Ecdysteroids

May combine two claims.

For sources containing ecdysteroid glycosides, aqueous alcohol provides good extraction yields; for sources containing non-polar conjugates, pure alcohol is more effective.

“For sources that contain ecdysteroid glycosides, aqueous alcohol provides good extraction yields, but for sources containing non-polar conjugates (acetates, acetonides, glucosylferulates; [109]), pure alcohol is more effective.”

• cites [109] Ho, 2008, Isolation of a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid chromatographic methods, J. Chromatogr. Sci. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Micro-extraction methods allow 100-200 samples to be processed by one person in a day.

“For the screening of large numbers of plant species for the presence of ecdysteroids, the development of micro-extraction methods has been particularly useful as they allow 100-200 samples to be processed by one person in a day.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Three solvent partitioning systems have proved particularly useful for the partial purification of phytoecdysteroids, either individually or sequentially.

“Three solvent partitioning systems have proved particularly useful for the partial purification of phytoecdysteroids, either individually or sequentially.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Heating at a moderate temperature with stirring or reflux improves extraction rates and yields without artefactual modification of ecdysteroids.

“Heating at a moderate temperature with stirring or reflux (Soxhlet extraction) improves extraction rates and yields without artefactual modification of ecdysteroids.”

May combine two claims.

Ecdysteroids and brassinosteroids differ in 3D structure: the distribution and orientation of hydroxyl groups are different, and the A/B-ring junction is cis in ecdysteroids and trans in brassinosteroids, resulting in a major difference in 3D shape.

“If one closely considers the 3D structure of ecdys-teroids and brassinosteroids (Figure 9), one sees that the distribution and orientation of the hydroxyl groups are different; the A/B-ring junction is cis in ecdysteroids and trans in brassinosteroids, resulting in a major difference in the 3D-shape of the molecules.”

Probably not a substantial claim: may not pass S3 (load-bearing).

RP-C18-SPE has become a standard workhorse of ecdysteroid partial purification.

“Without a doubt, RP-C18-SPE has become a standard workhorse of ecdysteroid partial purification.”

May combine two claims.

The equilibration of ecdysteroid acyl derivatives is enhanced by acid, which can also induce dehydration of the 14-OH.

“This equilibration is enhanced by the presence of acid and the same can also induce some dehydration of the 14-OH [106].”

• cites [106] Jacobson, M., and Crosby, D.G. (1971). The Ecdysones. Naturally Occurring Insecticides, Marcell Dekker, Inc. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

DCCC is not more frequently used because it requires specialised equipment and a separation may take several days.

“It is not more frequently used because it requires specialised equipment, which is not commonly available, and a separation may take several days.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CPC is fast, efficient and reproducible.

“CPC is a development of DCCC that uses a centrifugal process to speed up the separation process; it is thus fast, efficient and reproducible.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The more recent development of dedicated HPLC/DAD/MS equipment has made the analysis, identification and quantification (by MS/MS) of ecdysteroids in complex samples almost routine.

“The more recent development of dedicated HPLC/DAD/MS equipment has made the analysis, identification and quantification (by MS/MS) of ecdysteroids in complex samples almost routine.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In early electron-impact MS, it was not easy to obtain the molecular weight of ecdysteroids due to weak [M]+ ions and dehydration upon heating, resulting in losses of 18 amu.

“In the early days of the analysis of ecdysteroids by electron-impact MS, it was not easy to obtain the MW owing to the formation of weak [M] + ions and dehydration of the molecule upon heating of the probe, resulting in a series of losses of 18 amu, depending, in number, on the number of hydroxyl groups present in the ecdysteroid.”

May combine two claims.

Two major problems exist for in-line assessment of HPLC effluents by NMR: the customary use of protic solvents to elute HPLC columns and the sensitivity of NMR detection.

“Two major problems exist for in-line assessment of HPLC effluents by NMR: the customary use of protic solvents to elute HPLC columns and the sensitivity of NMR detection.”

May combine two claims.

NMR is the technique of choice for the identification of any new ecdysteroid analogue since as little as 50 μg of pure compound is often enough for full structural identification.

“The extensive improvements in the sensitivity, resolution and range of techniques available for NMR over the past 40 years have made it the technique of choice for the identification of any new ecdysteroid analogue since as little as 50 µg of pure compound is often enough to obtain a full structural identification, including the resolution of many of the stereochemical questions, such that it is”

May combine two claims.

This chromophore provides a major UV absorbance at 242 nm in MeOH or EtOH (Log ε = 4.103) and a minor absorbance at 310 nm.

“This provides a major UV absorbance at 242 nm in MeOH or EtOH (Log ε = 4.103) and a minor absorbance at 310 nm.”

May combine two claims.

The many hydroxyl groups and the 6-oxo group in ecdysteroids offer several possibilities for linking to a carrier protein, allowing various orientations, with the consequence that antibodies will preferentially recognise the part of the ecdysteroid molecule distal to the linkage site.

“The many hydroxyl groups and the 6-oxo group in ecdysteroids offer several possibilities for linking to a carrier protein, thus allowing various possible orientations of the steroid relative to the surface of the protein, with the consequence that antibodies will preferentially recognise the part of the ecdysteroid molecule that is distal to the linkage site.”

May combine two claims.

Ecdysteroids are not immunogenic in mammals because their molecular weights are normally <1000, so they need to be chemically coupled as hapten groups onto a suitable protein to raise an immune response.

“The ecdysteroids, by themselves, are not immunogenic in mammals as their MWs are normally <1000, so they need to be chemically coupled as hapten groups onto the surface of a suitable protein to raise an immune response.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Immunoassays allow the sensitive detection of small amounts of analyte in samples of limited purity, making them suitable for the quantification of levels and the comparison of many samples simultaneously.

“Immunoassays allow the sensitive detection of small amounts of analyte in samples of limited purity, so they are suitable for the quantification of levels and the comparison of many samples simultaneously.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Currently, only 20E is commercially available in large amounts.

“Currently, only 20E is commercially available in large amounts, so this will be used as the example in the discussion below:”

May combine two claims.

Most ecdysteroid-accumulating species contain between 0.01% and 0.1% of the dry weight as ecdysteroids, whereas the few identified high-accumulators contain 1% and above.

“Most ecdysteroid-accumulating species contain between 0.01% and 0.1% of the dry weight as ecdysteroids, whereas the few identified high-accumulators contain 1% and above.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The vast majority of currently available commercial sources of 20E are derived from Cyanotis spp., Pfaffia spp., Rhaponticum (Leuzea) spp. or Serratula spp.

“The vast majority of currently available commercial sources of 20E are derived from Cyanotis spp., Pfaffia spp., Rhaponticum (Leuzea) spp.or Serratula spp.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

Each antiserum raised differs in its sensitivity and specificity, and these have to be assessed in each case; cross-reactivities for a representative range of steroid analogues should be determined relative to the standard ecdysteroid.

“Each antiserum raised differs in its sensitivity and specificity, and these have to be assessed in each case; the cross-reactivities for a representative range of steroid analogues should be determined relative to the standard ecdysteroid (usually ecdysone or 20-hydroxyecdysone).”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Turkesterone (11α-hydroxy20E) from Ajuga turkestanica is commercially available.

“In addition, turkesterone (11α-hydroxy20E) from Ajuga turkestanica is commercially available.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

These first bioassays were very time-consuming and only semi-quantitative.

“These assays were exceedingly impor-tant at the time, but they were very time-consuming and only semi-quantitative.”

May combine two claims.

The hairy roots of some other ecdysteroid-producing species do not contain ecdysteroids, so this cannot be viewed as a general approach.

“Additionally, the hairy roots of some other ecdysteroid-producing species do not contain ecdysteroids, so this cannot be viewed as a general approach.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The quality of commercial ecdysteroid-containing supplements is not constant and may vary from batch to batch.

“Moreover, the quality is not constant and may vary from batch to batch (unpublished observations).”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The quality of dietary supplements containing 20E is not constant and may vary from batch to batch.

“Moreover, the quality is not constant and may vary from batch to batch (unpublished observations).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The first ecdysteroid was isolated in 1954 by Butenandt and Karlson from pupae of the silkworm Bombyx mori.

“The first ecdysteroid was isolated in 1954 by Butenandt and Karlson [5] from pupae of the silkworm Bombyx mori using a multi-step process (Figure 2), relying on an insect bioassay to detect moult-inducing activity.”

• cites [5] Butenandt, 1954, Über die Isolierung eines Metamorphose-Hormons der Insekten in kristallisierter Form, Z. Naturforsch. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Ecdysteroids are a family of invertebrate steroid hormones involved in the regulation of moulting, development and reproduction.

“Ecdysteroids are a family of invertebrate steroid hormones that are involved in the regulation of moulting, development and reproduction [1].”

• cites [1] Koolman, J. (1989). Ecdysone: From Chemistry of Mode of Action, Thieme Verlag.Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Phytoecdysteroid profiles may vary from simple to intermediate to complex.

“Phytoecdysteroid profiles may vary from simple (the presence of one or two major components) through intermediate (a mixture of major and minor components) to complex (a cocktail of many analogues) [3].”

• cites [3] Smagghe, G. (2009). Phytoecdysteroids: Diversity, biosynthesis and distribution. Ecdysone: Structures and Functions, Springer Science & Business Media B.V.Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The structure of ecdysone was established eleven years after its isolation.

“This compound was named”

• cites [6] Huber, 1965, Zur Chemie des Ecdysons, VII: Die Kristall- und Molekülstrukturanalyse des Insektenverpuppungshormons ecdyson mit der automatisierten Faltmolekülmethode, Chem. Ber. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Currently, 537 natural ecdysteroid analogues have been identified.

“Currently, 537 natural ecdysteroid analogues have been identified (Ecdybase [4])”

• cites [4] Lafont, R., Harmatha, J., Marion-Poll, F., Dinan, L., and Wilson, I.D. (2002). The Ecdysone Handbook, Cybersales. [3rd ed.]. Available online: http://ecdybase.org/.Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

In leaves of an even greater proportion of investigated plant species than seeds, ecdysteroids are present in detectable amounts.

“They are present in detectable amounts in the seeds of 5-6% of investigated plant species and in leaves of an even greater proportion of species [2].”

• cites [2] Dinan, 2001, On the distribution of phytoecdysteroids in plants, Cell Mol. Life Sci. sourceNot checked yet

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May combine two claims.

20E is produced from cholesterol through a multi-step process that is not yet fully elucidated.

“20E is produced from cholesterol through a multi-step process that is not yet fully elucidated [7,11,12] (Figure 4).”

• cites [7] Kannangara, 2021, Regulation of ecdysone production in Drosophila by neuropeptides and peptide hormones, Open Biol. sourceNot checked yet

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• cites [11] Gilbert, L.I. (2012). Ecdysteroid chemistry and biochemistry. Insect Endocrinology, Academic Press.Not checked yet

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• cites [12] Saleuddin, 2020, Molecular functions of ecdysteroids in insects, Advances in Invertebrate (Neuro)endocrinologyNot checked yet

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May combine two claims.

In adults, 20E controls reproduction and is produced by the gonads.

“in adults, it controls reproduction, but in this case, it is produced by the gonads [8,9].”

• cites [8] Gilbert, L.I. (2012). Insect Endocrinology, Academic Press.Not checked yet

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• cites [9] Belles, X. (2020). Insect Metamorphosis. From Natural History to Regulation of Developement and Evolution, Academic Press.Not checked yet

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May combine two claims.

The relatively high accumulation of ecdysteroids in plants facilitated the identification of further phytoecdysteroids and prompted the search for other ecdysteroid-accumulating species.

“This relatively high accumulation facilitated the identification of further phytoecdysteroids and prompted the search for other ecdysteroid-accumulating species in the plant world [27][28][29].”

• cites [27] Matsuoka, 1969, Studies on phytoecdysones—A review of our works, Ann. Rep. Takeda Res. LabNot checked yet

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• cites [28] Yen, 1974, Screening of Formosan ferns for phytoecdysones. I, Chem. Pharm. Bull. sourceNot checked yet

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• cites [29] Volodin, 2002, Screening plants of European North-East Russia for ecdysteroids, Biochem. Syst. Ecol. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Two early reports also identified close analogues: ponasterone A, ponasterone B, ponasterone C and inokosterone.

“two reports also identified close analogues-ponasterone A (25-deoxy-20-hydroxyecdysone; ponA), which is amongst the most biologically active ecdysteroids in arthropod systems, ponasterone B (2,3-di-epi-ponA), ponasterone C (5β,24S-dihydroxyponA) [18,25,26] and inokosterone [19].”

• cites [18] Nakanishi, K., Koreeda, M., Sasaki, S., Chang, M.L., and Hsu, H.Y. (1966). Insect hormones. The structure of ponasterone A, an insect moulting hormone from the leaves of Podocarpus nakaii Hay. J. Chem. Soc. Chem. Commun., 915–917. sourceNot checked yet

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• cites [25] Koreeda, M., and Nakanishi, K. (1970). 5β-Hydroxy-ecdysones and a revision of the structure of ponasterone C. Chem. Commun., 351–352. sourceNot checked yet

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• cites [26] Blunt, 1979, The absolute configuration at C-24 of the ecdysteroids dacrysterone, pterosterone and ponasterone C, Aust. J. Chem. sourceNot checked yet

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• cites [19] Takemoto, 1967, Isolation of the moulting hormones of insects from Achyranthes radix, Yakugaku Zasshi sourceNot checked yet

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May combine two claims.

All but one of the early reports of ecdysteroids in plants concerned the isolation of 20-hydroxyecdysone, which had also been identified, along with ecdysone, as a more minor component from Bombyx mori pupae.

“All but one of these reports concerned the isolation of 20-hydroxyecdysone, which had also been identified, along with ecdysone, as a more minor component from B. mori pupae [24]”

• cites [24] Hocks, 1966, 20-Hydroxyecdyson, isoliert aus Insekten, Tetrahedron Lett. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The extensive range of ecdysteroid analogues provides an excellent natural resource for SAR studies in arthropods and mammals.

“In addition to providing reference compounds for biochemical studies, the extensive range of ecdysteroid analogues provides an excellent natural resource for SAR studies in arthropods (reviewed in [37]) and mammals [38].”

• cites [37] (2003). Ecdysteroid structure-activity relationships. Studies in Natural Product Chemistry 29, Elsevier B.V.Not checked yet

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• cites [38] Hunyadi, 2008, Phytoecdysteroids and anabolic-androgenic steroids—Structure and effects on Humans, Curr. Med. Chem. sourceNot checked yet

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May combine two claims.

The huge diversity of phytoecdysteroid molecular structures indicates that biosynthesis in plants occurs as a combinatorial system of a network of enzymes without strong substrate specificity, which could result in over 1000 possible permutations of structure from a small number of early intermediates.

“The huge diversity of phytoecdysteroid molecular structures indicates that biosynthesis in plants occurs as a combinatorial system of a network of enzymes without strong substrate specificity, which could result in over 1000 possible permutations of structure from a small number of early intermediates (14α-hydroxy-6-oxo-7-ene C 27/28/29sterols [35]).”

• cites [35] Dinan, 2001, Phytoecdysteroids: Biological aspects, Phytochemistry sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The biosynthetic pathway(s) in plants has not yet been fully elucidated, but the biosynthesis appears to differ mechanistically from that in animals, and may also differ within types of plant (e.g., between angiosperms and ferns).

“The biosynthetic pathway(s) in plants, just like that in arthropods, has not yet been fully elucidated, but the biosynthesis appears to differ mechanistically from that in animals (Figure 7), and maybe also differ within types of plant (e.g., between angiosperms and ferns; [3,35])”

• cites [3] Smagghe, G. (2009). Phytoecdysteroids: Diversity, biosynthesis and distribution. Ecdysone: Structures and Functions, Springer Science & Business Media B.V.Not checked yet

  Not checked in the free run.

• cites [35] Dinan, 2001, Phytoecdysteroids: Biological aspects, Phytochemistry sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Most, if not all, plants retain the genetic capacity to synthesise ecdysteroids, but the expression of the biosynthetic pathway(s) is down-regulated in most species and at certain stages of development.

“These three lines of evidence strongly suggest that most, if not all, plants retain the genetic capacity to synthesise ecdysteroids but that the expression of the biosynthetic pathway(s) is down-regulated in most species and at certain stages of development.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Progesterone stimulates germination in maize (Zea mais).

“progesterone stimulates germination in maize (Zea mais; [51])”

• cites [51] Turk, H. (2021). Progesterone promotes mitochondrial respiration at the biochemical and molecular level in germinating maize seed. Plants, 10. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

20E affects germination and stimulates shoot elongation in young tomato (Lycopersicum esculentum) seedlings.

“20E affects germination and stimulates shoot elongation in young tomato (Lycopersicum esculentum) seedlings [50]”

• cites [50] Bakrim, 2007, Effect of plant hormones and 20-hydroxyecdysone on tomato (Lycopersicum esculentum) seed germination and seedlings growth, Afr. J. Biotechnol. sourceNot checked yet

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May combine two claims.

Ecdysteroids seem to stimulate the size and growth of the alga Chlorella vulgaris and growth and heterocyst formation in the cyanobacterium Nostoc.

“Ecdysteroids seem to stimulate the size and growth of the alga Chlorella vulgaris [52,53] and growth and heterocyst formation in the cyanobacterium Nostoc [54].”

• cites [52] Bajguz, 2001, Effect of ecdysone application on the growth and biochemical changes in Chlorella vulgaris cells, Plant Physiol. Biochem. sourceNot checked yet

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• cites [53] Bajguz, 2004, Effects of ecdysteroids on Chlorella vulgaris, Physiol. Plant. sourceNot checked yet

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• cites [54] Smith, 1992, The effect of ecdysone on the cyanobacterium Nostoc 6720, Z. Naturforsch.Not checked yet

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May combine two claims.

One alternative strategy that has been successfully commercially developed is the use of non-steroidal ecdysteroid analogues as insecticidal compounds (bisacylhydrazines), since these chemicals are easy to synthesise, non-polar and resistant to metabolism.

“One alternative strategy that has been successfully commercially developed is the use of non-steroidal ecdysteroid analogues as insecticidal compounds (bisacylhydrazines; [90]) since these chemicals are easy to synthesise, non-polar and resistant to metabolism.”

• cites [90] Dhadialla, 1998, New insecticides with ecdysteroidal and juvenile hormone activity, Ann. Rev. Entomol. sourceNot checked yet

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May combine two claims.

Phytoecdysteroids may act as feeding deterrents and/or endocrine disruptors for insects and soil nematodes.

“Phytoecdysteroids may act as feeding deterrents [56,57] and/or endocrine disruptors for insects [58][59][60][61][62][63][64][65][66] and soil nematodes [67].”

• cites [56] Regnault-Roger, C., Philogène, B.J.R., and Vincent, C. (2008). The role of phytoecdysteroids in the battle against phytophagous insects. Biopesticides of Plant Origin, Lavoisier.Not checked yet

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• cites [57] Taste, 2009, Chapter 4: Tasting toxicants as bitter: Phytoecdysteroids, SEB Experimental Biological SeriesNot checked yet

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• cites [58] Bergamasco, R., and Horn, D.H.S. (1983). Distribution and role of insect hormones in plants. Endocrinology of Insects, A. R. Liss Inc.Not checked yet

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• cites [67] Soriano, 2004, Phytoecdysteroids: A novel defense against plant-parasitic nematodes, J. Chem. Ecol. sourceNot checked yet

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May combine two claims.

The introduction of Sep-Pak cartridges in the early 1980s revolutionised the partial purification of ecdysteroid-containing samples and allowed their simple separation into different classes.

“The introduction of Sep-Pak cartridges in the early 1980s revolutionised the partial purification of ecdysteroid-containing samples, initially for relatively small samples but later also for preparative purposes as larger SPE columns became commercially available, and also allowed their simple separation into different classes (polar conjugates, free ecdysteroids, non-polar conjugates) by applying”

• cites [115] Lafont, 1982, Sample processing for high-performance liquid chromatography of ecdysteroids, J. Chromatogr. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The use of TLC in the analysis, quantification and purification of ecdysteroids has now largely been superseded by HPLC.

“Although important in the past, the use of TLC in the analysis, quantification and purification of ecdysteroids has now largely been superseded by HPLC.”

• cites [118] Waksmundska-Hajnos, M., Sherma, J., and Kowalska, T. (2008). TLC of sterols, steroids and related triterpenoids. Thin Layer Chromatography in Phytochemistry, CRC Press. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The most commonly used HPLC systems are variants on a C18-RP-column eluted with a linear gradient of methanol or acetonitrile in water.

“The most commonly used HPLC systems are variants on a C 18 -RP-column eluted with a linear gradient of methanol or acetonitrile in water.”

• cites [107] Lafont, 1994, Chromatographic procedures for ecdysteroids, J. Chromatogr. A sourceNot checked yet

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• cites [117] Lafont, R., Blais, C., Harmatha, J., and Wilson, I.D. (2013). Ecdysteroids: Chromatography. Module in Chemistry, Molecular Sciences and Chemical Engineering, Elsevier. sourceNot checked yet

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• cites [125] Lafont, 1979, Separation of ecdysteroids by using high-pressure liquid chromatography on microparticulate supports, J. Chromatogr. sourceNot checked yet

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• cites [126] Hofmann, J.A. (1980). Analysis of ecdysteroids by high-peformance liquid chromatography and coupled gas-liquid chromatography-mass spectrometry. Progress in Ecdysone Research, Elsevier/North Holland Biomedical Press.Not checked yet

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• cites [127] Lafont, 1994, Selectivity in the high-performance liquid chromatography of ecdysteroids, J. Chromatogr. A sourceNot checked yet

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• cites [128] Waksmundska-Hajnos, M., and Sherma, J. (2011). HPLC of steroids. High Performance Liquid Chromatography in Phytochemical Analysis, CRC Press. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Open-column chromatography used to be the standard workhorse for laboratory purification of ecdysteroids but has gradually been replaced by flash chromatography and preparative HPLC.

“Open-column chromatography on silica or alumina eluted with solvents of increasing percentages of methanol or ethanol in hexane, dichloromethane or chloroform used to be the standard 'workhorse' for the laboratory purification of ecdysteroids, as it was of other plant secondary compounds, but this has gradually been replaced by flash chromatography and preparative HPLC on NP-and RP-stationary”

• cites [107] Lafont, 1994, Chromatographic procedures for ecdysteroids, J. Chromatogr. A sourceNot checked yet

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• cites [117] Lafont, R., Blais, C., Harmatha, J., and Wilson, I.D. (2013). Ecdysteroids: Chromatography. Module in Chemistry, Molecular Sciences and Chemical Engineering, Elsevier. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

SFC separation of ecdysteroids provides shorter retention times, sharper peaks and greater sensitivity than HPLC.

“SFC separation of ecdysteroids, using critical CO 2 , which is non-polar, modified with methanol and silica/bonded (NP) silica HPLC columns or fused silica capillary columns, provides shorter retention times, sharper peaks and greater sensitivity than HPLC.”

• cites [107] Lafont, 1994, Chromatographic procedures for ecdysteroids, J. Chromatogr. A sourceNot checked yet

  Not checked in the free run.

• cites [117] Lafont, R., Blais, C., Harmatha, J., and Wilson, I.D. (2013). Ecdysteroids: Chromatography. Module in Chemistry, Molecular Sciences and Chemical Engineering, Elsevier. sourceNot checked yet

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• cites [129] Raynor, 1989, Packed column suercritical-fluid chromatography and linked supercritical-fluid chromatography-mass spectrometry for the analysis of phytoecdysteroids from Silene nutans and Silene otites, J. Chromatogr. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

SFC is compatible with UV/vis-, FT-IR and MS-detection.

“SFC is compatible with UV/vis-, FT-IRand MS-detection [107,117,129].”

• cites [107] Lafont, 1994, Chromatographic procedures for ecdysteroids, J. Chromatogr. A sourceNot checked yet

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• cites [117] Lafont, R., Blais, C., Harmatha, J., and Wilson, I.D. (2013). Ecdysteroids: Chromatography. Module in Chemistry, Molecular Sciences and Chemical Engineering, Elsevier. sourceNot checked yet

  Not checked in the free run.

• cites [129] Raynor, 1989, Packed column suercritical-fluid chromatography and linked supercritical-fluid chromatography-mass spectrometry for the analysis of phytoecdysteroids from Silene nutans and Silene otites, J. Chromatogr. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Girault and Lafont published the first complete proton-NMR assignments for ecdysone and 20-hydroxyecdysone in 1988.

“Girault and Lafont [133] published the first complete proton-NMR assignments for ecdysone and 20-hydroxyecdysone in 1988.”

• cites [133] Girault, 1988, The complete 1H-NMR assignment of ecdysone and 20-hydroxyecdysone, J. Insect Physiol. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

HPLC/DAD/MS has become almost routine for the analysis of ecdysteroid mixtures.

“As the technical problems have been overcome and reliable equipment is now commercially available, HPLC/DAD/MS has become almost routine for the analysis of ecdysteroid mixtures (e.g., [44]).”

• cites [44] Dinan, 2020, Small-scale analysis of phytoecdysteroids in seeds by HPLC-DAD-MS for the identification and quantification of specific analogues, dereplication and chemotaxonomy, Phytochem. Anal. sourceNot checked yet

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May combine two claims.

The protic solvent problem was overcome by using deuterated water in the mobile phase of RP-HPLC, and the sensitivity problem by using a stopped-flow method.

“The former was overcome by using deuterated water in the mobile phase of the RP-HPLC and the latter by using a stopped-flow method to allow adequate time to obtain adequate (if not complete) 1 H-spectra in 10 min of stopped-flow with ca. 100 µg of reference ecdysteroids [137].”

• cites [137] Wilson, 1998, High-performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy. Application to the ecdysteroids of Silene otites, J. Chromatogr. A sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Borst and O'Connor developed the first immunoassay for ecdysteroids.

“Borst and O'Connor [145] developed the first immunoassay for ecdysteroids, and, over the subsequent years, several others have been developed [146][147][148][149][150][151][152][153][154][155][156][157][158][159].”

• cites [145] Borst, 1972, Arthropod molting hormone: Radioimmune assay, Science sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Currently, isolation from an ecdysteroid-accumulating plant species is the only scientifically and financially viable option to source 20E or other ecdysteroid analogues.

“Several approaches could be envisaged to source 20E or other ecdysteroid analogues, but, currently, isolation from an ecdysteroid-accumulating plant species is the only scientifically and financially viable option.”

May combine two claims.

The Drosophila melanogaster Kc cell line assay had advantages of much greater reproducibility and no ecdysteroid metabolism but was still time-consuming.

“The next major development was to use the response of the Drosophila melanogaster Kc cell line to quantify the potency of ecdysteroid analogues [162], which had the advantages of much greater reproducibility and no ecdysteroid metabolism but was still time-consuming as it required the measurement of neurone-like cellular projections that were part of the response.”

• cites [162] Cherbas, 1980, The morphological response of Kc-H cells to ecdysteroids: Hormonal specificity, Wilhelm Roux’ Arch. sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

It is necessary to start with plant material that contains essentially only 20E.

“This underlines the need to start with plant material that contains essentially only 20E.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Given that a suitable plant source will contain typically 1-2% of its dry weight as 20E and that a pharmaceutical/medicinal dose of 20E is likely to be in the 100 mg-2 g/day range, it is necessary to be able to process tonnes of plant material.

“Given that a suitable plant source will contain typically 1-2% of its dry weight as 20E and that a pharmaceutical/medicinal dose of 20E is likely to be in the 100 mg-2 g/day range, it is necessary to be able to process tonnes of plant material.”

• AssumptionRests on an assumption: A pharmaceutical/medicinal dose of 20E is likely to be in the 100 mg-2 g/day range.
• AssumptionRests on an assumption: A suitable plant source will contain typically 1-2% of its dry weight as 20E.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Supercritical fluid extraction and bi-phasic extraction are generally more expensive than conventional extraction and would only be cost-effective for potent, high-value target molecules, which 20E is not.

“Other methods, such as super critical fluid extraction or bi-phasic extraction, could be used, but these are generally more expensive and would only be cost-effective if the target molecule is potent (low daily dose required) and high-value, which is not the case for 20E.”

• AssumptionRests on an assumption: 20E is not a potent (low daily dose) or high-value target molecule.

May combine two claims.

Chemical synthesis of 20-hydroxyecdysone is a complex, multi-step (ca. 18) process with an overall yield of <1%, starting from a precursor that is probably not available at the required scale.

“Although feasible [171], chemical synthesis of 20-hydroxyecdysone is a complex, multi-step (ca.18) process with an overall yield of <1%, starting from a precursor that is probably not available at the required scale.”

• cites [171] Koolman, J. (1989). Strategies for the synthesis of ecdysteroids. Ecdysone: From Chemistry to Mode of Action, Thieme Verlag.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Plant milking is appropriate for the production of complex, rare and costly metabolites.

“This approach is appropriate for the production of complex, rare and costly metabolites”

May combine two claims.

Applying genetically transformed yeast to produce ecdysteroids is a big challenge because of the complex multi-step process from cholesterol, the specific sequence involving different cellular compartments, and the early steps that still require elucidation.

“Whether this can be applied to produce ecdysteroids is, however, a big challenge owing to a complex multi-step process from cholesterol, the fact that it must proceed with a specific sequence involving different cellular compartments and because the early steps (the 'Black Box') still require elucidation”

Probably not a substantial claim: may not pass S3 (load-bearing).

Ecdysteroids have not been proven to be responsible for all the pharmacological activities of traditional medicinal plant extracts.

“but it must be borne in mind that ecdysteroids have not been proven to be responsible for all the pharmacological activities of these plant extracts.”

Probably not a substantial claim: may not pass S3 (load-bearing).

When ecdysteroids occur in plants, they can accumulate to high concentrations.

“but when they do occur, they can accumulate to high concentrations.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Much remains to be discovered about ecdysteroid mode of action in arthropods because of their enormous diversity.

“but, in view of the enormous diversity of arthropods, much certainly remains to be discovered.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

Final clarification of ecdysteroid biochemical pathways, enzymology and regulation remains a major challenge.

“Final clarification of the biochemical pathways, enzymology and regulation remains a major challenge”

May combine two claims.

Ecdysteroids are the most frequently occurring steroid hormones because of the predominance of arthropods and other invertebrates.

“and are, in view of the predominance of these animals in the world, the most frequently occurring steroid hormones”

Probably not a substantial claim: may not pass S3 (load-bearing).

New ecdysteroid analogues, particularly in plants, are continuing to be identified.

“and new analogues (particularly in plants) are continuing to be identified.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The control of development by ecdysteroids in other invertebrates is still poorly understood.

“The control of development by ecdysteroids in other invertebrates is still poorly understood.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based). May combine two claims.

The availability of reliable, pure preparations of ecdysteroids is essential for continuing clinical trials and for the ultimate commercialisation of medical drugs with ecdysteroids as their API.

“The availability of reliable, pure preparations of ecdysteroids is essential for continuing clinical trials and for the ultimate commercialisation of medical drugs with ecdysteroids as their API.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The biosynthesis of ecdysteroids in arthropods and plants remains to be fully elucidated.

“The biosynthesis of ecdysteroids in arthropods and plants remains to be fully elucidated.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The mode of action of ecdysteroids in some model arthropod species is now fairly well understood.

“The mode of action of ecdysteroids in some model arthropod species is now fairly well understood”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Data and literature concerning ecdysteroids have been compiled, are readily available online and are continuously updated at www.ecdybase.com.

“Perhaps uniquely, the data and literature concerning ecdysteroids have been compiled, are readily available online and are continuously updated (www.ecdybase.com).”

Probably not a substantial claim: may not pass S4 (based).

Low doses of ecdysteroids can have beneficial developmental and commercial consequences for the rearing of silkworms, bees, prawns and crabs.

“it is clear that low doses can have beneficial developmental and commercial consequences for the rearing of silkworms, bees, prawns and crabs.”

Probably not a substantial claim: may not pass S4 (based).

Certain plant species are high accumulators of ecdysteroids (mainly 20E).

“Certain plant species are high accumulators of ecdysteroids (mainly 20E)”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

High concentrations of exogenous ecdysteroids are generally deleterious to invertebrate development.

“Although high concentrations of exogenous ecdysteroids are generally deleterious to invertebrate development”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Ecdysteroids are the steroid hormones of arthropods and other invertebrates.

“They are the steroid hormones of arthropods and other invertebrates”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

The analysis, identification and quantification of ecdysteroids are highly advanced.

“The analysis, identification and quantification of ecdysteroids are highly advanced”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The modes of action by which ecdysteroids' pharmacological effects occur are now starting to be understood.

“The modes of action by which these effects occur are now starting to be understood.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Ecdysteroids are widespread in nature.

“Ecdysteroids are widespread in nature.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Ecdysteroids occur in plants, but their occurrence is not universal.

“They also occur in plants, where their occurrence is not universal”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The supercritical fluid extraction method is not generally used, probably because it requires extensive prior optimisation of parameters (pressure, temperature, % of modifier, extraction time and flow rate).

“The method is not generally used, probably because it requires the extensive prior optimisation of parameters (pressure, temperature, % of modifier, extraction time and flow rate).”

May combine two claims.

Deep eutectic solvents and ionic liquids are a recently introduced method for extracting phytoecdysteroids, with high extraction yields and fast.

“A further, recently introduced method for the extraction of phytoecdysteroids is the use of deep eutectic solvents and ionic liquids [114], which possess high extraction yields and are fast.”

• cites [114] Bajkacz, S., Rusin, K., Wolny, A., Adamek, J., Erfurt, K., and Chorbok, A. (2020). Highly efficient extraction procedures based on natural deep eutectic solvents of ionic liquids for determination of 20-hydroxyecdysone in spinach. Molecules, 25. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

In Table 4, the amounts are based on the amount announced by the dietary supplement supplier, not verified.

“Based on the amount announced by the dietary supplement supplier, not verified.**”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In Table 4, for some studies, the measured amount was used, but the absence of other anabolic substances in the dietary supplement used was not established.

“Measured amount, but the absence of other anabolic substances in the dietary supplement used was not established.”