Bio-guided isolation of a new sesquiterpene from Artemisia cina with anthelmintic activity against Haemonchus contortus L3 infective larvae

Haemonchus contortus is a blood-feeding gastrointestinal parasite that impacts grazing sheep, causing economic losses in animal production. Due to its anthelmintic resistance, alternative antiparasitic treatments like plant-based anthelmintics are necessary to explore. Artemisia cina (Asteraceae) is a plant whose n-hexane extract and ethyl acetate extract exhibit anthelmintic activity against H. contortus, the n-hexane more active. To discover additional bioactive metabolites, a chemical analysis was performed on ethyl acetate extract, which presented an LC90 of 3.30 mg/mL and allowed the isolation of 11-[(1R,5S,7R,8R,10S,)-1,8-dihydroxy-5,10-dimethyl-4-oxodecahydroazulen-7-yl] acrylic acid. This new sesquiterpene was identified through one and two-dimensional NMR. The compound was named cinic acid and displayed an LC50 of 0.13 (0.11–0.14) mg/mL and LC90 of 0.40 (0.37–0.44) mg/mL, which, compared with ethyl acetate extract larvicidal activity, was 256-fold more active at LC50 and 15.71-fold at LC90. In this study, a new sesquiterpene with larvicidal activity against H. contortus L3 infective larvae was isolated from the ethyl acetate extract of Artemisia cina.


Introduction
Haemonchus contortus is a highly pathogenic nematode that feeds on the blood of small ruminants and is a significant cause of economic losses worldwide.It possesses a particularly substantial threat in tropical, subtropical, and warm temperate regions where warm and moist conditions favor the free-living stages of the parasite [1].Females of H. contortus can produce up to 5,000 eggs per day, which are then excreted from the host animal through feces.After hatching, the larvae undergo several chitin molts, ultimately reaching an infective larval stage known as L 3 .Ruminants ingest this larva as they consume grass.Upon reaching the abomasum, the L 4 larva initiates its blood-feeding role, and the adult closes the cycle, eliminating eggs to grass and is also hematophagous.Haemonchosis potentially causes a range of issues, such as malnutrition, low feed conversion, anemia, loss of appetite, low fertility rates, and even death in both young and older animals.Chronic inflammation, weight loss, and continuous diarrhea may contribute to the deterioration of the animal's health and ultimately lead to its demise [2].
Chemical anthelmintics have been used to control H. contortus in small ruminants [3].However, their inadequate and irresponsible use has facilitated the emergence of parasites with resistance to anthelmintics in different countries, including Mexico, where small ruminant grazing is a significant economic activity [4,5].Therefore, exploring and proposing alternative control strategies for this parasite is imperative.Using plant extracts containing chemical compounds with anthelmintic activity holds promise among the various options.
The genus Artemisia comprises approximately 500 species distributed worldwide.Artemisia species are characterized as small herbs or shrubs with a distinctive bitter taste and an intense aroma attributed to terpenoids, primarily monoterpenes in the essential oil, and sesquiterpene lactones [6].They also comprise terpenoids, flavonoids, coumarins, caffeoylquinic acids, sterols, and acetylenes [7].Artemisia cina, also known as santonica or Levant wormseed, has been traditionally used as a vermifuge to expel intestinal worms [8].The efficacy of A. cina against H. contortus has been demonstrated both in vitro and in vivo.In vitro, the n-hexane (n-HE) extract of A. cina exhibited the highest larvicidal activity against transitional larvae L 3 -L 4 of H. contortus compared to methanol and ethyl acetate extracts (EAE), achieving percentages of 75% and 82.6% of larvicidal activity at concentrations of 1 mg/mL and 2 mg/mL, respectively [4].In an in vivo study conducted on naturally infected periparturient goats, the administration of an n-HE derived from A. cina resulted in a notable reduction in the fecal egg count of H. contortus and Teladorsagia circumcincta.This extract was found to contain two previously unidentified compounds for A. cina, namely isoguaiacin and norisoguaiacin [9].

Plant material
The fresh pre-flowering leaves and stems of A. cina O. Berg ex Poljakov (Asteraceae) (10 kg) were bought at Hunab1 laboratory.A voucher specimen was authenticated by Dr. Alejandro Torres-Montu ´far and was deposited at the herbarium of Facultad de Estudios Superiores Cuautitla ´n (FES-C) UNAM, Me ´xico under voucher no 11967.The plant was grown at 80% humidity, 24˚C, and soil with pH = 6.3.

Artemisia cina extract
The extracts were obtained by maceration.Dry A. cina leaves and stems (1 kg) were ground and placed in 1 L erlenmeyers.Extraction using leaves and stems was performed using n-hexane (HE), ethyl acetate (EAE), and methanol (ME) maintained for 72 h at room temperature (23-25˚C).The extraction was performed using different vegetal materials for each solvent, avoiding the exhaustive extraction method used by Higuera-Piedrahita et al. 2021 [4].Extracts were filtered using a Whatman No. 4 paper, and the solvent was removed by low-pressure distillation using a rotary evaporator (DLAB RE-100 Pro) at 40˚C and 100 rpm.The extracts were finally lyophilized and kept at four ˚C for phytochemical and biological assays.

In vitro assays with Haeomonchus contortus L 3
The lethal effect of the three A. cina extracts and fractions on L 3 H. contortus infective larvae was determined using 96-well microplates for 24 hours at 24˚C.Two control groups were used: (a) Distilled water and (b) ivermectin (5 mg/mL, Sigma-Aldrich).A. cina extracts were tested at five different concentrations (8, 4, 2, 1, and 0.5mg/mL).For the A. cina extract × H. contortus infective larvae confrontations, approximately 100 L 3 larvae in 100 μL of aqueous suspension were used per well (n = 4), with three replicates under the same conditions.The lethal effect was evaluated at 24 hours post-exposure, and lethality percentages were obtained following the report by Delgado-Nu ´ñez [10].The H. contortus strain was obtained from Facultad de Estudios Superiores Cuautitla ´n.The strain is characterized by benzimidazole susceptibility and resistant heterozygotic genes [11].

Bio-guided separation
Twenty-four grams of the (ethyl acetate extract) EAE were utilized for column 1 and separated using open-column chromatography.Regular silica gel 60 (Merck1, 0.015-0.040mm) served as the stationary phase, and n-hexane-ethyl acetate was employed as the solvent gradient system.Sixty-one samples were obtained and grouped in three fractions according to their chemical similarity, monitored using thin-layer chromatography.Samples were concentrated using a rotary evaporator.The resulting fractions were named C1F1 (4.242 g), C1F2 (11.187 g) and C1F3 (5.691 g).

TLC and HPLC analysis
Analytical TLC was performed on precoated Merck1 silica gel 60F254 or RP-18F254 plates.Ceric sulfate reagent was used to visualize terpenes.

GC-MS analysis
The GC-MS analysis was performed using an Agilent Technologies HP 6890 gas chromatograph coupled to a quadrupole mass detector MSD 5973 (HP Agilent) and an HP-5MS capillary column (length: 30 m; inner diameter: 0.25 mm; film thickness: 0.25 μM).A constant helium flow was set as the carrier gas to the column at 1 mL/min.The inlet temperature was fixed at 250˚C, while the oven temperature was initially kept at 40˚C for 1 min and increased to 280˚C at intervals of 10˚C/min.The mass spectrometer was used in positive electron impact mode with an ionization energy of 70 eV.Detection was performed in selective ion monitoring mode.The signals were identified and quantified using target ions.The compounds were identified by comparing their mass spectra with the NIST library version 1.7a.The relative percentages were determined by integrating the signals using GC Chem Station software, version C.00.01.The composition was reported as a percentage of the total signal area.

NMR experiments
One and two-dimensional Nuclear Magnetic Resonance (NMR) experiments ( 1 H, COSY, HSQC, HMBC, and DEPTq) were performed on a Bruker AVANCE III HD at 500 MHz.CD 3 COCD 3 was used as a solvent with tetramethylsilane (TMS) as an internal standard.Chemical shifts (δ) are reported in ppm values, and coupling constants are in Hz.

Near infrared spectroscopy analysis
NIR spectra were recorded on a Foss NIRSystems-6500 near infrared spectrophotometer (Raamsdonksveer, The Netherlands) following the methodology reported by Lo ´pez-Arellano et al. [13].

Melting point experiment
A Fisher-Johns melting point apparatus was used to determine the melting point.A small amount (less than 1 mg) of crystal was well spread and placed between two coverslips.The heating control was set at full power until 20 degrees of the theoretical melting point.Then, the power was set to increase by one ˚C per minute.The determination of the melting point was performed in triplicate.

Statistical analyses
The Duncan test (p < 0.05) compared differences among lethality percentage means.Lethal concentrations (LC 50 and LC 90 ) were determined using the PROBIT procedure included within the SAS statistic package.

Yield extraction
A maceration extraction was performed using different solvents such as methanol, ethyl acetate, and n-hexane.The ME showed a yield percentage of 4.10%, the EAE 3.86%, and the HE 1.09%.ME exhibited the highest yield, followed by EA and HE, respectively.

Anthelmintic activity of Artemisia cina crude extracts
Table 1 shows the in vitro lethal concentration of the crude extracts against Haemonchus contortus infective larvae (L3).
The HE had the best LC 50 , but no significant difference was found in LC 90 compared with EAE.I do not present a dose response, so LC 50 and LC 90 cannot be calculated.EAE extract was chosen to perform the bio-guided separation due to the LC 90 and the highest yield percentage of extraction, which is a crucial factor to consider in the formulation of pharmaceutic preparations.

Bio-guided separation of the EAE of A. cina monitoring larvicidal activity against Haemonchus contortus infective larvae L 3
EAE was separated into 61 samples and grouped into three fractions (C1F1, C2F2, and C3F3) based on their chemical similarity.C1F1, C2F2, and C3F3 were evaluated against L 3 H. contortus infective larvae.C1F1 exhibited the highest larvicidal activity (Table 2), followed by C1F2.C1F3 did not show at least 50% larvicidal activity; LC 50 and LC 90 were not calculated.Fraction C1F2 was chosen to continue the separation process due to its significantly higher yield percentage than C1F1.The separation of C1F2 was grouped into nine fractions, with C2F7 displaying the highest larvicidal activity.C2F7 was then selected for attempts to isolate the bioactive molecule.
Fig 1 is a schematic representation that illustrates the bio-guided separation methodology applied to the EAE, monitoring the larvicidal activity against the L 3 of H. contortus infective larvae in vitro.As a result, an unknown compound with anthelmintic activity was isolated, yielding 0.01% compared to the EAE.

Identification of compound 1
Compound 1 was obtained through the chromatographic separation of C2F7, using 1:1 n-hexane-ethyl acetate as the mobile phase.Colorless needle crystals were obtained, and according to UV and mass spectra, there was no information on the compound reported in Artemisia cina.It was necessary to perform one-and two-dimensional NMR spectroscopy to identify it.

Discussion
Haemonchus contortus is a hematophagous gastrointestinal parasite that threatens grazing sheep.Its feeding habits contribute to conditions such as anemia and poor digestion,   potentially leading to mortality in young individuals.Chronic inflammation, weight loss, and persistent diarrhea are joint in adults, resulting in significant losses in animal production worldwide [17].Furthermore, drug-resistant helminths a considerable challenge to the sustainability of current helminth control strategies [18].It is imperative to develop alternative antiparasitic treatments against H. contortus, including the exploration of plant-based anthelmintics through discovery and development efforts.Artemisia species have attracted significant research attention due to sesquiterpenoid lactones, coumarins, flavonoids, and phenolic acids.These compounds are responsible for many biological activities, including hepatoprotective, neuroprotective, antidepressant, cytotoxic, antitumor, digestion-stimulating, and antiparasitic effects [15].The n-hexane (n-HE) extract of Artemisia cina has been documented for its anthelmintic activity against H. contortus, targeting eggs and L 3 infective larvae [9], transitional larvae L 3 -L 4 [4], and naturally infected periparturient goats [3].Those activities are attributed to two lignans, 3 0 -Demethoxy-6-O-Demethylisoguaiacin and norisoguaiacin [9].However, the n-HE and the lignans' low yield percentage make it challenging to use for formulating or creating A. cina-based pharmaceutical preparations.This study chose the ethyl acetate extract (EAE) for its significantly higher extraction yield (3.86 times higher) than the n-HE while maintaining a similar larvicidal activity to n-HE.
The bio-guided isolation of the EAE from A. cina, led to separate and identify a new sesquiterpene that was called cinic acid.This compound exhibited great larvicidal activity and was 256 times more active at LC 50 and 15.71 times at LC 90 than EAE, likely responsible for a significant portion of the overall extract activity.The compound yields 0.01% relative to the extract, but there may be a presence of synergism between cinic acid and other compounds of the EAE, but this hypothesis had to be probed.
Sesquiterpenes are secondary metabolites with a 15-carbon skeleton built from three isoprene units.They are commonly cyclized, found in the Asteraceae family, and exhibit several pharmacological activities [19].The predominant sesquiterpenes isolated in Artemisia species are sesquiterpene lactones [20].Sesquiterpene lactones are categorized into four primary groups: germacranolides (with a 10-membered ring), eudesmanolides (6-6 bicyclic compounds), guaianolides, and pseudoguaianolides (5-7 bicyclic compounds).A distinguishing characteristic of sesquiterpene lactones (STLs) is a γ-lactone ring closed at C-6 or C-8.This γlactone often includes, in numerous instances, an exo-methylene group conjugated to the carbonyl group [21].Cinic acid is a novel sesquiterpene with a structure resembling a pseudoguaianolide-type sesquiterpene lactone, specifically an ambrosanolide.It features two methyl groups cis-orientated at C-5 and C-10 and an exocyclic methylene conjugated to a γ-carbonyl moiety necessary for biological activity.The notable distinction lies in the absence of lactonization at C8.The activity of sesquiterpene lactones is determined by the presence of the α-methylene, γ-lactone system, which acts as a Michael acceptor, allowing interaction with thiol groups of proteins [22].The α-methylene, γ-carbonyl system in cinic acid could explain it is in vitro anthelmintic activity against L 3 infective larvae of H. contortus [23].

Conclusion
The bio-guided separation of ethyl acetate extract (EAE) allowed the identification of a new sesquiterpene with anthelmintic activity against H. contortus (L 3 ) infective larvae.The EAE could be a promising candidate for a plant-based pharmaceutical preparation with anthelmintic activity from Artemisia cina.Also, cinic acid is a promissory compound that should be evaluated in eggs or H. contortus adults.Cinic acid sholud be registered in Scifinder1, and the lethal effect should be patented, and the studies with the molecule must be continued to know the in vivo effect.