Separation and Cytotoxicity of Enzymatic Transformed Prosaikogenins from Bupleurum falcatum

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

Publication History

Received: 22 April 2024

Accepted after revision: 16 April 2025

Article published online:
10 June 2025

• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 24 Zhang L, Wu S. Hydrophobic and hydrophilic interactions in countercurrent chromatography. J Chromatogr A 2020; 1611: 460576
• 25 Englert M, Vetter W. Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency. Anal Chim Acta 2015; 884: 114-123
• 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
• 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
• 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
• 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
• 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
• 31 Berthod A, Faure K. Revisiting resolution in hydrodynamic countercurrent chromatography: Tubing bore effect. J Chromatogr A 2015; 1390: 71-77
• 32 Englert M, Vetter W. Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency. Anal Chim Acta 2015; 884: 114-123
• 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
• 34 Koji S, Sakae A, Ogihara Y. New derivatives of saikosaponins. Chem Pharm Bull 1985; 33: 3349-3355
• 35 Nose M, Amagaya S, Takeda T, Ogihar Y. New derivatives of saikosaponin c. Chem Pharm Bull 1989; 37: 1293-1296
• 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
• 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
• 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