Impact of organic manure and chemical fertilizers on artemisinin content and yield in Artemisia annua L

Artemisinin isolated from the aerial parts of Artemisia annua L. is a promising and potent antimalarial drug. It posses remarkable activity against both chloroquinine resistant as well as chloroquinine sensitive strains of Plasmodium falciparum. It is also useful in the treatment of cerebral malaria. The relatively low content of artemisinin in A. annua and unavailability of cost effective and viable synthetic protocol however, are major obstacles to the commercial production of the drug. The enhanced production of artemisinin is hence, highly desirable, which can be achieved by adequate and judicious supply of plant nutrients. The present experiment was therefore, designed to study the effect of organic manure (15 tonnes ha⁻¹) and chemical fertilizers (N₄₀₊₄₀, P₄₀, K₄₀, S₁₅₊₁₅ kg ha⁻¹; nitrogen, phosphorus, potassium and sulphur) on the accumulation of artemisinin and biomass in various plant parts through the developmental stages of A. annua L. Artemisinin yield (kg ha⁻¹) was also determined through the developmental stages of A. annua L. Artemisinin content and artemisinin yield of dried leaves were increased significantly at pre-flowering stage in the plants treated with NPKS (27.3% and 53.6%) and NPK (18.2% and 33.5%), respectively, when compared with control. Maximum dry yield of leaf ranging from 2596 to 3141 kg ha⁻¹ was observed at pre-flowering stage with various treatments.     

Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L.

There has been much interest in artemisinin owing to its excellent activity against malaria, an infectious disease threatening the tropical world. However, the low artemisinin content (0.01-0.8%, DW) in Artemisia annua, which is the only commercial source of artemisinin, makes artemisinin expensive to produce and not yet available on a global scale. Here we show that foliar application of 100 mg l⁻¹ chitosan improved artemisinin biosynthesis in A. annua. The content of dihydroartemisinic acid and artemisinin in chitosan-treated leaves increased by 72% and 53% compared with control values, respectively. Chitosan induced the expression of ADS and DBR2, which could explain the increase in level of artemisinic metabolites. After chitosan treatment, the amounts of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) in leaves of A. annua were 1.4 and 3.0 times higher than those of the control, respectively. Accumulation of reactive oxygen species (ROS) probably accelerated the conversion of dihydroartemisinic acid to artemisinin. Foliar application of 100 mg l⁻¹ chitosan had no harmful effect on A. annua growth. The simple method described here could be an effective method to improve artemisinin production in A. annua field cultivation.     

Influence of planting date on growth, artemisinin yield, seed and oil yield of Artemisia annua L. under temperate climatic conditions

Artemisia annua L. is an annual aromatic antibacterial herb, with effective antimalarial properties due to the presence of artemisinin. The intention of the present study was to establish plant survival, growth attributes, yield attributes and artemisinin yield of A. annua cv CIM - Arogya with different transplanting months in two cropping seasons (March 2005-February 2006 and March 2006-February 2007) under temperate climatic conditions of Himalaya, India. Artemisinin yield in the dried leaves was found maximum amongst the plants that were transplanted in March (24.39 kg ha⁻¹) and minimum in those transplanted in November (3.39 kg ha⁻¹).     

Increases in leaf artemisinin concentration in Artemisia annua in response to the application of phosphorus and boron

Malaria resurgence particularly in the third world is considerable and exacerbated by the development of multi-drug resistances to chemicals such as chloroquinone. Drug therapies, as recommended by WHO include the use of antimalarial compounds derived from Artemisia annua L., i.e. artemisinin-based therapies. This work aims to determine how A. annua plant dry matter can be enhanced while maximising artemisinin concentration from understanding the plant's mineral requirements for P and B. Experiments with differing of P, from 5 to 120 mg L⁻¹ and B from 0.1 to 0.9 mg L⁻¹ were undertaken. Mineral nutrients were supplied in irrigation water to potted plants and after a period of growth, dry matter production and leaf artemisinin concentration were determined. Increases in P application enhanced plant growth and total dry matter production. An optimal application rate, with respect to dry matter, was apparent around 30 mg P L⁻¹. Despite increases in P application having no influence on leaf artemisinin concentration, optimal yields of artemisinin, on a per plant basis, were again achieved at supply rate around 30-60 mg L⁻¹. Increasing B application rate had little influence on dry matter production despite increases in B leaf tissue concentration promoting the total amount of B per plant. Leaf artemisinin concentration significantly increased with B application rate up to 0.6 mg B L⁻¹. The higher artemisinin concentrations when multiplied by total leaf dry matter at the higher B application rates produced an increase in total artemisinin production per plant. There was however no further significant effect on leaf artemisinin concentration when B supply concentrations increased further (0.9 mg L⁻¹). Artemisinin production varied between the two experiments to a greater extent than plant dry matter production and the reasons for this are discussed in relation to growing environments and their possible impacts on artemisinin biosynthesis.     

Optimisation of a solvent for the complete extraction of prolamins from heated foods

Wheat bread was lyophilised, ground, extracted and centrifuged. The supernatants were analysed for gluten content by RP-HPLC and a commercial sandwich ELISA. Prolamin extraction solvents contained tris(2-carboxyethyl)phosphine (TCEP; 1, 2, 5, 10, 20, 50 mmol/L), guanidine hydrochloride (GUA; 0 or 2 mol/L) and a buffered salt solution. A commercial cocktail solution (250 mmol/L mercaptoethanol (ME), 2 mol/L GUA, buffered salt solution) as well as 60% (v/v) ethanol were used as control solvents. Wheat flour was the control for the extractability of the native prolamins. 60% ethanol only extracted 37% of the prolamins from wheat bread (cocktail = 100%). When ME was replaced by TCEP the protein yield increased from 35% at the lowest TCEP-level to 95% when 20–50 mmol/L TCEP were used. The use of GUA was essential to extract prolamins quantitatively. Comparative protein analysis using RP-HPLC and ELISA showed that both methods provided comparable prolamin (gliadin) concentrations of the wheat flour (40.3–45.7 mg/g), when 60% ethanol was used as extraction solvent. The extraction yields from bread were considerably lower (16.7–24.7 mg/g). Cocktail and TCEP extracted almost the same amount of protein from flour and bread with TCEP showing slightly lower yields. Total extractable protein (gliadin + glutenin) as determined by RP-HPLC was 70.5–75.3 mg/g, and total gliadin as determined by ELISA was 42.7–44.2 mg/g. Thus, the study has shown that TCEP in combination with GUA extracts proteins from heated, gluten-containing foods as effective as the commercial cocktail solution.     

A Novel Lipid Extraction Method from Wet Microalga Picochlorum sp. at Room Temperature

A novel method using ethanol was proposed for extracting lipids from wet microalga Picochlorum sp. at room temperature and pressure. In this study, Central Composite design (CCD) was applied to investigate the optimum conditions of lipid extraction. The results revealed that the solvent to biomass ratio had the largest effect on lipid extraction efficiency, followed by extraction time and temperature. A high lipid extraction yield (33.04% of the dry weight) was obtained under the following extraction conditions: 5 mL solvents per gram of wet biomass for 37 min with gentle stirring at room temperature. The extraction yield was comparable to that obtained by the widely used Bligh-Dyer method. Furthermore, no significant differences in the distribution of lipid classes and fatty acid composition were observed according to different extraction methods. In conclusion, these results indicated that the proposed procedure using ethanol could extract lipids from wet biomass efficiently and had giant potential for lipid extraction at large scale.     

Effects of varying nitrogen doses on yield,yield components and artemisinin content of Artemisia annua L.

Artemisia annua L. is an aromatic-antibacterial herb that destroys malarial parasites, lowers fevers and checks bleeding, and of which the secondary compound of interest is artemisinin. The objective of the present study was to determine yield, yield components and artemisinin content of A. annua L. grown under four nitrogen applications (0, 40, 80 and 120 kg ha⁻¹) in the Çukurova region of Turkey in 2004 and 2005. Field trials were conducted at Çukurova University, Agricultural Faculty Field Crops Department. In the study, plant height, number of branches, fresh herbage yield, dry herbage yield, fresh leaf yield, dry leaf yield, essential oil content and artemisinin content (by high performance liquid chromatography, HPLC) were examined. By analysis of variance, nitrogen doses had no any statistical effect on the traits investigated except for artemisinin content. Artemisinin content of the dried leaves were significantly affected by nitrogen applications, which varied from 6.32 to 27.50 mg 100 g⁻¹. Contents were from 120 and 80 kg ha⁻¹ nitrogen for the years of 2004 and 2005, respectively.     

β-Glucan extraction from bran of hull-less barley by accelerated solvent extraction combined with response surface methodology

The objective of this study was to explore optimal extraction technology of β-glucan from bran of hull-less barley (Qingke in Chinese), and provide scientific basis for industrialization of β-glucan extraction from a commodity waste which is rich in β-glucan. β-Glucan extraction from bran of hull-less barley was performed with an accelerated solvent extraction (ASE) technique and compared with ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and reflux extraction. The best combination of extraction parameters was obtained through response surface methodology (RSM) with a three-variable-three-level Box-Behnken design (BBD). The optimum extraction parameters were as follow: extraction time for 9 min, extraction temperature at 70 °C, number of cycles at 4, and extraction pressure at 10 MPa. Under these conditions, the experimental extraction yield of β-glucan was 16.39 ± 0.3%, which agreed closely with the predicted value (16.33%). Compared with other extraction methods, ASE produced much higher β-glucan and more environmentally friendly extraction and solvent systems, less extraction discrimination and shorter time, and could be useful to the development of industrial extraction processes.     

Optimization of extraction techniques and quantification of Betulinic Acid (BA) by RP-HPLC method from Ancistrocladus heyneanus Wall. Ex Grah.

Simple, cost effective, quick and sustainable technique was investigated for determining Betulinic acid (BA) from samples of Ancistrocladus heyneanus. The methods comprised of continuous shaking, Soxhlet, ultra sonication and microwave assisted extraction techniques. RP-HPLC technique was used to quantify BA from varied samples and confirmation of the samples was done using TLC, FT-IR and FT-Raman methods. The study also evaluated productivity of operational parameters such as solvent composition and extraction time for three distinctive parts of the plant (green leaves, brown leaves and stem). Brown leaves showed response to continuous shaking extraction and ultrasonic extraction techniques with 95% Aq. MeOH as a good extraction solvent. BA was detected and quantified in continuous shaking method with 15, 30 and 45 min of exposure time. Comparison of 6 min with 12 min of ultra sonication, showed longer sonication diminished extraction efficiencies. Concluding, brown leaves in 95% MeOH and ultrasonic extraction technique to be the fastest, easiest and best method for detection and screening of BA from A. heyneanus.     

Comparison of Cell Disruption Methods for Improving Lipid Extraction from Thraustochytrid Strains

Lipid extraction is an integral part of biodiesel production, as it facilitates the release of fatty acids from algal cells. To utilise thraustochytrids as a potential source for lipid production. We evaluated the extraction efficiency of various solvents and solvent combinations for lipid extraction from Schizochytrium sp. S31 and Thraustochytrium sp. AMCQS5-5. The maximum lipid extraction yield was 22% using a chloroform:methanol ratio of 2:1. We compared various cell disruption methods to improve lipid extraction yields, including grinding with liquid nitrogen, bead vortexing, osmotic shock, water bath, sonication and shake mill. The highest lipid extraction yields were obtained using osmotic shock and 48.7% from Schizochytrium sp. S31 and 29.1% from Thraustochytrium sp. AMCQS5-5. Saturated and monounsaturated fatty acid contents were more than 60% in Schizochytrium sp. S31 which suggests their suitability for biodiesel production.     

Extraction of carotenoids from <Emphasis Type="Italic">Lycium ferocissimum</Emphasis> fruits for cotton dyeing: Optimization survey based on a central composite design method

Studies were carried out to assess the extraction yield of carotenoids from the African boxthorn (Lycium ferocissimum Miers, Solanaceae) fruits with different solvents and solvent mixtures, to optimize the extraction conditions for maximum recovery and to improve the extraction efficiency. Among other solvents, a mixture of hexane and acetone gave the highest carotenoid extraction. Extraction conditions, such as hexane percentage in hexane/acetone solvent mixture, solvent-solid ratio, and extraction time were optimized using a statistically designed experiment. A regression equation for predicting the carotenoid yield as a function of three extraction variables was derived by statistical analysis. The optimized conditions for maximum carotenoids yield were 45 % hexane in solvent mixture, solvent-solid ratio of 70 (ml/g) and extraction time 70 min. The dyeability of cotton with carotenoids extract has also been studied. Unmordant and postmordanted bleached cotton fabric with alum and ferrous sulphatewas dyed. Color measurements and fastness properties as light, rubbing and wash were tested.     

Biobased Solvents for Pressurized Liquid Extraction of Nannochloropsis gaditana Omega-3 Lipids

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal biomass.     

Chemical composition and antioxidant and antimicrobial activity of essential oil of Artemisia annua L. from Bosnia

Hydrodistilled volatile oil obtained from the aerial parts of Artemisia annua L., cultivated near Sarajevo, Bosnia, was analyzed by GC-MS. More than one hundred compounds were identi?ed, representing 95.5% of the total oil. The major constituents of essential oil were oxygenated monoterpenes, artemisia ketone (30.7%) and camphor (15.8%). Isolated essential oil was tested for radical-scavenging ability using the stable DPPH radical, the ABTS radical, for reducing power ability with a test based on the reduction of ferric cations, for reducing ability of hydroxy radical in ORAC assay, and for metal chelating ability using the ferrozine assay. In all tests oil did not show a prominent antioxidant activity, but still comparable with thymol, an already known antioxidant. The screening of antimicrobial activity of oil was individually evaluated against representatives of Gram-positive, Gram-negative bacteria and fungi, using the agar diffusion method. All tested microorganisms were inhibited by essential oil. To the best of our knowledge, this is the first report of antimicrobial activity of essential oil of A. annua against Haemophilus influenzae, Enterococcus faecalis, Streptococcus pneumoniae, Micrococcus luteus and Candida krusei microbial strains. The antioxidant, antibacterial and antifungal activity of essential oil of A. annua from Bosnia is presented here for the first time and extends our knowledge in the range of valuable biological activities and possible roles in therapy associated with this medicinal herb.     

Phenolic composition and antioxidant capacity of six artemisia species

The aim of this work was to establish the antioxidant capacities and the polyphenolic profile of six different Artemisia species that could potentially be used in the human diet. A high-performance liquid chromatographic method coupled with photodiode-array detector was used to identify and quantify individual phenolic compounds of the Artemisia leaves. A total of 18 polyphenolic compounds were identified and quantified in Artemisia leaves, including hydroxybenzoic acids (4), hydroxycinnamic acids (5), flavonols (3), and catechins (2). It was observed that total phenolic content of A. annua and A. stelleriana leaves were significantly lower than the other four species. Ferulic and caffeic conjugates acids were the most dominant hydroxycinnamic acid and gallic acid and catechin were the most dominant hydroxycinnamic acid and catechins respectively. According to DPPH assays, the antioxidant capacity of A. arborescens spp., A. ludoviciana spp., A. oleandica spp. and A. princepts spp., were found to be higher (reflecting a 2-fold difference) than that of the other two species. Compared with those of major commercial leafy vegetables, leaves of Artemisia contain a higher content of flavonoids and phenolic acids, which provide significant health benefits and may be used as natural colorants.     

Response Surface Methodology for Ultrasound-Assisted Extraction of Astaxanthin from Haematococcus pluvialis

Astaxanthin is a novel carotenoid nutraceutical occurring in many crustaceans and red yeasts. It has exhibited various biological activities including prevention or amelioration of cardiovascular disease, gastric ulcer, hypertension, and diabetic nephropathy. In this study, ultrasound-assisted extraction was developed for the effective extraction of astaxanthin from Haematococcus pluvialis. Some parameters such as extraction solvent, liquid-to-solid ratio, extraction temperature, and extraction time were optimized by single-factor experiment and response surface methodology. The optimal extraction conditions were 48.0% ethanol in ethyl acetate, the liquid-to-solid ratio was 20:1 (mL/g), and extraction for 16.0 min at 41.1 °C under ultrasound irradiation of 200 W. Under optimal conditions, the yield of astaxanthin was 27.58 ± 0.40 mg/g. The results obtained are beneficial for the full utilization of Haematococcus pluvialis, which also indicated that ultrasound-assisted extraction is a very useful method for extracting astaxanthin from marine life.     

Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity

Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI)+)/ion trap-MS) characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC) assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM). No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.     

Characterization of biodiesel and bio-oil from Sterculia striata (chicha) oil

This work describes the mechanical and solvent extraction of Sterculia striata seed oil. It was determined that the seeds contain up to 41% in oil, which has an unusual composition. Indeed, up to 50% of the fatty acid contain cyclopropenoid ring. The oil was used as raw material to produce bio-oil and biodiesel and their physical-chemical properties were evaluated. Some of the studied physical-chemical properties of the S. striata biodiesel are in acceptable range for use as biodiesel in diesel engines, showing a promising economic exploitation of this raw material in semi-arid regions. It was also observed that the cyclopropenoid ring remains after transesterification and is decomposed during pyrolysis.     

Rapid Solid-Liquid Dynamic Extraction (RSLDE): a New Rapid and Greener Method for Extracting Two Steviol Glycosides (Stevioside and Rebaudioside A) from Stevia Leaves

Stevioside and rebaudioside A are the main diterpene glycosides present in the leaves of the Stevia rebaudiana plant, which is used in the production of foods and low-calorie beverages. The difficulties associated with their extraction and purification are currently a problem for the food processing industries. The objective of this study was to develop an effective and economically viable method to obtain a high-quality product while trying to overcome the disadvantages derived from the conventional transformation processes. For this reason, extractions were carried out using a conventional maceration (CM) and a cyclically pressurized extraction known as rapid solid-liquid dynamic extraction (RSLDE) by the Naviglio extractor (NE). After only 20 min of extraction using the NE, a quantity of rebaudioside A and stevioside equal to 1197.8 and 413.6 mg/L was obtained, respectively, while for the CM, the optimum time was 90 min. From the results, it can be stated that the extraction process by NE and its subsequent purification developed in this study is a simple, economical, environmentally friendly method for producing steviol glycosides. Therefore, this method constitutes a valid alternative to conventional extraction by reducing the extraction time and the consumption of toxic solvents and favouring the use of the extracted metabolites as food additives and/or nutraceuticals. As an added value and of local interest, the experiment was carried out on stevia leaves from the Benevento area (Italy), where a high content of rebaudioside A was observed, which exhibits a sweet taste compared to stevioside, which has a significant bitter aftertaste.     

Comparative chemistry and insect antifeedant action of traditional (Clevenger and Soxhlet) and supercritical extracts (CO2) of two cultivated wormwood (Artemisia absinthium L.) populations

A comparison between traditional extraction techniques (hydrodistillation and organic solvent extraction) and supercritical fluid extraction was made for two different populations and crops of Artemisia absinthium L., cultivated in the field and aeroponically. The composition of the extracts, volatile and non volatile oils, was analyzed by GC-MS and HPLC-DAD, respectively. The antifeedant and phytotoxic activity of the extracts was tested on insect pests (Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi) and plants (Lactuca sativa and Lolium perenne). The supercritical extracts exhibited stronger antifeedant effects than the traditional ones (up to 8 times more active) with moderate selective phytotoxic effects on L. perenne root growth (<50% inhibition).     

Are traditional neem extract preparations as efficient as a commercial formulation of azadirachtin A?

Neem seeds contain many substances with insecticidal properties, the main insecticidal ingredient being azadirachtin A. In developing countries such as Mali, a neem seed water extract is prepared by soaking ground seeds in water for three or seven days. The aim of this study was to check the effectiveness of this extract in terms of azadirachtin A extraction yield and insecticidal activity. The yield of extraction was 0.19 g azadirachtin A/100 g seeds. The concentration of azadirachtin A in the seed extract was approximately 200 mg l⁻¹, eight times higher than the recommended concentration of commercial products (25 mg l⁻¹). A comparison of the extractive capacity of different solvents indicated that the best solvents were water and methanol. The azadirachtin A concentration declined in extracts stored for more than 3 days at a temperatures higher than 30 °C. Bioassays were performed on target insects (the leafhopper Macrosteles quadripunctulatus, the moth Spodoptera littoralis and the tobacco whitefly Bemisia tabaci) in order to compare the insecticidal activity of the neem extract with that of the commercial product Neemazal T/S and of a solution of pure azadirachtin A. The bioassays conducted on the leafhopper and the moth demonstrated that the neem extract at the recommended concentration (25 mg l⁻¹ active ingredient) was as effective as the azadirachtin-based commercial product at the same concentration, while for the control of the whitefly B. tabaci a higher concentration of the water extract was needed.