Gastrointestinal helminths are a big danger and cause signifi cant general health issues that can occasionally result in morbidity until death (Ranasinghe et al., 2023; Hassan et al., 2024). Although invisible to the human eye due to their small dimensions (250 μm to 12 mm in length and 15 to 35 μm in width), they pose a serious risk to human and animal health, which may result in growth retardation (Catani et al., 2023). Because helminth infections continuously contaminate the environment with their eggs and larvae, the persistent and asymptomatic character of the illness, especially in its early stages, maybe the cause of its neglect (Nalule et al., 2013). The soil-transmitted helminths, also known as intestinal nematodes, fi larial worms, schistosomes, and onchocerciasis worms are the most prevalent (Sayed et al., 2024). Contains several hosts for various phases. In addition, a major adaptive feature of a worm’s parasitism is its complex life cycle, which involves trophic transmission (Kasl et al., 2018). Certain soil-borne nematodes, including Strongyloides and hookworms, in their embryonic life cycle have two stages: the free-living stage, known as rhabditiform larvae, and the parasitic stage, known as fi lariform larvae, which may need a different host or habitat (Bruno et al., 2019). Gastrointestinal helminth and protozoa infections can affect a host’s capacity for survival and reproduction in two ways: directly, by causing pathological effects like tissue damage and blood loss; and indirectly, by lowering the host’s condition, making it more difficult for it to evade predators, and using up energy (Scantlebury et al., 2007).
First-stage larvae develop into third-stage larvae that bear to the colonic lumen and become adult larvae after a week of survival in the colon’s submucosa (Stepek et al., 2006). Mature females reside in the large intestine, where they spend forty to fifty days before ovulation. The stool pellets excreted at night are covered with a mucus layer by the eggs. It takes them 6 – 7 days at 24°C to become infectious, and they can survive for weeks outside of the host (Fox, 2015). Infections are usually asymptomatic. After the eaten eggs hatch, the larvae proceed to the middle colon, where they spend four to five days in crypts. They go on to the proximal colon after the host has been infected for around three weeks. The infection’s 10 – 12-day extended life cycle causes infestations to develop in slightly older mice; the worst infestation is predicted to occur 5 – 7 weeks after initial exposure (Omer et al., 2020). Significant variations in medication resistance and susceptibility between mouse strains of A. tetraptera are evident from observations of naturally occurring and experimental oxyuroid infections (Matté et al., 2023). Therefore, infection in lab mice cannot be avoided, and if treatment is not given, the infection will persist in the animals. Several researchers have demonstrated the influence of several parasites, including A. tetraptera, resulting in reduced immune-response, decreased hemoglobin, erythrocyte count, and serum albumin, which may negatively affect the results of experiments, even though infected animals cannot show clinical signs in immunocompetent experimental murine (Hernando et al., 2019; Hernando et al., 2025).
The use of deworming medications is a key component of helminth control efforts. The available synthetic medicines have side effects, and many parasites are resistant to medication, which means that the anthelmintic drugs now in use are not effective in controlling these organisms. (Oliveira et al., 2009). The growing interest in and awareness of the health benefits of natural resources, including herbs, spices, and fruits, has led to a heightened consumption of natural products as a safe and effective means of addressing illness and increasing overall health (Hegazy et al., 2023). Plants include various chemical compounds, such as polyphenols, flavonoids, and xanthones, many of which exhibit therapeutic properties or pharmacological advantages (Bagheri et al., 2024). In this context, pharmacological discovery has always come from nature, exploring potential plant-based antiparasitic medicines that are affordable, easily accessible, and promising substitutes. Thus, researching the anthelmintic properties of conventionally used plants could lead to more effective treatment alternatives (Ranasinghe et al., 2023). Plant secondary metabolites with biocidal activities, particularly essential oils (EOs), have been extensively studied during the past two decades (Fanelli et al., 2025).
C. verum bark is a plant species generally referred to as “sweet bay” or “bay laurel.” A fragrant angiosperm native laurel is a member of the camphor family (Lauraceae) (Paparella et al., 2022). In its native environment, this slow-growing plant can grow up to 60 feet (ca. 18 m) tall with a conical or pyramidal appearance. It is a member of the Lauraceae family of flowering plants (Paparella et al., 2022). What makes it unique is that it exhibits biological activity (Caputo et al., 2017). According to studies by Simić et al. (2004), Sırıken et al. (2018), Fernandez et al. (2020), Jemâa et al. (2012), and others, it has antifungal, antiviral, and antibacterial properties when combined with its extract and essential oils.
In this work, mice naturally infected with Aspiculuris tetraptera were used to test the anthelmintic efficiency of C. verum bark; this particular species of worm was also evaluated in vitro. It can therefore be applied to humans and other types of animals.
A taxonomist from the Faculty of Science (Plant Biology), University of King Saud, certified the botanical identity of C. verum bark collected from the spice markets in Riyadh. 400 g of bark was desiccated at 40 °C, it was milled in the parasitology laboratory using a grinder (Moline M-06, Italy). It was subsequently immersed for 24 hours at 4 °C in 70 % methanol. Thereafter, the resulting extract was dried and concentrated utilizing a rotary vacuum evaporator (Yamato RE300, Japan) at reduced pressure and 40 °C. The crude extracts were produced and subsequently kept at -20 °C until utilized in an experiment (Manikandan et al., 2008).
Using a Trace GC-ISQ Quantum mass spectrometer system (Thermo Scientific, Austin, TX, USA), the extract of C. verum bark was investigated. A flow rate of 1 mL/min was employed. A GC-MS was equipped with a TG-5MS column (30 m, 0.25 mm ID, 0.25 mm film thickness), and about 1 μL of the material was injected into it. Helium gas was the carrier at a constant flow rate of 1 mL/min. The observed mass spectra fell between 50 and 500 m/z. The temperature was initially set for 10 min at 50 °C, increased to 250 °C at a rate of 5 °C/min, held at 300 °C/2 min, and then held at 350 °C/10 min. The phytochemical components were identified by comparing the recorded mass spectra of each part with the information stored in earlier libraries, such as NIST, Adams, Terpenoids, and Volatile Organic Compounds libraries. Each component’s relative percentage was determined using the retention time index, and the average peak area was then contrasted with the total of all peak areas.
A total of 70 mice were examined with comparable weights and ages. Then, isolating each mouse in a cage to collect excrement, we found that 25 of the mice had A. tetraptera pinworm infections. Mice were anesthetized with CO2 and dissected. Mice’s intestines were removed and cleaned using a 0.9 % NaCl saline solution. From the infected mice’s colon and cecum, adult worms were removed. The active adults were separated into small plastic plates and distributed in 9 cm Petri dishes in about 7 cc of sterile physiological saline at 23 °C using a stereomicroscope (PX51, Olympus Co., Tokyo, Japan). The A. tetraptera was identified following the taxonomic characteristics (Omer et al., 2020). The worms that were chosen were healthy, motile, and had a standard microscopic structure. The experiment was then initiated as soon as the worms were gathered.
Aspiculus tetraptera worms were tested in vitro using six groups: control negative and positive, and contained four concentrations of C. verum bark extract (25, 50, 100, and 200 mg/mL). Next, ten mature worms that were actively moving and at room temperature were added to each Petri plate. As positive and negative controls, a saline solution and 10 mg/mL of albendazole were made. 10 worms were placed in each petri dish. Three replicates for each group. Following the course of therapy, observations were conducted by timing the worms’ deaths at 15, 30, 60, and 120 minutes. When a surgical needle is used to contact the worms’ body sections and the petri dish is rocked, the worms are deemed dead if they remain still for 30 seconds.
Larvae or adult worms were tested to evaluate the effectiveness of the extract by motility in vitro. Sensitivity analyses were conducted using the subculture method (Alimi et al., 2021). The percentage of inhibition and motility in treated worms was calculated (Murthy & Chatterjee, 1999) using a total of 30 worms (in replicates of 10 worms) for the motility assay per test concentration to assess the vitality of the treated worms. Under a microscope, parasite motility was assessed during test periods of exposure to all levels. Mortality percentage was recorded at 15, 30, 60, and 120 min after treatments, and it is expressed as a percentage of control. (Stepek et al., 2006). The parasite mortality rate for the extract was calculated according to the following equation:
Twenty-five adult C57BL/6 mice, weighing an average of 21 ± 2 g and naturally infected with A. tetraptera were utilized. The mice were between 11±2 weeks old. They were kept in pristine cages with 12-hour light-dark cycle and 20 °C ambient temperature as typical laboratory conditions. Animals were subjected to parasitological analyses using centrifugal sedimentation methods that were produced with salt water. The mice employed in the investigation were those that A. tetraptera naturally infected.
The mice that were naturally injured with A. tetraptera were split into six treated groups: all groups contained 5 mice: The 1st control (without treatment), the 2nd received 10 μg/mL of Albendazole (Veterinary Agriculture Products Company, Amman, Jordan (V.A.P.C.O.)) as a reference drug for 3 days, and the 3rd received 25 μg/mL, the 4th received 50 μg/mL, the 5th received 100 μg/mL, and the 6th received 200 μg/mL. The C. verum bark extract was administered for five days.
Following treatment, the mice were split into two stages: The first step was gathering each group’s excrement for three days following therapy. the eighth day following the five-day course of therapy. 1g of excrement was removed from each group after each mouse was put in its cage. Following an analysis of the worm count, three mice from each treatment were anesthetized, and the gut of each treated mouse was opened and cleaned with saline solution. We gathered and characterized parasites using a stereomicroscope. The worm burden in each group was compared.
Similarly, the feces were gathered and checked, and the worms were tallied in the second stage, which took place a week following the first. Following this, the remaining mice were all anatomically together. A comparison was made of the worm burden through the two stages and among the groups.
A completely randomized design (CRD) was employed for the bioassay analysis. In the adult bioassay of the C. verum bark extracts, a one-way analysis of variance (ANOVA) was performed using SAS 9.2 software (SAS, 2012). To compare mortality means, Duncan’s Multiple Range tests (P≤0.05) were employed. The presentation of all mortality data is as mean ± standard error (SE). The association between exposure time and death percentage was examined using a linear correlation.
The study complies with Saudi Arabia’s ethical guidelines for utilizing animals in research (Ethics Agreement ID: KSU-SE-21-86).
The GC-MS analysis and qualitative phytochemical investigation of C. verum bark extracts showed 20 biologically active phytochemical compounds that were distributed throughout various peak areas and retention times (Fig. 1). The chemicals with the highest quantity found in CNB extract by GC-MS were 2-methyl benzofuran, pentadecanoic acid 14-methyl ester, cinnamon aldehyde, (E)-2-propenal-3-phenyl, methoxyphenyl, and hexadecanoic acid-2-methyl ester (Table 1). As can be seen, each chemical together with its pharmacological relevance in the fight against parasitic diseases was discussed.
GCMS results of Cinnamomum verum bark extract.
Identification of phytochemical compounds by GC-Mass in Cinnamomum verum bark extracts.
| Retention time (min) | Bioactive phytochemicals | Molecular weight | Formula | Peak area % |
|---|---|---|---|---|
| 7.53 | 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- | 144 | C6H8O4 | 3.61 |
| 8.92 | 5-Hydroxymethylfurfural | 126 | C6H6O3 | 4.33 |
| 10.74 | Eugenol | 164 | C10H12O2 | 25.89 |
| 11.34 | Methyleugenol | 178 | C11H14O2 | 0.77 |
| 13.04 | 1,2,4-Cyclopentanetrione, 3-(2-pentenyl)- | 180 | C10H12O3 | 0.81 |
| 13.93 | Methoxyeugenol | 194 | C11H14O3 | 1.30 |
| 15.14 | 2-Cyclohexen-1-one, 4-(3-hydroxybutyl)-3,5,5-trimethyl- | 201 | C13H22O2 | 0.33 |
| 15.35 | Spathulenol | 220 | C15H24O | 0.28 |
| 16.15 | 5,5,8a-Trimethyl-3,5,6,7,8,8a-hexahydro-2H-chromene | 180 | C12H20O | 0.80 |
| 17.87 | n-Hexadecanoic acid | 256 | C16H32O2 | 5.97 |
| 19.68 | Linoleic acid | 280 | C18H32O2 | 32.84 |
| 19.72 | Oleic Acid | 282 | C18H34O2 | 14.61 |
| 19.87 | Octadecanoic acid | 284 | C18H36O2 | 1.72 |
| 20.62 | Palmitoyl chloride | 274 | C16H31ClO | 0.23 |
| 22.16 | Linoleoyl chloride | 298 | C18H31ClO | 1.35 |
| 25.34 | Squalene | 410 | C30H50 | 4.52 |
| 27.52 | γ-Tocopherol | 416 | C28H48O2 | 0.42 |
| 28.09 | Stigmastan-3,5-diene | 396 | C29H48 | 0.11 |
| 31.30 | Stigmasterol | 412 | C29H48O | 0.34 |
| 32.39 | Stigmast-7-en-3-ol | 414 | C29H50O | 0.17 |
The effect of the extract from C. verum bark on A. tetraptera worms was studied in vitro using different concentrations of the extract (25, 50, 100, and 200 mg/mL), alongside the standard drug, 10 mg/mL albendazole, and distilled water as a control. Which was tested during 15, 30, 60, and 120 min to determine vitality and mortality rate. The results indicated that the mortality rate was low in worms exposed to reduced concentrations of extract 25 and 50 mg/mL, with death rates of 22.3 % and 38.7 %, while they had comparatively higher mortality rates at a concentration of 100 mg/mL, which amounted to 56.33 %, respectively. On the other hand, the worms that were exposed to high levels of extract concentration (200 mg/mL) and reference drug Albendazole (10 mg/mL) experienced very high death rates during 120 min, with death rates of 96 % and 100 %, respectively (Table 2).
Means of mortality % (± SE) of Cinnamomum verum bark extract at various exposure duration (15, 30, 60, and 60 mins) against Aspiculuris tetraptera.
| Concentration | Mortality (%) | |||
|---|---|---|---|---|
| 15 min | 30 min | 60 min | 120 min | |
| Control | 0.0 ± 0.0 c | 0 ± 0.0 c | 0 ± 0.0 c | 0 ± 0.0 c |
| 25 mg/mL | 0.0 ± 0.0 c | 11± 3.5 abc | 4± 2.4 bc | 0 ± 0.0 c |
| 50 mg/mL | 14.3± 2.7 abc | 35± 2.5 a | 23.3± 4.4 abc | 3.3± 2.7 bc |
| 100 mg/mL | 26.7± 11.9 abc | 26.3± 22.4 ab | 45.7± 7.2 abc | 3.5± 2.5 bc |
| 200 mg/mL | 40± 4.7 a | 33.3± 16.6 ab | 23.3± 11 abc | 3.3± 2.7 bc |
| Albendazole 10 mg/mL | 41 ± 3.2 a | 30±13.4 ab | 22.3± 11.9 abc | 4.3± 1.9 abc |
There is a significant difference (p< 0.05) between the means in columns or rows with distinct lowercase letters. indicates that there is no significant difference (p > 0.05) between those with identical lowercase letters, based on Duncan’s Multiple Range Test, conducted after ANOVA. The mean ± standard error is used to express each value.
The results indicated that the mortality percentage ranged from 13.3 % after 30 min at the 25 mg/mL concentration and 15 % after 60 min to 100 % after 120 min at the 200 mg/mL concentration. The highest average mortality % reached 41.3 % at 200 mg/mL concentration of CVBE after 15 min and 36.7 % at 10 mg/mL Albendazole after 30 min (Figs. 2, 3). The mortality percentage ranged from 7.7 % at a 50 mg/mL concentration to 33.3 % at 200 mg/mL after 30 min. The control group did not experience any mortality during the entire exposure time or at a 25 % concentration for 15 min. In the present investigation, the efficiency of extract concentrations increased with increasing exposure time (Fig. 2). The A. tetraptera worms generally have a higher mortality rate when they have longer times and high doses of the extract from C. verum bark.
Impact of different dosages of Cinnamomum verum bark leaf extracts on Aspiculuris tetraptera mortality rates at 15, 30, 60, and 120 minutes (*Significant relative to the untreated group, p ≤0.01), # Significance of the untreated group statistically (p ≤0.05).
Mortality % of Aspiculuris tetraptera assayed with Cinnamomum verum bark extract (25, 50, 100, and 200 mg/mL) and the reference medication (10 mg/ml Albendazole), at various exposure durations (15, 30, 60, and 120 min).
Significant differences (P < 0.05) were obtained in the A. tetraptera adult worm mortality percentage among bark extract concentrations (50, 100, and 200 mg/mL) compared to 25 mg/mL of CNB and the control. Overall, no significant differences were observed between 200 mg/mL concentrations with the albendazole 10 mg/mL (Fig. 4).
Mean mortality % of Aspiculuris tetraptera adults at 200 mg/mL concentration for various exposure times (hrs).
The mortality % was highly negatively correlated with exposure times [R2 = -0.98, P = .0001, (y = 16x + 19; R2 = 0.8477; p < 0.05)] at 200 mg/mL of CNB (Fig. 4). The overall results of the current study suggest that the extract of CNB may possess potential anthelmintic properties, which could potentially be employed in worm management.
The concentrations of the bark extract were evaluated to determine the optimal concentration that results in a high worm mortality rate. The concentration of 200 mg/kg showed the most lethal effects for worms (Fig. 5). Three days after giving the treatment, the murine was dissected. The results showed that the mortality rate of the worms counted from the murine’s intestines was 96 % at a concentration of 200 mL/kg of the extract and 89 % for the murine treated with a dose of 10 mL/kg of albendazole. Whereas the mortality rate of the worms taken from the gut of the treated mice that were dissected six days after giving the treatment was 100 % at a concentration of 200 mL/kg of the extract and 97 % for the mice treated with a dosage of 10 mL/kg of albendazole (Fig. 6). It is shown in Table 3 that mortality in the group treated with 200 mg/kg of CVBE and 10 mg/kg of albendazole decreased the count of eggs in the stool and even arrived at zero on the sixth day, as well as when dissected in the gut (Table 3). The concentration of 200 mL/kg demonstrated the most influence on worm mortality (Fig. 6).
The general impacts of different dosages of Cinnamomum verum bark extracts (25,50, 100, 200 mg/ml), and reference drug (10 mg/ml Albendazole) on the mortality rate of A. tetraptera of 15 to 120 minutes.
(*Significant relative to the untreated group, p ≤0.01), # Significance of the untreated group statistically (p ≤0.05).
Principal effects of extracts from the bark of Cinnamomum verum at several dosages on the mortality rate of Aspiculuris tetraptera at 20, 40, 80, 120, and 180 minutes (*Significant relative to the untreated group, p ≤0.01). # Significance of the untreated group statistically (p ≤0.05).
The average number of A. tetraptera worms obtained by fecal and intestinal centrifugation flotation per cage per mouse at necropsy in a strain of mice treated with Cinnamomum verum bark extract and the anthelmintic drug.
| Groups | n | Average no. of worms after 3 days of treatment | Average no. of worms after 6 days of treatment | ||
|---|---|---|---|---|---|
| In feces | In intestinal | In feces | In intestinal | ||
| Infected | 5 | 245±45 | 395±57 | 284±25 | 367±45 |
| 25 mg/kg | 5 | 213±15 | 275±15 | 220±5 | 156±11 |
| 50 mg/kg | 5 | 75±11 | 121±15 | 110±5 | 112±9 |
| 100 mg/kg | 5 | 48±11 | 90±15 | 20±5 | 25±9 |
| 200 mg/kg | 5 | 2±1.1 | 7±1 | 0 | 0 |
| Albandazol 10 mg/kg | 5 | 3±1 | 11±2 | 0 | 0 |
Plant products possess significant medicinal impacts for treating numerous contagious diseases (Beshbishy et al., 2019). This property makes medicinal plants an attractive choice as a source of new therapeutic compounds (Verdú et al., 2023). Despite its antiparasitic effect, researchers have not evaluated CNB against A. tetraptera. So, this study looks at how well CNB extract can kill A. tetraptera worms in lab tests and in living organisms. In this study, we noticed an increase in the death rate of the worm as exposure time progressed. Mortality ratios of 56.33 % – 100 % were noticed following treatment durations of 120 minutes for A. tetraptera worms with concentrations of 100 and 200 mg/mL of CNB extract and 96 % at 10 mL/mL albendazole, compared to the control (distilled water). The mortality rate was low for the low-concentration treatments (25 and 50 mg/mL). The results indicated that the CNB extract had a strong impact on A. tetraptera worms at all the concentrations tested in the lab (Mares et al., 2024). This study suggests that the CNB extract could be a potential alternative treatment for parasitic infections caused by A. tetraptera. We need further studies to explore the mechanism of action and evaluate the efficacy and safety of CNB extract in vivo.
The current results align with those reported by other researchers using various types of parasites. Sebai et al., (2022) discovered that 4 mg/mL of CNB essential oil exposed adult worms to 87.5 % immobility after 8 hours, exhibiting an inhibitory impact against H. contortus egg hatching with an inhibition value of 1.72 mg/mL. The plant’s yield C. verum bark essential oil, which contains D-limonene, linalool, and monoterpenes 1,8-cineol. Previous research has demonstrated the promising biological activity of plant linalool-rich extracts, including antibacterial, antiparasitic, and cytotoxic effects. Discovered that the most prevalent component in extracts from Cinnamomum camphora bark was linalool. Yang et al. (2014) reported that linalool showed effects on Schistosoma japonicum cercaria and snails in vitro. The C. verum bark extract demonstrated both larval effectiveness and acaricidal activity at various doses and times (Alimi et al., 2021).
The study revealed that the chemical composition of CNB essential oil works to inhibit both the antibacterial and antibiofilm activities against clinical Staphylococcus aureus strains (Merghni et al., 2016). In vivo, on the sixth day from the beginning of the experiment, all the rats were slaughtered, and the intestines, especially the caecum, were taken and opened in a Petri dish in the middle of saline solution. The worms in each group were counted and compared between all groups. We noticed that the number of worms obtained in the group injected with a concentration of 200 mg/mL CNB extract was deficient and almost nonexistent compared to the untreated infected group. Also, in the other groups dosed orally with concentrations of 100, 50, and 25 mg/kg, the percentage of worms obtained after examination under the microscope was low compared to the infected control group. Where he noticed that the effectiveness of the 200mg/kg concentration of the CNB extract was more effective at evicting worms than the albendazole treatment used commercially to eliminate worms. The results of this study are in line with numerous earlier investigations that confirmed the efficacy of CNB leaves in combating a range of parasites. After 7 days of treatment and a 79.2 % decrease in the overall number of worms, Sebai et al., 2022, discovered that the in vivo anthelmintic potential of CNB extract abolished the egg output of Heligmosomoides polygyrus. Furthermore, after a challenge infection, the amount of recovered schistosomulum from mouse skin was significantly decreased by the linalool found in the CNB plant. According to Yang et al. (2014), it reduced the worm load in infected animals. Linalool, which is essential for getting rid of worms in sick mice’s intestines, is abundant in the bark of the CNB plant (Sebai et al., 2022). Strong antibacterial, antimicrobial, and antioxidant properties have been reported for CNB (Koike et al., 2015). Traditionally, CNB has been used to treat gastrointestinal symptoms, such as eructation, epigastric bloating, impaired digestion, and flatulence (Kivçak et al., 2022).
The suitable anti-parasitic properties of C. verum bark were found in this study and others, suggesting that they could replace chemical medications in parasite control initiatives.
This page lists the components of medicinal plants that are effective against gastrointestinal parasites both in vivo and in vitro. The results of these investigations indicate that herbal remedies hold great promise for creating new medications to treat parasitic illnesses, and the plant derivatives found in them provide useful structures for drug manufacturing and bioactivity optimization. Furthermore, the products of these plants provide useful compounds for the manufacture of pharmaceuticals. Plant medicament also hold great promise for the development of innovative therapies to treat parasite illnesses.