3.1 Ingredient changes during Astragalus fermentation
3.1.1 Content determination
After seven days of Bacillus subtilis fermentation, the content of total polysaccharides was decreased and the content of total flavonoids and total saponin were increased by using a colorimetric method. The contents of ononin, formononetin, astragaloside IV, and astragaloside Ⅱ were significantly increased and the contents of calycosin glycoside, calycosin, and astragaloside Ⅰ were decreased (Table 1). Overall, the global content of astragaloside was increased, corresponding to the total saponin content.
Table 1 Content determination of fermented Astragalus (μg/mL)
|
Pre-fermentation
|
Post-fermentation
|
Total flavonoids
|
1.2381
|
1.9533
|
Total polysaccharide
|
23.7273
|
16.9669
|
Total saponin
|
2.8825
|
3.1263
|
Calycosin glycoside
|
0.3598
|
0.2392
|
Calycosin
|
0.0831
|
0.0596
|
Ononin
|
0.1066
|
0.1274
|
Formononetin
|
0.0107
|
0.0222
|
Astragaloside IV
|
5.0325
|
7.859
|
Astragaloside I
|
6.4309
|
0.5254
|
Astragaloside Ⅱ
|
1.5029
|
2.4253
|
3.1.2 UHPLC-TOF-MS ingredient determination
The ingredient alteration before and after fermentation was determined by the mass spectrometry approach. After Astragalus fermentation, many macromolecules such as glycosides were catabolized to small molecules such as aglycones. Moreover, numerous new ingredients appeared such as lariciresinol. Detailed data were shown in Table 2.
The m/z 283 in all samples had [M-H]- ions less than 5 ppm at 14.37m in. In the ESI-MS/MS spectrum, the [M-H]- ions at m/z 283 and [M-H-CH3]- ions at m/z 268 were also produced. Compared with the reference standard, m/z 283 was positive. At 10.83m in, C23H23O12 (error < 5 ppm) is in the negative ion mode. In the ESI-MS/MS spectrum, [M-H-Glc-HCOOH]- ions at m/z 283 and [M-H-Glc-HCOOH-CH3]- ions at m/z 268 were also generated. M/z 491=m/z 445[m-H-Glc]+m/z 46[HCOOH] was inferred as calycosin-7-glucoside+HCOOH.
Table 2 MS analysis of Astragalus fermentation
Sample
|
tR
|
Formula [M+H]+
|
Theoretical Mass m/z
|
Experimental Mass m/z
|
Error
|
MS2/MS2 fragment ions
|
Identification/
|
name
|
/min
|
(ppm)
|
Reactions
|
UA1
|
1.37
|
C5H5N5
|
134.047
|
134.0477
|
3.7
|
MS2[134]:132(8),117(12),107(23),92(38),90(18),65(100)
|
Adenine
|
UA2
|
1.65
|
C9H12N2O6
|
243.062
|
243.0628
|
-1.8
|
MS2[243]:164(8),150(8),122(15),110(37),94(22),82(100),66(80),68(44)
|
Uridine
|
UA3
|
17.66
|
C16H14O4
|
269.082
|
269.0826
|
2.1
|
MS2[269]:253(46),225(100),91(61)
|
(-)-Medicarpin
|
UA4
|
21.88
|
C18H30O2
|
277.217
|
277.2175
|
2.1
|
MS2[277]:127(29),71(100),58(57)
|
Linolenic acid
|
UA5
|
22.25
|
C18H32O2
|
279.233
|
279.2332
|
-0.2
|
MS2[279]:279(100),96(98)
|
Isolinolic acid
|
UA6
|
14.37
|
C16H12O5
|
283.061
|
283.0612
|
-0.5
|
MS2[283]:268(26),267(22),239(83),211(100),240(13),251(8),195(46),148(45)
|
Calycosin
|
UA7
|
17.94
|
C17H16O5
|
299.092
|
299.0943
|
-0.2
|
MS2[299]:269(44),254(17),241(100),213(38),185(22),199(14),171(18),133(12)
|
3-Hydroxy-9,10-Dimethoxy-pterocarpan
|
UA8
|
18.13
|
C17H18O5
|
301.108
|
301.1102
|
2.1
|
MS2[301]:271(25),256(39),253(21),149(100),201(22),135(54),121(58)
|
Isomucronulatol
|
UA9
|
14.82
|
C17H14O6
|
313.072
|
313.0732
|
0.1
|
MS2[313]:283(24),255(100),227(79),211(42),199(53),183(32),171(35),143(9)
|
1,7-Dihydroxy-3,9-dimethoxy pterocarpene
|
UA10
|
11.88
|
C22H24O10
|
447.130
|
447.1324
|
-1
|
MS2[447]:329(7),285(16),270(100),269(14),255(8)
|
Licoagroside D
|
UA11
|
9.05
|
C29H38O16
|
641.209
|
641.2120
|
0.2
|
MS2[641]:479(7),317(100),302(11),285(18),195(16),121(24)
|
5'-Hydroxy-isomucronulatol-2',5'-di-O-glucoside
|
UA12
|
17.85
|
C41H68O14
|
783.454
|
783.4572
|
-4.9
|
MS2[783]:783(100),489(16),161(18),159(9),101(17)
|
Astragaloside IV
|
UA13
|
17.79
|
C41H68O14.HCOOH
|
829.459
|
829.4585
|
-4.2
|
MS2[829]:829(100),783(47),651(7),179(22),161(17),119(22),89(25)
|
Astragaloside IV+HCOOH
|
Bsl
|
14.37
|
C16H12O5
|
283.061
|
283.0600
|
0
|
MS2[283]:239(53),211(100),148(20),195(23),267(13)
|
Adenine
|
Bs2
|
17.94
|
C17H16O5
|
299.092
|
299.0916
|
0
|
MS2[299]:269(49),241(100),254(12),213(35),185(21),171(19),133(12)
|
Calycosin
|
Bs3
|
11.89
|
C22H24O10
|
447.130
|
447.1282
|
0.7
|
MS2[447]:285(10),270(100),269(10),74(8)
|
3-Hydroxy-9,10-Dimethoxypterocarpan
|
Bs4
|
9.95
|
C22H22O11
|
461.109
|
461.1080
|
0.7
|
MS2[461]:299(26),284(100),255(18),240(7),283(12)
|
Isomucronulatol
|
Bs5
|
9.95
|
C22H22O11
|
461.109
|
461.1080
|
3.5
|
MS2[461]:299(24),284(100),255(13),240(7),135(5)
|
1,7-Dihydroxy-3,9-dimethoxy-pterocarpene
|
Bs6
|
13.97
|
C23H28O10
|
463.161
|
463.1594
|
4.5
|
MS2[463]:301(100),286(47),271(76),135(53),179(21),164(30),149(23),121(71)
|
(+)-Lariciresinol
|
Bs7
|
13.97
|
C23H28O10
|
463.161
|
463.1594
|
4.5
|
MS2[463]:301(100),286(47),271(76),135(53),179(21),164(30),149(23),121(71)
|
Licoagroside D
|
Bs8
|
13.95
|
C23H28O10
|
463.161
|
463.1594
|
4.5
|
MS2[463]:301(100),286(47),271 (76),135(53),179(21),164(30),149(23),121(71)
|
Rhamnolidin-3-O-beta-D-glucoside
|
Bs9
|
13.95
|
C23H28O10
|
463.161
|
463.1594
|
4.5
|
MS2[463]:301(100),286(47),271(76),135(53),179(21),164(30),149(23),121(71)
|
9,10-Dimelhoxy-pterocarpane-3-O-β-D-glucoside
|
Bs10
|
10.83
|
C22H22O10.HCOOH
|
491.119
|
491.1176
|
4.5
|
MS2[491]:283(63),268(100),267(3),211(3),239(5)
|
Isomucronulatol-7-O-g lucoside
|
Bs11
|
12.53
|
C29H38O15
|
625.214
|
625.2131
|
-0.8
|
MS2[491]:625(12),301(100),286(16),135(11),271(7),179(6),121(6)
|
Calycosin-7-o-glucoside+HCOOH
|
Bs12
|
17.79
|
C41H68O14.HCOOH
|
829.459
|
829.4565
|
-1.6
|
MS2[829]:829(100),783(51),651(9),400(6),489(13),179(27),161(32),89(37),621(8)
|
Isomucronulatol-7,2'-di-O-glucoside
|
Bs13
|
18.38
|
C43H70O15.HCOOH
|
871.470
|
871.4642
|
4.3
|
MS2[871]:871(100),825(55),765(34),783(14),603(6),179(17),161(12),59(45),89(13)
|
5'- Hydroxy-isomucronulatol-2',5'-di-O-glucoside
|
Bs14
|
1928
|
C45H72O16.HCOOH
|
913.480
|
913.4745
|
3
|
MS2[913]:913(100),867(37),825(25),807(15),765(13),179(12),161(11),119(13),59(33)
|
Astragaloside IV
|
Bs15
|
18.34
|
C48H78O18
|
941.512
|
941.5067
|
3
|
MS2[941]:941(100),923(3),528(4)
|
Astragaloside IV+HCOOH
|
UA: unfermented Astragalus; Bs: fermented Astragalus by Bacillus subtilis.
3.2 The weight and food intake changes in all groups
During the experiment, the mice in the control group presented a good mental state, glossy fur, and normal stool. The mice in the hyperuricemia group were and exhibited sparse fur. The mice in all fermented Astragalus groups presented significantly better general condition than in the hyperuricemia group. The mice in the fermented Astragalus (0.5 g/kg) group worked best among all groups. The mice in the benzbromarone and unfermented Astragalus groups exhibited dull fur, but to a lesser degree compared with the hyperuricemia group. The situation of the Bacillus subtilis group is similar to the hyperuricemia group. No mice died during the experimental procedure.
The weight of the mice increased with time (Fig. 1). After 6 weeks of hyperuricemia feedstuffs feeding, the weight of the mice was lower than that in the control group (P<0.05). After fermented Astragalus intervention, the mice exhibited a significant increase in body weight compared to the hyperuricemia group (P<0.05). Similar weight changes were observed in the benzbromarone and unfermented Astragalus groups (P<0.05). Bacillus subtilis intervention alone had no effect on body weight. Food intake per unit body weight was not significantly different among the groups (F=0.56, P=0.78).
3.3 Fermented Astragalus reduced the uric acid level in mice
After 6 weeks, the SUA level in the hyperuricemia group increased significantly (P<0.05) compared with the control group. Compared with the hyperuricemia group, the SUA level was decreased to varying degrees in all fermented Astragalus, benzbromarone, unfermented Astragalus groups (P<0.05). SUA in the fermented Astragalus (0.25, 0.5, and 1 g/kg) groups presented a dose-response relationship and was reduced more significantly than unfermented Astragalus.
The BUN and SCr levels increased significantly after hyperuricemia feedstuffs feeding compared to the control, which were decreased significantly by fermented Astragalus (0.5 and 1 g/kg) (P<0.05). Compared with the hyperuricemia group, the BUN and SCr levels were decreased in the benzbromarone group (P<0.05). No differences were found among the hyperuricemia group, unfermented Astragalus group and Bacillus subtilis group (Fig. 2B, C).
3.4 Fermented Astragalus relieved pathological changes of the kidney in hyperuricemic mice
Compared with the control group, the glomeruli were swollen, the glomerular basement membrane was thickened, and the mesangial area was widened in the hyperuricemia group. Furthermore, renal tubules were dilated, renal tubular epithelial cells were disorderly, and scattered infiltration of inflammatory cells was found in the local renal interstitial tissue of hyperuricemic mice. Compared with the hyperuricemia group, glomerular capillary swelling was improved, mesangial matrix was normal, and the capsular space stenosis was improved in the fermented Astragalus (0.25 g/kg) group. The structures of glomeruli and renal tubules were normal, with the epithelial cells arranging neatly in the fermented Astragalus (0.5 and 1 g/kg) group, close to that of the control group. In the benzbromarone group, the dilatation of the renal tubules was reduced, and the epithelial cells of the renal tubules were arranged neatly with occasional vacuolar degeneration. In the unfermented Astragalus group, glomerular structure was unclear, and the interstitial regions exhibited occasionally inflammatory cell infiltration. In the Bacillus subtilis group, renal tubular epithelial cells were disorganized, scattered infiltration of inflammatory cells was found (Fig. 3).
3.5 Fermented Astragalus lowered the liver XOD activity and improved liver function in mice
After 6 weeks, the liver XOD activity in the hyperuricemia group increased significantly compared with the control group (P<0.05). Compared with the hyperuricemia group, the liver XOD activity was decreased to varying degrees in the fermented and unfermented Astragalus groups (P<0.05) and fermented Astragalus intervention worked better.
At week 6, both serum ALT and AST levels were significantly elevated in the hyperuricemia group (P<0.05). Fermented Astragalus had certain protective effects on the liver in mice (P<0.05). However, benzbromarone poorly ameliorated liver function in hyperuricemic mice. Compared with the hyperuricemia group, the ALT and AST were decreased in the unfermented Astragalus group (P<0.05). Compared with the hyperuricemia group, the ALT level was decreased in the Bacillus subtilis group (P<0.05), while the AST level had no significant difference as compared with the hyperuricemia group (Fig. 4B).
3.6 Fermented Astragalus improved inflammatory levels in mice kidney
Hyperuricemic mice displayed increased levels of inflammatory factors IL-1β, IL-6 and TNF-α in the kidneys compared to the control (P<0.05), among which IL-1β and IL-6 were significantly attenuated by fermented Astragalus treatment (P<0.05). Compared with the hyperuricemia group, IL-1β level was decreased in the benzbromarone group (P<0.05). Unfermented Astragalus also exhibited a negative regulatory effect on the expressions of IL-1β and IL-6 in hyperuricemic mice (P<0.05). There was no significant difference in IL-1β, IL-6, and TNF-α levels between the hyperuricemia and Bacillus subtilis group (Table. 3).
TLR4, p38 MAPK, p-p38 MAPK, NF-κB, p-NF-κB, and NLRP3 protein levels were observed by immunoblot analysis, and results are expressed as the ratio of the optical density of corresponding protein (Fig. 5). Expression of TLR4 in hyperuricemic mice was increased, which was attenuated by fermented Astragalus treatment. In addition, TLR4 expression was reduced in the benzbromarone and unfermented Astragalus groups. Although the total p38 MAPK level was not increased, the relative phosphorylation level of p38 MAPK obviously increased in the hyperuricemia group compared to the control (P <0.05). Fermented Astragalus treatment significantly diminished p38 MAPK phosphorylation (P <0.05). Although benzbromarone and unfermented Astragalus decreased the levels of p38 MAPK phosphorylation, these did not reach the degree of fermented Astragalus. The hyperuricemia group showed a significant increase in NF-κB phosphorylation, while obviously decreased in the fermented Astragalus group compared with the hyperuricemia group (P<0.05). There was no significant difference in NLRP3 protein expression in each group.
Table 3 Pro-inflammatory cytokines (n=10) of hyperuricemia mice and treated for 6 weeks with fermented Astragalus.
Groups
|
IL-1β (pg/mg)
|
IL-6 (pg/mg)
|
TNF-α (pg/mg)
|
Control
|
163.85±52.86
|
29.81±7.78
|
165.58±22.45
|
Hyperuricemia
|
442.46±72.71*
|
132.89±29.74*
|
309.43±68.93*
|
Benzbromarone
|
200.33±53.36#
|
85.10±26.43*
|
282.61±62.29*
|
Fermented Astragalus
|
141.17±26.95#
|
36.67±18.52#
|
282.61±48.48*
|
Unfermented Astragalus
|
287.88±31.60*#
|
50.79±4.27*#
|
236.19±49.23
|
Bacillus subtilis
|
438.83±96.88*
|
96.00±20.88*
|
273.96±72.54*
|
Values are expressed as mean ± SD. * indicates that the difference is statistically significant compared with the control group (P<0.05); # indicates that the difference is statistically significant compared to the hyperuricemia group (P<0.05).