Jorge F.S. Ferreira

Analysis of underivatized artemisinin and related sesquiterpene lactones by high-performance liquid chromatography with ultraviolet detection

INTRODUCTION Although high-performance liquid chromatography with ultraviolet detection (HPLC-PAD) is widely available, it has not been used for artemisinin (1) analysis because of the lack of UV absorption reported for this lactone. Increased Artemisia annua cultivation for production of 1 requires an affordable and reliable method to analyse 1 and its precursors dihydroartemisinic acid (2) and artemisinic acid (3) simultaneously from underivatized plant extracts. OBJECTIVE To validate HPLC-PAD for the quantification of underivatized artemisinin from A. annua and artemisinin-based drugs. METHODOLOGY Dried A. annua leaves were extracted with petroleum ether, dried, reconstituted in acetonitrile, and analysed by HPLC-PAD at 192 nm using an isocratic mobile phase (60:40, acetonitrile:0.1% acetic acid). HPLC-PAD was evaluated through accuracy, precision, recovery and comparison with HPLC with evaporative light scattering detection (HPLC-ELSD). RESULTS HPLC-PAD proved accurate, precise and reproducible for the direct quantification of 1 and related compounds, and was more sensitive than ELSD for most of the compounds tested. The limit of quantification of 1-3 from plants was 0.048, 0.024 and 0.008 g/100 g dry weight, respectively. Recoveries were over 98%, with good intra- and inter-day repeatability. HPLC-PAD correlated significantly (r(2 )= 0.99, p < 0.001) with HPLC-ELSD for artemisinin analysis. HPLC-PAD was also reliable for the analysis of dihydroartemisinin, artesunate and artelinic acid. CONCLUSION HPLC with ultraviolet detection was validated for the quantification of underivatized 1, 2, and 3 from crude plant samples, and is readily applicable for the quality control of herbals and artemisinin-related pharmaceutical compounds.

Anthelmintic activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita essential oils evaluated in four different in vitro tests

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC(50) value of 0.045 mg/ml in EHA, 0.063 mg/ml in LDA, 0.009 mg/ml in LFIA, and 24.66 mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil.

Water deficit effect on the accumulation of biomass and artemisinin in annual wormwood(Artemisia annua L., Asteraceae)

Despite the importance of Artemisia annua L. as the only source of the anti-parasitic drug artemisinin, little is known on the effects of biotic and abiotic stress on artemisinin accumulation. Water deficit is the most limiting factor on plant growth, however it can trigger secondary metabolite accumulation, depending on the plant growth stage and intensity. A. annua cultivated in growth chambers was submitted to five water deficit treatments (watered, 14, 38, 62 e 86 hours without irrigation). Water deficits of 38 and 62 hours (Yw = -1.39 and -2.51 MPa, respectively) increased leaf artemisinin content, but only 38 hours led to a significant increase in both leaf and plant artemisinin (29%), with no detriment to plant biomass production. The other treatments had no effect on, or decreased artemisinin accumulation. A. annua plants tolerated well water deficit treatments, including the most severe water deficit applied (Yw -3.97 MPa or 86 hs without irrigation) and recovered their turgor pressure after rehydration. These results suggest that moderate water deficit prior to harvesting the crop may not only reduce time and costs in drying the crop, but can also induce artemisinin accumulation, both of which increase crop profit margins. Results also suggest that artemisinin could be part of A. annua chemical system of defense against water deficit.

Anthelmintic effect of plant extracts containing condensed and hydrolyzable tannins on Caenorhabditis elegans, and their antioxidant capacity

Although tannin-rich forages are known to increase protein uptake and to reduce gastrointestinal nematode infections in grazing ruminants, most published research involves forages with condensed tannins (CT), while published literature lacks information on the anthelmintic capacity, nutritional benefits, and antioxidant capacity of alternative forages containing hydrolyzable tannins (HT). We evaluated the anthelmintic activity and the antioxidant capacity of plant extracts containing either mostly CT, mostly HT, or both CT and HT. Extracts were prepared with 70% acetone, lyophilized, redissolved to doses ranging from 1.0mg/mL to 25mg/mL, and tested against adult Caenorhabditis elegans as a test model. The extract concentrations that killed 50% (LC(50)) or 90% (LC(90)) of the nematodes in 24h were determined and compared to the veterinary anthelmintic levamisole (8 mg/mL). Extracts were quantified for CT by the acid butanol assay, for HT (based on gallic acid and ellagic acid) by high-performance liquid chromatography (HPLC) and total phenolics, and for their antioxidant activity by the oxygen radical absorbance capacity (ORAC) assay. Extracts with mostly CT were Lespedeza cuneata, Salix X sepulcralis, and Robinia pseudoacacia. Extracts rich in HT were Acer rubrum, Rosa multiflora, and Quercus alba, while Rhus typhina had both HT and CT. The extracts with the lowest LC(50) and LC(90) concentrations, respectively, in the C. elegans assay were Q. alba (0.75 and 1.06 mg/mL), R. typhina collected in 2007 (0.65 and 2.74 mg/mL), A. rubrum (1.03 and 5.54 mg/mL), and R. multiflora (2.14 and 8.70 mg/mL). At the doses of 20 and 25mg/mL, HT-rich, or both CT- and HT-rich, extracts were significantly more lethal to adult C. elegans than extracts containing only CT. All extracts were high in antioxidant capacity, with ORAC values ranging from 1800 μmoles to 4651 μmoles of trolox equivalents/g, but ORAC did not correlate with anthelmintic activity. The total phenolics test had a positive and highly significant (r=0.826, p ≤ 0.01) correlation with total hydrolyzable tannins. Plants used in this research are naturalized to the Appalachian edaphoclimatic conditions, but occur in temperate climate areas worldwide. They represent a rich, renewable, and unexplored source of tannins and antioxidants for grazing ruminants, whereas conventional CT-rich forages, such as L. cuneata, may be hard to establish and adapt to areas with temperate climate. Due to their high in vitro anthelmintic activity, antioxidant capacity, and their adaptability to non-arable lands, Q. alba, R. typhina, A. rubrum, and R. multiflora have a high potential to improve the health of grazing animals and must have their anthelmintic effects confirmed in vivo in both sheep and goats.

Caenorhabditis elegans as a model to screen plant extracts and compounds as natural anthelmintics for veterinary use.

The most challenging obstacles to testing products for their anthelmintic activity are: (1) establishing a suitable nematode in vitro assay that can evaluate potential product use against a parasitic nematode of interest and (2) preparation of extracts that can be redissolved in solvents that are miscible in the test medium and are at concentrations well tolerated by the nematode system used for screening. The use of parasitic nematodes as a screening system is hindered by the difficulty of keeping them alive for long periods outside their host and by the need to keep infected animals as sources of eggs or adults when needed. This method uses the free-living soil nematode Caenorhabditis elegans as a system to screen products for their potential anthelmintic effect against small ruminant gastrointestinal nematodes, including Haemonchus contortus. This modified method uses only liquid axenic medium, instead of agar plates inoculated with Escherichia coli, and two selective sieves to obtain adult nematodes. During screening, the use of either balanced salt solution (M-9) or distilled water resulted in averages of 99.7 (± 0.73)% and 96.36 (± 2.37)% motile adults, respectively. Adult worms tolerated DMSO, ethanol, methanol, and Tween 80 at 1% and 2%, while Labrasol (a bioenhancer with low toxicity to mammals) and Tween 20 were toxic to C. elegans at 1% and were avoided as solvents. The high availability, ease of culture, and rapid proliferation of C. elegans make it a useful screening system to test plant extracts and other phytochemical compounds to investigate their potential anthelmintic activity against parasitic nematodes.

Effects of artemisinin and Artemisia extracts on Haemonchus contortus in gerbils (Meriones unguiculatus).

Haemonchus contortus is a blood-sucking abomasal parasite of small ruminants that is responsible for major losses to producers worldwide. Resistance of this nematode to commercial anthelmintics has produced a demand for alternative control methods. Plants in the genus Artemisia have traditionally been used as anthelmintics and whole plants and plant extracts have demonstrated activity against gastrointestinal nematodes in several studies. In addition, Artemisia annua is the sole commercial source of artemisinin, the raw material used to produce drugs effective against the hemoprotozoan malaria parasites (Plasmodium species). Artemisinin derivatives have also shown efficacy against some trematodes, including Fasciola hepatica and Schistosoma species. In this study, artemisinin was tested for efficacy against H. contortus in a gerbil model of infection. Also tested in the gerbil model were an aqueous extract, an ethanolic extract and the essential oil of A. annua, and an ethanolic extract of Artemisia absinthium. In all experiments, gerbils were infected with 600 third-stage H. contortus larvae. In experiment 1, gerbils were treated orally with 400 milligrams per kilogram body weight (mg/kg BW) artemisinin once or 200mg/kg BW artemisinin daily for 5 days (Days 4-8 post-infection). In experiment 2, gerbils were treated daily for 5 days with 600 mg/kg BW of A. annua ethanolic or aqueous extract. In Experiment 3, gerbils were treated with 1000 mg/kg BW of A. annua or A. absinthium ethanolic extract or with 300 mg/kg BW of A. annua essential oil daily for five consecutive days (Days 4-8 post-infection). No significant effects of treatment were seen with artemisinin or any of the Artemisia species extracts at the dosages studied. The non-ionic surfactant Labrosol(®) was an effective nontoxic solvent for delivery of hydrophilic plant extracts and the lipophilic essential oil used in the study.

Affordable and sensitive determination of artemisinin in Artemisia annua L. by gas chromatography with electron-capture detection.

Artemisinin demand has increased sharply since the World Health Organization recommended its use as part of the artemisinin combination therapies in 2001. The area for the crop cultivation has expanded in Africa and Asia and simpler and affordable methods for artemisinin analysis are needed for crop quality control. This work presented a novel chromatographic method of artemisinin analysis using gas chromatography with electron-capture detection. The sample extraction and preparation involved a single-solvent one-step extraction, with samples being analyzed in the extraction solvent directly after extraction. This method was accurate and reproducible with over 97% recoveries. The limit of detection was less than 3 microg/mL and the limit of quantification was less than 9 microg/mL, allowing samples as low as 100mg dry weight to be analyzed for artemisinin. The method can be applied to quality control of commercial plant extracts and to artemisinin-derived pharmaceuticals.

Variable salinity responses of 12 alfalfa genotypes and comparative expression analyses of salt-response genes

Twelve alfalfa genotypes that were selected for biomass under salinity, differences in Na and Cl concentrations in shoots and K/Na ratio were evaluated in this long-term salinity experiment. The selected plants were cloned to reduce genetic variability within each genotype. Salt tolerance (ST) index of the genotypes ranged from 0.39 to 1. The most salt-tolerant genotypes SISA14-1 (G03) and AZ-90ST (G10), the top performers for biomass, exhibited the least effect on shoot number and height. SISA14-1 (G03) accumulated low Na and Cl under salinity. Most genotypes exhibited a net reduction in shoot Ca, Mg, P, Fe, and Cu, while Mn and Zn increased under salinity. Salinity reduced foliar area and stomatal conductance; while net photosynthetic rate and transpiration were not affected. Interestingly, salinity increased chlorophyll and antioxidant capacity in most genotypes; however neither parameter correlated well to ST index. Salt-tolerant genotypes showed upregulation of the SOS1, SOS2, SOS3, HKT1, AKT1, NHX1, P5CS1, HSP90.7, HSP81.2, HSP71.1, HSPC025, OTS1, SGF29 and SAL1 genes. Gene expression analyses allowed us to classify genotypes based on their ability to regulate different components of the salt tolerance mechanism. Pyramiding different components of the salt tolerance mechanism may lead to superior salt-tolerant alfalfa genotypes.

Evaluation of Cymbopogon schoenanthus essential oil in lambs experimentally infected with Haemonchus contortus

Hematophagous gastrointestinal parasites cause significant economic losses in small ruminant grazing systems. The growing reports of multi-drug resistant parasites call for intensive research on alternative treatments for anthelmintics to help small ruminants cope with these parasites. Two-month-old lambs with mean body weight (BW) of 22.5 kg were experimentally infected with a multidrug-resistant Haemonchus contortus strain. Infected animals were dosed orally with Cymbopogon schoenanthus essential oil to evaluate its anthelmintic potential. Eighteen animals were allocated into three groups of six animals, and each received one of the following treatments: Group 1 - control (10 mL of water), Group 2 - C. schoenanthus essential oil (180 mg/kg BW); and Group 3 - C. schoenanthus essential oil (360 mg/kg BW). Animals received the oil once a day for 3 consecutive days. Lambs were evaluated clinically for blood biochemistry before, at 1, 5, 10, 15 and 20 days after treatment, and then were euthanized to assess the total worm burden. No statistically significant reduction in fecal egg count, packed cell volume or total worm count was observed after treatments. Also, no statistical difference among group means for blood levels of urea, creatinine, albumin, alkaline phosphatase, aspartate aminotransferase and gamma glutamyl transferase was found. Larval development assay (LDA) and egg hatch assay (EHA) were performed from feces of treated animals at 1, 5, 10 and 15 days after essential oil administration. An inhibition in LDA was observed 1 day after the 3-day treatment in larvae from feces of animals treated with 360 mg/kg essential oil. In conclusion, the essential oil at the doses of 180 mg/kg and 360 mg/kg was safe to sheep, but failed as an anthelmintic treatment when applied to young sheep artificially infected with a multidrug-resistant H. contortus strain.

The effects of combining Artemisia annua and Curcuma longa ethanolic extracts in broilers challenged with infective oocysts of Eimeria acervulina and E. maxima.

Due to an increasing demand for natural products to control coccidiosis in broilers, we investigated the effects of supplementing a combination of ethanolic extracts of Artemisia annua and Curcuma longa in drinking water. Three different dosages of this herbal mixture were compared with a negative control (uninfected), a positive control (infected and untreated), chemical coccidiostats (nicarbazin+narazin and, later, salinomycin), vaccination, and a product based on oregano. Differences in performance (weight gain, feed intake, and feed conversion rate), mortality, gross intestinal lesions and oocyst excretion were investigated. Broilers given chemical coccidiostats performed better than all other groups. Broilers given the two highest dosages of the herbal mixture had intermediate lesion scores caused by Eimeria acervulina, which was higher than in broilers given coccidiostats, but less than in broilers given vaccination, oregano and in negative controls. There was a trend for lower mortality (P = 0·08) in the later stage of the growing period (23-43 days) in broilers given the highest dosage of herbal mixture compared with broilers given chemical coccidiostats. In conclusion, the delivery strategy of the herbal extracts is easy to implement at farm level, but further studies on dose levels and modes of action are needed.