Pharmaceutical Potential of Remedial Plants and Helminths for Treating Inflammatory Bowel Disease

Research is increasingly revealing that inflammation significantly contributes to various diseases, particularly inflammatory bowel disease (IBD). IBD is a major medical challenge due to its chronic nature, affecting at least one in a thousand individuals in many Western countries, with rising incidence in developing nations. Historically, indigenous people have used natural products to treat ailments, including IBD. Ethnobotanically guided studies have shown that plant-derived extracts and compounds effectively modulate immune responses and reduce inflammation. Similarly, helminths and their products offer unique mechanisms to modulate host immunity and alleviate inflammatory responses. This review explored the pharmaceutical potential of Aboriginal remedial plants and helminths for treating IBD, emphasizing recent advances in discovering anti-inflammatory small-molecule drug leads. The literature from Scopus, MEDLINE Ovid, PubMed, Google Scholar, and Web of Science was retrieved using keywords such as natural product, small molecule, cytokines, remedial plants, and helminths. This review identified 55 important Aboriginal medicinal plants and 9 helminth species that have been studied for their anti-inflammatory properties using animal models and in vitro cell assays. For example, curcumin, berberine, and triptolide, which have been isolated from plants; and the excretory-secretory products and their protein, which have been collected from helminths, have demonstrated anti-inflammatory activity with lower toxicity and fewer side effects. High-throughput screening, molecular docking, artificial intelligence, and machine learning have been engaged in compound identification, while clustered regularly interspaced short palindromic repeats (CRISPR) gene editing and RNA sequencing have been employed to understand molecular interactions and regulations. While there is potential for pharmaceutical application of Aboriginal medicinal plants and gastrointestinal parasites in treating IBD, there is an urgent need to qualify these plant and helminth therapies through reproducible clinical and mechanistic studies.


Introduction
Inflammation is a defense response to injuries, chemicals, or microorganisms, involving by tissue damage, as well as metabolic and microcirculation disorders.It involves the release of mediators such as nitric oxide, prostaglandins, and cytokines, which are essential for removing harmful stimuli, restoring normal physiology, and controlling inflammation [1].Different cytokines, markedly, interleukin-1 (IL-1) and tumor necrosis factor (TNF), regulate inflammation.These chemical mediators, primarily triggered by bacterial lipopolysaccharide [2], are released by monocytes, macrophages, and other cells [3].Antiinflammatory drugs have been used to address severe inflammation and pain.Presently, the United States Food and Drug Administration (USFDA) has approved nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin, ibuprofen, cyclooxygenase-2 enzyme (COX-2) inhibitors like celecoxib and naproxen, for managing inflammation.However, these NSAIDs are linked to side effects on various bodily systems [4].For instance, Pharmaceuticals 2024, 17, 819 2 of 33 the long-term use of NSAIDs increases the risk of ulcers and complications in the upper gastrointestinal tract by about four times, while in the lower gastrointestinal tract, they cause mucosal ulcers or erosions and alter the diversity of microbiota, thereby modulating their toxicity [5].
Among the various inflammatory disorders, the chronic-relapsing condition of IBD, which mainly affects the gastrointestinal tract, has become a global health burden [5].The etiology and pathogenesis of IBD are multifaceted, and it is difficult to determine a single causative agent [6].Overall, these disorders are marked by persistent inflammation, often resulting in complications such as a heightened likelihood of hospitalization, surgical intervention, colorectal cancer, and disability.Acknowledging the chronic and advancing characteristics of IBD, commencing effective treatment during the early phases of the disease is crucial to minimize relapses and prevent complications [7].In this view, NSAIDs such as 5-aminosalicylic acids (5-ASA), corticosteroids, and immunomodulators such as 6-mercaptopurine and azathioprine are used for managing IBD [7].These medications can induce and sustain remission [8]; unfortunately, they are linked to numerous side effects [9,10].For instance, 5-ASA and corticosteroids, often prescribed for patients with mild-to-moderate ulcerative colitis, have exhibited only moderate efficacy.Long-term medication use is associated with side effects such as stunted growth, depression, hypertension, and osteoporosis [11].
Similarly, immunomodulators such as 6-mercaptopurine and azathioprine are also associated with pancreatic inflammation, decreased hematopoiesis, and liver toxicity [12].Among the biological agents or biologics, infliximab is commonly utilized for IBD treatment.However, biologics are expensive and associated with adverse infusion lupus-like syndrome and infections, including sepsis [11,13].Historically, natural products (NPs) have been a vital source of structurally diverse and pharmacologically important antiinflammatory small molecules (SMs, <10 kDa molecular weight) [14][15][16][17][18].For instance, in the period spanning from 1981 to 2019, the USFDA approved 33.5% (1394 SMs) of the small-molecule (SM) drugs derived from natural products, underscoring the significant role of NPs in shaping the global healthcare landscape [18].A total of 1881 new drugs were approved, which included 71 drugs sourced from unaltered natural products and 14 from botanicals.Among the 1602 new chemical entities, 53 were identified to exhibit anti-inflammatory properties [18].
Overall, the treatment of inflammatory disorders such as IBD has seen a transition from focusing on symptomatic control to emphasizing more objective endpoints [19].This has resulted in the development of large-molecule biologics and synthetic small-molecule drugs (SMDs).However, these drugs have efficacy, safety, cost, and management time frame limitations.Considering this, more cost-effective oral drugs that offer improved efficacy and tolerability are in demand.Consequently, there is rising interest in developing new, targeted anti-inflammatory SMDs for IBD and other immune-mediated inflammatory conditions.Due to their low molecular weight, SMDs can quickly diffuse through cell membranes.The variance in size notably influences aspects such as the route of administration, target site, pharmacokinetics, antigenicity, and drug interactions.When administered orally, these drugs resist gastric degradation and quickly enter into the systemic circulation.The short half-life of SMDs can prove advantageous, especially in settings where instant drug elimination occurs, such as in infections, during surgical procedures, or during pregnancy.Hence, herbal and natural product compounds have been screened for therapeutic potential in anti-inflammatory-related diseases due to their chemical diversity, lower toxicity, and costeffectiveness.However, biocompatibility and toxicity concerns have impeded clinical trial progress [20].To address issues like poor bioavailability, solubility, and targeted delivery, advanced drug delivery systems such as nanoparticles, bioadhesive microspheres, chitosanbased hydrogels, liposomes, and phytosomes are being developed [21][22][23][24][25][26].Nanoparticles can encapsulate or attach therapeutic drugs, ensuring precise targeting and controlled release, enhancing drug delivery and efficacy.FDA-approved nanoparticle-based therapies, such as liposomes and micelles, improve drug delivery by preventing gastrointestinal Pharmaceuticals 2024, 17, 819 3 of 33 degradation and enhancing the delivery of poorly soluble drugs [27][28][29].Similarly, the phytophospholipid complex technique converts water-soluble herbal extracts into lipidcompatible complexes, improving absorption and protecting compounds from degradation, thus enhancing therapeutic efficacy [30].Examples of nanoformulated phytochemicals include silymarin, hypericin, and curcumin [31].
While general anti-inflammatory drugs have been reviewed elsewhere in more detail, we examined emerging the SMDs with anti-inflammatory properties derived from natural sources for treating IBD.Specifically, we explored anti-inflammatory SMs in medicinal plants and gastrointestinal parasites, offering insights into their potential future roles and impact.Databases such as Scopus, MEDLINE Ovid, PubMed, Google Scholar and Web of Science were queried using keywords like 'Aboriginal remedial plants', 'helminths therapy', and 'anti-inflammatory small molecules' to uncover the literature discussing the biological activities, phytochemistry, and ethnomedical applications of both Aboriginal plants and helminths against inflammatory-related disorders.

Plant-Derived Anti-Inflammatory Compounds
Plants have been a healing source since time immemorial [32].Since their chemical analysis in the 19th century, bioactive compounds, referred to as phytochemicals in plants, animals, and living organisms, which influence physiological processes and have therapeutic potential and offer health benefits [33,34], have been crucial in advancing drug development [35].Nearly 150,000 plant species, including medicinal plants, have been studied, with numerous harboring valuable therapeutic compounds, particularly anti-inflammatory compounds [36,37].For example, the Eucalyptus and Melaleuca species are herbal remedies used by Australian Aboriginal people to treat inflammatory ailments such as muscular aches, sores, internal pains, and painful joints [38].Likewise, Bhutanese traditional medicine involves the use of Aconitum laciniatum Stapf and the entire plant of Aconitum orochryseum Stapf to treat chronic parasitic and microbial infections, inflammatory diseases, and bilious fever [39].Considering these traditional practices, researchers isolated the compound 14-O-acetylneoline, which exhibited anticolitis activity in TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis [40,41].This practice emphasizes the long-standing recognition of the anti-inflammatory properties of NPs.Patients with IBD often turn to botanicals due to their perceived safety and effectiveness.Popular herbal remedies include Tripterygium wilfordii Hook.f., Plantago ovata Phil., Artemisia absinthium L., Aloe vera L., Curcuma longa L., Boswellia serrata Roxb., and Cannabis sativa L. [42].A review of 27 studies and 1874 patients with IBD found seven herbal remedies were beneficial for ulcerative colitis (UC) and four induced remissions in cystic fibrosis diseases [42].The oral administration of Aloe vera gel and Plantago ovata Phil.seeds showed promising antiinflammatory results in UC [42].Overall, ethnopharmacology insight has been a guiding force in the biodiscovery of anti-inflammatory SMs from NPs.
Inflammatory mediators play a crucial role in modulating IBD through various mechanisms.For example, nitric oxide (NO) is a signaling molecule produced by inducible nitric oxide synthase (iNOS) during inflammation and plays a significant role in vasodilation, modulation of blood flow, and immune defense.However, excess NO production can contribute to inflammation and tissue damage [43].Both crude extracts and compounds isolated from natural sources exhibit anti-inflammatory effects by regulating key cytokines [44,45].Pro-inflammatory cytokines like IL-6 promote the immune response by stimulating acute-phase protein production and influencing B-cell differentiation, while TNF and IL-1β, produced mainly by macrophages, mediate inflammation by promoting leukocyte recruitment, fever, and apoptosis in infected cells.Conversely, anti-inflammatory cytokines such as IL-10, IL-4, and IL-13, released by T helper 2 (Th2) cells, inhibit proinflammatory responses, modulating immune functions and inflammation [46].However, excess cytokines are associated with chronic inflammation and autoimmune diseases.
The crude extracts and a few isolated compounds from NPs showing anti-inflammatory activity in diverse colitis animal models are summarized in Table 1.Representative chemical structures are shown in Figure 1.
Similar work on A. caninum by Wangchuk et al. [188] showed the protection of mice from colitis by low-molecular-weight metabolites of somatic extracts, ESPs, and the latter when used to treat mice resulted in a significant reduction in inflammatory cytokines such as IL-23, TNF, and IL-1β.Similarly, different concentrations of the hexane-dichloromethaneacetonitrile somatic fraction of A. caninum exhibited notable reductions in TNF, IL-1β, IL-6, and monocyte chemoattractant protein-1 (MCP-1) production [188].Hence, helminth therapy is gaining attention in IBD treatment.For example, preliminary investigations into the positive impacts of helminths on IBD revealed that the oral administration of Trichuris muris whipworm eggs notably decreased TNBS-induced colitis in IL-10 −/− mice [189].Similarly, Trichuris suis ova showed promise in human trials, inducing remission in 21 out of 29 patients with active Crohn's disease (CD) [184].Hence, identifying and characterizing helminth molecules offers a unique opportunity to create nature-inspired, effective, safe, and minimally immunogenic drugs.
Wangchuk et al. [190] identified 54 SMs in the ESP of Trichuris muris and N. brasiliensis, of which 17 SMs had exhibited pharmacological activities in other studies.Similarly, in the case of Dipylidium caninum, 49 SMs were characterized by gas chromatography/mass spectrometry (GC-MS), with succinic acid as the chief constituent of its ESP [191].The study also highlighted that among the 35 polar metabolites, lactic acid, malic acid, methionine, glycerol, and fructose were previously reported to exhibit anti-inflammatory and proinflammatory activities [191].Furthermore, Wangchuk et al. [192] carried out the initial metabolomic and lipidomic examination of the infective third-stage filariform larvae of the human hookworm Necator americanus, utilizing liquid chromatography-mass spectrometry.The study unveiled 645 SMs, with 495 metabolites exclusive to the somatic tissue extract and 34 found solely in the ESP component.Of these, 45 SMs were identified as polar metabolites; 26 SMs had previously been documented for their anti-inflammatory properties [192].For example, in a study conducted to investigate the impact of L-arginine on the inflammatory response and casein expression, the findings indicated that arginine mitigated the LPS-induced production of inflammatory markers such as IL-1β, IL-6, TNF, and iNOS [193].Among the SMs identified, lactic acid, malic acid, methionine, glycerol, and fructose, sourced from various NPs, have demonstrated anti-inflammatory properties [194][195][196][197][198].However, the immunomodulatory properties of nonprotein SMs secreted or excreted by helminths remain relatively underexplored for medicinal applications [199].Therefore, based on these promising preliminary results, the somatic tissues and ESPs of helminths could be sources of anti-inflammatory SMs that can be used for treating IBD and other inflammatory conditions.The chemical structure of common anti-inflammatory SMs identified through metabolomic studies from Trichuris muris and Ancylostoma caninum are shown in Figure 2.
acid, methionine, glycerol, and fructose, sourced from various NPs, have demonstrated anti-inflammatory properties [194][195][196][197][198].However, the immunomodulatory properties of nonprotein SMs secreted or excreted by helminths remain relatively underexplored for medicinal applications [199].Therefore, based on these promising preliminary results, the somatic tissues and ESPs of helminths could be sources of anti-inflammatory SMs that can be used for treating IBD and other inflammatory conditions.The chemical structure of common anti-inflammatory SMs identified through metabolomic studies from Trichuris muris and Ancylostoma caninum are shown in Figure 2.

Anti-Inflammatory Agents in Clinical Trials
Several plant species and their allied small molecules are being examined for their potential in treating IBD, specifically UC and CD.The small molecule epigallocatechin-3gallate derived from Camellia sinensis is being investigated for its safety in patients with mild to moderately active UC during clinical remission and maintenance therapy, with the study currently in phase II.Another anti-inflammatory SM, curcumin, is also being investigated at various clinical phases.In pediatric patients with IBD, a phase I study is aiming to assess the tolerability of curcumin.In patients with UC, a phase I trial is investigated the effectiveness of a combined therapy involving curcumin and 5-ASA compared to that of 5-ASA alone (Table 3).
In contrast, a phase III trial is exploring the impact of combining curcumin with thiopurines to prevent postoperative recurrence.The bioactive compound berberine, isolated from Coptis chinensis, is the focus of a phase I study evaluating its safety in patients with UC in clinical remission and undergoing maintenance therapy.Finally, triptolide, reported as a bioactive compound in Tripterygium wilfordii, is being studied to assess its impact and safety in inducing remission in CD, comparing its efficacy with that of mesalamine, with ongoing trials in both phase II and III.Overall, studies underscore the potential of natural compounds in treating BD, offering insights into safety, efficacy, and therapeutic strategies at the various stages of clinical development (Table 3).
Following the positive tolerance and absence of side effects observed in the open trial involving patients with active UC and CD, researchers conducted clinical trials primarily using Trichuris suis, the pig whipworm, and Necator americanus, a hematophagous hookworm [169].In an open-label trial (phase 1) at the University of Iowa, seven patients with IBD received 2500 TSO, resulting in remission for six patients per the IBD Quality of Life Index [169,214].Subsequently, a randomized, double-blind, placebo-controlled trial (NCT01433471) involved 54 active UC patients treated with 2500 TSO or placebo every two weeks for 12 weeks.The study showed significant improvement in 43.3% of patients treated with TSO compared to 16.7% of placebo recipients [169,214].Similarly, a trial with 29 patients with active CD demonstrated a 79.3% decrease in the CD activity index (CDAI) and a 72.4% remission rate after 24 weeks of TSO treatment (Table 3).N. americanus was also tested in patients with CD, showing clinical improvement and remission in eight of nine patients after 20 weeks despite mild side effects.Additionally, helminth-derived products, such as P28 S-glutathione transferase (P28SGT), demonstrated promise, reducing disease activity and inflammatory markers in patients with CD in clinical trials (NCT02281916) [169] (Table 3).Considering the reported immunomodulatory properties, helminths show significant potential for treating inflammatory bowel disease (IBD).However, further research is needed to fully understand their mechanisms and ensure their safe and effective application in clinical settings.Assess the effectiveness in regulating immunologic and inflammatory markers in blood and tissue, as well as determining the occurrence of clinical recurrence using the disease activity index

Advances and Recent Approaches in SM Drug Lead Discovery
In drug discovery, including SMs, drug leads demand the physical screening of large chemical libraries for biological targets, which is common but time-consuming and expensive.As such, there have been substantial advancements in techniques for SM drug discovery, including high-throughput screening, structure-based drug design, virtual screening, and the refinement of lead compounds.High-throughput screening (HTS) analyzes over a million compounds biochemically, requiring substantial time and investment [215].To address this, virtual high-throughput screening was developed as a cost-effective computational method that is extensively applied in early drug discovery.It aims to identify novel, active small molecules by searching vast compound libraries, supporting the goals of HTS while reducing costs by evaluating only selected compounds for pharmacological activity [216,217].
Furthermore, molecular docking has proved to be a crucial approach in drug discovery in predicting the interaction patterns between proteins and small molecules and indicating the presence of bioactive compounds in natural products.For instance, GC-MS-identified bioactive ingredients and molecular docking against key targets revealed 3,5-dehydro-6-methoxy, ethyl iso-allocholate cholest-22-ene-21-ol, alpha-cadinol, and pivalate of Phyllanthus nivosus as promising compounds for UC drug development [218].Computational methods, particularly in silico discovery, enhance traditional drug development, ensuring sustainable and cost-effective drug discovery with increased efficacy [219].Modern techniques like pharmacophore modeling are also crucial for virtual screening, utilizing advanced compound databases and computing power to find small molecules of lead compounds [17].
To expedite the discovery of bioactive NPs in extracts, metabolomics data have been subjected to chemometric methods like multivariate data analysis, which correlate measured activity with nuclear magnetic resonance (NMR) and MS spectra signals, facilitating the tracking of active compounds in complex mixtures without additional bioassays [168,220].Recent advancements in analytical technologies, particularly higher-field NMR instruments and probe technology, have allowed for precise structure determination of NPs even from limited quantities (<10 µg) [221].Microcrystal electron diffraction, a cryo-electron-microscopy-based technique, is being increasingly applied for unambiguous structure determination of SMs in NP research [222].Bioactivity-guided fractionation techniques with NMR-based methods have recently been utilized for screening, identifying, and isolating anti-inflammatory bioactive compounds from natural products.For instance, a methanolic extract of Uraria crinite (L.) roots was screened to isolate the immunomodulatory isoflavone genistein.This compound exhibited immunomodulatory activity against producing proinflammatory cytokines (IL-6 and TNF) [223].

Challenges and Future Directions
Exploring the small anti-inflammatory molecules derived from remedial plants and helminths represents a promising frontier in pharmacopoeia research.These naturally sourced compounds often exhibit unique mechanisms of action, lower toxicity, and fewer side effects.However, challenges exist in identifying and isolating bioactive compounds from diverse natural products.The use of HTS, computational approaches like molecular docking and virtual screening, and integrating artificial intelligence (AI) and machine learning (ML) algorithms can enhance the accuracy and efficiency of identifying promising candidates from natural compound libraries.It is crucial to understand the molecular mechanisms through which these natural compounds exert their anti-inflammatory effects.Research should focus on investigating how these compounds interact with key inflammatory mediators.Advanced techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing and RNA sequencing can provide better insights into these interactions.Additionally, addressing challenges related to bioavailability and unfavorable pharmacokinetic profiles is essential.Techniques like nanoencapsulation, liposomal delivery, and phytosome tech-nology should be incorporated to improve small anti-inflammatory molecules' absorption, stability, and targeted delivery.Research has shown that helminths are therapeutic in treating inflammatory disorders due to their mechanisms evolved for modulating host immune responses.Further research should explore isolating and characterizing the small molecules from helminths to assess their therapeutic potential in inflammatory diseases.Techniques like proteomics and metabolomics can be instrumental in identifying bioactive helminth-derived compounds.Continued interdisciplinary research and collaboration will unlock the full therapeutic potential of these natural compounds.

Conclusions
In conclusion, our comprehensive analysis of the anti-inflammatory properties inherent in natural products, encompassing both crude extracts and isolated SMs, underscores their remarkable capacity to modulate a spectrum of inflammatory pathways.The antiinflammatory efficacy of natural products is manifested by inhibiting key inflammatory mediators such as NO, Cox-2, and proinflammatory cytokines, alongside the stimulation of anti-inflammatory cytokine production.While certain reported extracts or SMs operate through singular or dual mechanisms, others exhibit a more diverse array of actions.Furthermore, emerging research highlights the therapeutic promise of helminths and their secretory products (ESPs) in coordinating host immune responses and alleviating inflammatory maladies.Helminth-induced immune modulation fosters a milieu conducive to Th2-, IL10-, and TGFβ-dependent immune regulation, effectively attenuating Th1/Th17 inflammatory responses.Several helminth species, including Schistosoma mansoni, Hymenolepis diminuta, Trichinella spiralis, and Trichuris suis, demonstrate significant protective effects against colitis in preclinical models and human trials, highlighting their potential as therapeutic agents.
In light of these findings, NPs remain a fertile ground for identifying and discovering diverse structures of anti-inflammatory SMs.These structures can be either directly developed or serve as initial frameworks or scaffolds for further optimization into innovative anti-inflammatory drugs.Despite the challenges associated with drug development, such as high attrition rates and obstacles related to accessibility, sustainable supply, and intellectual property constraints, we remain optimistic that ongoing scientific and technological advancements will establish a robust foundation for NP-based drug discovery and harness the vast potential of nature's pharmacopeia.

Figure 1 .
Figure 1.Selected common chemical structures of plant-derived anti-inflammatory SMs (the halfmaximal inhibitory concentration (IC50) measures a small molecule's efficacy by indicating the amount needed to inhibit inflammation by 50%, thus reflecting its potency in treating inflammatoryrelated disorders) [167].

Figure 1 .
Figure 1.Selected common chemical structures of plant-derived anti-inflammatory SMs (the halfmaximal inhibitory concentration (IC50) measures a small molecule's efficacy by indicating the amount needed to inhibit inflammation by 50%, thus reflecting its potency in treating inflammatoryrelated disorders) [167].

Figure 2 .
Figure 2. Chemical structure of anti-inflammatory SMs identified through metabolomic studies of Trichuris muris and Ancylostoma caninum (common to both).

Figure 2 .
Figure 2. Chemical structure of anti-inflammatory SMs identified through metabolomic studies of Trichuris muris and Ancylostoma caninum (common to both).

Table 1 .
Anti-inflammatory activity of crude extract and bioassay-guided isolated SMs from Aboriginal medicinal plants.

Table 2 .
Anti-inflammatory activities of different stages of helminths and their components in various colitis animal models and cell lines.

Table 3 .
Nature-derived anti-inflammatory compounds and helminth products in clinical trials for treating inflammatory bowel diseases.