Synergistic Interaction Effect of Artemisia cina n-hexane Extract and Tagetes lucida Ethyl Acetate Extract on Haemonchus Contortus

Purpose We analysed the possible synergistic activity among active extracts from Artemisia cina and Tagetes lucida combinations on Haemonchus contortus, a nematode parasitising sheep. Methods The work was carried out in vitro on eggs and infective larvae (L3) of H. contortus. The results were analysed with SAS 9.1, applying the ANOVA and Tukey test, and the lethal concentration (LC) values LC50 and LC90 were determined with regression analysis, employing Proc Probit of SAS 9.1. Additionally, the lethal concentration (LC) was calculated with LC50 and LC90 to determine the synergistic effect. Results The results demonstrated a high efficacy of the two plants studied on both nematode eggs and L3 larvae as well as of their combinations. The highest egg hatching inhibition was obtained with a 50/50 combination, and the best larvae mortality was obtained with 25% A. cina and 75% T. lucida at 10 mg/mL. Additionally, this combination showed a synergistic effect. Conclusion The two plant species studied here can be applied as natural anthelmintic alternatives due to their high bioactive effect and synergistic response. Supplementary Information The online version contains supplementary material available at 10.1007/s11686-024-00839-6.


Introduction
Gastroenteric helminths are the cause of major parasitic diseases in grazing ruminants.These disorders cause damage to animal health and welfare and reduce productive performance such as milk production, weight gain, carcass quality and fertility, among others [1].Haemonchosis is a parasitic disease with high importance in small ruminants.Haemonchus contortus feeds on the blood of its host, resulting in anaemia, weight loss, hyperacute infections and sudden death [2].Due to the rapid development and increase of anthelmintic resistance (AR) in nematode populations worldwide and to diminish the damage caused by these parasites in production systems, the search for pharmacological and non-pharmacological tools is necessary [3,4].Within these management strategies, the use of selective deworming with the aid of indicators such as the body condition score [5] and the FAMACHA colour chart [5].Other practices are focused on population resistance, such as using secondary metabolites from biological organisms, including nematophagous fungi [6] or ruminal bacteria [7].
The family Asteraceae contains numerous species with benefits for human and animal health.These plants contain several secondary compounds such as terpenoids, flavonoids and other molecules with anticancer, antimalarial and anthelmintic properties [19][20][21].Artemisinin is a sesquiterpene lactone obtained from Artemisia annua with demonstrated efficacy against malaria, which has been resistant to chloroquine since 1950, along with other properties [19,21,22].
Artemisia cina, commonly known as Levant wormseed, is a herbaceous plant that has been traditionally used as an anthelmintic agent in many parts of the world, especially in the Mediterranean and Middle Eastern regions.Several studies have shown the efficacy of A. cina in controlling various parasitic infections.For instance, a study found that the aqueous and ethanolic extracts of A. cina significantly reduced the number of eggs of gastrointestinal nematodes in sheep [23,24].Similarly, another study reported that the ethanolic extract of A. cina was highly effective in controlling gastrointestinal nematodes in goats [25].
Moreover, A. cina has also been found to be effective against other parasitic infections such as malaria, leishmaniasis and schistosomiasis [26,27].The active compounds present in A. cina, such as santonin, artemisinin and their derivatives, are the main contributors to its anthelmintic activity [28].A study with an A. cina aqueous extract, which was administered to sheep naturally infected with Moniezia spp., recorded a 100% efficacy faecal egg count reduction) on at 9th day post-administration [29].Additionally, an A. cina n-hexane extract was assessed against H. contortus infective larvae and caused a mortality of 80% at 2 mg/mL.In this same research, the authors administered the A. cina n-hexane at 2 mg/kg to gerbils artificially infected with H. contortus, resulting in a 100% efficacy [23].
Tagetes lucida is another genus (Asteraceae) extensively studied as an anthelmintic since it is rich in some phenolic acids and other metabolites such as coumarins and terpenes [30].This plant, commonly known as Mexican "Pericón," is a flowering plant traditionally used for various medicinal purposes, including its cytotoxic activity, which can result in anthelmintic potential [31].
Studies have reported the efficacy of T. lucida in controlling parasitic infections.For example, the authors of one study found that the ethanolic extract of T. lucida significantly reduced the number of eggs of gastrointestinal nematodes in sheep [32].Similarly, another study [33] reported that the methanolic extract of T. lucida had high anthelmintic activity against the gastrointestinal nematode H. contortus in sheep.
Moreover, T. lucida has also been effective against other parasitic infections such as malaria and leishmaniasis [34].The active compounds in T. lucida, such as target one, piperine and their derivatives, have been identified as the main contributors to its anthelmintic activity [32].
Combinations of chemical anthelmintics (albendazole and levamisole) with extracts from plants (terpenes) showed an improved nematocidal effect on gastrointestinal nematodes (GIN), suggesting a possible synergistic effect [35].Combining natural products with chemical anthelmintics could contribute to controlling GIN.Natural plant compounds have a wide range of biological activities, including antiparasitic activity.However, most studies focus on isolated compounds or extracts from single plants, which may not be sufficient to achieve optimal therapeutic effects.Therefore, studying the synergistic effects of different plants could lead to the development of more effective and safer antiparasitic treatments [36].
Several studies have demonstrated the potential synergistic activity of combinations of plant extracts on parasites.For instance, the antileishmanial and antischistisomal activity of four plant extracts and their combinations are promising [26,28].
A recent review by Athanasiadou [37] summarised the potential of plant synergies for antiparasitic activity.The authors highlight that plant synergies can improve efficacy, reduce toxicity and prevent the development of resistance in parasites.In the present study, using in vitro assays, we evaluated the interaction effects of A. cina (n-hexane extract) and T. lucida (Ethyl acetate extract) against H. contortus.

Plant Material
We purchased 10 kg of fresh pre-flowering leaves and stems of A. cina O. Berg ex Poljakov (Asteraceae) from the Hunab laboratory.For identification, a voucher specimen was examined by Dr. Alejandro Torres-Montúfar and deposited at the herbarium of the Facultad de Estudios Superiores Cuautitlán (FES-C) UNAM, México.under voucher no 11,967.The plant was cultivated under specific conditions, including a humidity of 80%, a temperature of 24 °C and a soil pH of 6.

Artemisia cina Extracts
To obtain the extract, 5 kg of dried A. cina leaves and stems were ground and placed in crystal containers with a capacity of 1,000 mL.To extract the compounds, n-hexane was used, and the mixture was kept at room temperature (23-25 °C) for 48 h.The extract was filtered using Whatman No. 4 paper, and the solvent was removed by low-pressure distillation through a rotary evaporator (Heidolph Laborota 4000, Heidolph Instruments, Schwabach, Germany).Finally, the extract was lyophilised and stored at 4 °C for phytochemical and biological assays.

Tagetes Lucida Extract
To obtain the extract, 5 kg of dried T. lucida were macerated in ethyl acetate for 48 h.The extract was filtered using Whatman No. 4 paper, and the solvent was removed by low-pressure distillation through a rotary evaporator (Heidolph Laborota 4000, Heidolph Instruments, Schwabach, Germany).
An aliquot of 50 µL of an aqueous suspension containing 100 ± 20 H. contortus eggs was deposited in each well (experimental unit).Then, 50-µL aliquots of the treatments or controls were deposited, giving a total volume of 100 µL.The plates were incubated at room temperature (25-28 °C) for 48 h, and subsequently, hatching was stopped using 10 µL of Lugol´s solution.The total eggs and larvae (first and second stage) of each well were counted, and the egg hatching inhibition percentage (EHI %) for each treatment was determined using the following formula: number of eggs number of eggs + number of larvae x100 (1)
The plates were incubated at room temperature (18-25 °C) for 24 h.Subsequently, the total larvae, alive or dead, in evaluation of T. lucida ethyl acetate extract showed the best EHI percentage, close to 90%, at 2.5 mg/mL.At the same time, the A. cina n-hexane extract exhibited the lower EHI with the three concentrations used.The combination of 50/50 presented an IEH at almost 100% with all concentrations used (1, 2.5 and 5 mg/mL).

Larval Mortality
A higher larvae mortality was obtained using the individual extracts with a concentration of 10 mg/mL (Table 2).This same response was achieved with the combination of 25/75% of A. cina and T. lucida, respectively, using 10 mg/ mL.The 1 mg/mL concentration resulting in a lower mortality in all treatments except for T. lucida.This indicates a dose-response relationship.Ivermectin showed 100% of larvae mortality.

Lethal Concentration (LC) 50 and 90
The LC 50 and LC 90 values calculated for the A. cina and T. lucida extracts are shown in Table 3.The T. lucida extract showed the LC 50 with s minor concentration, and the A. cina extract alone presented the major LC 50 .The LC 90 , by contrast, was obtained for the A. cina extract.The proportion of LC 50 and LC 90 attributed for each extract according to the proportion added in the combination is presented in the following table; based on this information, the isobolograms were drawn (Fig. 1).

Synergistic Activity
The synergistic activity is shown in Fig. 1.According to the estimates for LC 50 , the extracts are more lethal when combined; the isobologram reflects that the concentration needed to cause a 50% mortality of H. contortus larvae is lower in all combinations than in the extracts alone; the lines of the three combinations are below the line of the LC 50 of the extracts alone, suggesting synergistic activity.
The TF (total fraction) for each combination is presented in Table 4.This table shows the effect in all combinations, and both LC 50 and LC 90 are sub-additive (antagonism).Moreover, for the 25:75 A. cina: T. lucida combination showed a synergistic effect was obtained.

Discussion
Artemisia cina n-hexane extract contains lignans with potent antiparasitic activity, such as isoguaiacine and norisoguaiacine [40].The major compounds identified in T. lucida are caffeic acid, ferulic acid, coumaric acid and chlorogenic each well were counted under an optic microscopy Hinotek Lab model L1200, and the mortality percentages were estimated based on the following formula: Mortality Larvae % = number dead larvae numbers of dead larvae + number of alive larvae x100 (2)

Statistical Analysis
The IEH and mortality percentage data were analysed through ANOVA based on a completely randomised design by the general linear model in the SAS programme.The dependent variables were the combinations and controls.
Tukey´s test was performed to identify significant differences among treatments at a 0.05 significance level.The treatments dependent on the concentration were subjected to regression analysis to estimate the lethal concentrations (LC) 50 and 90 using a Probit analysis (SAS®, 2014).

Synergic Interaction Analysis
To characterise the interactions among the combinations of the extracts, A. cina and T. lucida, and isobolographic analysis was used following the methodology described by Tallarida et al. [39].According to this method, the lethal concentrations of 50 and 90 of each extract (assay individual) and their combinations were considered to determine the synergic effect.Likewise, the interaction magnitude of the combinations, the total fraction of LC of the A. cina extract and T. lucida extract and their combinations were determined according to the following formula: where: TF = total fraction, LC = lethal concentration.
Values close to 1 indicate a possible additive effect, and values below 1 indicate a super-additivity or synergistic effect.

Ovicidal Activity
The ovicidal activity of both extracts in individual form and their combinations is shown in Table 1.The individual Synergistic effects of metabolites from plants of the genus Artemisia and secondary metabolites from other plants have been previously evaluated and identified.Essential oils of A. herba alba and Lavandula angustifolia have an antibacterial effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus at 20 µL/mL when used individually and at 0.5 and 0.1 µL/mL when used in combination, indicating synergistic activity [46].The activity was calculated with volume concentrations [46].In addition, a trial conducted on antibiotic-resistant Staphylococcus aureus strains with a combination of several flavonoids from A. rupestris and norfloxacin showed a TF of less than 0.5 (synergistic effect) [47].In another study, two of the significant methoxylated compounds from the dichloromethane fraction of the T. lucida plant showed a possible synergistic effect of coumarins and flavonoids against fungi [48].These findings may explain the results obtained in the present work.
Artemisia cina is active against H. contortus.In a previous study, A. cina 30 CH (centesimal Hahnemannians) administered to sheep naturally infected with gastrointestinal helminths reduced egg counts per gram (69%) and inhibited egg hatching by 100% in vitro [49].In another study, n-hexane extract was active against H. contortus acid [41].The synergistic effects of plant extracts have gained considerable attention as in alternative treatments of parasitic infections in animals [42].Plants contain several bioactive compounds, and their combination may enhance their efficacy as antiparasitic agents.Several studies have reported the benefits of using plant extracts in combination to treat parasitic infections in animals.For instance, Khanolkar [42] demonstrated that the combination of Piper longum and Tinospora cordifolia extracts showed a synergistic effect against gastrointestinal nematodes in sheep.Similarly, Bhosale et al. [43] found that the combination of extracts from various plants, including Achyranthes aspera, Vernonia amygdalina and Lantana camara, had a synergistic effect against gastrointestinal nematodes in goats.
The synergistic effects of plant extracts may be due to their ability to target different stages of the parasite's life cycle or to other physiological mechanisms.In addition, combining compounds may lead to increased bioavailability and better absorption, resulting in a higher efficacy [44].Moreover, the use of plant extracts in combination may help to reduce the development of resistance to the treatment as it is less likely for parasites to develop resistance to multiple compounds simultaneously [45].whereas in our study, a similar effect was found using 10 mg/mL.
The aerial parts of the plant T. lucida contain several metabolites, mainly coumarins, flavonoids and terpenes [30], with anti-inflammatory effects [30] as well as antimicrobial effects at 450 mg/mL using petroleum ether and chloroform extracts on bacteria such as Escherichia coli, Enterobacter alcalifaciens and Staphylococcus aureus [50].In other studies, its effect against fungi such as Aspergillus larvae [23].Based on these reports, this type of extract was selected in the present study.Recently, for this extract, two lignan compounds responsible for the activity against H. contortus have been identified, namely 3'-demethoxy-6-Odemethylisoguaiacine and norisoguaiacine [40].However, the anthelmintic effect observed for the n-hexane extract was more significant than that found in the present study as it resulted in a larval mortality of 80% at 4 mg/mL [40], 024-00839-6.

Acknowledgements
The authors acknowledge Consejo Mexiquense de Ciencia y Tecnología COMECyT for the financial support to the first author through a Cathedra with number CAT2022-54 under the direction of Dr. Rosa Isabel Higuera-Piedrahita and Dr. Agustín Olmedo-Juárez.
Funding Project PAPIIT IA204822 titled 'Evaluation of the toxic effect of n-hexane extract from Artemisia cina and cinaguaiacina on biochemical parameters in blood and anatomopathological alterations in Wistar rats after oral administration' from the National Autonomous University of Mexico.
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3. Tagetes lucida aerial parts were harvested in El Cerrillo Piedras Blancas, Toluca Municipality, Mexico State (19.42984N, -99.669345W).One plant specimen was deposited in the Herbarium of the Centre for Research in Biodiversity and Conservation, Universidad Autónoma del Estado de Morelos (UAEM-Mexico), under voucher code 33,784.

Table 1
Haemonchus contortus egg hatch inhibition percentages (EIH%) after 48 h exposure to Artemisia cina and Tagetes lucida extracts and Means with same letter are similar statistically P ≤ 0.05, s.d.= standard deviation