Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 20, December 2022, pages 80-89

Pharmacological bioactivity of enzymatically bio-transformed ginsenosides

Figures

Figure 1.
Figure 1. LC-MS chromatograph of ginsenoside samples (a) GR0, (b) GR8, (c) GL0, and (d) GL8. Chromatographic separation was performed on an RP18 (2.1 mm ID × 100 mm, 1.7 μm). Peaks: 1, Re; 2, digoxin (as internal standard); 3, Rb1; 4, Rc; 5, Rb2; 6, Rd; 7, CK.
Figure 2.
Figure 2. Culture of A. niger for the production of β-glucosidase enzyme. (a) First activation of A. niger (50 μL) from stock solution, (b) second activation, and (c) spore inoculation in malt extract.
Figure 3.
Figure 3. SDS-PAGE analysis of protein extract from A. niger on 10% separating gel. Lane assignment: lane M, protein marker; lanes 1?8, different batches of proteins produced by A. niger.
Figure 4.
Figure 4. Effect of (a) temperature and (b) time at 37 °C on the enzymatic activity of β-glucosidase from A. niger.
Figure 5.
Figure 5. HCT-116 cell viability after treatment with GR0, GR8, GL0, and GL8 at concentrations of 100, 200, and 400 μg/mL for (a) 24 hours and (b) 48 hours. Cell survival was determined by MTT assay and was calculated as a ratio of the control. Results were statistically analyzed using Student’s t-test. Data are presented as mean ± SD from triplicate wells and three independent experiments.
Figure 6.
Figure 6. RAW 264.7 murine macrophage cell viability after treatment with GR0, GR8, GL0, and GL8 at concentrations of 100, 200, and 400 μg/mL for 24 hours. Cell survival was determined by MTT assay and was calculated as a ratio of the control. Data are presented as mean ± SD from triplicate wells and three independent experiments.
Figure 7.
Figure 7. Effect of RAW 264.7 murine macrophages on nitric oxide production after treatment with GR0, GR8, GL0, and GL8 at concentrations of 100, 200, and 400 μg/mL for 24 hours. Results were statistically analyzed using Student’s t-test. Data are presented as mean ± SD from triplicate wells and three independent experiments.

Tables

Table 1. Eluent gradient used for LC-MS analysis
 
Time (min)Eluent A (%), 0.1% F.A. in H2OEluent B (%), 0.1% F.A. in Acetonitrile
0.08416
2.08416
5.07822
15.07525
20.06832
40.06733
55.04060
57.00100
60.00100
62.08416
65.08416

 

Table 2. Summary of ginseng powder samples by methanol extraction
 
SamplesInitial weight (g)After methanol extraction (g)After freeze drying (g)Extraction yield (%)
Values presented in the table are averages of separately conducted experiments.
Ginseng roots150121.770.444.52
Ginseng leaves and stems150130.2356.6335.26

 

Table 3. Summary of enzyme production from A. niger
 
Preparation stepTotal protein volume (mL)Protein concentration (mg/mL)Activity total (U)Specific Activity (U/mg)
Values presented in the table are averages of separately conducted experiments.
Culture filtrate138.27–––
Ammonium sulfate precipitation3.441.61711.7278.13

 

Table 4. General data for target ginsenosides
 
AnalyteRetention Time (min)FormulaExact MassQuantitative MS2 Fragment
Ginsenoside Rb123.9C54H92O231,153.6011945.5416
Ginsenoside Rb225.6C53H90O221,123.5905945.5436
Ginsenoside Rc24.9C53H90O221,123.5905945.5436
Ginsenoside Rd28.1C48H82O18991.5483783.4888
Compound K51.2C36H62O8667.4426459.3848
Digoxin (as IS)16.9C41H64O14825.4278649.3067

 

Table 5. Content of the ginsenosides
 
Sample GRO (μg/mL)GR0GR8GL0GL8
Values are presented as mean ± SD. ND, not detectable. Trace, below linear range of calibration curve. *Ginsenoside Rc was calculated as Rb2 equivalents.
Ginsenoside Rd11.38±0.430.91±0.0517.84±0.45Trace
Ginsenoside Rb125.55±0.42ND3.84±0.53ND
Ginsenoside Rb214.31± 0.29Trace9.69±0.72ND
Compound KTrace26.35±1.28Trace26.70±1.24
Ginsenoside Rc* (µg Rb2 eq/mL)19.69±0.26Trace9.31±0.48ND