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Microbiology and Biotechnology Letters

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Microbial Biotechnology (MB)  |  Whole Cell Biocatalysis and Bioprocess Engineering

Microbiol. Biotechnol. Lett. 2022; 50(2): 218-227

https://doi.org/10.48022/mbl.2203.03002

Received: March 10, 2022; Revised: April 13, 2022; Accepted: April 14, 2022

Synthesis of α-cichoriin Using Deinococcus geothermalis Amylosucrase and Its Antiproliferative Effect

Keumok Moon1, Hyun Su Park2, Areum Lee3, Jugyeong Min3, Yunjung Park3, and Jaeho Cha1,3*

1Microbiological Resource Research Institute, Pusan National University, Busan 46241, Republic of Korea
2Manufacturing Divisional Group, Celltrion, Inc., Incheon 22014, Republic of Korea
3Department of Microbiology, Pusan National University, Busan 46241, Republic of Korea

Correspondence to :
Jaeho Cha,       jhcha@pusan.ac.kr

Glycosylation of aesculetin was performed using amylosucrase from the hyperthermophilic bacterium Deinococcus geothermalis DSM 11300 to improve the solubility and biological activity of aesculetin. A newly synthesized aesculetin glycoside was identified as α-cichoriin (aesculetin 7-α-D-glucoside) by nuclear magnetic resonance analysis. The solubility of α-cichoriin was 11 times higher than that of aesculetin because of the attached glucose moiety. Aesculetin and α-cichoriin had no significant effect on the proliferation of normal cells, such as RAW 264.7, but they showed a cell proliferation inhibitory effect on B16F10 melanoma cells. Unlike treatment with aesculetin and α-cichoriin, aesculin (aesculetin 6-β-D-glucoside) showed no antiproliferative activity in B16F10 cells. Based on the molecular structures of aesculin and α-cichoriin, the position where glucose binds to aesculetin and the anomeric configuration between glucose and aesculetin are thought to be important for exerting an antiproliferative effect on the B16F10 cell line. Based on these results, we propose that α-cichoriin, the α-glycosylated form of aesculetin, may serve as a model for developing phytochemical analogs with therapeutic potential for the treatment of diseases associated with tumor cell proliferation without cytotoxicity to normal cells.

Keywords: Aesculetin, α-cichoriin, amylosucrase, transglycosylation, antiproliferative activity

Graphical Abstract


Aesculetin is a coumarin derivative that is abundantly present in the roots, leaves, and bark of plants, especially in the families Umbelliferae and Rutaceae. Coumarin is currently reported to have various pharmacological and biochemical properties, including antioxidant, antiinflammatory, antiallergic, platelet aggregation, protein kinase inhibition, apoptotic, antiviral, anti-differentiation, and antimutagenic effects [1-3]. Aesculetin is 6,7-dihydrocoumarin used for anti-inflammatory and allergy treatment in folk medicine and is also known as a secondary metabolite of plants [4, 5]. This component has various pleiotropic mechanisms [6] and has been shown to inhibit the growth of vascular smooth muscle cells through mitogen signaling mediated by Ras protein and kinase inhibition [7].

Among the biological effects and molecular mechanisms of aesculetin, antioxidant activity plays a crucial role in decreasing the levels of reactive oxygen species (ROS) and is potentially related to its antiproliferative, anti-inflammatory, antiphospholipid syndrome, and other pharmacological activities. Aesculetin and its derivative aesculin (aesculetin 6-β-D-glucoside) have been specifically identified for their antiproliferative properties in cancer cells [8-10] and are potential anticancer agents [11]. Cichoriin (aesculetin 7-glucoside), another aesculetin derivative, is a natural product found in Koelpinia linearis and Lactuca intricate and known as potential therapeutical agent against drug-induced benign breast tumor in rats [12].

Although aesculetin and aesculin have been reported to exhibit a variety of biological activities, they have limited pharmaceutical use because of their low solubility in water and ethanol, extensive phase II metabolism, and rapid elimination from the human body [13-15]. Aesculetin derivatives with substituted alkyl, aryl, or phenyl groups have been reported to overcome this limitation [16-18]. However, the drawbacks of the chemical methods of synthesis of these derivatives include compromised product purity, difficulty in preparation, and environmental pollution.

The addition of a sugar moiety to a compound by enzymatic glycosylation can alleviate these disadvantages, making enzymes powerful tools [19, 20]. In recent years, significant progress has been achieved in the enzymatic glycosylation of natural and artificial compounds, including flavone and anthraquinone [21-28]. The enzymatic glycosylation, which is mediated by bacterial glycosidases and glycosyltransferases, has been reported to have various merits such as increased water solubility, oxidative stability, and bioavailability and decreased cytotoxicity.

Amylosucrase (AS, E.C. 2.4.1.4) is a versatile sucrose hydrolase belonging to the GH family 13 [29]. AS can catalyze the synthesis of α-1,4-glucan using sucrose as its sole substrate. It can also mediate glycosylation of various small molecules and sucrose. Therefore, many studies have been conducted on the synthesis of industrially useful glycosides using AS derived from various microorganisms and sucrose. The recombinant Deinococcus geothermalis amylosucrase (DGAS) was used to glycosylate isoquercitrin and isoflavone [30, 31]. Rha et al. (2019) also reported that DGAS can glycosylate daidzein glucoside daidzin (daidzein-7-O-β-glucoside) to form daidzein diglucoside (daidzin-4′′-O-α-glucopyranoside) [32]. However, biocatalytic glycosylation of coumarin derivatives has not been reported before.

In this study, we analyzed whether glycosylation of aesculetin enhances its solubility and antiproliferative effect. Glycosylation of aesculetin was performed using DGAS. The molecular structure of the transglycosylation product of aesculetin was determined, and its water solubility and antiproliferative effects were examined. As aesculetin is known to be effective in treating several types of cancer, we examined whether glycosylated aesculetin, other than aesculin, is more effective in suppressing the proliferation of B16F10 melanoma cells.

Chemicals and reagents

Aesculetin, aesculin, sucrose, fructose, maltooligosaccharides, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (USA). Water and methanol [high-performance liquid chromatography (HPLC)-grade] were purchased from Honeywell Burdick & Jackson (Muskegon, MI) for purification of the transfer products. All other chemicals were of reagent grade and were purchased from Sigma-Aldrich. Recombinant DGAS was prepared in E. coli as previously described [33].

Animal cell culture

The murine macrophage cell line, RAW 264.7, and B16F10 melanoma cells were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated FBS (cDMEM; Thermo Fisher, USA), 100 U/ml penicillin, and 100 μg/ ml streptomycin (Gibco, USA). The cells were cultured at 37℃ under 5% CO2 in fully humidified air.

Synthesis of α-cichoriin using DGAS

To synthesize α-cichoriin, a substrate solution containing 5 mM aesculetin and 10 mM sucrose in 50 mM Tris-HCl buffer (pH 8) was pre-incubated at 35℃ for 10 min. After preincubation, DGAS (0.1 mg/ml) was added to the reaction mixture, and the enzymatic synthesis was carried out at 35℃ for 3 h [33]. The transfer reaction was stopped by heating the mixture in boiling water for 30 min and placing the mixture tube in ice. The reaction mixture was centrifuged at 3,000 ×g for 20 min to remove insoluble substances. The supernatant fraction was filtered using a 0.22 μm syringe filter (Satorius, Germany). The chemical structure of aesculetin, aesculin and cichoriin as well as the enzymatic reaction process is shown in Scheme 1.

Figure 6.Scheme 1. The chemical structure of aesculetin, aesculin, cichoriin and the enzymatic reaction process of DGAS with aescueltin and sucrose.

Purification of α-cichoriin

The transferred products were separated using a C18-T cartridge (100 mg/ml, StrataR) and a recycling preparative HPLC (Prep LC) equipped with a refractive index detector (JAI, Japan). A C18-T cartridge, which was previously activated using methanol and water, was used to absorb aesculetin glycoside from the transglycosylation reaction mixture and remove any sugars and salts. The transglycosylation reaction mixture was filtered using a 0.45 μm syringe filter (Sartorius) and added to the C18-T cartridge. After washing twice with water, the transferred products were eluted with methanol. The main transfer product in methanol was purified using a W-252/W-251 polymeric gel filtration column (2 × 50 cm, JAI) in a recycling preparative HPLC system. The mobile phase was deionized water at a flow rate of 3 ml/ min. Fractions corresponding to the detected peaks were collected and freeze-dried. The purity of each sample was confirmed using thin-layer chromatography (TLC).

TLC analysis

The purified transfer products were spotted on Whatman K5F silica gel plates (Whatman, Maidstone, UK) and activated at 110℃ for 30 min. The plates were developed in a developing solution containing n-butanol/ethanol/ water (5:3:2, v/v/v) for the aesculetin glycoside. The developed TLC plates were dried completely at room temperature and visualized using a UV lamp in combination with a UV viewing box (Camag, Switzerland) at 366 nm. The transfer products were visualized by dipping in a solution containing 0.3% (w/v) N-(1-naphthyl)-ethylenediamine and 5% (v/v) H2SO4 in methanol, followed by heating at 110℃ for 10 min.

HPLC analysis

The transfer products or purified aesculetin glycoside were centrifuged at 3000 ×g for 20 min and filtered using a 0.22 μm syringe filter. HPLC analysis was performed using the Shiseido Nanospace SI-2 HPLC system (Shiseido, Japan) equipped with a UV detector (Shodex, Japan). Separations were performed by reverse-phase HPLC using a C18 (4.6 × 250 mm) column (Shodex) at a flow rate of 1.0 ml/min. The column temperature was 40℃, and the mobile phase consisted of 0.5% acetic acid in water (buffer A) and methanol (buffer B). Chromatography was conducted isocratically in a 22% buffer B. The injection volume was 30 μl. Aesculetin and aesculetin glycoside were observed at 340 nm, which corresponds to the maximum UV absorbance of aesculetin. Three injections were performed for each sample and the standard. The conversion yield was defined as the ratio of the amount of synthesized aesculetin or aesculetin glycosides to the initial amount of aesculetin added.

Nuclear magnetic resonance analysis

Approximately, 5.5 mg of each aesculetin and purified aesculetin glycoside were dissolved in 0.5 ml of pure deuterated methanol (CD3OD) and placed in 5 mm NMR tubes. 1H and 13C NMR spectra of aesculetin and purified aesculetin glycosides were obtained using the Varian Inova AS 400 MHz NMR spectrometer (Varian, Palo Alto, CA). The bond between aesculetin and glucose in aesculetin glycoside was confirmed by examining the heteronuclear multiple bond correlation (HMBC) spectrum. The sample was dissolved in DMSO-d6 at 24℃, with tetramethylsilane (TMS) as an internal standard. Chemical shifts were reported as s (singlet), d (doublet), t (triplet), m (multiplet), or br s (broad singlet). The coupling constants were reported in Hz. Chemical shifts were reported as parts per million (δ) relative to the solvent peak.

Solubility determination

Excess amounts of aesculetin, aesculin, and α-cichoriin were suspended in 1 ml distilled water in a microfuge tube at 25℃. A JAC-4020 ultrasonic cleaner (Kodo, Korea) was used to maximize the solubility of aesculetin glycosides. After sonication at room temperature for 1 h with intermittent pauses, the samples were centrifuged at 12,000 ×g for 20 min. The supernatant of each sample was filtered through a 0.45 μm membrane filter, and the concentration of the water-soluble compound in the supernatant was estimated by measuring its absorbance at 340 nm using the Gene Quant pro UV/Vis spectrophotometer (Amersham Biosciences, UK).

Stability of oxidative degradation

Aesculetin, aesculin, and α-cichoriin were dissolved in phosphate-buffered saline (PBS) of pH 7.4 and DMEM to a final concentration of 10 mM. The solutions were then incubated in a humidified incubator at 37℃ under 5% CO2, and an aliquot (300 μl) of the reaction mixture was extracted at different intervals up to 72 h, filtered, and analyzed using HPLC.

Cell Viability assay

RAW264.7 cells or B16F10 melanoma cells were maintained in DMEM supplemented with 10% FBS, and the cell viability was determined using an MTT-based colorimetric assay. The cells were seeded in 6-well or 24-well plates at a density of 1.5 × 104 cells/well and cultured for 18 h. The cells were treated with fresh DMEM containing aesculetin (10 μM), aesculin (10 μM), and α-cichoriin (10-3, 10-1, and 10 μM). After 24 h, the cells were washed with PBS twice, and 200 μl of MTT (0.5 mg/ml) was added to the medium and incubated for 4 h. The supernatant was removed, and the formazan crystals were dissolved in 200 μl of DMSO. The Wallac 1420 microplate reader was used to determine absorbance at 595 nm. All assays were conducted in triplicate for each sample.

Antiproliferative activity assay

The cell antiproliferative activity was determined using an MTT-based colorimetric assay. RAW264.7 cells or B16F10 melanoma cells were plated at a density of 4 × 103 cells/well into 6-well or 24-well plates to investigate the antiproliferative effects of aesculetin, aesculin, and α-cichoriin. Following overnight incubation, the cultures were fed with fresh medium containing 10 μM aesculetin or aesculin and various concentrations of α-cichoriin. After 72 h, the cells were washed with PBS twice, and 200 μl of MTT (0.5 mg/ml) was added to the medium and incubated for 4 h. The supernatant was removed, and the formazan crystals were dissolved in 200 μl of DMSO. Absorbance was read at 595 nm using the Wallac 1420 microplate reader. All assays were conducted in triplicate for each sample. To confirm the antiproliferative effects of α-cichoriin on B16F10 cells, the cells were observed at 100 × magnification using the Zeiss Axioskop 2 plus microscope (Jena, Germany).

Statistical analysis

All experiments were performed in triplicate. Data were analyzed using SigmaPlot software (version 12.5, Systat, USA) and expressed as the mean ± standard deviation. The data were analyzed by one-way analysis of variance, and the mean values were considered significantly different at p < 0.05, p < 0.005, and p < 0.001.

Synthesis of α-cichoriin by DGAS

Although aesculetin is a well-known antiproliferative and anti-inflammatory compound and has other pharmacological activities, its applications are limited owing to its low solubility and cytotoxicity. Because enzymatic glycosylation enhances the physicochemical properties of various phytochemicals, we tested whether glycosylation of aesculetin would increase the bioavailability of aseculetin without affecting its antiproliferative activity. DGAS successfully glycosylated aesculetin in the presence of sucrose as the donor molecule, and the reaction products were detected using TLC and Prep LC analyses (Scheme 1). On the TLC plate, a spot corresponding to aesculetin and two newly produced aesculetin glycosides appeared after dipping the TLC plate into a sulfuric acid solution (Fig. 1A, inset). Prep LC analysis of the reaction products showed that aesculetin and the two aesculetin glycosides were eluted at 147, 110, and 102 min, respectively (Fig. lA). The first eluted peak is a solvent peak. The two aesculetin glycosides were separated and purified using a W-251 and W-252 polymeric gel column through Prep LC, and the purified aesculetin glycosides were visualized under UV light (Fig. 1A, inset). This suggested that they originated from aesculetin, as aesculetin was the only chromophore in the reaction. These results suggest that DGAS was successfully used to produce aesculetin glycosides. The newly produced aesculetin glycosides were believed to contain glucosyl and maltosyl units attached to aesculetin based on the reaction mechanism of DGAS. The conversion yield of the major aesculetin glycoside, which is thought to be glucosyl-aesculetin, was 17.0% through enzymatic synthesis by DGAS, as estimated using HPLC (Fig. 1B).

Figure 1.TLC and HPLC analyses of the aesculetin transfer products synthesized using recombinant DGAS with aesculetin and sucrose as the acceptor and donor, respectively. (A) TLC analysis: lane M, glucose to maltotriose; lane 1, sucrose; lane 2, fructose; lane 3, aesculetin; lane 4, reaction products; lane 5, purified Glc-α-cichoriin; lane 6, purified α-cichoriin. In Prep LC, aesculetin, α-cichoriin, and Glc-α-cichoriin were eluted at 147 min, 110 min, and 102 min, respectively. (B) Reversed phase HPLC analysis of the aesculetin transfer products synthesized using DGAS.

Structural analysis of α-cichoriin

The purified major aesculetin glycoside was concentrated using a rotary vacuum evaporator and confirmed as a single peak in HPLC analysis. The molecular structure of the newly synthesized aesculetin glycoside was determined using NMR analysis. The 1H and 13C NMR spectra of the transfer products were compared to those of aesculetin. The anomeric proton signal at δH 5.5, with a coupling constant of 3.2 Hz, indicated the α-linkage between the glucose moiety and aglycone aesculetin in the 1H NMR spectrum (Fig. 2A). The 13C NMR spectrum showed carbon signals of aesculetin and glucose. Glucose was identified as an α-D-glucopyranose based on the carbon chemical shifts indicated in blue, which included an anomeric carbon (δC 100.8). In the HMBC spectrum, the anomeric proton signal (δH 5.5, H-1′) correlated with the olefin quaternary carbon signal (δC 150.3, C-7) (Fig. 2B), suggesting that the glycosidic bond was at C-7. From the combination of the above-described data, the aesculetin glycoside was identified as aesculetin 7-O-α-D-glucopyranoside (α-cichoriin).

Figure 2.1H-NMR, 13C-NMR (A), and HMBC (B) spectra of α-cichoriin. Red line indicates that the anomer proton (H-1’) of sugar moiety shows a cross peak with C-7 of coumarin molecule.

Effects of glucosylation on water solubility and chemical stability

The solubility of α-cichoriin in water was evaluated by comparing it with that of aesculetin and aesculin, which were determined to be 0.1 mg/ml and 2.59 mg/ml, respectively (Table 1). The water solubility of α-cichoriin was 1.12 mg/ml, which was approximately 11 times higher than that of natural aesculetin but approximately 2.3 times lower than that of aesculin. This implies that the attachment of a glucosyl residue to aesculetin enhances the water solubility of the original compound. The enhanced solubility observed in our study is consistent with the findings of other researchers. It has been reported that glycosylation of various phenolic compounds, such as epigallocatechin gallate, naringin, puerarin, quercetin, baicalein, and caffeic phenethyl ester, with amylases or amylosucrases increased the water solubility of the unglycosylated flavonoids by 14-200 times [34-37].

Table 1 . Water solubilities of aesculetin and its glucosides.

SampleSolubility in water (mg/ml)Relative solubility
Aesculetin0.101
Aesculin2.5925.9
α-cichoriin1.1211.2


Acero et al. [38] studied the chemical oxidation of aesculetin using single oxidants (ozone, UV radiation, and hydroxyl radicals). Aesculetin and its derivatives undergo rapid oxidative degradation upon exposure to radiation. It is known that transient protection of the oxidative group with a promoiety would stabilize the phenolic compounds against oxidative degradation. The stability of aesculetin, aesculin, and α-cichoriin was determined by their half-lives, t1/2 (h), in PBS buffered solution and a cell culture medium cDMEM. All three compounds were stable in PBS after 72 h of incubation. Aesculetin was somewhat stable in cDMEM, with a halflife of approximately 24 h. As anticipated, glucosylated compounds of aesculetin, aesculin and α-cichoriin, resulted in stable aesculetin derivatives in cDMEM, even after 72 h of incubation. The stability of phenolic compounds in biological fluids, such as plasma, is important for their absorption in the gut. It is speculated that dietary intake of these glycosylated compounds is effective in enhancing the bioavailability of aesculetin in the human body owing to its hydrophilic property in the intestinal fluid. It is known that the glycosides linked with various sugars in plant foods enhance the absorption of dietary phenolic compounds in the gut [39]. Pharmacokinetic studies, on the maximum plasma concentration achieved after dosing and the values for the area under the plasma concentration-time curve in serum, revealed that glycosylated purarin and hesperidin were absorbed more rapidly and efficiently than their aglycones [40, 41]. Therefore, the absorption of aesculetin and its derivatives in the gut depends on the solubility and stability of the compounds used for administration.

Effect of α-cichoriin on cytotoxicity in RAW 264.7 cells and B16F10 melanoma cells

The effect of α-cichoriin on cell viability was evaluated in RAW 264.7 cells and B16F10 melanoma cells. The cells were treated with aesculetin, aesculin, and various concentrations of α-cichoriin for 24 h. There was no significant difference (p < 0.05) in the cytotoxicity of the three compounds toward RAW 264.7, in cDMEM (Fig. 3). However, in B16F10 cells, aesculetin significantly diminished cell viability (p < 0.005) at a concentration of 10 μM, whereas α-cichoriin did not show cytotoxicity up to 10 μM. It is thought that it can be used as an alternative to aesculetin because α-chicorin synthesized via a transglycosylation reaction of aesculetin did not show cytotoxicity to normal or cancer cells.

Figure 3.Effect of α-cichoriin on cell viability of RAW 264.7 cells (A) and B16F10 melanoma cells (B). MTT assay was performed to determine cytotoxicity of aesculetin (10 μM), aesculin (10 μM) and α-cichoriin. Each cell line was treated with indicated amounts of compounds for 24 h. Data were derived from three independent experiments and expressed as mean ± SE. *p < 0.05, ** p < 0.01, and ***p < 0.005 indicate significant differences from the control group.

Antiproliferative effect of α-cichoriin on B16F10 melanoma cells

Clinical studies have reported the antitumor activity of coumarin in several cancer types [42-44]. Coumarin acts as a prodrug to create active metabolites that could be responsible for the observed effects [45]. Experimental analysis showed that coumarin exerted an antiproliferative effect in tumor cell lines. For example, the dihydroxycoumarin derivative aesculetin has shown efficacy in inflammatory and allergic diseases [4, 5]. The antiproliferative effect of aesculetin has been reported to be greater in tumor cells than in non-malignant cells [8-10]. To examine the effects of aesculetin and its aesculetin glucosides (i.e., aesculin and α-cichoriin) on the cell proliferation in RAW264.7 cells and B16F10 cells, we conducted an MTT assay to evaluate the viability. When RAW264.7 cells were treated with α-cichoriin, the cell viability was not affected as the control cells, while the cell viability was decreased by 25% when treated with aesculetin (Fig. 4A). However, the viability of B16F10 cells was 16.6% and 36.7% after treatment with aesculetin and α-cichoriin at 10 μM, respectively (Figs. 4B and 5). α-Cichoriin only inhibited the growth of B16F10 cells, whereas aesculetin affected the viability of both the cell lines. Interestingly, no antiproliferative activity was observed in cells treated with aesculin (Figs. 4 and 5). α-Cichoriin (20 μM) also inhibited the growth of SKMEL-2 cells, a human skin melanoma cell line, by 41.3% (data not shown). Based on the molecular structures of aesculin and α-cichoriin, the position where glucose binds to aesculetin and the anomeric configuration between glucose and aesculetin are thought to be important for exerting an antiproliferative effect on the B16F10 cell line. The effects of α- and β-arbutin on the activity of tyrosinase in mushroom and mouse melanomas were examined [46]. This suggests that manipulation of the configuration of the anomeric carbon in the sugar of polyphenol glycoside could change the functions of the glycoside. Based on these results, we suggest that α-cichoriin, a soluble aesculetin derivative, may provide a model for developing derivative analogs of various phytochemicals with therapeutic potential for the treatment of diseases associated with tumor cell proliferation without exerting cytotoxicity on normal cells.

Figure 4.Antiproliferative activity of α-cichoriin in RAW 264.7 cells (A) and B16F10 melanoma cells (B). MTT assay was performed to determine cytotoxicity of aesculetin (10 μM), aesculin (10 μM) and α-cichoriin. Each cell line was treated with indicated amounts of compounds for 72 h. Data were derived from three independent experiments and expressed as mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.005 indicate significant differences from the control group.
Figure 5.Photomicrograph of confluent cultures of B16F10 cells. Cells were inoculated into culture dishes and treated with each compound, and then the cells were grown to confluence in the medium as described in the text for 72 h.

In conclusion, this study provides evidence for the enhancement of water solubility and stability of aesculetin by glycosylation. We propose that glycosylated aesculetin increases the bioavailability of aesculetin by protecting it from chemical or enzymatic oxidation, thereby extending its half-life in the cell and exhibiting beneficial antiproliferative properties.

This work was supported by a 2-Year Research grant from the Pusan National University.

The authors have no financial conflicts of interest to declare.

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