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Planta Med
DOI: 10.1055/a-2592-0968
Original Papers

Separation and Cytotoxicity of Enzymatic Transformed Prosaikogenins from Bupleurum falcatum

Hyejin Ko 
1   College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul, Republic of Korea
2   Natural Product Drug Development Division, Korea Institute of Science and Technology (KIST) Gangneung Institute of Natural Products, Gangneung, Republic of Korea
,
Kyoung Jin Lee
1   College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul, Republic of Korea
,
Kwangho Song
1   College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul, Republic of Korea
3   Korean Medicine Clinical Trial Center, Kyung Hee University Korean Medicine Hospital, Seoul, Republic of Korea
,
In Jin Ha
3   Korean Medicine Clinical Trial Center, Kyung Hee University Korean Medicine Hospital, Seoul, Republic of Korea
,
Yeong Shik Kim 
1   College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul, Republic of Korea
› Author Affiliations
This research was supported by the Korea Institute of Science and Technology Intramural Research Grant (2E32702) and the National Research Foundation of Korea (NRF-2021R1F1A1049427). H. K. is supported by the ‘National Research Council of Science & Technology (NST)’ – ‘Korea Institute of Science and Technology (KIST)’ Postdoctoral Fellowship Program for Young Scientists at KIST in the Republic of Korea.
› Further Information
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Abstract

Saikosaponins, bioactive compounds derived from Bupleurum falcatum roots, have limited applications due to their low bioavailability and the absence of efficient large-scale separation methods. To address this, an enzymatic transformation in vitro with cellulase was employed to remove glucose at the C-3 position, producing lipophilic prosaikogenins. These metabolites were separated using countercurrent chromatography (CCC) and preparative HPLC. The optimal CCC solvent system was determined to be dichloromethane/methanol/water (4 : 3 : 2, v/v/v). Prosaikogenin F and prosaikogenin G (PSG G) were isolated from the deglycosylated fraction, and the effect of rotation speed on compound retention was examined. Further enzymatic biotransformation using α-L-rhamnosidase and cellulase resulted in the isolation of prosaikogenins E1 and E3. The efficient separation of these four prosaikogenins was achieved through a combination of enzymatic transformation and CCC. Of these, PSG G demonstrated the strongest anticancer activity against the cancer cell lines MDA-MB-468, HepG2, and HCT116, while exhibiting lower toxicity in normal cells, supporting its potential as an effective anticancer agent. This study presents a highly efficient enzymatic transformation and separation strategy that can aid in the pharmaceutical development of saikosaponin derivatives.

Keywords

Bupleurum falcatum - Apiaceae - Saikosaponin - Prosaikogenin - enzymatic transformation - countercurrent chromatography

Supporting Information

    Optimized conditions for the enzymatic transformation and countercurrent separation of prosaikogenins, MS and 1D NMR analysis of prosaikogenins, and cytotoxicity of PSG G against normal human cell lines.

  • Supporting Information


Publication History

Received: 22 April 2024

Accepted after revision: 16 April 2025

Article published online:
10 June 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

 

  • References

  • 1 Ashour ML, Wink M. Genus Bupleurum: A review of its phytochemistry, pharmacology and modes of action. J Pharm Pharmacol 2011; 63: 305-321
    PubMedSearch in Google Scholar
  • 2 Yao RY, Zou YF, Chen XF. Traditional use, pharmacology, toxicology, and quality control of species in genus Bupleurum L. Chin Herb Med 2013; 5: 245-255
    PubMedSearch in Google Scholar
  • 3 Huang HQ, Zhang X, Lin M, Shen YH, Yan SK, Zhang WD. Characterization and identification of saikosaponins in crude extracts from three Bupleurum species using LC-ESI-MS. J Sep Sci 2008; 31: 3190-3201
    PubMedSearch in Google Scholar
  • 4 Li P, Wu M, Xiong W, Li J, An Y, Ren J, Xie Y, Xue H, Yan D, Li M, Zhong G. Saikosaponin-d ameliorates dextran sulfate sodium-induced colitis by suppressing NF-κB activation and modulating the gut microbiota in mice. Int Immunopharmacol 2020; 81: 106288
    PubMedSearch in Google Scholar
  • 5 Liu M, Zhang G, Naqvi S, Zhang F, Kang T, Duan Q, Wang Z, Xiao S, Zheng Y. Cytotoxicity of Saikosaponin A targets HEKa cell through apoptosis induction by ROS accumulation and inflammation suppression via NF-κB pathway. Int Immunopharmacol 2020; 86: 106751
    PubMedSearch in Google Scholar
  • 6 Pan Y, Ke Z, Ye H, Sun L, Ding X, Shen Y, Zhang R, Yuan J. Saikosaponin C exerts anti-HBV effects by attenuating HNF1α and HNF4α expression to suppress HBV pgRNA synthesis. Inflamm Res 2019; 68: 1025-1034
    PubMedSearch in Google Scholar
  • 7 Li X, Li X, Huang N, Liu R, Sun R. A comprehensive review and perspectives on pharmacology and toxicology of saikosaponins. Phytomedicine 2018; 50: 73-87
    PubMedSearch in Google Scholar
  • 8 Wang C, Zhang R, Chen X, Yuan M, Wu J, Sun Q, Miao C, Jing Y. The potential effect and mechanism of Saikosaponin A against gastric cancer. BMC Complement Med Ther 2023; 23: 295
    PubMedSearch in Google Scholar
  • 9 Liu G, Guan Y, Liu Y, Wang Y, Zhang J, Liu Y, Liu X. Saikosaponin D inducing apoptosis and autophagy through the activation of endoplasmic reticulum stress in glioblastoma. Biomed Res Int 2022; 2022: 5489553
    PubMedSearch in Google Scholar
  • 10 Du J, Song D, Cao T, Li Y, Liu J, Li B, Li L. Saikosaponin-A induces apoptosis of cervical cancer through mitochondria- and endoplasmic reticulum stress-dependent pathway in vitro and in vivo: involvement of PI3K/AKT signaling pathway. Cell Cycle 2021; 20: 2221-2232
    PubMedSearch in Google Scholar
  • 11 Lai M, Ge Y, Chen M, Sun S, Chen J, Cheng R. Saikosaponin D inhibits proliferation and promotes apoptosis through activation of MKK4-JNK signaling pathway in pancreatic cancer cells. Onco Targets Ther 2020; 13: 9465-9479
    PubMedSearch in Google Scholar
  • 12 Shyu KG, Tsai SC, Wang BW, Liu YC, Lee CC. Saikosaponin C induces endothelial cells growth, migration and capillary tube formation. Life Sci 2004; 76: 813-826
    PubMedSearch in Google Scholar
  • 13 Liu G, Tian Y, Li G, Xu L, Song R, Zhang Z. Metabolism of saikosaponin a in rats: Diverse oxidations on the aglycone moiety in liver and intestine in addition to hydrolysis of glycosidic bonds. Drug Metab Dispos 2013; 41: 622-633
    PubMedSearch in Google Scholar
  • 14 Yu KU, Jang IS, Kang KH, Sung CK, Kim DH. Metabolism of saikosaponin c and naringin by human intestinal bacteria. Arch Pharm Res 1997; 20: 420-424
    PubMedSearch in Google Scholar
  • 15 Yousof Ali M, Jannat S, Mizanur Rahman M. Ginsenoside derivatives inhibit advanced glycation end-product formation and glucose-fructose mediated protein glycation in vitro via a specific structure-activity relationship. Bioorg Chem 2021; 111: 104844
    PubMedSearch in Google Scholar
  • 16 Chun J, Ha IJ, Kim YS. Antiproliferative and apoptotic activities of triterpenoid saponins from the roots of Platycodon grandiflorum and their structure-activity relationships. Planta Med 2013; 79: 639-645
    PubMedSearch in Google Scholar
  • 17 Hwang CR, Lee SH, Jang GY, Hwang IG, Kim HY, Woo KS, Lee J, Jeong HS. Changes in ginsenoside compositions and antioxidant activities of hydroponic-cultured ginseng roots and leaves with heating temperature. J Ginseng Res 2014; 38: 180-186
    PubMedSearch in Google Scholar
  • 18 Kim MH, Lee YC, Choi SY, Cho CW, Rho J, Lee KW. The changes of ginsenoside patterns in red ginseng processed by organic acid impregnation pretreatment. J Ginseng Res 2011; 35: 497-503
    PubMedSearch in Google Scholar
  • 19 Shin KC, Kil TG, Kang SH, Oh DK. Production of deglucose-apiose-xylosylated platycosides from glycosylated platycosides by crude enzyme from Aspergillus tubingensis. J Microbiol Biotechnol 2022; 32: 430-436
    PubMedSearch in Google Scholar
  • 20 Jocquel C, Muzard M, Plantier-Royon R, Rémond C. An integrated enzymatic approach to produce pentyl xylosides and glucose/xylose laurate esters from wheat bran. Front Bioeng Biotechnol 2021; 9: 647442
    PubMedSearch in Google Scholar
  • 21 Rha CS, Kim HG, Baek NI, Kim DO, Park CS. Using amylosucrase for the controlled synthesis of novel isoquercitrin glycosides with different glycosidic linkages. J Agric Food Chem 2020; 68: 13798-13805
    PubMedSearch in Google Scholar
  • 22 Han T, Cao X, Pei H, Fan C. Overview of solvent system selection strategies for countercurrent chromatography. Sep Purif Rev 2023; 52: 305-325
    PubMedSearch in Google Scholar
  • 23 Kostanyan AA, Voshkin AA, Belova VV. Analytical, preparative, and industrial-scale separation of substances by methods of countercurrent liquid-liquid chromatography. Molecules 2020; 25: 6020
    PubMedSearch in Google Scholar
  • 24 Zhang L, Wu S. Hydrophobic and hydrophilic interactions in countercurrent chromatography. J Chromatogr A 2020; 1611: 460576
    PubMedSearch in Google Scholar
  • 25 Englert M, Vetter W. Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency. Anal Chim Acta 2015; 884: 114-123
    PubMedSearch in Google Scholar
  • 26 Wu D, Cao X, Wu S. Overlapping elution-extrusion counter-current chromatography: A novel method for efficient purification of natural cytotoxic andrographolides from Andrographis paniculate. J Chromatogr A 2012; 1223: 53-63
    PubMedSearch in Google Scholar
  • 27 Lee KJ, Xu MY, Shehzad O, Seo EK, Kim YS. Separation of triterpenoid saponins from the root of Bupleurum falcatum by countercurrent chromatography: The relationship between the partition coefficients and solvent system composition. J Sep Sci 2014; 37: 3587-3594
    PubMedSearch in Google Scholar
  • 28 Svrkota B, Krmar J, Protić A, Otašević B. The secret of reversed-phase/weak cation exchange retention mechanisms in mixed-mode liquid chromatography applied for small drug molecule analysis. J Chromatogr A 2023; 1690: 463776
    PubMedSearch in Google Scholar
  • 29 Mattarozzi M, Riboni N, Maffini M, Scarpella S, Bianchi F, Careri M. Reversed-phase and weak anion-exchange mixed-mode stationary phase for fast separation of medium-, long- and very long chain free fatty acids by ultra-high-performance liquid chromatography-high resolution mass spectrometry. J Chromatogr A 2021; 1648: 462209
    PubMedSearch in Google Scholar
  • 30 Ovchinnikov DV, Pokrovskiy OI, Kosyakov DS, Bogolitsyn KG, Ulʼyanovskii NV, Falev DI. Evaluation of temperature and pressure effects on retention in supercritical fluid chromatography on polar stationary phases. J Chromatogr A 2020; 1610: 460600
    PubMedSearch in Google Scholar
  • 31 Berthod A, Faure K. Revisiting resolution in hydrodynamic countercurrent chromatography: Tubing bore effect. J Chromatogr A 2015; 1390: 71-77
    PubMedSearch in Google Scholar
  • 32 Englert M, Vetter W. Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency. Anal Chim Acta 2015; 884: 114-123
    PubMedSearch in Google Scholar
  • 33 Qizhen D, Caijuan W, Guojun Q, Pingdong W, Ito Y. Relationship between the flow-rate of the mobile phase and retention of the stationary phase in counter-current chromatography. J Chromatogr A 1999; 835: 231-235
    PubMedSearch in Google Scholar
  • 34 Koji S, Sakae A, Ogihara Y. New derivatives of saikosaponins. Chem Pharm Bull 1985; 33: 3349-3355
    PubMedSearch in Google Scholar
  • 35 Nose M, Amagaya S, Takeda T, Ogihar Y. New derivatives of saikosaponin c. Chem Pharm Bull 1989; 37: 1293-1296
    PubMedSearch in Google Scholar
  • 36 Tran TNA, Son JS, Awais M, Ko JH, Yang DC, Jung SK. β-glucosidase and its application in bioconversion of ginsenosides in Panax ginseng. Bioengineering 2023; 10: 484
    PubMedSearch in Google Scholar
  • 37 Ōmura S, Asami Y, Crump A. Staurosporine: New lease of life for parent compound of todayʼs novel and highly successful anti-cancer drugs. J Antibiot 2018; 71: 688-701
    PubMedSearch in Google Scholar
  • 38 Ko H, Lee JH, Kim HS, Kim T, Han YT, Suh YG, Chun J, Kim YS, Ahn KS. Novel galiellalactone analogues can target STAT3 phosphorylation and cause apoptosis in triple-negative breast cancer. Biomolecules 2019; 9: 170
    PubMedSearch in Google Scholar