|Year : 2015 | Volume
| Issue : 42 | Page : 225-230
Phytoecdysteroids of the East Asian Caryophyllaceae
Elena Novozhilova1, Viacheslav Rybin2, Petr Gorovoy1, Irina Gavrilenko1, Roman Doudkin3
1 Laboratory of Plant Chemotaxonomy, G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia
2 Laboratory of Comparative Biochemistry, A.V. Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia
3 Far East Federal University, Vladivostok, Russia
|Date of Submission||13-Jan-2014|
|Date of Acceptance||24-Feb-2014|
|Date of Web Publication||27-May-2015|
Dr. Elena Novozhilova
Laboratory of Plant Chemotaxonomy, G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences, 159 Prospect 100-let Vladivostoku, Vladivostok 690022
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Occurrence of integristerone A (1), 20-hydroxyecdysone (2), ecdysone (3), 2-deoxy-20-hydroxyecdysone (4) has been analyzed in 64 species of the East Asian Caryophyllaceae. Materials and Methods: Ecdysteroid content was determinate by high-performance liquid chromatography (HPLC). HPLC with a high-resolution mass spectrometry was performed on Shimadzu LCMS-IT-TOF (Japan) system equipped with a LC-20A Prominence liquid chromatograph, a photodiode array detector SPD-M20A and ion-trap/time-of-flight mass spectrometer. Results: New sources of phytoecdysteroids: Melandrium sachalinense and Melandrium firmum have been revealed. It is the 1 st time that two has been identified in M. sachalinense and M. firmum; 1 in the species: Lychnis fulgens, Silene repens, Silene foliosa, Silene stenophylla, Silene jenisseensis and M. sachalinense; 3 in Lychnis cognata; 4 in L. fulgens, S. stenophylla and S. jenisseensis (the tribe Lychnideae, the subfamily Caryophylloideae). Ecdysteroid-negative taxa are Spergularia rubra of the tribe Sperguleae; species of the genera Minuartia, Honckenya, Eremogone, Arenaria, Moehringia, Pseudostellaria, Fimbripetalum, Stellaria and Cerastium of the tribe Alsineae; Scleranthus annuus of the tribe Sclerantheae, as well as the East Asian representatives of the genera Gypsophila, Psammophiliela, Dianthus and Saponaria of the tribe Diantheae; Oberna and Agrostemma of the tribe Lychnideae. Conclusion: This investigation shows the most promising sources of ecdysteriods are species of genera Silene and Lychnis.
Keywords: Caryophyllaceae, ecdysteroids, high-performance liquid chromatography
|How to cite this article:|
Novozhilova E, Rybin V, Gorovoy P, Gavrilenko I, Doudkin R. Phytoecdysteroids of the East Asian Caryophyllaceae. Phcog Mag 2015;11, Suppl S1:225-30
|How to cite this URL:|
Novozhilova E, Rybin V, Gorovoy P, Gavrilenko I, Doudkin R. Phytoecdysteroids of the East Asian Caryophyllaceae. Phcog Mag [serial online] 2015 [cited 2019 Oct 23];11, Suppl S1:225-30. Available from: http://www.phcog.com/text.asp?2015/11/42/225/157746
| Introduction|| |
Plants of the family Caryophyllaceae are perspective sources of phytoecdysteroids-hormones which control molting in insects and crustaceans. Phytoecdysteroids possess stimulating and adaptogenic action, causing considerable decrease in cholesterol content in blood serum and displaying anabolic activity in relation to human and mammals; in this case, unlike anabolic steroids, ecdysteroids, possessing pronounced anabolic activity, do not manifest androgen effect to make possible their lengthy use. Their use in medicinal preparations of adaptogenic, cardiotropic, antiatherosclerotic, counter-ulcer, would-healing and antimicrobial action was shown to be promising. ,,
First studies on phytoecdysteroids of the Caryophyllaceae in the flora of the former Soviet Union were conducted by the researchers of the Institute of the Chemistry of Plant Substances of the Academy of Sciences of the Uzbek SSR (Tashkent city) in the 1980s. ,,,,,
New sources of phytoecdysteroids are actively sought. Distribution pattern of integristerone A (1), 20-hydroxyecdysone (2), ecdysone (3), 2-deoxy-20-hydroxyecdysone (4) in plants is studied. ,,,
| Materials and Methods|| |
Standards of compounds 2, 3, and 4 were obtained from Sigma and Aldrich Company (St. Louis, MO, USA). Compound 1 was obtained from U. A. Baltaev (Institute of Petroleum Chemistry and Catalysis, Ufa, Russian Federation). All solvents and chemicals were of analytical grade.
The plant material from wild flora was collected during 2005-2012 years in the Far East, Russian Federation. Specimens have been deposited in the herbarium of the laboratory of plant chemotaxonomy of G. B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences. Ecdysteroid content was analyzed in the aerial part of plants collected during their flowering period.
Analysis of ecdysteroids
Air-dried samples (about 200 mg, accurately weighed, residual moisture <8.2%) were extracted with ethanol-water (7:3, 10 mL) at room temperature for 3 days. The resulting extracts were filtered. A portion (0.9 mL) was treated with water (12 mL). Solid-phase extraction was carried out with Supelclean C18 columns (Supelco, St. Louis, USA) using ethanol-water (3:2) as eluent.
High-performance liquid chromatography (HPLC) with a high-resolution mass spectrometry was performed on LCMS-IT-TOF (Shimadzu, Kyoto, Japan) system equipped with a LC-20A Prominence liquid chromatograph, a photodiode array detector SPD-M20A (Shimadzu, Kyoto, Japan) and ion-trap/time-of-flight mass spectrometer. Separation occurred over a column - Ascentis C 18 100 × 2.1 mm i.d.; 3.0 μm part size, Supelco, USA) at 40°C. Elution rate was 0.25 mL/min. The elution gradient was as follows: Acetonitrile-water (1:9, v/v) 5 min, acetonitrile-water (1:1, v/v) 15 min, acetonitrile 15 min. The range of detection was m/z 200-800 (atmospheric pressure chemical ionization [APCI], positive ion detection). The potential in the ion source was - 4.5 kV. The drying gas (N 2 ) pressure was 25 kPa. The nebulizer gas (N 2 ) flow rate was 2 L/min. The interface temperature was 350°C. Phytoecdysteroids were identified using data of ultraviolet (UV) detection and mass-spectral data as well as standard retention times. Quantification was carried out on the basis of standard calibration curves using LC Solution version 1.24 (Shimadzu, Kyoto, Japan).
| Results and Discussion|| |
The Caryophyllaceae is a large family, with 86 genera and some 2200 species mostly distributed in the Northern Hemisphere in extra-tropical regions. 
Until date, approximately 323 species of 66 genera have been analyzed. No positive species have been detected in the genera: Arenaria, Cerastium, Gypsophila, Minuartia, Spergula, Spergularia, and Stellaria. Many positive species occur in other genera: Lychnis, Petrocoptis and Silene. Most data regarding ecdysteroid distribution are available for the Silene/Lychnis complex, into which Melandrium has been subsumed. Among 700 species of Silene of the world flora, 160 has been analyzed and include 96 species containing ecdysteroids, 52 species lacking them and there are controversial data for 12 species. The genus Lychnis numbers about 89 species in the world flora, 24 species of those have been tested for the presence of ecdysteroids, of which 18 are positive, 5 are negative and 1 is uncertain. 
2, 3 and their 2-deoxy-derivatives are the most frequently present in the Caryophyllaceae, , 2 is usually a predominant component, and the other phytoecdysteroids are in minor amounts.
The experimental data on ecdysteroid distribution in the plant world will possibly bring scholars closer to solving an important ecological problem - clarifying the functions of ecdysteroids in plants as well as allowing us to reveal possible uses of this group of compounds in plant chemotaxonomy.
In this investigation, we have carried out screening of the East Asian representatives of the Caryophyllaceae for 1, 2, 3, and 4 contents [Figure 1].
We have improved the described recently method of simultaneous identification of these four ecdysteroids  that belong to the same metabolic branch. 
Standards of each compound, as well as their mixture, were studied by HPLC with UV sequence and mass-selective detection. We improved conditions for HPLC analysis of the mixture of the four compounds and found that reliable information on ecdysteroid content can be obtained in a short time [13 min, [Figure 2].
|Figure 2: High-perfomance liquid chromatography profile of ecdysteroid standard mixture (a); detection: UV 246 nm. Integristerone A (1), 20-hydroxyecdysone (2), ecdysone (3) and 2-deoxy-20-hydroxyecdysone (4) and extract from the aerial part Lychnis fulgens (b)|
Click here to view
While applying standards of the individual ecdysteroids, their elution sequence from nonpolar sorbents was determined as 1 → 2 → 3 → 4 [Figure 2]a. The last two compounds are isomers in their hydroxyl group location, which leads to their good resolvability in the chromatogram. The structure differences of the investigated ecdysteroids [Figure 1] result in some similarities and differences in their mass-spectral patterns. The intensity of the quasi-molecular ions of 2 and 4 comprised 74 and 100%, respectively, and their fragmentation was similar. Contrary to this finding, in the mass spectra of 1 and 3, the intensity of the quasi-molecular ions was 4.5 and 10%, respectively. This can be explained by the instability of the polyhydroxylated intermediates produced by APCI, with a rapid loss of water. ,
Mass-spectral pattern of 1-4 coincided with data described recently. , The using a high-resolution mass-spectrometry allowed to determine the composition of all fragment ions of each investigated components [Table 1].
|Table 1: Chromatographic (HPLC) and spectral (UV, MS) characteristics of ecdysteroids 1 - 4 |
Click here to view
For extensive screening, it was necessary to apply a sensitive and specific method. We have been employed HPLC for study on the presence or absence of phytoecdysteriods in 64 species (21 genera), which are allocated to 3 subfamilies: Paronychioideae, Alsinoideae and Caryophylloideae. The results of the research on the East Asian ecdysteroids positive Caryophyllaceae are summarized in [Table 2].
|Table 2: Contents of integristerone A (1), 20-hydroxyecdysone (2), ecdysone (3) and 2-deoxy-20-hydroxyecdysone (4) in the Caryophyllaceae aerial parts |
Click here to view
Spergularia rubra (L.) J. et C. Presl., (the subfamily Paronychioideae, the tribe Sperguleae Vierh.) has turned out to be ecdysteroid-negative.
In the subfamily Alsinoideae Vierh. no ecdysteroids have been detected in Scleranthus annuus L. (the tribe Sclerantheae Vierh.) and in representatives of the tribe Alsineae Pax: Arenaria redowskii Cham. et Schlecht., Cerastium holosteoides Fries, Cerastium pauciflorum Stev. ex Ser., Cerastium fischerianum Ser., Cerastium beeringianum Cham. et Schlecht., Cerastium arvense L., Eremogone juncea (Bieb.) Fenzl, Eremogone capillaris (Poir.) Fenzl, Eremogone tschuktschorum (Regel) Ikonn., Fimbripetalum radians (L.) Ikonn., Honckenya oblongifolia Torr. et Gray, Minuartia arctica (Stev. ex Ser.) Graebn., Minuartia macrocarpa (Pursh) Ostenf., Minuartia biflora (L.) Schinz et Thell., Minuartia verna (L.) Hiern, Minuartia stricta (Sw.) Hiern, Minuartia sibirica (Regel et Til.) N.S. Pavlova, Moehringia lateriflora (L.) Fenzl, Pseudostellaria sylvatica (Maxim.) Pax, Pseudostellaria rigida (Kom.) Pax, Stellaria bungeana Fenzl, Stellaria graminea L., Stellaria media (L.) Vill., Stellaria eschscholtziana Fenzl, Stellaria fischeriana Ser., Stellaria ruscifolia Pall. ex Schlecht., Stellaria discolor Turcz., Stellaria humifusa Rottb., Stellaria cherleriae (Fisch. ex Ser.) F. Williams, Stellaria longifolia Muehl. ex Willd., Stellaria uliginosa Murr., Stellaria calycantha (Ledeb.) Bong., Stellaria filicaulis Makino and Stellaria dichotoma L.
In the subfamily Caryophylloideae, ecdysteroids have not been revealed in the species of the tribe Diantheae Pax: Dianthus chinensis L., Dianthus repens Willd., Dianthus barbatus L., Dianthus superbus L., Gypsophila pacifica Kom., Gypsophila violacea (Ledeb.) Fenzl, Gypsophila patrinii Ser., Gypsophila davurica Turcz. ex Fenzl, Psammophiliella muralis (L.) Ikonn. As well as in the species of the tribe Lychnideae A. Br.: Oberna behen (L.) Ikonn., Saponaria officinalis L., Melandrium album (Mill.) Garcke, Melandrium olgae Maxim., Melandrium apricum (Turcz. ex Fisch. et Mey.) Rohrb., Agrostemma githago L., Lychnis ajanensis (Regel et Til.) Regel and Silene acaulis (L.) Jacq.
Screening the plants growing in the East Asian have confirmed the data on the absence of ecdysteroids in the species of Arenaria, Cerastium, Gypsophila, Honckenia, Minuartia, Scleranthus, Spergularia, Agrostemma published previously by other researchers. ,,,
In a number of genera ecdysteroid-negative as well as ecdysteroid-positive species. Ecdysteroids are detected in Dianthus deltoides and Saponaria bellidifolia,  but no ecdysteroids have been found in the East Asian representatives of Dianthus and Saponaria, which shows their patchy distribution within the genus.
We have not detected any ecdysteroids in O. behen, which confirms some other researchers' data , on the lack of ecdysteroids in this species; nevertheless, ecdysteroids have been revealed in the individuals of this species growing in the European part of the Russian Federation. 
Ecdysteroid-containing species have been detected in the subfamilies Alsinoideae and Caryophylloideae. However, among 34 studied species of 9 genera of the tribe Alsineae of the subfamily Alsinoideae, there is only one ecdysteroid-containing representative-Sagina maxima A. Gray. Ecdysteroids 1, 3, and 4 have not been found [Table 2].
Ecdysteroids have been detected in 10 species of the genera Lychnis, Silene and Melandrium of the tribe Lychnideae, the subfamily Caryophylloideae [Table 2].
Lychnis is represented with five species in the Russian Far East. We analyzed all five species of which four species have turned out to be ecdysteroid-containing ones: Lychnis wilfordii (Regel) Maxim., Lychnis cognata Maxim., Lychnis fulgens Fisch. ex Curt. and Lychnis sibirica L. In Lychnis ajanensis (Regel et Til.) Regel ecdysteroids have not been revealed. This species was described as Melandrium biflorum β. ajanense Regel et Til.  Later Regel and Tiling raised taxon to the species.  V. N. Voroshilov transferred L. ajanensis to Silene genus (Silene ajanensis (Regel et Tiling) Worosh). 
In all four species the predominant compound is 2.
It is the 1 st time that 1 has been found in L. cognata and L. fulgens, 3 in L. cognata, 4 in L. fulgens.
The previous data , on the presence of 1, 2 in L. wilfordii and 2 in Lychnis cognate have been confirmed.
Earlier 2, 3 and polypodine B were identified in the Far Eastern L. fulgens. The results of our research have also detected 2 and 3 in this taxon.
The genus Silene is represented with nine species in the Russian Far East. We have investigated five species, of which 4 have turned out to be ecdysteroid-containing: Silene foliosa, Silene stenophylla, Silene jenisseensis (the section Chloranthae (Rohrb.) Schischk.) and Silene repens (the section Spergulifoliae). In S. acaulis (the section Nanosilene Otth.), ecdysteroids have not been detected, although there is some information on the presence of ecdysteroids in this species, in a number of papers. ,,
It is the 1 st time that 1 has been revealed in S. repens Patrin, S. foliosa Maxim., S. stenophylla Ledeb. and S. jenisseensis Willd., maximum content of which reaches 0.49 μg/mg in the aerial part in S. stenophylla during its blooming period. In the aerial part of S. stenophylla and S. jenisseensis, 3 have been detected for the 1 st time.
The predominant component in all the investigated East Asian species of the genus Silene is 2, its content in the aerial part if the investigated species varies from 0.83 μg/mg in S. repens to 4.7 μg/mg in S. stenophylla.
The genus Melandrium is represented with seven species in the Russian Far East. Five species have been analyzed, of which two species turned out to be ecdysteroid-containing. It is the 1 st time that 2 has been found in Melandrium firmum (Siebold et Zucc.) Rohrb. and M. sachalinense (Fr. Schmidt) Schischk., in the latter 1 has been detected as well.
| Conclusion|| |
Ecdysteroid-containing species have been detected in two subfamilies of the Caryophyllaceae: Paronychioideae and Caryophylloideae. Most species containing ecdysteroids are representatives of the tribe Lychnideae of the subfamily Caryophylloideae. In the tribe Diantheae (the subfamily Caryophylloideae), no sources of ecdysteroids have been found. It can be asserted that ecdysteroid-containing taxa are confined to the tribe Lychnideae, however, the distribution of ecdysteroids in the genera of the tribe Lychnideae is patchy. Along with genera containing ecdysteroids there are ecdysteroid-negative taxa.
The analysis of ecdysteroid content in the species of Silene, Lychnis and Melandrium revealed that a genus may include species containing ecdysteroids as well as ones in which they have not been identified. In the genus Silene, ecdysteroids have been revealed in the species, which belong to the sections Chloranthae and Spergulifoliae. The plants of these sections are the ones in which finding of new ecdysteroid sources can be prognosed. Data on the quantity of ecdysteroids are available as for several species of Caryophyllaceae of particular interest. According to our results the ecdysteroid content is 4-5 μg/mg in aerial part of Silene species.
Most perspective sources of ecdysteroid are species of the Silene. Perspective sources of ecdysteroids are species from Silene.
| References|| |
Sláma K, Koudela K, Tenora J, Mathová A. Insect hormones in vertebrates: Anabolic effects of 20-hydroxyecdysone in Japanese quail. Experientia 1996;52:702-6.
Todorov IN, Mitrokhin YI, Efremova OI, Sidorenko LI. Impact of extracts from Leuzea carthamoides
on biosynthesis of RNA and proteins in mouse organs. Khimico-Farmatsevticheskii Zhurnal 2000;34:24-6.
Volodin VV, Shirshova TI, Burtseva SA, Melnik MV. Biological activity of 20-hydroxyecdysone and its acetates. Rastitel′nye Resursy 1999;35:76-81.
Abubakirov NK. Ecdysteroids of the flowering plants (Angiospermae
). Khimiya Prirodnykh Soedinenii 1981;6:685-702.
Baltaev UA, Rashkes YA, Darmogray VN, Belov YP, Abubakirov NK. Phytoecdysteroids of the Silene nutans.
II. 22-Deoxyecdysterone and its mass spectral characteristics. Khimiya Prirodnykh Soedinenii 1985;1:62-6.
Baltaev UA, Darmograi VN, Abubakirov NK. Phytoecdysteroids of plants of the genus Silene.
XIV. Ecdysterone 20-O-benzoate from Silene tatarica.
Khimiya Prirodnykh Soedinenii 1987;6:850-2.
Saatov Z, Usmanov BZ, Abubakirov NK. Phytoecdysones of Silene praemixta
. I. Silenosterone. Khimiya Prirodnykh Soedinenii 1979;6:793-7.
Saatov Z, Gorovits MB, Abdullae ND, Usmanov BZ, Abubakirov NK. Phytoecdysteroids from Silene
plants. VIII. 2-Deoxyecdysterone 3-acetate from Silene praemixta
. Khimiya Prirodnykh Soedinenii 1985;1:60-2.
Saatov Z, Gorovits MB, Abubakirov NK. Phytoecdysteroids in plants of the genus Silene
. Khimiya Prirodnykh Soedinenii 1993;5:627-35.
Lafont R, Horn DH. Phytoecdysteroids: Structures and occurrence. In: Koolman J, editor. Ecdysone: From Chemistry to Mode of Action. Stuttgart: Georg Thieme Verlag; 1989. p. 39-64.
Revina TA, Revushkin AS, Rakitin AV. Ecdysteroid-containing species in flora of the Altai mountains. Rastitel′nye Resursy 1988;34:565-70.
Volodin V, Chadin I, Whiting P, Dinan L. Screening plants of European North-East Russia for ecdysteroids. Biochem Syst Ecol 2002;30:525-78.
Zibareva LN, Dinan L, Eryomina VI. Screening of Caryophyllaceae
species for phytoecdysteroids presence. Rastitel′nye Resursy 2007;43:66-75.
Brummitt RK. Vascular Plant Families and Genera. Kew: Royal Botanic Gardens; 1992. p. 804.
Zibareva L, Volodin V, Saatov Z, Savchenko T, Whiting P, Lafont R, et al
. Distribution of phytoecdysteroids in the Caryophyllaceae
. Phytochemistry 2003;64:499-517.
Bergamasco R, Horn DH. Distribution and role insect hormones in plants. In: Downer RG, Laufer H, editors. Endocrinology of Insects. New York: A.R. Liss; 1983. p. 627-57.
Rybin V, Boltenkov E, Novozhilova E. Application of high-performance liquid chromatography for simultaneous identification of integristerone A, 20-hydroxyecdysone, ecdysone and 2-deoxy-20-hydroxyecdysone. Nat Prod Commun 2007;2:1101-4.
Rees HH, Mendis AH. The occurrence and possible significance of ecdysteroids during nematode and cestode development. In: Hoffmann JA, Porchet P, editors. Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones. Berlin: Springer-Verlag; 1984. p. 338-45.
Stevens JF, Reed RL, Morré JT. Characterization of phytoecdysteroid glycosides in Meadowfoam (Limnanthes alba
) seed meal by positive and negative ion LC-MS/MS. J Agric Food Chem 2008;56:3945-52.
Wainwright G, Prescott MC, Lomas LO, Webster SG, Rees HH. Trace analysis of arthropod hormones by liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry. Biochem Soc Trans 1996;24:476.
Destrez B, Pinel G, Bichon E, Monteau F, Lafont R, Le Bizec B. Detection of 20-hydroxyecdysone in calf urine by comparative liquid chromatography/high-resolution mass spectrometry and liquid chromatography/tandem mass spectrometry measurements: Application to the control of the potential misuse of ecdysteroids in cattle. Rapid Commun Mass Spectrom 2008;22:4073-80.
Darmogray VN, Serova TG. Phytochemical studies on the plants of the genus Oberna
Adans. of the family Caryophyllaceae
Juss. Sovremennye naukoyomkie tekhnologii 2004;5:111-2.
Regel E, Tiling SH. Florula Ajanenesis. Nouveaux Memoires de la Soc. Nat. de Moscou 1859;11:1-128.
Regel E. Aufzählung der von Radde in Baikalien, Dahurien und am Amur gesammelten Pflanzen. Bull Nat Moscou 1861;34:458-578.
Voroshilov VN. Key of the Plants of Soviet Far East. Moscow: Nauka; 1982. p. 253.
Zibareva LN, Baltaev UA, Sviridova TP, Saatov Z, Abubakirov NK. Species of the genus Lychnis L. - Potential sources of ecdysteroids. Rastitel′nye Resursy 1995;31:1-9.
Baltaev UA, Gorovoi PG, Abubakirov NK. Phytoecdysteroids of Lychnis fulgens. Khimiya Prirodnykh Soedinenii 1986;6:794-5.
Zibareva L. Distribution and levels of phytoecdysteroids in plants of the genus Silene
during development. Arch Insect Biochem Physiol 2000;43:1-8.
[Figure 1], [Figure 2]
[Table 1], [Table 2]