Separation and Cytotoxicity of Enzymatic Transformed Prosaikogenins from Bupleurum falcatum

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Reagents and plant materials
• Apparatus
• Preparation of saponin-enriched fraction for enzymatic transformation
• Optimization of enzymatic transformation of saponin-enriched fraction
• Large-scale transformation of saponin-enriched fraction
• Determination of the partition coefficients (K D)
• Countercurrent chromatography separation
• Preparative HPLC separation
• Identification of isolated compounds
• Cytotoxicity assay against cancer cells
• Contributorsʼ Statement
• References

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.

Introduction

Saikosaponins are bioactive constituents of Bupleurum falcatum L. (Apiaceae) widely utilized in traditional East Asian medicine. The roots of the Bupleurum species have been prescribed for over 2000 years to treat fever, pain, inflammation, chronic hepatitis, and autoimmune disease [1], [2]. These compounds belong to the triterpenoid saponin group and are synthesized via the mevalonic acid pathway. Various derivatives have been identified, including saikosaponins, prosaikogenins, saikogenins, malonylsaikosaponins, hydrosaikosaponins, and chikusaikosids [3]. The primary bioactive components–saikosaponin A (SSA), saikosaponin C (SSC), and saikosaponin D (SSD)–exhibit hepatoprotective, antitumor, immunomodulatory, and anti-inflammatory properties [4], [5], [6], [7]. Notably, saikosaponins have demonstrated cytotoxicity against cancer cells, suggesting their potential as anticancer agents [8], [9], [10], [11], [12]. However, their low bioavailability and the absence of efficient large-scale separation techniques hinder their pharmaceutical applications.

In vivo, saikosaponins are metabolized by intestinal microbiota and hepatic enzymes, yielding bioactive intermediates like prosaikogenins and sapogenins [13], [14]. SSC is converted by gut bacteria into prosaikogenin E₁ (PSG E₁), E₂, and E₃, as well as saikogenin, while SSA undergoes hepatic phase I metabolism, producing prosaikogenins and sapogenins. However, these metabolic processes are inefficient and inconsistent, limiting their direct utilization. Although these compounds are of significant pharmaceutical interest, efficient methodologies for their large-scale separation and purification remain underdeveloped. Deglycosylation plays a crucial role in altering the bioactivity of saponins. For example, the removal of sugar moieties from ginsenosides enhances their anti-aging and antidiabetic properties, whereas glycoside cleavage in platycosides significantly reduces cytotoxicity, underscoring the importance of sugar groups in the structure-activity relationship of saponins [15], [16]. Among various deglycosylation approaches, enzymatic biotransformation has emerged as the most selective and effective method, providing more precise control over glycosidic bond cleavage compared to heat treatment, acid hydrolysis, or fermentation [17], [18], [19], [20], [21].

Countercurrent chromatography (CCC) is an effective liquid–liquid extraction technique that offers significant advantages over conventional column chromatography. Several strategies, including solvent system optimization and elution mode selection, have been explored to improve CCC efficiency [22], [23], [24], [25], [26]. Selecting an appropriate solvent system and elution method is critical for effective separation, particularly in the preparative-scale isolation of bioactive compounds from natural sources. Despite its advantages, large-scale CCC separation of enzymatically transformed saikosaponin derivatives has not been systematically studied.

In this study, the saikosaponin-enriched fraction was enzymatically transformed using cellulase and α-L-rhamnosidase to eliminate O-linkages between β-D-glucopyranose and α-L-rhamnopyranose. Three cytotoxic prosaikogenins were successfully separated from the enzyme-treated fraction using CCC and preparative high-performance liquid chromatography (HPLC). Furthermore, CCC rotation speed was optimized to balance stationary-phase retention, resolution, and retention time. This strategy establishes a preparative-scale method for efficiently isolating bioactive metabolites from natural sources, facilitating further pharmacological investigations.

Results and Discussion

Several pharmacokinetic studies have examined the metabolic pathways involved in the action of saikosaponins. Saikosaponins undergo hydrolysis, leading to the formation of prosaikogenins and saikogenins via the elimination of sugar moieties ([Fig. 1]). In a previous study, we reported a separation method for separating saikosaponins from a saponin-enriched fraction of B. falcatum by CCC [27]. Based on HPLC analysis, the major components of the fraction, including SSA, SSC, and SSD, were detected, each possessing sugar moieties at the C-3 position ([Fig. 2 a]).

Fig. 2SA (Supporting Information) illustrates the enzymatic sensitivity of saikosaponins to cellulases (a schematic representation of the overall experimental workflow is provided in Fig. 1S, Supporting Information). SSA and SSD were effectively transformed by cellulase and β-D-glycosidase, respectively, whereas SSC remained unconverted, suggesting differential enzymatic sensitivity due to structural variations at the C-3 position. The presence of α-L-rhamnopyranose in SSC might interfere with cellulase activity on the 6 → 1 linkage of β-D-glucopyranose, preventing its conversion. Therefore, the removal of the 4 → 1 linkage by α-L-rhamnosidase is essential for effective transformation. Under optimized conditions (using cellulase from Tichoderma reesei (liquid), as shown in Fig. 2SA, Supporting Information), 10 g of the saponin-enriched fraction was enzymatically transformed. The optimization results are presented in Fig. 2SB−2SC (Supporting Information).

The converted fraction was analyzed using HPLC and LC-MS. LC-MS analysis revealed the molecular weights of the two major peaks. In negative mode, the observation of m/z 617 indicated the elimination of the glucose moiety from SSA and SSD (Fig. 3S, Supporting Information). The structures were further confirmed using ¹³C nuclear magnetic resonance (NMR) spectroscopy after CCC separation. Following the separation of prosaikogenin F (PSG F) and prosaikogenin G (PSG G), the residue in the stationary phase of CCC separation (fraction S) was treated with α-L-rhamnosidase to eliminate α-L-rhamnopyranose (fraction SR). Subsequently, fraction SR was treated with cellulase to cleave the 6 → 1 linkage of β-D-glucopyranose (fraction SRC) ([Fig. 2 b]−e).

The separation of target compounds by CCC depends mainly on the selection of a suitable biphasic solvent system. Owing to the elimination of the glucose moiety from saikosaponins, the target compounds are converted into more hydrophobic byproducts. Based on this hypothesis, we revised our previously reported two-phase solvent system [27]. The chloroform in the chloroform–methanol–water solvent system was replaced with dichloromethane, and the composition of each solvent was adjusted based on the measured partition coefficients (K _D). Solvent systems composed of hexane-ethyl acetate–methanol–water, as listed in [Table 1], were deemed unsuitable because PSG G and PSG F exhibited low separation resolutions in these solvent systems. A solvent system composed of dichloromethane–methanol–water (4 : 3 : 2 v/v) was selected for CCC separation. This choice facilitated the separation of PSG G and PSG F more efficiently than preparative HPLC.

The resolution of the peaks in the CCC separation was significantly influenced by the retention of the stationary phase. Several investigations on the relationship between the retention of the stationary phase and various factors of CCC have been reported [28], [29], [30], [31], [32], [33]. In particular, the relationship between the flow rate of the mobile phase and stationary phase retention [33] and the relationship between the resolution and stationary phase retention [31] have been studied. Specifically, a linear relationship exists between the square root of the flow rate (F) and retention of the stationary phase (S_f ). The relationship between the rotation speed of the CCC device and the retention of the stationary phase was also investigated. As shown in [Fig. 3 a], rotational speed and retention of the stationary phase are closely related. The dots were plotted based on several experimental equilibrium results for the two phases. At speeds higher than 500 rpm, the retention of the stationary phase and rotation speed were relatively consistent with our expectations. However, at speeds lower than 500 rpm, the actual retention of the stationary phase decreased more dramatically than predicted. The centrifugal force at relatively low rotational speeds was sensitively affected by the rotational speed.

As illustrated in [Fig. 3 b], the retention time of each compound varied depending on the retention time of the stationary phase. CCC separation at different rotational speeds is shown in Fig. 4S (Supporting Information). While the retention time of PSG F (K _D > 1) decreased due to the loss of the stationary phase (135 – 107 min), the purity in all cases remained higher than 99%. However, in the case of PSG G (K _D < 1), both the amount and purity were affected by stationary phase retention. The retention time increased slightly owing to the loss of the stationary phase (60 – 62 min), and the purity decreased (97 – 88%). As illustrated in [Fig. 4], the optimum conditions (considering the purities and retention times of PSG G and PSG F) were confirmed to be a rotational speed of 650 rpm (71% stationary phase retention). The recovery and purity of each prosaikogenin were determined and assessed using HPLC-ELSD: PSG G, 86.3 mg (purity 94%); PSG F, 126.5 mg (purity 99%) from 300 mg of the saponin-enriched fraction. As shown in Fig. 3S and Table 1S (Supporting Information), the chemical structures were identified using ESI-MS and ¹³C NMR spectroscopy and compared with literature data [34].

The SR and SRC fractions as shown in [Fig. 2 d]–e were further separated using preparative HPLC. Five milligrams of PSG E₁ (purity > 97%) and PSG E₃ (purity > 97%) were obtained from the SR and SRC fractions, respectively. The structures of PSG E₁ and PSG E₃ were identified with LC-MS data compared to the scheme of enzymatic transformation of saikosaponins in [Fig. 1] and previous literature [14], [35]. The [M – H]⁻ ion of PSG E₁ at m/z 779.4 and the [M – H + HCOOH]⁻ ion of PSG E₃ at m/z 663.3 in negative ion mode as shown in Fig. 3S (Supporting Information) were identified with the loss of rhamnosyl (146 Da) and glucosyl (162 Da) moiety by the removal of terminal glucose or/and rhanmnose from the [M – H]⁻ ion of SSC at m/z 925.5.

The enzymatically transformed compounds exhibited similar structural features, each with a mono sugar moiety at the C-3 position and allyl oxide linkages at the C-13 and 28 positions. In this study, we investigated the cytotoxicity of prosaikogenins in human cancer cells and compared it to that of their parent compounds. The results are shown in [Table 2], with staurosporine as a positive control. Following enzymatic transformation, the cytotoxicity of the saikosaponin fractions increased across multiple human cancer cell lines. PSG G was identified as the key cytotoxic compound, exhibiting a lower 50% inhibition concentration (IC₅₀) than its parent compound (SSD). The IC₅₀ values of PSG G for six cancer cell lines–MDA-MB-468 (breast adenocarcinoma), A549 (lung carcinoma), HepG2 (hepatocellular carcinoma), AGS (gastric adenocarcinoma), PANC-1 (pancreas epithelioid carcinoma), and HCT116 (colorectal carcinoma)–were 22.38, 32.15, 22.58, 25.12, 24.89, and 22.46 µM, respectively ([Table 2]). Notably, compared to its precursor SSD, PSG G exhibited the lowest IC₅₀ values in MDA-MB-468, HepG2, and HCT116, while exerting a weaker effect on normal cell lines (MCF-10A, NKNT-3, and FHC; Fig. 5S (Supporting Information)). These results indicate its strong anticancer potential against breast, liver, and colorectal cancer cells.

Although the enzymatically transformed prosaikogenins share similar structural features, each possessing a mono sugar moiety at the C-3 position and an allyl oxide linkage at the C-13 and C-28 positions, variations in their cytotoxic activities suggest that stereochemical factors, such as the hydroxyl group configuration at C-16, may play a critical role. Further investigations are required to clarify the relationship between structure and cytotoxicity in cancer cells; however, these findings highlight PSG G as a strong anticancer candidate.

In this study, four prosaikogenins (PSG E₁, E₃, F, and G) were successfully isolated through the enzymatic transformation of major saikosaponins from B. falcatum roots. The elimination of glucose at the C-3 position of saikosaponins was achieved through enzymatic transformation using cellulase and α-L-rhamnosidase, leading to the production of lipophilic prosaikogenins. The enzymatically converted fraction was effectively separated using CCC with an optimized solvent system (dichloromethane–methanol–water at 4 : 3 : 2, v/v/v). Furthermore, key parameters such as rotation speed and stationary phase retention were optimized to enhance separation efficiency. Among the isolated compounds, PSG G exhibited the most potent cytotoxic activity, with notable effects on MDA-MB-468 (breast adenocarcinoma), HepG2 (hepatocellular carcinoma), and HCT116 (colorectal carcinoma), demonstrating its potential as a promising anticancer agent.

Although PSG G exhibited significant cytotoxicity activity, its precise molecular mechanism remains unclear, necessitating further studies to elucidate its mode of action and molecular targets. In vivo validation is required to evaluate its bioavailability, pharmacokinetics, and therapeutic efficacy. Additionally, a structure–activity relationship analysis should be conducted to determine how specific structural modifications affect bioactivity.

Despite these challenges, the enzymatic transformation and CCC-based separation approach presented in this study offer a promising strategy for enhancing the bioavailability and therapeutic potential of saikosaponins. The successful isolation of PSG derivatives, along with their cytotoxicity, underscores their potential as anticancer drug candidates. Continued research will be key to advancing these compounds toward clinical applications, bridging the gap between traditional herbal medicine and modern oncology.

Materials and Methods

Reagents and plant materials

Dried roots of B. falcatum were purchased from Omniherb. A voucher specimen (SNU-16-014) was deposited in the herbarium of the Natural Products Research Institute, College of Pharmacy, Seoul National University. The specimen was identified as B. falcatum by Professor Young Bae Suh (College of Pharmacy, Seoul National University). All organic solvents (industrial grade) used for the CCC separation were purchased from Dae Jung Science. HPLC analysis was performed using HPLC-grade acetonitrile (J. T. Baker) and distilled water (NANO Pure Diamond). β-D-glucosidase originating from almonds (solid/liquid types) and cellulase isolated from T. reesei (solid/liquid types) were purchased from Sigma–Aldrich. α-L-rhamnosidase from a prokaryote was purchased from Megazyme. The polystyrene adsorption resin, Diaion HP-20, was purchased from Mitsubishi Chemicals. Cholera toxin, epidermal growth factor, hydrocortisone, insulin, staurosporine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich.

Apparatus

The CCC instrument for preparative separation was a TBE-300B (Shanghai Tauto Biotech., coil I. D.: 1.6 mm, coil volume: 280 mL including a 20 mL sample loop) and the rotation speed ranged from 0 to 1000 rpm. The solvent was pumped into the CCC column using a Hitachi L-7100 quaternary pump, and the eluted solution was monitored using an evaporative light-scattering detector (Sedex 75 ELSD).

Preparation of saponin-enriched fraction for enzymatic transformation

The dried roots of B. falcatum L. (1 kg) were pulverized and extracted with 5 L of methanol by sonication for 3 h, and this process was repeated two more times. The extract was filtered and concentrated using a rotary vacuum evaporator. The methanolic extract (120.5 g) was dissolved in water and partitioned using n-butanol. The n-butanol fraction (64 g) was loaded onto an open column packed with Diaion HP-20 resin (100 × 10 cm; the volume, 7 L). The three fractions were eluted with water, 30% aqueous methanol, and methanol. After washing off the hydrophilic components, a saponin-enriched fraction (10.4 g) was obtained by methanol elution. The fraction was evaporated for subsequent enzymatic transformations.

Optimization of enzymatic transformation of saponin-enriched fraction

To select an appropriate enzyme for β-D-glucose removal, the saponin-enriched fraction was incubated with candidate enzymes in sodium phosphate buffer (pH 7.0) at 37.5 °C for 12, 24, 36, and 48 h. Preliminary screening was conducted using glycosidase (solid, liquid) and cellulase (solid, liquid). Previous studies have reported that β-D-glycosidases, including cellulase, effectively hydrolyze glucose from saponins [36]. Following enzyme screening, cellulase from T. reesei (liquid) was selected as the most efficient enzyme for enzymatic transformation. The enzyme concentration was optimized by testing different amounts: 0 (control), 1, 2, 3, 4, 5, and 6 units. Based on these results, the amount of cellulase per gram of saponin-enriched fraction was set at 300 U/g. Twenty milligrams of the saponin-enriched fraction was dissolved in sodium phosphate buffer (pH 7.0), and the designated enzyme amount was added. The solutions were incubated at 37.5 °C for specified time points, and reactions were terminated by heating at 90 °C for 3 min. The optimal reaction time was determined as 36 h based on the relative abundance of PSG F and PSG G, enzymatically converted from SSA and SSD, respectively.

Large-scale transformation of saponin-enriched fraction

The 10 g of the saponin-enriched fraction and 3000 units of cellulase were incubated in sodium phosphate buffer (pH 7.0) at 37.5 °C for 36 h with stirring. The solution was placed in 90 °C water to terminate the reaction and then loaded onto a Diaion HP-20 open column to eliminate cellulase and cleaved sugar moieties. The prosaikogenin-enriched fraction was obtained from the 100% methanol eluent after washing with water and 50% aqueous methanol. The fractions were further separated by CCC. The residue in the stationary phase of CCC separation was collected (fraction S), and 130 mg of the fraction S was treated with 100 units of α-L-rhamnosidase and then incubated in sodium phosphate buffer (pH 7.0) at 37.5 °C for 36 h with agitation. Afterwards, they were placed in a water bath at 90 °C to terminate the reaction (fraction SR). Fraction SR was converted to fraction SRC by incubation with 50 units of cellulase to eliminate β-D-glucose of prosaikogenin E₁ (PSG E₁). The SRC fraction was then separated by preparative HPLC to obtain prosaikogenin E₃ (PSG E₃).

Determination of the partition coefficients (K _D)

A suitable two-phase solvent system was selected based on the K _D values of the target prosaikogenins. Three milligrams of the enzymatically transformed fraction were dissolved in 1 mL of the upper and lower phases (1 : 1, v/v). The mixture was shaken, and the two equilibrated phases were separated and evaporated. The residue was dissolved in methanol and analyzed using HPLC. The K _D value is expressed as the peak area of prosaikogenins in the upper phase divided by that in the lower phase.

Countercurrent chromatography separation

A two-phase solvent system comprising of dichloromethane–methanol–water (4 : 3 : 2, v/v/v) was used for CCC separation. The prosaikogenin-enriched fraction (300 mg) was dissolved in the lower phase (20 mL) and filtered with PTFE filter before sample injection. The CCC capillary column was filled with the upper phase and the apparatus was rotated at 650 rpm. The lower phase was pumped into the column at a flow rate of 4 mL/min and the sample solution was injected after hydrodynamic equilibrium was achieved. Stationary phase retention was measured based on the volume of the replaced stationary phase. The eluent was continuously monitored by connecting the tail outlet of the capillary column to the ELSD system using a split valve. The ELSD system was set to a probe temperature of 50 °C and the nebulizer N₂ gas was adjusted to 3.0 bar. The separated compounds were collected according to the chromatographic profiles and further analyzed using HPLC-ELSD.

Preparative HPLC separation

PSG E₁ and PSG E₃ were separated from the SR and SRC fractions, respectively, using preparative HPLC. The column used for the preparative separation was an INNO C₁₈ column (250 mm × 10 mm, 10 µm particle size) of YoungJin Biochrom (Seongnam, Korea). The preparative HPLC separation used water as eluent A and acetonitrile as eluent B, and the elution was performed as follows: 0 – 15 min (25 – 30% B); 15 – 30 min (30 – 50% B); 30 – 35 min (50 – 100% B) at a flow rate of 4.5 mL/min. The UV detection wavelength was set at 205 nm.

Identification of isolated compounds

The saponin-enriched fractions and separated compounds were analyzed using HPLC-ELSD. An INNO C₁₈ column (150 mm × 4.6 mm, 5 µm particle size) of YoungJin Biochrom and a Hitachi L-6200 instrument equipped with a Sedex 75 ELSD were used. The analysis using water as eluent A and acetonitrile as eluent B was conducted using the following elution program: 0 – 5 min (25% B); 5 – 30 min (25 – 50% B); 30 – 35 min (50 – 100% B). The column was equilibrated with 25% B for 5 min at a flow of 1 mL/min. The separated compounds were further identified using ESI-MS, ¹H NMR, and ¹³C NMR spectroscopy. All ESI-MS experiments were performed by using a Finnigan LCQ ion trap mass spectrometer (Thermo Finnigan) and an Agilent Technologies 1200 series coupled with an Agilent Technologies 6130 quadrupole mass spectrometer. NMR spectra were recorded by using a Bruker AVANCE 500 NMR spectrometer with tetramethylsilane as an internal standard.

Cytotoxicity assay against cancer cells

MDA-MB-468 (breast cancer), A549 (lung cancer), HepG2 (hepatocellular carcinoma), AGS (gastric cancer), PANC-1 (pancreatic cancer), HCT116 (colon cancer), MCF-10A (a commonly used normal breast epithelial cell line), and FHC (a normal colon cell line) were purchased from the ATCC. NKNT-3 (a highly differentiated cell line derived from normal adult human hepatocytes) was obtained from Professor Park Sunghyouk (Seoul National University). MDA-MB-468, HepG2, PANC-1, and NKNT-3 cells were cultured in DMEM supplemented with 10% FBS and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL). A549, AGS, and HCT116 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and the same antibiotics. MCF-10A cells were cultured in DMEM/F12 with 10% FBS, 0.1 µg/mL cholera toxin, 20 µg/mL epidermal growth factor, 0.5 µg/mL hydrocortisone, 5 µg/mL insulin, and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL). FHC cells were maintained in DMEM/F12 medium with 10% FBS and the same antibiotics. All cells were maintained in a humidified incubator with 5% CO₂ at 37 °C. The cells were seeded at a density of 1 × 10⁴ cells/well in 96-well plates and incubated at 37 °C for 24 h. The cells were treated with vehicle (medium with 0.1% DMSO) or various sample concentrations for 24 h. Staurosporine (a well-known apoptosis inducer) was used as a positive control to validate the cytotoxicity assays [37]. Cytotoxicity was evaluated using the MTT assay [38]. Three independent repeated evaluations were conducted for each compound and cell line (n = 3 – 4). The IC₅₀ values and 95% confidence intervals were estimated using GraphPad Prism 6.01 (GraphPad Software).

Contributorsʼ Statement

H. Ko: Methodology, Formal analysis, Data Curation, Visualization, Writing–original draft and review & editing; K. J. Lee: Methodology, Formal analysis, Data Curation, Visualization, Writing–original draft; K. Song: Methodology, Investigation. I. J. Ha: Funding acquisition, Writing–review & editing. Y. S. Kim: Conceptualization, Supervision, Funding acquisition, Writing–review & editing.

Conflict of Interest

The authors declare that they have no conflict of interest.

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• 31 Berthod A, Faure K. Revisiting resolution in hydrodynamic countercurrent chromatography: Tubing bore effect. J Chromatogr A 2015; 1390: 71-77
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• 34 Koji S, Sakae A, Ogihara Y. New derivatives of saikosaponins. Chem Pharm Bull 1985; 33: 3349-3355
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• 35 Nose M, Amagaya S, Takeda T, Ogihar Y. New derivatives of saikosaponin c. Chem Pharm Bull 1989; 37: 1293-1296
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• 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
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Correspondence

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

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  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

• Supplementary Material