It is not just artemisinin: Artemisia sp. for treating diseases including malaria and schistosomiasis.

Artemisia sp., especially A. annua and A. afra, have been used for centuries to treat many ailments. While artemisinin is the main therapeutically active component, emerging evidence demonstrates that the other phytochemicals in this genus are also therapeutically active. Those compounds include flavonoids, other terpenes, coumarins, and phenolic acids. Artemisia sp. phytochemicals also improve bioavailability of artemisinin and synergistically improve artemisinin therapeutic efficacy, especially when delivered as dried leaf Artemisia as a tea infusion or as powdered dry leaves in a capsule or compressed into a tablet. Here results from in vitro, and in vivo animal and human studies are summarized and critically discussed for mainly malaria, but also other diseases susceptible to artemisinin and Artemisia sp. including schistosomiasis, leishmaniasis, and trypanosomiasis.

SARS-CoV-2 omicron variants are susceptible in vitro to Artemisia annua hot water extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has >2000 yr of history in treating fever a symptom common to many infectious diseases including viruses. The plant is widely used as a tea infusion in many areas of the globe to thwart many infectious diseases. AIM OF THE STUDY: The SARS-CoV-2 (COVID-19) virus continues to infect millions while rapidly evolving new variants that are more transmissible and evade vaccine-elicited antibodies, e.g., omicron and its subvariants. Having shown potency against all previously tested variants, A. annua L. extracts were further tested against highly infectious omicron and its recent subvariants. MATERIALS AND METHODS: Using Vero E6 cells, we measured the in vitro efficacy (IC50) of stored (frozen) dried-leaf hot-water A. annua L. extracts of four cultivars (A3, BUR, MED, and SAM) against SARS-CoV-2 variants: original WA1 (WT), BA.1 (omicron), BA.2, BA.2.12.1, and BA.4. End point virus titers of infectivity in cv. BUR-treated human lung A459 cells overexpressing hu-ACE2 were determined for both WA1 and BA.4 viruses. RESULTS: When normalized to the artemisinin (ART) or leaf dry weight (DW) equivalent of the extract, the IC50 values ranged from 0.5 to 16.5 µM ART and from 20 to 106 µg DW. IC50 values were within limits of assay variation of our earlier studies. End-point titers confirmed a dose-response inhibition in ACE2 overexpressing human lung cells to the BUR cultivar. Cell viability losses were not measurable at leaf dry weights ≤50 µg for any cultivar extract. CONCLUSIONS: A. annua hot-water extracts (tea infusions) continue to show efficacy against SARS-CoV-2 and its rapidly evolving variants and deserve greater attention as a possible cost-effective therapeutic.

SARS-CoV-2 omicron variants succumb in vitro to Artemisia annua hot water extracts.

The SARS-CoV-2 (COVID-19) global pandemic continuous to infect and kill millions while rapidly evolving new variants that are more transmissible and evading vaccine-elicited antibodies. Artemisia annua L. extracts have shown potency against all previously tested variants. Here we further queried extract efficacy against omicron and its recent subvariants. Using Vero E6 cells, we measured the in vitro efficacy (IC 50 ) of stored (frozen) dried-leaf hot-water A. annua L. extracts of four cultivars (A3, BUR, MED, and SAM) against SARS-CoV-2 variants: original WA1 (WT), BA.1.1.529+R346K (omicron), BA.2, BA.2.12.1, and BA.4. IC 50 values normalized to the extract artemisinin (ART) content ranged from 0.5-16.5 µM ART. When normalized to dry mass of the extracted A. annua leaves, values ranged from 20-106 µg. Although IC 50 values for these new variants are slightly higher than those reported for previously tested variants, they were within limits of assay variation. There was no measurable loss of cell viability at leaf dry weights ≤50 µg of any cultivar extract. Results continue to indicate that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective approach to help stave off this pandemic virus and its rapidly evolving variants.

Artemisia annua L. hot-water extracts show potent activity in vitro against Covid-19 variants including delta.

ETHNOPHARMACOLOGICAL RELEVANCE: For millennia, Artemisia annua L. was used in Southeast Asia to treat "fever". This medicinal plant is effective against multiple pathogens and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria caused by Plasmodium parasites. AIM OF THE STUDY: The SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants. MATERIALS AND METHODS: Using Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua L. extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1). RESULTS: In addition to being effective against the original wild type (WT) WA1, A. annua cultivars A3, BUR, MED, and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3 to 8.4 µM and 1.4-25.0 µM, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0 to 67.7 µg DW and 59.5-160.6 µg DW, respectively. Cell toxicity was insignificant at a leaf dry weight of ≤50 µg in the extract of any cultivar. CONCLUSIONS: Results suggest that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.

Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti SARS-CoV-2 activity against fully infectious virus of dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as the concentrations that inhibited viral replication by 50%; CC50s were also calculated and defined as the concentrations that kill 50% of cells. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet its hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.

Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet the hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.

Aeroponics for the culture of organisms, tissues and cells.

Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.

Mist deposition onto hairy root cultures: aerosol modeling and experiments.

We analyzed the applicability of the standard models for aerosol deposition in randomly packed fibrous filter beds to mist deposition across a bed of hairy roots in the nutrient mist bioreactor. Although the assumptions inherent in the models are met on a local level, the overall structure of the root bed introduces some uncertainty into the correct choice of root packing fraction and gas velocity required by the model. For reasonable parameter values, the minimum in the deposition efficiency curves is close to the peak in the mist number and mass distributions, and good penetration of the root bed is possible. We then measured the deposition of mist across a packed bed of Artemisia annua transformed roots as a function of droplet size, bed length, and gas flow rate at a root packing fraction alpha = 0.5. We compared the experimental measurements with the predictions of the aerosol deposition model and found good agreement between the measured and predicted values for the diameter where the deposition efficiency across the bed is 50%, D0.5. Agreement between the model and the experiments broke down when the flow rate was increased to the point where the creeping flow assumptions were no longer valid.

Sugars proportionately affect artemisinin production.

Little is known about the effect of sugars in controlling secondary metabolism. In this study, sugars alone or in combination with their analogs were used to investigate their role in the production of the antimalarial drug, artemisinin, in Artemisia annua L. seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. Different ratios of fructose to glucose yielded artemisinin levels directly proportional to increases in relative glucose concentration. When the glucose analog, 3-O-methylglucose, was added with glucose, artemisinin production was dramatically decreased, but hexokinase activity was significantly increased compared to glucose alone. In contrast, neither mannose nor mannitol had any significant effect on artemisinin yield. In comparison with 30 g/l sucrose, artemisinin levels were significantly reduced by 80% in the presence of 27 g/l sucrose + 3 g/l palatinose, which cannot be transported into cells through the sucrose transporter. Together these results suggest that both monosaccharide and disaccharide sugars are likely acting not only as carbon sources but also as signals to affect the downstream production of artemisinin, and that the mechanism of these effects appears to be complex.

N(2)O evolution by green algae.

Evidence is presented here that axenic cultures of Chlorella, Scenedesmus, Coelastrum, and Chlorococcum spp. evolve N(2)O when grown on NO(2), showing that the Chlorophyceae are a source of N(2)O in aquatic systems.

Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308.

Cyanophycin granules in the unicellular cyanobacterium Aphanocapsa 6308 were examined with the electron microscope in both thin section and by freeze-fracture techniques. Purified granules were examined with the electron microscope, by arginine determinations, by chromatography, and by elemental analysis. They are similar in ultrastructure and composition to those isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica, consisting of equal molar quantities of L-arginine and L-aspartic acid.

Variations in short term products of inorganic carbon fixation in exponential and stationary phase cultures of Aphanocapsa 6308.

Aphanocapsa 6308 metabolizes both NaHCO3 and Na2CO3. The short term incorporation (5-s) metabolic pattern and the patterns of incorporation of bicarbonate for exponential versus stationary phase cultures differ, however. Cells were equilibrated for 10 min in air and distilled water prior to injection of either NaH14CO3 at pH 8.0, or Na214CO3 at pH 11.0. Hot ethanol extracts were analyzed via paper chromatography and autoradiography for products of CO2 fixation. At 5 s, malate (51.5%) predominates slightly as a primary bicarbonate fixation product over 3-phosphoglycerate (40.3%); 3-phosphoglycerate is the primary product of carbonate fixation. At 60 s, the carbonate and bicarbonate labelling patterns are similar. Cells in stationary phase fix in 5 s a greater proportion of bicarbonate into malate (36% vs. 14% for 3-phosphoglycerate) than do cells in exponential growth. Likewise, 60 s incorporations show a large amount of bicarbonate fixed into aspartate (30.9%) in stationary phase cells over that of exponential phase (11.6%). These data suggest an operative C4 pathway for purposes not related to carbohydrate synthesis but rather as compensation for the incomplete tricarboxylic acid cycle in cyanobacteria. The enhancement of both aspartate fixation and CO2 fixation into citrulline in stationary phase correlates with an increase in cyanophycin granule production which requires both aspartate and arginine.

One-step acclimatization of plantlets using a mist reactor.

A mist reactor was used to grow and acclimatize carnation plants in vitro without using ex vitro acclimatization techniques. The acclimatization protocol in the reactor consisted of altering the mist-on period during the course of the culture period and a stepwise reduction in the relative humidity surrounding the plants from 98% to 70% relative humidity (RH) during the final week of in vitro growth. After transfer and further growth in a greenhouse for 5 weeks, survival was 91% for plants grown in reactors, 81% from vented boxes, and 50% from unvented boxes. Ex vitro survival directly correlated with increased in vitro rooting and decreased hyperhydration. In vitro rooting also correlated with high-quality plants, but did not significantly correlate with low hyperhydration, as normal plants often lacked roots. After 5 weeks in the greenhouse, the quantity of mid- and high-quality plants obtained from reactors and ventilated boxes was similar. Conditions in the mist reactor can be manipulated to produce plants that are readily acclimatized and are equal or better in quality and yield than plants produced using conventional methods.

Controlling hyperhydration of carnations (Dianthus caryophyllus L.) grown in a mist reactor.

Carnations (Dianthus caryophyllus L.) grown in vitro often develop physiological abnormalities such as hyperhydration. The amount of hyperhydration and growth was compared between carnations grown in mist reactors and conventional semisolid micropropagation systems (vented or unvented GA7 culture boxes). Plants grown in the mist reactor with long misting times (10 min h(-1)) produced more dry mass than those grown with <10 min h(-1); however, more misting also produced more hyperhydrated plants (70% hyperhydration). Control of hyperhydration in the mist reactor involved either reducing the overall nutrient mist supply or altering the mist supply throughout the culturing period. Stepped decreases in the mist supply throughout the 3-week period or an overall decrease in the duration of misting reduced hyperhydration to 13% and 5%, respectively. However, for both misting regimes, the biomass of normal (healthy) plants (fresh and dry weights) was limited. Further analysis suggested that, although normal plant biomass increased with longer mist exposure, hyperhydration levels also increased while the water content, based on percent dry weight, approached that of hyperhydrated plants. Sufficient normal plant development (fresh weight, leaf and shoot numbers, height, and rooting) with < 50% hyperhydration was obtained by weekly, stepped increases in the nutrient mist supply.

Arginine catabolism in Aphanocapsa 6308.

The catabolic products of arginine metabolism were observed in Aphanocapsa 6308, a unicellular cyanobacterium, by thin layer chromatography of growth media, by limiting growth conditions, and by enzymatic analysis. Of the organic, nitrogenous compounds examined, only arginine supported growth in CO2-free media. The excretion of ornithine at a concentration level greater than citrulline suggested the existence in Aphanocapsa 6308 of the arginine dihydrolase pathway which produced ornithine, CO2,NH4,+ adenosine 5'-triphosphate. Its existence was confirmed by enzymatic analysis. Although cells could not grow on urea as a sole carbon source a very active urease and subsequently an arginase were also demonstrated, indicating that Aphanocapsa can metabolize arginine via the arginase pathway. The level of enzymes for both pathways indicates a lack of genetic control. It is suggested that the arginase pathway provides only nitrogen for the cells wheras the arginine dihydrolase pathway provides not only nitrogen, but also CO2 and adenosine 5'-triphosphate.

The growth of single roots of Artemisia annua in nutrient mist reactors.

To better characterize the development and growth of hairy roots in a mist-fed root bed, a single root aerosol reactor was developed. Growth kinetics studies were conducted on hairy roots of Artemisia annua as a function of the mist cycle, carrier gas, and nutrient compositions. Sustained rapid growth was only observed when conditioned medium was fed to the roots. The presence of 1% CO(2) in the carrier gas did not enhance the growth kinetics but it did prevent necrosis of the tissue at the highest mist cycle.

Changes in Hechtian strands in cold-hardened cells measured by optical microsurgery.

Optical microsurgical techniques were employed to investigate the mechanical properties of Hechtian strands in tobacco (Nicotiana tabacum) and Ginkgo biloba callus cells. Using optical tweezers, a 1. 5-microm diameter microsphere coated with concanavalin A was inserted though an ablated hole in the cell wall of a plasmolyzed cell and attached to a Hechtian strand. By displacing the adhered microsphere from equilibrium using the optical trapping force, the tensions of individual strands were determined. Measurements were made using both normal and cold-hardened cells, and in both cases, tensions were on the order of 10(-12) N. Significant differences were found in the binding strengths of cold-hardened and normal cultured cells. An increased number density of strands in cold-hardened G. biloba compared with normal cultured cells was also observed. Although no Hechtian strands were detected in any Arabidopsis callus cells, strands were present in leaf epidermal cells. Finally, the movement of attached microspheres was monitored along the outside of a strand while cycling the osmotic pressure.

Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308.

The effect of a number of conditions on the amount of cyanophycin granule polypeptide [multi-L-arginyl poly(L-aspartic acid)] formed in the unicellular cyanobacterium Aphanocapsa 6308 was determined. Light, CO2, sulfur, and phosphorus starvation as well as the addition of arginine to culture media increased the amount of cyanophycin granule polypeptide in cells when compared with that in cells grown under conditions optimal for growth. Nitrogen limitation and reduction of growth temperature to 30 degrees C decreased the amount of cyanophycin granule polypeptide on a dry-weight basis. Shift-up and shift-down experiments suggest cyanophycin granule polypeptide may be a reserve nitrogen polymer in Aphanocapsa 6308.