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Cited by 20Chromatography in Natural ProductsPhytochemical Studies and BioactivitiesPhytochemistry and Biological Activities

Isolation and purification of three ecdysteroids from the stems of Diploclisia glaucescens by high-speed countercurrent chromatography and their anti-inflammatory activities in vitro

Open access
Fang, Lei, Li, Jialian, Zhou, Jie, Wang, Xiao, Guo, Lanping·2017·Molecules
DOI 10.3390/molecules22081310PMC PMC6152221
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Abstract

High-speed counter-current chromatography was used to separate and purify ecdysteroids for the first time from the stems of Diploclisia glaucescens using a two-phase solvent system composed of ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v). Three ecdysteroids were obtained from 260 mg of ethyl acetate extract of the residue obtained after evaporation of the crude ethanolicextractof D. glaucescens in one-step separation, which were identified as paristerone (I, 30.5 mg), ecdysterone (II, 7.2 mg), and capitasterone (III, 8.1 mg) by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Their anti-inflammatory activities were evaluated by measuring the inhibitory ratios of β-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor. Compounds I–III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 μM, respectively.

AI snapshot

Written from the paper’s full text.

TL;DR

High-speed counter-current chromatography (HSCCC) was used to isolate three ecdysteroids from the stems of *Diploclisia glaucescens* in one step. The isolated paristerone, ecdysterone, and capitasterone showed anti-inflammatory activity in a rat polymorphonuclear leukocyte assay.

Key findings

  • From 260 mg of ethyl acetate extract, HSCCC with ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v) yielded paristerone (I, 30.5 mg, 99.1% purity), ecdysterone (II, 7.2 mg, 98.3% purity), and capitasterone (III, 8.1 mg, 98.1% purity).
  • Stationary phase retention was 74.0%, and separation time was within 5 h per run.
  • Compounds I–III inhibited β-glucuronidase release in rat polymorphonuclear leukocytes induced by platelet-activating factor, with IC50 values ranging from 1.51 to 11.68 μM.
  • Compound I was more potent than the positive control, while compound II was similar but slightly less potent than the positive control.

Methods

  • Stems of *D. glaucescens* (2.0 kg) were extracted with 95% ethanol, partitioned successively with n-hexane, ethyl acetate, and n-butanol; the dried ethyl acetate extract (19 g) was used for HPLC and HSCCC.
  • HSCCC used a two-phase solvent system of ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v), with the upper phase as stationary phase, lower phase as mobile phase, flow rate 2 mL/min, rotation 850 rpm, and UV detection at 254 nm.
  • Structures were identified by ESI-MS and 1H-/13C-NMR; anti-inflammatory activity was assayed by measuring inhibitory ratios of β-glucuronidase release in rat PMNs induced by platelet-activating factor, using ginkgolide B as positive control.

Limitations

  • The paper states no limitations.
  • Evident from the text: anti-inflammatory activity was evaluated only in vitro using rat PMNs, with no in vivo or clinical data.
  • Evident from the text: no statistical analysis, replicate numbers, or per-compound IC50 values are reported in the provided text; Table 2 is referenced but not shown.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/molecules22081310

Cites

17 of 17 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 17 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    HSCCC was used to separate and purify ecdysteroids for the first time from the stems of Diploclisia glaucescens using a two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water (3:0.2:0.8:3, v/v).

    “High-speed counter-current chromatography was used to separate and purify ecdysteroids for the first time from the stems of Diploclisia glaucescens using a two-phase solvent system composed of ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v).”
    • Also stated in: § 1. Introduction · plain
      “In the present paper, an efficient HSCCC method was developed for the separation and purification of ecdysteroids from D. glaucescens.”
    • Also stated in: § 4. Conclusions · plain
      “An efficient HSCCC method was developed and successfully applied to the separation and purification of three ecdysteroids from ethyl acetate extract of the residue obtained after evaporation of the crude ethanolic extract of D. glaucescens using ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v) as the two-phase solvent system.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Few reports have been published on the separation and purification of ecdysteroids from D. glaucescens by HSCCC.
  • Findingown data

    Three ecdysteroids were obtained from 260 mg of ethyl acetate extract of the residue obtained after evaporation of the crude ethanolic extract of D. glaucescens in one-step separation.

    “Three ecdysteroids were obtained from 260 mg of ethyl acetate extract of the residue obtained after evaporation of the crude ethanolicextractof D. glaucescens in one-step separation, which were identified as paristerone (I, 30.5 mg), ecdysterone (II, 7.2 mg), and capitasterone (III, 8.1 mg) by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR).”
    • Also stated in: § 2.3. Purification of Three Ecdysteroids by HSCCC · plain
      “The ethyl acetate extract (260 mg) was separated and purified in one step by the preparative HSCCC with ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v) as a solvent system (Figure 3).”
    Quote in the paperSubstantial claim
  • Findingown data

    Compound I exerts a more potent anti-inflammatory activity than the positive control used.

    “Compound I exerts a more potent anti-inflammatory activity than the positive control used.”
    Quote in the paperSubstantial claim
  • Findingown data

    Compound II showed a similar but slightly less potent anti-inflammatory activity than the selected positive control.

    “Furthermore, compound II showed a similar (but slightly less potent) activity than the selected positive control.”
    Quote in the paperNot a substantial claim
  • Findingown data

    The K-values in the two-phase solvent system ethyl acetate-n-butanol-ethanol-water (3:0.2:0.8:3, v/v) were suitable for separation of the target compounds.

    “It can be seen that the K-values in the solvent systems with the volume ratio of 3:0.2:0.8:3 (v/v) were suitable for separation of the target compounds.”
    Quote in the paperSubstantial claim
  • May combine two claims. 9
  • The quote only partly states this claim. 4
  • Classified as probably not a substantial claim. 2

  • May combine two claims. The quote only partly states this claim.

    Findingcitation

    Compound II was identified as ecdysterone, with 7.2 mg and 98.3% purity determined by HPLC.

    “The separation produced 30.5 mg of paristerone with 99.1% purity, 7.2 mg of ecdysterone with 98.3% purity, and 8.1 mg of capitasterone with 98.1% purity determined by HPLC analysis.”
    • Also stated in: § 2.4. Structure Identification of the Separated Compounds · plain
      “According to literature [11], compound II was identified as ecdysterone.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Bandara, 1989, Ecdysterone from stem of Diploclisia glaucescens, Phytochemistry source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The K-values of the three ecdysteroids in the two-phase solvent system ethyl acetate-n-butanol-water (3:1:3, v/v) were greater than 2, which led to band broadening and excessive time for elution.

    “As shown in Table 1, the K-values of three ecdysteroids in the two-phase solvent systems composed of ethyl acetate-n-butanol–water (3:1:3, v/v) were greater than 2, which led to band broadening and excessive time for elution.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Replacing n-butanol with ethanol in the two-phase solvent system decreased the K-values of the three ecdysteroids, with the value of compound I smaller than 0.5, resulting in poor resolution of the targeted compounds.

    “While n-butanol in above two-phase solvent systems was replaced by ethanol, the K-values of three ecdysteroids decreased, especially the value of compound I was smaller than 0.5, which result in poor resolution of the targeted compounds.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingcitation

    Compound I was identified as paristerone, with 30.5 mg and 99.1% purity determined by HPLC.

    “The separation produced 30.5 mg of paristerone with 99.1% purity, 7.2 mg of ecdysterone with 98.3% purity, and 8.1 mg of capitasterone with 98.1% purity determined by HPLC analysis.”
    • Also stated in: § 2.4. Structure Identification of the Separated Compounds · plain
      “According to literature [15], compound I was identified as paristerone.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] Sinh, 1982, Structure and stereochemistry of paristerone, a novel phytoecdysteroids from the tubers of Paris polyphylla, Tetrahedron source

      Not checked in the free run.

  • May combine two claims. The quote only partly states this claim.

    Findingcitation

    Compound III was identified as capitasterone, with 8.1 mg and 98.1% purity determined by HPLC.

    “The separation produced 30.5 mg of paristerone with 99.1% purity, 7.2 mg of ecdysterone with 98.3% purity, and 8.1 mg of capitasterone with 98.1% purity determined by HPLC analysis.”
    • Also stated in: § 2.4. Structure Identification of the Separated Compounds · plain
      “According to literature [16], compound III was identified as capitasterone.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Takemoto, 1968, Structure of capitasterone, a novel C29 insect-moulting substance from Cyathulacapitata, Tetrahedron Lett. source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The retention of the stationary phase was 74.0% and the separation time was within 5 h in each separation run.

    “The retention of the stationary phase was 74.0%, and the separation time was within 5 h in each separation run.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    Compounds I-III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 μM in the β-glucuronidase release assay in rat PMNs induced by platelet-activating factor.

    “Compounds I–III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 μM, respectively.”
    • Also stated in: § 2.5. Anti-Inflammatory Activities of Three Ecdysteroids · plain
      “As shown in Table 2, compounds I–III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 μM, respectively.”
    • Also stated in: § 4. Conclusions · plain
      “Three ecdysteroids, paristerone, ecdysterone, and capitasterone, showed significant anti-inflammatory activities by measuring the inhibitory ratios of β-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] Dai, 2004, Bioactive diels-alder type adducts from the stem bark of morus macroura, Planta Med. source

      Not checked in the free run.

    • Rests on an assumption: The β-glucuronidase release assay in rat PMNs induced by platelet-activating factor is a valid model for anti-inflammatory activity relevant to the antirheumatic activity of D. glaucescens in traditional medicine.

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

    Premisecitation

    D. glaucescens leaves and stems have been used as a folk medicine in the treatment of rheumatism, snake venom, biliousness, and venereal diseases.

    “The leaves and stems of the plant have been used as a folk medicine in the treatment of rheumatism, snake venom, biliousness, and venereal diseases [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Nanjing University of Chinese Medicine (2006). Dictionary of Chinese Herbs, Shanghai Science and Technology Press. [2nd ed.].

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Conventional methods for isolating ecdysteroids from D. glaucescens consumed large quantities of organic solvents and often offered low recoveries of the target products.

    “Unfortunately, the conventional methods consumed large quantities of organic solvents, and often offer low recoveries of the target products.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] Miller, 1985, Phytoecdysteroids of Diploclisia glaucescens, Planta Med. source

      Not checked in the free run.

    • cites [11] Bandara, 1989, Ecdysterone from stem of Diploclisia glaucescens, Phytochemistry source

      Not checked in the free run.

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

    Premisecitation

    Only 20-hydroxyecdysone was successfully isolated by HSCCC from Radix Serratulae Chinensis.

    “Only 20-hydroxyecdysone was successfully isolated by means of HSCCC from Radix Serratulae Chinensis [14].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Huang, 2014, Isolation and purification of 20-hydroxyecdysone from Radix SerratulaeChinensis by high-speed counter-current chromatography, J. Liq. Chromatogr. Relat. Technol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Ecdysteroids exhibit pharmacological effects on mammals, such as stimulation of protein synthesis, antidiabetic activity, and antirheumatic activity.

    “Ecdysteroids exhibit interesting pharmacological effects on mammals, such as the stimulation of protein synthesis, antidiabetic activity, and antirheumatic activity [5,6,7].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] Maclean, 2008, Phytoecdysteroids increase protein synthesis in skeletal muscle cells, J. Agric. Food Chem. source

      Not checked in the free run.

    • cites [6] Kizelsztein, 2009, 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model, Am. J. Physiol. Endocrinol. Metab. source

      Not checked in the free run.

    • cites [7] Dinan, 2001, Phytoecdysteroids: Biological aspects, Phytochemistry source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    HSCCC eliminates irreversible adsorption of analytes on the solid phase in conventional column chromatography and offers full recovery of target compounds.

    “High-speed counter-current chromatography (HSCCC), being a kind of liquid–liquid partition chromatography, eliminates the irreversible adsorption of analytes on the solid phase in conventional column chromatography and offers full recovery of target compounds.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [12] Fang, 2011, Separation of alkaloids from herbs using high-speed counter-current chromatography, J. Sep. Sci. source

      Not checked in the free run.

    • cites [13] Sutherland, 2009, Role of counter-current chromatography in the modernisation of Chinese herbal medicines, J. Chromatogr. A source

      Not checked in the free run.

12 of 12

  • The paper relies on this without support.

    Few reports have been published on the separation and purification of ecdysteroids from D. glaucescens by HSCCC.

    “To the best of our knowledge, few reports have been published on the separation and purification ecdysteroids from D. glaucescens by HSCCC.”

  • The paper relies on this without support.

    The β-glucuronidase release assay in rat PMNs induced by platelet-activating factor is a valid model for anti-inflammatory activity relevant to the antirheumatic activity of D. glaucescens in traditional medicine.

    “the anti-inflammatory activities of the three ecdysteroids have been evaluated herein by measuring the inhibitory ratios of β-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor.”
    “In the present study, the anti-inflammatory activities of the three ecdysteroids were evaluated by measuring the inhibitory ratio of β-glucuronidase release in rat PMNs induced by PAF in vitro [17].”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Paristerone, ecdysterone, and capitasterone isolated from Diploclisia glaucescens showed significant anti-inflammatory activity with IC50 values ranging from 1.5 to 11.6 μmol/L, measured by β-glucuronidase release in rat PMNs induced by platelet-activating factors.

    “Some of the known PEs, such as paristerone, ecdysterone, and capitasterone isolated from the stems of Diploclisia glaucescens, have shown significant anti-inflammatory activity with half maximal inhibitory concentration (IC50) values ranging from 1.5 to 11.6 μmol/L, respectively; this was discovered by measuring the inhibitory ratios of β-glucuronidase release in rat polymorphonuclear leukocytes (”

    From The Phytochemical, Biological, and Medicinal Attributes of Phytoecdysteroids: An Updated Review (2021) · cites this paper as [156]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • HPLC flow rate has conflicting units

    Minor

    The optimized HPLC flow rate is stated as 1.0 mL/min, but the HPLC analysis procedure gives 1.0 m/min. The latter is a length-per-time unit, not a volumetric flow-rate unit.

  • Two different carbon shifts are assigned to C-9 in compound III

    Minor

    The compound III 13C-NMR list assigns both 33.6 and 38.0 to C-9.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Nanjing University of Chinese Medicine (2006). Dictionary of Chinese Herbs, Shanghai Science and Technology Press. [2nd ed.].

Cited by

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Related

Authors
Fang, L., Li, J., Zhou, J., Wang, X., & Guo, L.
Affiliations
  • University of Jinan (CN)
  • Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Jinan 250014, China
  • China Academy of Chinese Medical Sciences (CN)
Corresponding author
  • Zhou, Jie
  • Guo, Lanping
ORCID
Venue
Molecules
Publisher
MDPI AG
Published
2017-08-07
Volume
22
Issue
8
Pages
1310
ISSN
1420-3049 (ISSN-L)
DOI
10.3390/molecules22081310
PMC
PMC6152221
OpenAlex
W2744732753
Cited by
20
References
17
Citations by year
2026: 3 · 2025: 2 · 2024: 1 · 2023: 3 · 2022: 2 · 2021: 4 · 2020: 2 · 2019: 1 · 2018: 2
FWCI
0.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecules (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Natural Science Foundation of China (81673527)
  • the Project for Key research Fields of Chinese Academy of Traditional Chinese Medicine (ZZ10-27)
  • the Construction Project for Sustainable Utilization of Valuable Traditional Chinese Medicine Resources (2060302)
  • the Science and Technology Program of ShanDong Province (2015GSF119026,2017GSF19115)
  • the Science Foundation of University of Jinan (XKY1608)
Topics
  • Physical Sciences › Chemistry › Analytical Chemistry › Chromatography in Natural Products
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Phytochemical Studies and Bioactivities
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Phytochemistry and Biological Activities
Keywords
ecdysteroids, 20-hydroxyecdysone, anti-inflammatory activity, platelet-activating factor, beta-glucuronidase release, polymorphonuclear leukocytes, high-speed countercurrent chromatography, natural product isolation, plant stems
MeSH terms
Animals, Anti-Inflammatory Agents, Chromatography, High Pressure Liquid, Neutrophils, Plant Extracts, Solvents, Molecular Structure, Biomarkers, Inhibitory Concentration 50, Ecdysteroids, Menispermaceae, Rats

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