TY - JOUR
AU - López-Arellano, Raquel
AB - 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 L3. Ruminants ingest this larva as they consume grass. Upon reaching the abomasum, the L4 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 L3-L4 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]. According to previous findings, the EAE presents anthelmintic activity against H. contortus L3 infective larvae [9]. However, the bioactive compounds remain unidentified. Therefore, the objective of this study was to isolate and identify a compound with anthelmintic activity against H. contortus L3 infective larvae from the EAE of A. cina through bio-guided separation. This is the first time that the 11-[(1R,5S,7R,8R,10S,)-1,8-dihydroxy-5,10-dimethyl-4-oxodecahydroazulen-7-yl] acrylic acid (cinic acid) and its anthelmintic activity against L3 H. contortus infective larvae is reported. Materials and methods Plant material The fresh pre-flowering leaves and stems of A. cina O. Berg ex Poljakov (Asteraceae) (10 kg) were bought at Hunab® laboratory. A voucher specimen was authenticated by Dr. Alejandro Torres-Montúfar and was deposited at the herbarium of Facultad de Estudios Superiores Cuautitlán (FES-C) UNAM, México 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 L3 The lethal effect of the three A. cina extracts and fractions on L3 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 L3 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-Núñez [10]. The H. contortus strain was obtained from Facultad de Estudios Superiores Cuautitlán. 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 (Merck®, 0.015–0.040 mm) 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). C1F2 achieved the highest yield percentage with larvicidal activity, so it was used for column 2. Column 2 was performed using the same chromatographic conditions as Column 1. 34 samples were grouped into nine fractions: C2F1 (0.093 g), C2F2 (0.088 g), C2F3 (0.090 g), C2F4 (0.137 g), C2F5 (1.727 g), C2F6 (2.656 g), C2F7 (2.780 g), C2F8 (0.769 g), and C2F9 (1.142 g). C2F1 to C2F4 were not evaluated due to low yield percentages Instead, C2F5 to C2F7 were analyzed at C1F2 LC50 and LC90. C2F7 displayed the highest larvicidal activity, and LC50-90 values were calculated. C2F7 was selected to carry out Column 3 and isolate a bioactive molecule. Column 3 had the same chromatographic conditions as Columns 1 and 2. Column 3 was separated into 26 samples; samples 13–15, dissolved in a 3:2 n-hexane-ethyl acetate mixture, crystallized into needle crystals. These crystals were decanted, and the resulting crystals were washed with n-hexane, yielding 256 mg. TLC and HPLC analysis Analytical TLC was performed on precoated Merck® silica gel 60F254 or RP-18F254 plates. Ceric sulfate reagent was used to visualize terpenes. HPLC separations were performed on a Waters 2695 separations module equipped with a Waters 2996 photodiode array detector, and HPLC analysis was carried out using a LiChrospher® 100 RP-18 column (4 mm × 250 mm, five μm) (Merck, Kenilworth, NJ, USA). The mobile phase consisted of two solvent reservoirs, A (H2O-Trifluoroacetic acid 0.05%) and B (CH3CN). The gradient system was as follows: 0–8 min, 100–0% B; 9–12 min, 90–10% B; 13–15 min, 80–20% B; 16–20 min, 70–30%, 21–25 min, 0–100% B, and 26–28 min 100–0% B. The flow rate was set at 1 mL/min, with a 2 mg/mL sample concentration and an injection volume of 10 μL [12]. The absorption was measured at λ = 205 nm 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 (1H, COSY, HSQC, HMBC, and DEPTq) were performed on a Bruker AVANCE III HD at 500 MHz. CD3COCD3 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 López-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 (LC50 and LC90) were determined using the PROBIT procedure included within the SAS statistic package. Plant material The fresh pre-flowering leaves and stems of A. cina O. Berg ex Poljakov (Asteraceae) (10 kg) were bought at Hunab® laboratory. A voucher specimen was authenticated by Dr. Alejandro Torres-Montúfar and was deposited at the herbarium of Facultad de Estudios Superiores Cuautitlán (FES-C) UNAM, México 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 L3 The lethal effect of the three A. cina extracts and fractions on L3 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 L3 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-Núñez [10]. The H. contortus strain was obtained from Facultad de Estudios Superiores Cuautitlán. 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 (Merck®, 0.015–0.040 mm) 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). C1F2 achieved the highest yield percentage with larvicidal activity, so it was used for column 2. Column 2 was performed using the same chromatographic conditions as Column 1. 34 samples were grouped into nine fractions: C2F1 (0.093 g), C2F2 (0.088 g), C2F3 (0.090 g), C2F4 (0.137 g), C2F5 (1.727 g), C2F6 (2.656 g), C2F7 (2.780 g), C2F8 (0.769 g), and C2F9 (1.142 g). C2F1 to C2F4 were not evaluated due to low yield percentages Instead, C2F5 to C2F7 were analyzed at C1F2 LC50 and LC90. C2F7 displayed the highest larvicidal activity, and LC50-90 values were calculated. C2F7 was selected to carry out Column 3 and isolate a bioactive molecule. Column 3 had the same chromatographic conditions as Columns 1 and 2. Column 3 was separated into 26 samples; samples 13–15, dissolved in a 3:2 n-hexane-ethyl acetate mixture, crystallized into needle crystals. These crystals were decanted, and the resulting crystals were washed with n-hexane, yielding 256 mg. TLC and HPLC analysis Analytical TLC was performed on precoated Merck® silica gel 60F254 or RP-18F254 plates. Ceric sulfate reagent was used to visualize terpenes. HPLC separations were performed on a Waters 2695 separations module equipped with a Waters 2996 photodiode array detector, and HPLC analysis was carried out using a LiChrospher® 100 RP-18 column (4 mm × 250 mm, five μm) (Merck, Kenilworth, NJ, USA). The mobile phase consisted of two solvent reservoirs, A (H2O-Trifluoroacetic acid 0.05%) and B (CH3CN). The gradient system was as follows: 0–8 min, 100–0% B; 9–12 min, 90–10% B; 13–15 min, 80–20% B; 16–20 min, 70–30%, 21–25 min, 0–100% B, and 26–28 min 100–0% B. The flow rate was set at 1 mL/min, with a 2 mg/mL sample concentration and an injection volume of 10 μL [12]. The absorption was measured at λ = 205 nm 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 (1H, COSY, HSQC, HMBC, and DEPTq) were performed on a Bruker AVANCE III HD at 500 MHz. CD3COCD3 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 López-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 (LC50 and LC90) were determined using the PROBIT procedure included within the SAS statistic package. Results 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). Download: PPT PowerPoint slide PNG larger image TIFF original image Table 1. The lethal concentration of Artemisia cina extracts on Haemonchus contortus infective larvae L3. https://doi.org/10.1371/journal.pone.0305155.t001 The HE had the best LC50, but no significant difference was found in LC90 compared with EAE. I do not present a dose response, so LC50 and LC90 cannot be calculated. EAE extract was chosen to perform the bio-guided separation due to the LC90 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 L3 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 L3 H. contortus infective larvae. C1F1 exhibited the highest larvicidal activity (Table 2), followed by C1F2. C1F3 did not show at least 50% larvicidal activity; LC50 and LC90 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. Download: PPT PowerPoint slide PNG larger image TIFF original image Table 2. In-vitro mortality percentage of A. cina fractions from bio-guided isolation of an anthelminthic compound against L3 H. contortus infective larvae. https://doi.org/10.1371/journal.pone.0305155.t002 In Column 3, where C2F7 was separated (Table 3), a colorless needle crystal was observed in samples 13–15. The larvicidal activity of compound 1 (C313-15P) was determined. The LC50 was 0.01 (0.01–0.02) mg/mL, and the LC90 was 0.21 (0.19–0.25) mg/mL. In comparison with EAE larvicidal activity (LC50 = 2.56 (2.45±2.65) mg/mL and LC90 = 3.30 (3.26±3.66) mg/mL), compound 1 (C313-15P) exhibited considerably more lethal activity against infective larvae (L3). Download: PPT PowerPoint slide PNG larger image TIFF original image Table 3. In-vitro mortality percentage of colorless needle crystal C313-15P obtained of C2F7 separation (column 3) against L3 H. contortus infective larvae. https://doi.org/10.1371/journal.pone.0305155.t003 Fig 1 is a schematic representation that illustrates the bio-guided separation methodology applied to the EAE, monitoring the larvicidal activity against the L3 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. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Bio-guided separation scheme of the ethyl acetate crude extract of Artemisia cina monitoring larvicidal activity on L3 Haemonchus contortus infective larvae. https://doi.org/10.1371/journal.pone.0305155.g001 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. This compound was soluble in dichloromethane-methanol 1:1. TLC showed a weak florescent band when observed under λ = 254 nm UV light and no fluorescence at λ = 365 nm. HPLC analysis showed a peak at 15.775 min and an absorption spectrum λ = 211.0 nm, typically of terpenes (Fig 2), and a [M+H]+ = 283 m/z (S1 Fig) The NIRS spectra (S2 Fig) exhibited 2060 nm R-OH and 2116 nm C-C combinations. The presence of 1118 nm CH3 third overtone region, 1406 CH3 and CH2 second overtone region, 1690 nm, 1714 nm, and 1738 nm CH3, CH2 and CH first overtone region. 2270 nm, 2290 nm, 2310 nm CH3, CH2 and CH combination area [14]. It also displayed a 213°C melting point. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Chromatogram and UV-VIS spectra of C3 13-15P, obtained by HPLC-DAD and viewed at 215 nm. https://doi.org/10.1371/journal.pone.0305155.g002 1H-NMR spectrum (Table 4 and S3 Fig) indicated the de presence of two methyl groups (δ 1.20 d, J = 6.83 Hz) and (δ 1.06 s), one CH-O proton (δ 4.27 m, br) and Ha (δ 6.21 t, J = 1.01, 1.01 Hz), Hb (δ 5.72 dd, J = 0.91, 1.70 Hz) of a terminal alkene. The 13C -NMR spectrum (Table 4 and S4 Fig) showed 15 signals, typically of sesquiterpenes. This was correlated with the absorption spectrum. The δ 216.56 signal (C-4) corresponds to a ketone carbonyl and δ 168.20 to an ester or carboxylic acid carbonyl. The δ 90.38 and 74.84 correspond to C-O signals. The δ 141.36 (C-11) and δ 125.07 (C-13) signals were alkene type, and the aromatic signal was discarded due to the absence of signals between δ 7.00 and δ 8.00 in the 1H -NMR spectrum. Methyl groups were observed at δ 21.15 (C-14) and δ 15.06 (C-15). Download: PPT PowerPoint slide PNG larger image TIFF original image Table 4. 1H -NMR and 13C -NMR spectroscopy data of compound 1 (CD3COCD, 500 MHz). https://doi.org/10.1371/journal.pone.0305155.t004 Sesquiterpene lactones are commonly found in the Artemisia genus [15]. The presence of ketone (δ 216. 56), ester carbonyl (δ 168.20), and alkene (δ 141. 36 and δ 125.07) signals are frequently of guaianolides and pseudoguaianolides (5–7 bicyclic compounds) [16]. The presence of pseudoguaianolide could be confirmed due to a methyl group at the C-5 ring junction (C-14 δ 21.15) and C-10 (C-15 δ 15.06). The signals δ 34.11, and δ 34.44 correspond to CH2 with a similar electronic environment corresponding to C-6 and C9 sesquiterpene lactone lactonized towards C7-C8. Surprisingly, HMBC analysis did not show correlation between C-12 and H-8 (δ 4.27 m, br) but correlated with H13a (δ 6.21 t, J = 1.01, 1.01 Hz) H13b (δ 5.72 dd, J = 0.91, 1.70 Hz)) and H7 (δ 2.85 m, br) (S5 Fig), so it was not lactonized and the ring is open. COSY and NOESY also show the correlation between H7 (δ 2.85 m, br) and H8 (δ 4.27 m, br) consistent with cis-orientation of the protons (S6 and S7A Figs). According to the NOESY, the two methyl groups (δ 1.20 d, J = 6.83 Hz) and (δ 1.06 s)) are cis-orientated too (S7B Fig). The proposed structure is shown in Fig 3. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. New sesquiterpene isolated from aerial parts of Artemisia cina (a) Structure of cinic acid (b) Correlations of cinic acid (HMBC, COSY and NOESY at 500MHz). https://doi.org/10.1371/journal.pone.0305155.g003 Compound 1 was identified 11-[(1R,5S,7R,8R,10S,)-1,8-dihydroxy-5,10-dimethyl-4-oxodecahydroazulen-7-yl] acrylic acid, not previously reported and was named cinic acid. Due to the structural similarity to the pseudoguainolides, the compound was numbered accordingly. 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). Download: PPT PowerPoint slide PNG larger image TIFF original image Table 1. The lethal concentration of Artemisia cina extracts on Haemonchus contortus infective larvae L3. https://doi.org/10.1371/journal.pone.0305155.t001 The HE had the best LC50, but no significant difference was found in LC90 compared with EAE. I do not present a dose response, so LC50 and LC90 cannot be calculated. EAE extract was chosen to perform the bio-guided separation due to the LC90 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 L3 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 L3 H. contortus infective larvae. C1F1 exhibited the highest larvicidal activity (Table 2), followed by C1F2. C1F3 did not show at least 50% larvicidal activity; LC50 and LC90 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. Download: PPT PowerPoint slide PNG larger image TIFF original image Table 2. In-vitro mortality percentage of A. cina fractions from bio-guided isolation of an anthelminthic compound against L3 H. contortus infective larvae. https://doi.org/10.1371/journal.pone.0305155.t002 In Column 3, where C2F7 was separated (Table 3), a colorless needle crystal was observed in samples 13–15. The larvicidal activity of compound 1 (C313-15P) was determined. The LC50 was 0.01 (0.01–0.02) mg/mL, and the LC90 was 0.21 (0.19–0.25) mg/mL. In comparison with EAE larvicidal activity (LC50 = 2.56 (2.45±2.65) mg/mL and LC90 = 3.30 (3.26±3.66) mg/mL), compound 1 (C313-15P) exhibited considerably more lethal activity against infective larvae (L3). Download: PPT PowerPoint slide PNG larger image TIFF original image Table 3. In-vitro mortality percentage of colorless needle crystal C313-15P obtained of C2F7 separation (column 3) against L3 H. contortus infective larvae. https://doi.org/10.1371/journal.pone.0305155.t003 Fig 1 is a schematic representation that illustrates the bio-guided separation methodology applied to the EAE, monitoring the larvicidal activity against the L3 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. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Bio-guided separation scheme of the ethyl acetate crude extract of Artemisia cina monitoring larvicidal activity on L3 Haemonchus contortus infective larvae. https://doi.org/10.1371/journal.pone.0305155.g001 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. This compound was soluble in dichloromethane-methanol 1:1. TLC showed a weak florescent band when observed under λ = 254 nm UV light and no fluorescence at λ = 365 nm. HPLC analysis showed a peak at 15.775 min and an absorption spectrum λ = 211.0 nm, typically of terpenes (Fig 2), and a [M+H]+ = 283 m/z (S1 Fig) The NIRS spectra (S2 Fig) exhibited 2060 nm R-OH and 2116 nm C-C combinations. The presence of 1118 nm CH3 third overtone region, 1406 CH3 and CH2 second overtone region, 1690 nm, 1714 nm, and 1738 nm CH3, CH2 and CH first overtone region. 2270 nm, 2290 nm, 2310 nm CH3, CH2 and CH combination area [14]. It also displayed a 213°C melting point. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Chromatogram and UV-VIS spectra of C3 13-15P, obtained by HPLC-DAD and viewed at 215 nm. https://doi.org/10.1371/journal.pone.0305155.g002 1H-NMR spectrum (Table 4 and S3 Fig) indicated the de presence of two methyl groups (δ 1.20 d, J = 6.83 Hz) and (δ 1.06 s), one CH-O proton (δ 4.27 m, br) and Ha (δ 6.21 t, J = 1.01, 1.01 Hz), Hb (δ 5.72 dd, J = 0.91, 1.70 Hz) of a terminal alkene. The 13C -NMR spectrum (Table 4 and S4 Fig) showed 15 signals, typically of sesquiterpenes. This was correlated with the absorption spectrum. The δ 216.56 signal (C-4) corresponds to a ketone carbonyl and δ 168.20 to an ester or carboxylic acid carbonyl. The δ 90.38 and 74.84 correspond to C-O signals. The δ 141.36 (C-11) and δ 125.07 (C-13) signals were alkene type, and the aromatic signal was discarded due to the absence of signals between δ 7.00 and δ 8.00 in the 1H -NMR spectrum. Methyl groups were observed at δ 21.15 (C-14) and δ 15.06 (C-15). Download: PPT PowerPoint slide PNG larger image TIFF original image Table 4. 1H -NMR and 13C -NMR spectroscopy data of compound 1 (CD3COCD, 500 MHz). https://doi.org/10.1371/journal.pone.0305155.t004 Sesquiterpene lactones are commonly found in the Artemisia genus [15]. The presence of ketone (δ 216. 56), ester carbonyl (δ 168.20), and alkene (δ 141. 36 and δ 125.07) signals are frequently of guaianolides and pseudoguaianolides (5–7 bicyclic compounds) [16]. The presence of pseudoguaianolide could be confirmed due to a methyl group at the C-5 ring junction (C-14 δ 21.15) and C-10 (C-15 δ 15.06). The signals δ 34.11, and δ 34.44 correspond to CH2 with a similar electronic environment corresponding to C-6 and C9 sesquiterpene lactone lactonized towards C7-C8. Surprisingly, HMBC analysis did not show correlation between C-12 and H-8 (δ 4.27 m, br) but correlated with H13a (δ 6.21 t, J = 1.01, 1.01 Hz) H13b (δ 5.72 dd, J = 0.91, 1.70 Hz)) and H7 (δ 2.85 m, br) (S5 Fig), so it was not lactonized and the ring is open. COSY and NOESY also show the correlation between H7 (δ 2.85 m, br) and H8 (δ 4.27 m, br) consistent with cis-orientation of the protons (S6 and S7A Figs). According to the NOESY, the two methyl groups (δ 1.20 d, J = 6.83 Hz) and (δ 1.06 s)) are cis-orientated too (S7B Fig). The proposed structure is shown in Fig 3. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. New sesquiterpene isolated from aerial parts of Artemisia cina (a) Structure of cinic acid (b) Correlations of cinic acid (HMBC, COSY and NOESY at 500MHz). https://doi.org/10.1371/journal.pone.0305155.g003 Compound 1 was identified 11-[(1R,5S,7R,8R,10S,)-1,8-dihydroxy-5,10-dimethyl-4-oxodecahydroazulen-7-yl] acrylic acid, not previously reported and was named cinic acid. Due to the structural similarity to the pseudoguainolides, the compound was numbered accordingly. 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 pose 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 L3 infective larvae [9], transitional larvae L3-L4 [4], and naturally infected periparturient goats [3]. Those activities are attributed to two lignans, 3′-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 LC50 and 15.71 times at LC90 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 γ-lactone 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 L3 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 (L3) 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 Scifinder®, and the lethal effect should be patented, and the studies with the molecule must be continued to know the in vivo effect. Supporting information S1 Fig. Mass spectra of cinic acid obtained by CG-MS. https://doi.org/10.1371/journal.pone.0305155.s001 (DOCX) S2 Fig. Cinic acid near-infrared spectroscopy spectra. https://doi.org/10.1371/journal.pone.0305155.s002 (DOCX) S3 Fig. 1H NMR spectra of cinic dissolved in CD3COCD3 and obtained at 500 MHz. https://doi.org/10.1371/journal.pone.0305155.s003 (DOCX) S4 Fig. DEPTq spectra of cinic acid dissolved in CD3COCD3 and obtained at 500 MHz. https://doi.org/10.1371/journal.pone.0305155.s004 (DOCX) S5 Fig. HMBC spectra of cinic acid dissolved in CD3COCD3 and obtained at 500 MHz. https://doi.org/10.1371/journal.pone.0305155.s005 (DOCX) S6 Fig. COSY experiment of cinic acid dissolved in CD3COCD3 and obtained at 500 MHz: a) COSY spectra of cinic acid and b) COSY spectra of the correlation between H7 (δ 2.85 m, br) and H8 (δ 4.27 m, br), consistent with the cis-orientation of the protons. https://doi.org/10.1371/journal.pone.0305155.s006 (DOCX) S7 Fig. NOESY experiment of cinic acid dissolved in CD3COCD3 and obtained at 500 MHz: a) NOESY spectra of cinic acid and b) NOESY spectra of the correlation between the two methyl groups (δ 1.20 d, J = 6.83 Hz) and (δ 1.06 s)), consistent with the cis-orientation of the protons. https://doi.org/10.1371/journal.pone.0305155.s007 (DOCX) Acknowledgments To the social service students who helped obtain biological material: Eduardo Rico-Mejía, Fernanda Lazcano Cárdenas, and Lian Guillermo Cortés Campos.
TI - Bio-guided isolation of a new sesquiterpene from Artemisia cina with anthelmintic activity against Haemonchus contortus L3 infective larvae
JO - PLoS ONE
DO - 10.1371/journal.pone.0305155
DA - 2024-06-12
UR - https://www.deepdyve.com/lp/public-library-of-science-plos-journal/bio-guided-isolation-of-a-new-sesquiterpene-from-artemisia-cina-with-s0sBOMFIdV
SP - e0305155
VL - 19
IS - 6
DP - DeepDyve
ER -