2.2. Metabolites of Sibirioside A: SM1–SM4
The negative quasi-molecular ion of SM1 at m/z 229.0153 ([M − H]−) was detected in feces and urine samples, and speculated as C9H10O5S. Its degrees of unsaturation were five, meaning there was only one unsaturated bond except for the phenyl group in the molecule. The MS2 spectrum showed fragment at m/z 147.0459 (speculated to be [C9H8O2 − H]−), and its MS3 fragment ion at m/z 103.0348 ([C9H8O2 − CO2 − H]−) indicated that C9H8O2 might be cinnamic acid. Cinnamic acid was the product of SM1 with 82 Da lost, which was considered to be H2SO3. It is well-known that the sulphated compounds could easily lose 80 Da radical group (SO3) instead of 82 Da, but the neutral molecule H2SO3 might be lost if there was active-H in the phenylpropionic acid structure. Then we could conjecture SM1 as phenylpropionic acid sulphate. The cinnamic acid first reacted with glutathione and then hydrolyzed into the cysteine adduct by peptidase, and the cysteine β-liase gave a thiol immediately after, and finally oxidized to be SM1.
SM2 detected in feces samples showed quasi-molecular ion peak at m/z 259.0293 ([M − H]−), indicating C10H12O6S as the predicted formula. The characteristic fragment ion peaks were shown as m/z 177.0540 ([M − H2SO3 − H]−) and m/z 133.0983 ([M − H2SO3 − CO2 − H]−). Its trends of neutral loss conformed with those of SM1, and all the fragment ion peaks were 30 Da (predicted to be CH2O) larger than the correspondent peaks of SM1, which suggested the substitution of methoxyl group. The methoxyl group was the best possibility to substitute at the aromatic ring owing to the absence of methyl (14 Da) or methoxyl (30 Da) loss in MS2 spectrum. Then it could be speculated that SM2 might be 4-methoxyphenylpropanoic acid sulphate in consideration of the substitutive resistance.
The different chromatographic peak of SM3 was observed in feces samples, and showed quasi-molecular ion peak at m/z 393.0755 ([M − H]−). The fragment ion at m/z 311.1019 ([M − H2SO3 − H]−) suggested SM3 to be a sulphate, and it could be dissociated to get m/z 163.0583 and m/z 147.0454. There was no fragment ion at m/z 103.03 rooted from m/z 147.0454, which indicated that the ion at m/z 147.0454 was not cinnamic acid. According to the elemental composition C9H8O2, this fragment was most likely to be β-oxobenzenepropanal. In accordance with the above analysis, SM3 might be β-hydroxybenzenepropanoic anhydride sulphate.
The molecular formula of SM4 detected in stomach samples was predicted to be C27H38O17 according to the quasi-molecular ion peak at m/z 633.2014 ([M − H]−). Its MS2 spectrum showed fragment ion peaks at m/z 485.1416 ([M − cinnamoyl − H]−) and m/z 323.0932 ([sucrose − H]−). Neutral loss of 148 Da (cinnamic acid) occurred from the parent ion to obtain m/z 485.1416, then neutral loss of 162 Da (glucosyl) occurred from m/z 485.1416 to get m/z 323.0932, which was the main fragment ion of sibirioside A predicted to be [sucrose − H]−. To sum up, SM4 was conjectured to be a sibirioside A glucose conjugation.
The metabolites and metabolic pathways of sibirioside A in rats are shown in Figure 3
2.3. Metabolites of Angoroside C: AM1–AM25
AM1 showed [M − H]− ion peak at m/z 233.0119 ([M − H]−) in the negative MS1 spectrum, which was predicted as C8H10O6S. The fragment ion peaks at m/z 153.0583 ([M − SO3 − H]−) and m/z 123.0492 ([M − SO3 − CH2O − H]−) were observed in negative MS2 spectrum, and m/z 153.0583 should be the phenylethanoid unit derived from angoroside C. Therefore, AM1 was speculated to be 4-(2-hydroxyethyl)-1,2-benzenediol sulphate.
The formula of AM2 was determined to be C9H8O6S based on its quasi-molecular ion peak at m/z 242.9965 ([M − H]−). The characteristic fragment ion peaks were observed as m/z 163.0415 ([M − SO3 − H]−) and m/z 119.0532 ([M − SO3 − CO2 − H]−). Therefore, the ion at m/z 163.0415 was thought to be the demethoxylation product of ferulic acid, which was the hydrolysate of angoroside C. And AM2 was thus determined as p-coumaric acid sulphate. It was easy to confirm that AM3 was 4-hydroxybenzenepropanoic acid sulphate, the hydrogenation product of AM2 based on the quasi-molecular ion peak at m/z 245.0109 ([M − H]−) and its MS2 fragment ion peaks at m/z 165.0574 ([M − SO3 − H]−) and m/z 121.0680 ([M − SO3 − CO2 − H]−).
For AM4, AM5 and AM6, the exact formula C9H12O6S was calculated based on the quasi-molecular ion peak at m/z 247.03 ([M − H]−). The ion at m/z 167.07 ([M − SO3 − H]−) was detected in MS2 spectrum, which suggested that they were 3,4-hydroxyphenylpropanol sulphate. And there were three reaction sites in 3,4-hydroxyphenylpropanol for sulphating, so the three metabolites were considered as isomers.
The quasi-molecular ion peak of AM7 at m/z 259.0271 ([M − H]−) suggested the formula as C10H12O6S. And the fragment ion peaks at m/z 179.0726 ([M − SO3 − H]−), m/z 163.0385 ([M − SO3 − CH2 − H]−), m/z 147.0473 ([M − SO3 − CH4O − H]−, namely [cinnamic acid − H]−) and m/z 119.0507 ([M − SO3 − CH2 − CO2 − H]−) indicated the metabolite might be methoxybenzenepropanoic acid sulphate, which was a isomer of SM2.
AM8 and AM9 were speculated to be C9H10O7S owing to the quasi-molecular ion peak at m/z 261.01 ([M − H]−) in negative ion mode MS1 spectrum. The multistage fragment ion peaks at m/z 217.0175 ([M − CO2 − H]−), m/z 181.0522 ([M − SO3 − H]−) and m/z 137.0633 ([M − SO3 − CO2 − H]−) indicated that they were 3,4-dihydroxybenzenepropanoic acid-O-sulphate.
The quasi-molecular ion peak at m/z 275.02 ([M − H]−) observed in the negative MS1 spectrum indicated the formula C10H12O7S. Then, it was speculated that AM10 was 3-methoxyl-4-hydroxybenzenepropanoic acid sulphate and that AM11 was isomer of it owing to the fragment ion peaks at m/z 195.0665 ([M − SO3 − H]−), m/z 151.0799 ([M − SO3 − CO2 − H]−) and m/z 177.0777 ([M − SO3 − H2O − H]−) in the MSn spectrum.
The [M − H]− peaks of AM12, AM13 and AM14 were observed at m/z 329.09, suggesting the formula as C14H18O9. The parent ion was cleaved into two parts m/z 175.0282 ([glucuronic acid − H2O − H]−) and m/z 153.0568. And the ion at m/z 153.0568 was determined to be 3,4-dihydroxyphenylethanol similar to AM1, which has three reaction sites for gluconylation. The above data suggested AM12, AM13 and AM14 to be 4-(2-hydroxyethyl)-1,2-benzenediol glucuronide.
AM15, speculated to be C15H18O9, was detected in urine samples with quasi-molecular ion peak at m/z 341.0848 ([M − H]−). The MS2 fragment ion peaks at m/z 165.0520 ([M − glucuronic acid − H]−) and m/z 121.0628 ([M − glucuronic acid − CO2 − H]−) were just similar to AM3, and in combination with m/z 175.0244, AM15 was determined to be 4-hydroxybenzenepropanoic acid glucuronide. And the MS1 chromatographic peaks of AM16 and AM17 were extracted in urine sample at m/z 343.1017 ([M − H]−). Their fragment ion peaks at m/z 175.0265 and m/z 167.0713 indicated that they were 3,4-hydroxyphenylpropanol glucuronide, the reduzates of AM15.
The [M − H]− peak of AM18 at m/z 369.0794 (C16H18O10) was detected in urine samples. The MS2 fragment ion at m/z 175.0169 suggested the existence of glucuronic acid, and m/z 193.0508 (C9H10O4) was consistent with the phenylpropanoid unit of angoroside C. Therefore, AM18 might be 3-methoxyl-4-hydroxycinnamic acid glucuronide. And the quasi-molecular ion peak at m/z 371.0988 ([M − H]−) and the MS2 fragment ion peaks at m/z 175.0265 and m/z 195.0658 of AM19 suggested that it could be the hydrogenation product of AM18, namely 3-methoxyl-4-hydroxybenzenepropanoic acid glucuronide.
The ion peaks at m/z 637.21 were the quasi-molecular ions of AM20 and AM21, which was the characteristic fragment ion of angoroside C. And the MS2 fragment ion peaks at m/z 461.1636, m/z 311.1151, m/z 193.0548, m/z 167.1269 and m/z 137.0759 were also in conformity with angoroside C. Then they might be the isomers of deglycosylated angoroside C. For AM22 and AM23, the quasi-molecular ion peaks at m/z 959.30 and main MS2 fragment ion peak at m/z 783.26 ([angoroside C − H]−) were observed, which indicated AM22 and AM23 to be isomers of angoroside C glucuronide.
The formula of AM24 detected in feces samples was predicted to be C20H30O12 based on the quasi-molecular ion peak at m/z 461.1701. The fragment ion peaks at m/z 443.1711 and m/z 329.1180 could also be observed in the MS2 spectrum of angoroside C. So it could be speculated that AM24 was the hydrolysate of deferuloyl-angoroside C, namely 2-(3-hydroxy-4-methoxyphenyl)ethyl 6-O-arabinopyranosyl glucopyranoside.
The negative quasi-molecular ion of AM25 at m/z 247.0266 ([M − H]−) was detected in feces and plasma samples, and speculated as C9H12O6S. The fragment ion peaks at m/z 167.0750 ([M − SO3 − H]−), m/z 137.0627 ([M − SO3 − CH2O − H]−) and m/z 121.0264 ([M − SO3 − CH2O − CH4 − H]−) suggested that AM25 was 4-(2-hydroxyethyl)-1,2-benzenediol sulphate.
The metabolites and metabolic pathways of angoroside C in rats are shown in Figure 4
2.5. Biological Activity Prediction of the Metabolites of Sibirioside A and Angoroside C
The bio-activities of the metabolites of sibirioside A and angoroside C were predicted through “PharmMapper Server”, which based on a massive pharmacophore database containing relatively well-developed target information over 7000 pharmacophore models recorded in TargetBank, BindingDB, DrugBank, and other drug target databases [30
]. It automatically matches the pending molecules with all targets in the database and then calculates the fit degree to list the best scoring targets. The first 50 results on human targets were chosen and then the disease-related outcomes for every metabolite were picked out. These are shown in Table S1
. In total, 29 targets were picked out, of which RASH, INSR, KPYR, ANGI, BTK, FA7, NDKB, G6PI and CAH2 were identified over 10 times. Their functions include anti-cancer, anti-diabetes, anti-anemia, neuroprotection, anti-leukemia, anti-coagulopathy, and osteogenic activity. The most common target is RASH, which has GTPase activity and is considered to be related to inhibition of growth and metastasis of tumors. Defects in INSR, an insulin binding receptor with tyrosine kinase activity, could cause insulin resistance, leprechaunism, noninsulin-dependent diabetes mellitus, and familial hyperinsulinemic hypoglycemia. This target was identified as a potential target of 17 metabolites of sibirioside A and angoroside C. Defects in INSR cause insulin resistance, leprechaunism, noninsulin-dependent diabetes mellitus, and familial hyperinsulinemic hypoglycemia. Thus, SM1
might be used to cure diabetes, which was one of the main pharmacological activities of SR in traditional Chinese medicine. In addition, SM3
may be available to target chronic leukemia; AM2
may have osteogenic actions. The predicted outcomes were in good agreement with the actual activities reported in the literatures [11
The metabolites of sibirioside A and angoroside C have a variety of potential pharmacological actions, which require further research.