, since it markedly decreased the C. parvum oocyst count in infected mice with better activities at 200 mg/kg than the reference drug, NTZ, and noticeably improved and retained the normal structure of the small intestine and liver tissue. To sum up, H. hemistemon could be considered as a potential candidate for further evaluation as an antiparasitic agent, food supplement, and livestock feed. Further experiments are needed to determine its toxicity and explore its individual components as well the involved mechanisms.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files.