This study was performed (1) to investigate the combined effect of germination and high hydrostatic pressure (HHP) treatment on the conversion of phenolics, protein and soyasaponin in black soybean, (2) to investigate changes of physiological activities of phenolics, protein and soyasaponin rich extracts in conversion of functional compound, (3) to identify maker ingredient according to isolation of bioactive components in functional compound rich extracts, and (4) to clarify the cause converted maker compound of soybean with germination and HHP treatment. Black soybean was germinated at 25°C for two- or four-days, and subjected to HHP at 0.1, 50, 100, or 150 MPa for 24 h.
1. Conversion and physiological characteristics of phenolic compound in black soybean after germination and high hydrostatic pressure treatment were investigated. Total free polyphenol, flavonoid, and phenolic acid contents in raw black soybean increased from 1.03 mg GAE/g, 0.29 mg CE/g, and 315.67 μg/g to 1.44 mg GAE/g, 0.64 mg CE/g, and 511.01 μg/g, respectively, by 4 days after germination. Changes to phenolic acid compositions can be divided into four groups, and the germination process can convert compounds to phenolic acid via anabolism and catabolism. The highest total free isoflavone content in germinated black soybean of 3,724.40 μg/g was observed at 4 days. Bound polyphenol, flavonoid, phenolic acid, and isoflavone contents decreased as the germination period increased. The highest total polyphenol content of 3.90 mg GAE/g, flavonoid content of 0.79 mg CE/g, phenolic acid content of 938.34 μg/g, and isoflavone content of 2,562.77 μg/g were observed after germination for four days and HHP treatment at 100 MPa for 24 h. In terms of isoflavone composition, the malonyl glycoside, β-glycoside, and acetylglycoside contents decreased, while the aglycone content increased as increasing germination periods and applied pressure. The highest proliferative effect of 147.43% is observed at four days germination and HHP treatment at 100 MPa for 24h. In the results of correlation analysis, correlation coefficients between some phenolic compound (gallic acid, homogentisic acid, gentisic acid, p-coumaric acid, ferulic acid, naringin, cinnamic acid, naringenin, biochanin, daidzein and genistein), and estrogenic activity of phenolic compound extract has the high positive correlation. The highest estrogenic activity in single phenolic compound of high positive correlation was observed at genistein of 127.54% treated by low concentration of 0.5 μg/mL. Therefore, maker compound of phenolic compound extracts in black soybean treated by germination and HHP was selected to genistein. Germinated black soybean had a estrogenic activity and that HHP treatment improved this activity, enhanced genistein and conversion of isoflavone may positively impact estrogenic activities.
2. Conversion, physiological characteristics and identification of bioactive peptide in black soybean after germination and high hydrostatic pressure treatment were investigated. The highest soluble protein content was in soybeans subjected to HHP (100 MPa) after germination (4 days). Germination and HHP treatment caused the degradation of high molecular weight proteins; we detected the highest content of <3 and <10 kDa peptides and free amino acids in extracts from pre-germinated soybeans that received HHP (150 MPa). The ABTS radical scavenging activity of soluble protein extracted by germinated black soybean (4 days) increase linearly between 1.21 and 3.58 mg equivalent ascorbic acid/g, and 0.69 and 2.02 mg equicalent Trolox/g with increasing applied pressure until 150 MPa. Pre-germinated (4 days) soybean extracts treated with HHP (150 MPa) significantly inhibited LPS-induced expression of inflammatory markers, nitric oxide of 25.01%, TNF-α of 76.78%, IL-1β of 58.99%, and IL-6 of 84.48% by RAW 264.7 macrophages. Therefore, anti-inflammatory active peptide was isolated and identified from protein extracts of high hydrostatic pressure treated and germinated black soybean (HGBP extracts) of pre-germinated (4 days) soybean treated with HHP (150 MPa). The HGBP extracts was first passed through the 30 kDa MWCO membranes and then 10 kDa, 3 kDa MWCO membranes, and the strong inhibitory effect was observed at PM 2 fraction (3~10 kDa fraction). The fraction PM1 and PM2 obtained from ultrafiltration with prominent anti-inflammatory activity was further separated in to six fraction (PM-S1 ~ PM-S6) using gel permeation column chromatography and MPLC, the highest NO inhibitory effects were dose-dependent observed in S4 fraction. The active fraction HGB-PM-S4 obtained from gel permeation chromatography was futher purified using 1st semi-preparative HPLC using C18 column chromatography, and the highest NO inhibitory effects were dose-dependent observed in S4-P1 fraction. The active fraction HGB-PM-S4-P1 obtained from 1st semi-preparative HPLC was father purified using 2nd semi-preparative HPLC using C18 column chromatography, and the highest NO inhibitory effects were dose-dependent observed in PM-S4-P1-p3 fraction. After isolation and purification of active peptide, anti-inflammatory activity on raw 264.7 was measured. The isolated and puri?ed anti-inflammatory compound was analyzed by UV, LC-MS, 1H and 13C NMR, COSY, HMBC and HSQC. The isolated compound was identified as tripeptide (Arg-Asp-Gly).
3. Conversion, physiological characteristics and identification of bioactive soyasaponin in black soybean after germination and high hydrostatic pressure treatment were investigated. The highest crude soyasaponin contents of 57.85 mg/g was in black soybean subjected to HHP (100 MPa for 24 h) after germination (4 days). Soyasaponin Bb was detected as major soyasaponin among B group soyasaponin of raw black soybean, soyasaponin Bb contents of raw black soybean and germinated black soybean increase from 94.55 and 111.98 mg/100g to 87.46 and 128.95 mg/100g after HHP at 100 MPa for 24 h. The highest anti-proliferative effect on all human cancer cell is observed at four days germination and HHP treatment at 150 MPa for 24 h, particularly, in human gastric cancer cell (AGS) cell line, crude soyasaponin extracts derived from germinated black soybean with HHP (150 MPa) at different concentration of 50~400 μg/mL showed stronger anticancer effect of 78.17~23.94% than other human cancer cell line. The lipid accumulation rate (% positive control) during 3T3-L1 adipocyte differentiation in germinated black soybean treated by HHP are in range of 93.25 ~ 65.95% at concertation of 400 μg/mL. The highest anti-obesity effect is observed at four days germination and HHP treatment at 150 MPa for 24 h. Also, in case of anti-inflammatory activity, germinated (4 days) soybean extracts treated with HHP (150 MPa) significantly inhibited LPS-induced expression of inflammatory markers, TNF-α of 51.90%, IL-1β of 30.93%, and IL-6 of 64.02% by RAW 264.7 macrophages. Therefore, anti-inflammatory active soyasaponin was isolated and identified from crude soyasaponin extracts of high hydrostatic pressure treated and germinated black soybean (HGBS extracts) of pre-germinated (4 days) soybean treated with HHP (150 MPa). The HGBS was separated to nine fractions (HGBS-C1~C9) and washing fraction (HGBS-C10) using 1st C18 column chromatography and MPLC, and The highest NO inhibitory effects were dose-dependent observed in C9 fraction. The fraction HGBS-C9 obtained from 1st MPLC with prominent anti-inflammatory activity was further separated to four fractions (HGBS-C9-c1~HGBS-C9-c4) and washing fraction (HGBS-C9-c5) using 2nd C18 column chromatography with MPLC, he highest NO inhibitory effects were dose-dependent observed in C9-c2 fraction. The HGBS-C9-c2 fraction was further purified to isolate the active compound with anti-inflammatory activity using semi-preparative HPLC, compared with unpurified fraction (C9-c2), the obtained two fractions (C9-c2-P5 and P6) more potent NO inhibitory activity, with NO concertation of 7.10 and 12.84 μM. The two-active fraction HGBS-C9-c2-P5 and P6 separated from 1st semi-preparative HPLC was further purified using 2nd semi-preparative HPLC C18 column chromatography, and anti-inflammatory active compound of HGBS were isolated to compound 1 (HGBS-C9-c2-P5-p2) and compound 2 (HGBS-C9-c2-P6-p3). After isolation and purification of active soyasaponin, anti-inflammatory activity on RAW 264.7 was measured. The isolated and puri?ed anti-inflammatory compound was analyzed by UV, LC-MS, 1H and 13C NMR, COSY, HMBC and HSQC. The isolated compound was identified as soyasaponin Bb (compound 1) and soyasaponin Bc (compound 2)
4. Identification on factor affecting bioactive compound conversion of black soybean after germination and high hydrostatic pressure treatment were investigated. Genistein, tripeptide (RGD) and soysaponin Bc content increase linearly with increasing HHP treatment time (0, 6, 12, 18 and 24 h) in control. However, enzyme inactivation so affected the change of genistein, tripeptide (RGD) and soyasaponin Bc content with HHP treatment time that did not increase or slightly increase in genistein content. Therefore, the cause enhanced physiological activity and marker compound (genistein, tripeptide (RGD) and soysaponin Bc) of germinated black soybean with HHP treatment are postulated that biological factor and enzyme activation play a major factor.
In conclusion, these results showed that germinated black soybean had an estrogenic and anti-inflammatory activity and that high hydrostatic pressure (HHP) treatment improved this activity, enhanced maker compound (genistein, tripeptide and soyasaponin Bc) and conversion of isoflavone, protein, and soyasaponin may positively impact physiological activity. Also, factor affecting maker compound conversion is enzyme activation of biosynthesis by germination and HHP treatment. This study provides valuable information on the application of HHP in combination with pre-germination for improving the utilization of phenolic compound, protein, saponin rich ingredient extracted black soybean as an effective physiological agent.