Research Article

Efficacy of Chamomile Extract Loaded Chitosan Nanoparticles as a Therapeutic Agent against Entamoeba histolytica

Noor Adel Jasim1, Samah Ali Al-Lateef1, Hussam H. Kadhum2, Laith A. Yaaqoob3, Ahmed Flayyih Hasan1,4*

1Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq; 2Department of Pharmacy and Toxicology, College of Pharmacy, Al-Farabi University, Baghdad, Iraq; 3Department of Biotechnology, College of Science, University of Baghdad, Baghdad, Iraq; 4Department of Medical Laboratory Techniques, College of Health and Medical Technology, Al-Farabi University, Baghdad, Iraq.

Received | January 04, 2026; Accepted | February 16, 2026; Published | April 22, 2026

*Correspondence | Ahmed Flayyih Hasan, Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq; Email: [email protected]

Citation | Jasim NA, Al-Lateef SA, Kadhum HH, Yaaqoob LA, Hasan AF (2026). Efficacy of chamomile extract loaded chitosan nanoparticles as a therapeutic agent against Entamoeba histolytica. J. Anim. Health Prod. 14(2): 648-653.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.2.648.653

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

A common cause of health problems in developing countries is the protozoan parasite Entamoeba histolytica (E. histolytica), the causative agent of amoebic dysentery. Intestinal infections caused by E. histolytica can lead to bloody and mucus-containing diarrhea, with acute amoebic dysentery being the most prevalent clinical manifestation. E. histolytica has two life cycle stages: trophozoites and cysts. In the large intestine, trophozoites may exist asymptomatically; however, they can invade the intestinal mucosa and disseminate via the bloodstream, resulting in amoebic colitis. Severe complications include intense abdominal pain, amoebic liver abscess, pleuropulmonary abscess, and extraintestinal lesions, particularly in the liver (Kantor et al., 2018). The most commonly used antimicrobial agents for the treatment of amebiasis are 5-nitroheterocyclic drugs, especially metronidazole and, more recently, nitazoxanide (Farthing, 2005).

Herbal plants have recently gained increasing attention for the treatment of intestinal disorders, including infectious diseases (Kadhim and Hasan, 2025; Sandri et al., 2026). Matricaria chamomilla (MC), commonly known as German chamomile, is one of the most widely used medicinal herbs. It is distributed in various regions, including Asia, Australia, and South America. Two species Chamaemelum nobile and Matricaria chamomilla are commonly used in herbal medicine. Chamomile contains several bioactive constituents, including α-bisabolol, terpenoids, polysaccharides, essential oils, minerals, fatty acids, flavonoids, and phenolic compounds. It exhibits numerous pharmacological properties, such as antibacterial, anti-inflammatory, antifungal, anti-ulcer, antispasmodic, antiviral, and sedative effects. Internally, chamomile is used to aid digestion and treat diarrhea, nausea, urinary tract disorders, and dysmenorrhea. Externally, it is applied to promote wound healing and treat skin rashes, infections (such as shingles and boils), sore throat, hemorrhoids, and eye inflammation (Ali et al., 2013).

Chitosan, a polysaccharide partially deacetylated from chitin, has been widely utilized in medical applications for many years (Hadi et al., 2024). It is an important material for nanoparticle synthesis due to its biodegradability and non-toxic nature. Numerous studies have investigated chitosan nanoparticles (NPs) for the treatment of Giardia lamblia infection (Said et al., 2012). Additionally, chitosan NPs have been shown to enhance the efficacy of ivermectin as an antifilarial therapy (Ali et al., 2013).

The present study focuses on evaluating the ability of chamomile extract and chitosan nanoparticles to promote regeneration of small intestinal tissue following E. histolytica infection in a mouse model.

Materials and Methods

Plant extraction

Flowers of M. chamomilla were obtained locally from various locations in Kirkuk. The dried flowers were ground into a fine powder. Fifty grams of the powdered plant material were mixed with 500 mL of distilled water, and the mixture was concentrated to dryness using a rotary evaporator. The resulting extract was stored at 4 °C until use (Hajjaj et al., 2013).

Synthesis of chitosan nanoparticles

Nanomaterials were prepared using a chemical approach via the sol–gel process (Jasim et al., 2021). Chitosan was dissolved in deionized distilled water and subjected to ultrasonic treatment for 30 minutes. The pH of the solution was adjusted to 12 using 1 N NaOH, and the mixture was stirred with a magnetic stirrer for one hour at room temperature. The pH was then adjusted to 4 using 1 N HCl and stirred for an additional hour at ambient temperature. Finally, the pH was adjusted to 7 using 1 N HCl and stirred for 60 minutes at room temperature. The solution was subsequently centrifuged, and the precipitate was collected for further use.

Characterization of prepared nanoparticles

Atomic force microscopy (AFM) was used to generate two- and three-dimensional surface topography of the nanoparticles (Lyles et al., 2013). The Fourier-transform infrared (FT-IR) spectra of the chitosan nanoparticles were analyzed after drying the samples overnight in an incubator (Augustine et al., 2005).

Collection of stool sample

Diarrheal stool samples were collected from infected individuals during the study. The specimens were examined microscopically using a direct wet saline smear. Fresh samples were preserved in 2.5% potassium dichromate solution at 4 °C until use. The fecal material was inoculated, suspended, and subsequently centrifuged. The sediment was washed three times and resuspended in phosphate-buffered saline (PBS, pH 7.4) containing the antibacterial agents penicillin and streptomycin. Approximately 10³ viable cysts were orally administered to mice to induce acute infection (Hamad, 2021).

Experimental design

Twenty-five male Swiss white mice, aged 4–6 weeks and weighing 18–22 g, were obtained from the Iraqi Center for Cancer Research. The animals were housed in plastic cages, provided with a standard diet from the same source, and given sterile water in special bottles under controlled temperature and ventilation conditions. The mice were divided into five groups, each containing five animals.

The first four groups were orally inoculated with 0.1 mL of prepared Entamoeba inoculum using a micropipette, and fecal samples were examined daily to confirm the presence of the parasite. The fifth group was not infected and served as a healthy control to compare with infected tissues.

• Group 1 (Positive control): Mice received 0.1 mL of Entamoeba inoculum orally.
• Group 2: Mice received chamomile extract at a dose of 50 mg/kg orally for 14 consecutive days, administered once daily.
• Group 3: Mice received chitosan nanoparticles at a dose of 50 mg/kg orally for 14 consecutive days, administered once daily.
• Group 4: Mice received chitosan nanoparticles loaded with chamomile extract orally for 14 consecutive days, administered once daily.

Histopathological examination

Histological samples of the small intestine were collected from both treated and untreated mice at the end of the experiment and placed in sterile containers containing 10% formalin for preservation. The tissues were then processed for histological examination using the hematoxylin and eosin (H & E) staining method. Briefly, samples were dehydrated in a graded series of alcohols, cleared in xylene, embedded in paraffin, and sectioned at 5 µm thickness. The sections were mounted on glass slides, stained with hematoxylin to visualize nuclei, counterstained with eosin to highlight cytoplasm and extracellular matrix, and examined under a light microscope to assess intestinal tissue morphology and pathological changes.

Result and Discussions

Characterization of chitosan nanoparticles

Atomic force microscopy

Atomic Force Microscopy (AFM) was used to examine the surface morphology and topography of the nanoparticles (Figure 1). This technique provides two- and three-dimensional images of nanoparticle surfaces at the atomic level (Fadhil et al., 2015), allowing estimation of the average particle diameter at the nanoscale. Chitosan nanoparticles prepared using the sol–gel method were analyzed using AFM (Table 1). Careful surface analysis is required, as factors such as contamination can influence the results (Figure 2).

 

 

 

Fourier transform infrared spectroscopy

Fourier-Transform Infrared Spectroscopy (FTIR) was used to identify the main functional groups involved in the synthesis and capping of the nanoparticles. Spectra were recorded in the range of 400–4000 cm⁻¹ to detect chemical bonds and functional groups in the compounds. The FTIR spectra of chamomile extract and chitosan nanoparticles loaded with chamomile extract are presented in Table 2 and Figure 3.

 

 

For the chitosan nanoparticles with chamomile extract, peaks at 2978.21–2904.06 cm⁻¹ corresponded to O–H stretching, indicating the presence of alcohol groups. A broad peak at 1597.40 cm⁻¹ was attributed to N–H bending and C=C stretching, representing amine and alkene groups. Peaks at 1400.74 cm⁻¹ were associated with C–H bending of alkanes, while peaks at 1251.49 cm⁻¹ and 1054.71 cm⁻¹ indicated C–O stretching, characteristic of alcohols, carboxylic acids, esters, and ethers. Peaks observed at 614.43–438.50 cm⁻¹ corresponded to C–Br stretching, representing alkyl halides.

Similarly, the FTIR spectrum of chamomile extract showed a strong peak at 3334.19 cm⁻¹, corresponding to C=C stretching of alkenes. Peaks at 2983.12–2905.06 cm⁻¹ indicated O–H stretching of alcohol molecules, while a broad peak at 1635.99 cm⁻¹ represented N–H bending and C=C stretching (amines and alkenes). Peaks at 1452.55–1400.69 cm⁻¹ were associated with C–H bending of alkanes, and peaks at 1250.69 cm⁻¹ and 1056.53 cm⁻¹ corresponded to C–O stretching of alcohols, carboxylic acids, esters, and ethers. A peak at 588.52 cm⁻¹ was observed for C–Br stretching, indicating alkyl halides.

These results confirm the presence of characteristic functional groups, demonstrating the successful incorporation of chamomile extract into the chitosan nanoparticles (Figure 4).

 

Parasitological results

On day four post-infection, mice in the experimental groups began to excrete Entamoeba cysts in their stool. Several clinical signs were observed starting four days before the end of the experiment, including hair loss and reduced activity.

As shown in Figure 5, histological examination revealed that mice treated with chamomile extract showed partial improvement in intestinal morphology, including moderate to mild villous thickening and slight mucoid degeneration, compared with the infected untreated group. These findings are consistent with Khayyal et al. (2019), who reported protective effects of chamomile extract against tissue damage. Chamomile extract has been shown to preserve most histological structures by modulating inflammatory markers, oxidative stress, and apoptosis (Sabatke et al., 2022). Polysaccharides in chamomile, when combined with antiparasitic drugs, may inhibit parasite attachment to intestinal cells and enhance drug efficacy, as reported in studies on Giardia lamblia treatment.

 

Mice treated with chitosan nanoparticles displayed mild mucosal healing, normal villous thickness, and preserved cytoarchitecture of villus cells. These results align with Ahmed et al. (2016), who demonstrated that chitosan nanoparticles exert substantial effects on local gastrointestinal disorders and intestinal disinfection, and Wardani et al. (2018), who observed improved epithelial cell recovery in the gastrointestinal tract following nanoparticle treatment. However, Hu et al. (2011) reported that chitosan nanoparticles could induce cellular oxidative stress, increasing reactive oxygen species and causing cytotoxicity in mucosal epithelial cells.

Notably, mice treated with chitosan nanoparticles loaded with chamomile extract exhibited greater histological improvement than those treated with chamomile extract alone. Sections of the small intestine from this group showed restoration of normal duodenal mucosa, normal villous thickness, and intact villus cell cytoarchitecture. This enhanced effect is supported by previous studies, such as El-Gendy et al. (2021), who reported that loading metronidazole onto chitosan nanoparticles improved the therapeutic efficacy of both chitosan nanoparticles and the drug in hamsters experimentally infected with Giardia.

Overall, these results indicate that the combination of chitosan nanoparticles and chamomile extract provides superior protection and regenerative effects on intestinal tissue following E. histolytica infection compared with either treatment alone.

Conclusion

In summary, the combined therapy was more effective than the single treatments, as mice treated with chamomile extract loaded onto chitosan nanoparticles exhibited pronounced repair of the intestinal mucosa.

Acknowledgement

Thanks to the authors for their cooperation and support in completing the manuscript.

Novelty Statement

Our study has shown that chitosan nanoparticles loaded with chamomile extract have high therapeutic and preventive efficacy against amoebic pathogens in rats.

Author’s Contribution

NAJ,SAA:.Writing and Methodology.HHK, LAY:.Writing, Statistics, Analysis.AFH:.Corresponding Author and Reviewer Responses.

Generative AI and AI-assisted technology statement

The authors pledge not to use artificial intelligence tools in the article.

Ethical approval

Ethical approval to conduct this study and to use animals in the experiments was obtained from the Biotechnology Research Center, Al-Nahrain University.

Conflict of interest

The authors have declared no conflict of interest.

References

Ahmed TA, Aljaeid BM (2016). Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery. Drug design, development and therapy, pp. 483-507. https://doi.org/10.2147/DDDT.S99651

Ali AA, Alattar SA (٢٠١٨). Study the protective effect of Matricaria chamomilla flower extract against the toxicity of Entamoeba histolytica induces liver and renal dysfunctions in adult albino male rats. Iraqi J. Sci., pp. 832-838.

Ali M, Afzal M, Verma M, Misra-Bhattacharya S, Ahmad FJ, Dinda A (2013). Improved antifilarial activity of ivermectin in chitosan–alginate nanoparticles against human lymphatic filarial parasite, Brugia malayi. Parasitol. Res., 112(8): 2933-2943. https://doi.org/10.1007/s00436-013-3466-4

Augustine SK, Bhavsar SP, Kapadnis BP (2005). A non-polyene antifungal antibiotic from Streptomyces albidoflavus PU 23. J. Biosci., 30(2): 201-211. https://doi.org/10.1007/BF02703700

El-Gendy AML, Mohammed MAA, Ghallab MMI, Aziz MOA, Ibrahim SM (2021). Therapeutic effect of chitosan nanoparticles and metronidazole in treatment of experimentally giardiasis infected hamsters. Iran. J. Parasitol., 16(1): 32. https://doi.org/10.18502/ijpa.v16i1.5509

Fadhil FA, Hadi IH (2015). Preparation and characterization of zinc oxide nanoparticles by laser ablation of zinc in isopropanol. Eng. Technol. J., 33(5): 791-798. https://doi.org/10.30684/etj.33.5B.4

Farthing MJ (٢٠05). Treatment options for the eradication of intestinal protozoa. Nat. Clin. Pract. Gastroenterol. Hepatol., 3(8): 436-445. https://doi.org/10.1038/ncpgasthep0557

Hadi SA, Gandahi JA, Shah MG, Masood S, Gandahi NS (2024). Protective response of zinc oxide nanoparticle with chitosan oligosaccharide on intestinal integrity, goblet cell count, and meat quality of broiler chicken under heat stress. J. Anim. Health Prod., 12(4): 621-629. https://doi.org/10.17582/journal.jahp/2024/12.4.621.629

Hajjaj G, Amina B, Mouna T, Yahia C. and Amina Z (2013). Anti-inflammatory evaluation of aqueous extract of Matricaria chamomilla L (asteraceae) in experimental animal models from Morocco. J. Pharma. Res. 2(5): 1218-1228.

Hamad HK (2021). Evaluation of paromomycin loaded chitosan nanoparticles and oxidative stress activities against Entamoeba histolytica. J. Phys. Conf. Ser., IOP Publishing. 2114(1): 012085. https://doi.org/10.1088/1742-6596/2114/1/012085

Hu YL, Qi W, Han F, Shao JZ, Gao JQ (2011). Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model. Int. J. Nanomed., pp. 3351-3359. https://doi.org/10.2147/IJN.S25853

Jasim NA, Hamad HK (2021). Efficacy of paromomycin-loaded chitosan nanoparticles as therapeutic agent against Giardia lamblia. Group, 2, 1.

Kadhim HJ, Hasan IJ (2025). Investigating the Impact of dietary supplementation on mRNA expression of growth-related peptides and gut health in broilers. J. Anim. Health Prod., 13(1). https://doi.org/10.17582/journal.jahp/2025/13.1.51.58

Kantor M, Anarella A, Andrea E, Alan S, Jose T, Jose G, Robert H, Christopher O (2018). Entamoeba histolytica: updates in clinical manifestation, pathogenesis, and vaccine development. Canad. J. Gastrol. Hep. 1-6: 2018.

Khayyal MT, Kreuter MH, Kemmler M, Altmann P, Abdel-Naby DH, El-Ghazaly MA (2019). Effect of a chamomile extract in protecting against radiation-induced intestinal mucositis. Phytother. Res., 33(3): 728-736. https://doi.org/10.1002/ptr.6263

Lyles VD, Serem WK, Yu JJ, Garno JC (2013). Surface characterization using atomic force microscopy (AFM) in liquid environments. In: (eds. G. Bracco and B. Holst). Surface Science Techniques. Germany: Springer; pp. 599-620. https://doi.org/10.1007/978-3-642-34243-1_20

Sabatke B, Chaves PFP, Cordeiro LM, Ramirez MI (2022). Synergistic effect of polysaccharides from chamomile tea with nitazoxanide increases treatment efficacy against Giardia intestinalis. Life, 12(12): 2091. https://doi.org/10.3390/life12122091

Said DE, Elsamad LM, Gohar YM (2012). Validity of silver, chitosan, and curcumin nanoparticles as anti-Giardia agents. Parasitol. Res., 111: 545-554. https://doi.org/10.1007/s00436-012-2866-1

Sandri D, Fajri F, Agasi SY, Maulana F, Nurmawan AL, Yaman HI (2026). Effects of different levels of Cassia alata L. leaf extract on the production performance of laying quail. J. Anim. Health Prod., 14(1): 231-237. https://doi.org/10.17582/journal.jahp/2026/14.1.231.237

Wardani G, Eraiko K, Sudjarwo SA (2018). Protective activity of chitosan nanoparticle against cadmium chloride induced gastric toxicity in rat. J. Young Pharma., 10(3): 303. https://doi.org/10.5530/jyp.2018.10.67