Detection and Characterization of the Metabolites of Ciwujianoside B in Rats Based on UPLC-Fusion Lumos Orbitrap Mass Spectrometry

We previously conducted a systematic study on the metabolic process and products of hederasaponin B in rats. We hypothesized that the sugar chain structures play a key role in the metabolism of triterpenoid saponins. To verify this hypothesis, we conducted metabolic research on ciwujianoside B ascribed to the same sugar chains and a distinct aglycone and compared it with hederasaponin B. Specifically, we collected feces, urine, and plasma of rats after gavage with ciwujianoside B and identified 42 metabolites by UPLC-Fusion Lumos Orbitrap mass spectrometry. Finally, ciwujianoside B metabolism and hederasaponin B metabolism were compared, reaching the following conclusions: (i) more than 40 metabolites were identified in both, with the majority of metabolites identified in feces; (ii) the corresponding metabolic pathways in vivo were basically similar, including deglycosylation, acetylation, hydroxylation, glucuronidation, oxidation, and glycosylation; and (iii) deglycosylation was considered the main metabolic reaction, and its metabolites accounted for approximately 50% of all metabolites. Overall, this study provides a foundation for further research on the metabolism of triterpenoid saponins.


Introduction
Acanthopanax senticosus is a small woody shrub that belongs to the Araliaceae family.It is mainly distributed in the northeastern region of China, Korea, and Japan.Te rhizome and root of A. senticosus, also known as "Siberian ginseng," have been widely used as a tonic and antifatigue agent for the treatment and prevention of various diseases including cancer, diabetes, ischemic stroke, rheumatism, depression, and Parkinson's disease [1][2][3].However, from the perspective of resource utilization, the resources of A. senticosus leaves have gradually received attention from the medical and pharmaceutical felds.Pharmacological studies have indicated that A. senticosus leaves have multiple bioactivities, e.g., glycosidase inhibition, antiaging, antioxidant, and antitumor efects [4][5][6].It has been confrmed that the presence of saponins in the roots, stems, and leaves of A. senticosus is responsible for these major efects [7].However, saponins generally have low bioavailability due to the large chemical polarity and poor oral absorption, and their metabolism has been poorly studied [8,9].Terefore, it is essential to elucidate the metabolic fate of triterpene saponins in A. senticosus leaves for further exploitation and utilization of its leaf resources.
We previously conducted a systematic study on the metabolic process and products of hederasaponin B, a triterpenoid saponin obtained from A. senticosus leaves [10].We proposed that the sugar chain structures play a key role in the metabolism of triterpenoid saponins.Ciwujianoside B is also a triterpenoid saponin isolated from A. senticosus leaves, which has been shown to be able to penetrate and work in the brain, enhance memory function, and confer radioprotective efects [11,12].Compared with hederasaponin B, ciwujianoside B is ascribed to the same sugar chains and a distinct aglycone.According to our hypothesis, the metabolism of both should have similar results and searchable rules.
Terefore, in this study, we profled the in vivo metabolic fate of ciwujianoside B in rats based on the proposed strategy and compared it with that of hederasaponin B. Specifcally, we established a UPLC-Fusion Lumos Orbitrap mass spectrometry method for the rapid identifcation of metabolites of ciwujianoside B in plasma, urine, and feces samples [13,14].Te metabolite identifcation results were obtained by using Compound Discoverer 3.0 software combined with manual screening [15,16].Subsequently, the possible metabolic pathways of ciwujianoside B were analyzed.Additionally, we compared the results of ciwujianoside B with those of hederasaponin B to summarize the metabolic laws of both.Tese possible laws could provide valuable reference to further elucidate the metabolism of other similar triterpenoid saponins.

Materials and Methods
2.1.Chemicals and Reagents.A. senticosus leaves were collected at Muling County (Heilongjiang Province, China).Mass spectrometry-grade methanol and acetonitrile were obtained from Termo Fisher (Geel, Belgium).HPLC-grade formic acid was obtained from Dikma (Lake Forest, USA).Purifed water was obtained from Watsons (China).Other reagents were purchased from local sources and of analytical grade.

Preparation of Ciwujianoside B. Te 3 kg of A. senticosus
leaves were crushed and extracted with 30 L of 70% ethanol under refux conditions for three hours.Te ethanolic solution was fltered after standing, and extraction procedure was repeated three times.Te fltrates were combined and then concentrated by a rotary evaporator.Te extract was separated by an AB-8 macroporous resin column (9 cm i.d.×100 cm) and eluted with water (2.0 BV), 30% ethanol (4.0 BV), 60% ethanol (4.0 BV), and 95% ethanol (4.0 BV).Subsequently, the fraction obtained with 60% ethanol were subjected to silica gel column chromatography (dichloromethane-methanol-water (10 : 1 : 0.1) ⟶ methanol) to obtain six fractions (A-F).Fraction B was subjected to reversed-phase silica gel chromatography (70% methanolwater ⟶ methanol) to obtain three fractions (B 1 -B 3 ).Ten, fraction B 3 was purifed on a SHIMADZU C18 column (20 × 250 mm, 5 μm) using preparative liquid chromatograph equipped with a refractive index detector.Te mobile phase was acetonitrile/water (4 : 6), and the fow rate was 5 mL/min.Te retention time of ciwujianoside B was 10.5-10.8min.Finally, the collected preparation solution was concentrated and freeze-dried to obtain purifed ciwujianoside B. Its purity was greater than 98% as determined by HPLC-ELSD.

Animal Experiments. Specifc pathogen-free-grade
Sprague Dawley male rats (200 ± 20 g) were purchased from the Animal Experiment Center of Heilongjiang University of Chinese Medicine (SYXK (hei) 2021-010).Animals were raised in an environmentally controlled animal room with a temperature of 24 ± 2 °C and a 12-h dark/12-h light cycle for a week.And they had free access to water and food during the adaptation period.Ten, the rats were randomly divided into three groups (three rats per group): administration group A (collect plasma), administration group B (collect urine and feces), and blank control group C.After fasting for 12 h, groups A and B were given ciwujianoside B dissolved in physiological saline (150 mg/kg) orally, and blank group C was given physiological saline orally.Rats drunk water freely during the experiment.Te experimental procedures were approved by the Ethics Committee of Heilongjiang University of Chinese Medicine.

Collection and Preparation of Biosamples
2.4.1.Plasma Samples.Plasma samples were collected from administration groups A. Venous blood samples in the orbit were collected into heparinized tubes at diferent times (0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h) after oral administration.Te blood samples were centrifugated at 3000 rpm.Te obtained plasma was transferred and stored at −80 °C for further analysis.Before the preparation of biosamples, plasma samples were completely thawed.Each plasma sample (100 μL) was mixed with 700 μL of methanol, swirled for 1 min, and then centrifuged at 12000 rpm for 10 min (4 °C).Subsequently, a series of supernatants were merged and dried with nitrogen stream.Te residue was redissolved with methanol (100 μL) and centrifuged to obtain the supernatant for analysis.Blank plasma samples were collected and processed as described above.

Feces and Urine
Samples.Each rat in groups B was housed in separate metabolic cages, and the feces and urine samples were collected at diferent time periods (0-4 h, 4-8 h, 8-12 h, 12-24 h, 24-36 h, and 36-48 h) after oral administration.Before the preparation of biosamples, the feces samples were freeze-dried and ground into fne powder.Te serial fecal powder samples (0.5 g per serving) were extracted by ultrasound for 30 min using 3 mL of methanol and centrifuged at 12,000 rpm for 10 min (4 °C).Ten, each supernatant sample (100 μL) was mixed with 700 μL of methanol to precipitate the protein.After centrifugation again, the supernatants were pooled and dried with nitrogen stream.Te residue was redissolved with methanol (100 μL) and centrifuged to obtain the supernatant for analysis.
Urine samples were completely thawed at room temperature and then purifed by activated SPE cartridges [17].Te purifed urine samples (1.0 mL) were combined and dried with nitrogen stream, and the residue was redissolved with methanol (100 μL).After centrifugation and fltration, the supernatant was taken for analysis.Blank feces and urine samples were collected and processed as described above.
Te mobile phase was water (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B).Te UPLC system was eluted with a gradient program as follows: 10-90% B at 0-25 min, 90 − 10% B at 25-25.1 min, 10% B at 25.1-30 min.Te fow rate was 0.3 mL/min.MS analysis was performed using an Orbitrap Fusion Lumos tribrid mass spectrometer equipped with a heating electrospray ionization source (ESI).Te following ESI source parameters were used: an ion spray voltage of 3.2 kV, a capillary temperature of 350 °C, an ion transfer tube temperature of 320 °C, a sheath gas (N 2 ) fow rate of 42 arb, a sweep gas (N 2 ) fow rate of 1 arb, and an auxiliary gas (N 2 ) fow rate of 12 arb.MS spectra were acquired at the mass range of 350-2000 m/z.High collision-induced dissociation (HCD) was adopted with normalized collision energy setting of 40 eV in ESI − mode and 20 eV in the ESI + mode.MS 2 spectra were acquired by the data-dependent acquisition (DDA) scan mode, and the primary ions with ionic strength greater than 2.5e4 were broken into secondary fragments.Dynamic exclusion was set to 6.00 s.
2.6.Data Analysis.Te data were recorded in RAW fle (.raw) and could be processed using Termo Scientifc Xcalibur 4.2 workstation software.Te peaks with intensities above 50,000 were selected for analysis.Te Xcalibur fles of the blank and administration groups were added into Termo Scientifc Compound Discoverer 3.0 to identify the metabolites of ciwujianoside B, and all data fles were analyzed with the same parameter settings.Workfow selected "known compound detection" mode under processing, and the results were exported to a Microsoft Excel spreadsheet.Te parameters were set as follows: the degree of unsaturation was 0∼15; the maximum tolerance of mass error was 5 ppm; the elements' composition was C, H, O, N, S, etc.; and other parameters were default values.

Structural Characterization of Ciwujianoside B by NMR.
Te  13 C-NMR (C 5 D 5 N, 150 MHz) spectrum.In addition, the following data provide information on aglycone: two double-bond carbon signals at δ C � 122.9 and 143.2 (due to C-12 and C-13), a double-bond carbon signal at δ C � 107.1 (due to C-29), and a carboxyl carbon signal at δ C � 175.5 (due to C-28) were observed.Based on the above data, relevant literature [18], and mass spectrometry, we determined that the compound was ciwujianoside B (Figure 1).Table S1 provides the 13 C-NMR data and literature comparison for ciwujianoside B.

Structural Characterization of Ciwujianoside B by UPLC-MS
and [A − H] − ions were generated in sequence (A, aglycone).It was speculated that fragmentation of the ester bond was produced at the C-28 position of [M − H] − at m/z 1187.6, and then, [Y 0α − H] − at m/z 717.4 continued to break the C-3 glycan chain, thereby showing the above ion peaks.In the positive ion mode, an ion fragment was observed at m/z 423.3, which could be attributed to [A − H 2 O + H] + , and the aglycone was assumed to be akebonoic acid [19].According to the above evidences, it can be inferred that the main structure of M 0 was Rha ⟶ Ara-A-Glc ⟵ Glc ⟵ Rha.Te main cleavage characteristics are shown in Figure 2, which helped to identify other metabolites in vivo.

Structural Characterization of Ciwujianoside B
Metabolites.By analyzing the mass detection results of the treated biological samples and the corresponding blank samples, 42 metabolites (M 0 -M 41 ) were preliminarily identifed (Figure 3).Te corresponding metabolic pathways were proposed, including deglycosylation, acetylation, hydroxylation, oxidation, glycosylation, and glucuronidation reactions.Table 1 provides the detailed UPLC-MS 2 data of ciwujianoside B metabolites.In the following text, typical examples of diferent metabolic pathways are discussed in detail.O 11 , and their molecular weight is 718 Da, which is 470 Da less than that of M 0 .Te oligosaccharide chains of the prototype drug are prone to break from the outside to the inside, resulting in the metabolites that lose diferent glycosyls.Tis can manifest as a diference in the molecular weight-132, 146, and 162 Da-corresponding to the loss of arabinose (Ara), rhamnose (Rha), and glucose (Glc).Tus, it could be speculated that M 12 , M 18 , M 28 , and M 30 are generated through the removal of the C-28 sugar chain (Glc ← Glc ← Rha) during the metabolic process of the prototype drug.reactions to produce M 1 and M 3 .According to the detailed MS 2 data of M 1 and M 3 (Table 1), it can be assumed that the two hydroxylation reactions of M 1 occur one on aglycone and one on rhamnose at C-

Deglycosylated Metabolites (M
and [A − H] − ion fragments were generated in turn.It could be speculated that the hydroxylation of M 6 occurs on rhamnose at C-3.Based on the above data, the structures of M 4 , M 41 , and M 6 were speculated to be Rha ⟶ Ara-A (OH)-Glc ⟵ Glc ⟵ Rha and Rha (OH) ⟶ Ara-A-Glc ← Glc ← Rha (Figure S4).

Acetylated Metabolite (M 16 ). [M
m/z 439.3).It was preliminarily determined that the structure of M 16 is (Ac) Rha ⟶ Ara − A − Glc ⟵ Glc ⟵ Rha (as shown in Figure 5).Journal of Analytical Methods in Chemistry in plasma.It is noticeable that M 0 , M 28 , and M 32 were simultaneously found in rat plasma, urine, and feces.A few metabolites were found only in urine (i.e., M 38-40 ) or plasma (i.e., M 41 ), and most of them were detected in fecal samples (i.e., M 0-37 ) (Figure S8).It may be speculated that the main excretion route of ciwujianoside B was through feces.Deglycosylation products M 30 and M 33 were the most abundant components found in rat feces and plasma, indicating that deglycosylation is an important metabolic reaction of ciwujianoside B. Other metabolic pathways were similar to those of hederasaponin B, including acetylation, hydroxylation, glucuronidation, oxidation, and glycosylation.Due to the similar metabolic pathway, it may also be speculated that the deglycosylation of ciwujianoside B was likely to be infuenced by the gut microbiota to produce a series of more easily absorbable secondary glycosides, and then further hydroxylation and redox reactions through CYP 450 to product more metabolites, which are eventually discharged out of the body.Among them, there are 31 metabolites of phase I, seven metabolites of phase II, and three metabolites involved in both phase I and phase II metabolism.Te classifcation of all metabolites and possible metabolic pathways is shown in Figure 6.
On the basis of our previous research on the metabolism of hederasaponin B in vivo, some interesting points could be found by comparison.Firstly, more than 40 metabolites were found in both studies, with the majority found in feces.Secondly, as shown in Figure 7, the metabolic pathways of hederasaponin B and ciwujianoside B were basically similar, including phase I reactions such as deglycosylation, hydroxylation, demethylation, and oxidation, phase II reactions such as mainly acetylation, glycosylation, and glucuronidation, and   the number of metabolites produced by diferent metabolic pathways was also basically the same.Tirdly, deglycosylated metabolites account for approximately 50% of all metabolites, which implies that deglycosylation was the main metabolic pathway of both.Presumably, due to the poor absorption of saponins after oral administration, they undergo deglycosylation in the gut microbiota to produce secondary glycosides for better absorption [20,21].Under the action of CYP 450, further reactions such as hydroxylation and redox occur.In addition, by analyzing the cleavage behaviors of products and diferent metabolic pathways, it was found that the deglycosylation reaction mainly removes the C-28 sugar chain.Glycosylation and glucuronidation mainly occurred at the C-3 sugar chain, while hydroxylation tended to occur on the rhamnosyl and aglycone.

Conclusion
Te metabolism of ciwujianoside B in vivo was systematically studied for the frst time, and the main research results are as follows.Te metabolic pathways of ciwujianoside B involve acetylation, hydroxylation, glucuronidation, oxidation, and glycosylation reactions.Deglycosylation was considered the main metabolic reaction.A total of 42 metabolites (M 0 -M 41 ) were preliminarily identifed, and 38, 17, and 11 metabolites were found in feces, urine, and plasma.Tey include 31 phase I metabolites and seven phase II metabolites, and three products are involved in both phase I and phase II metabolism.In addition, ciwujianoside B metabolism and hederasaponin B metabolism were compared, which confrmed our hypothesis.In short, this study systematically explored the metabolic fate of ciwujianoside B and provided a valuable reference for elucidating the postadministration metabolism of other triterpene saponins.

3. 3 . 3 .
Deglycosylated and Acetylated Metabolites (M 17 , M 23 , and M 31 ).M 17 , M 23 , and M 31 are generated through the acetylation and deglycosylation of M 0 .Here, M 23 (t R �13.66 min) is taken as an example.Its molecular structural formula is C 49 H 77 O 18 , and the molecular weight is 952 Da, which is 236 Da less than that of M 0 .It could be speculated that M 23 was generated through the removal of Rha ⟶ Ara at the C-3 position and the addition of an acetyl group during the metabolic process of the prototype drug.Te ESI − -MS spectrum of M 23 showed the fragment ions of [M − H] − at m/z 951.4993 and [M − HCOOH-H] − at m/z 997.5049.Te MS 2 spectrum of M 23 showed the fragment ions at m/z 439.4 ([A − H] − ), which refected aglycone information.Tese data indicate that M 23 is produced by acetylation at a certain location in the C-28 sugar chain after the removal of the C-3 sugar chain (Rha ⟶ Ara) of the prototype drug, or by the acetylation of the metabolite M 21 .Terefore, M 23 was preliminarily identifed as A-Glc ⟵ Glc ⟵ Rha (Ac) (FigureS2).

Figure 3 :
Figure 3: UPLC-MS extracted ion chromatograms (EICs) of the metabolites in rat feces (a), urine (b), and plasma (c) of ciwujianoside B in the negative ion mode.

Figure 6 :
Figure 6: Proposed metabolic pathways of ciwujianoside B in rats.

Figure 7 :
Figure 7: Te numbers of metabolites of hederasaponin B (up direction) and ciwujianoside B (down direction) by diferent metabolic pathways.
3.3.8.Glycosylated Metabolites (M 9 , M 11 ).Tis section provides the data of the glycosylation metabolite M 9 .Te formula of M 9 (t R � 9.51 min) is C 64 H 102 O 30 , and its molecular weight is 162 Da greater than that of M 0 .It may be speculated that M 9 has an additional glucose group.In the negative ion mode, the [M − H] − ion of M 9 was detected at m/z 1349.6433.In the ESI − -MS 2 spectrum, the characteristic fragmentation ions sequence was m/z 879.5([Y 0α − H] − ) ⟶ m/z 717.4 ([Y 0α − Glc − H] − ) ⟶ m/z 571.3 ([Y 0α − Glc − Rha − H] − ) ⟶ m/z 439.3 ([A − H] − ) (Δm � 162, 146, and 132 Da in the sequence).Tis indicates that the sugar chain structure (C-3) of the metabolite is Glc ⟶ Rha ⟶ Ara.Based on these data, it was preliminarily determined that M 9 is Glc ⟶ Rha ⟶ Ara-A-Glc ⟵ Glc ⟵ Rha (FigureS6).Similarly, the formula of M 11 (t R �10.03 min) is C 63 H 100 O 29 , and its molecular weight is 132 Da greater than that of M 0 .It may be speculated that M 9 has an additional arabinose group.In the negative ion mode, [M − H] − ion of M 11 was detected at m/z 1319.6329.Te molecular weight of M 7 (t R � 9.16 min) and M 8 (t R � 9.46 min) is 1364 Da, which is 176 Da greater than that of the prototype drug.It may be speculated that M 7 and M 8 are the metabolites of glucuronidation of M 0 .Te ESI − -MS 2 spectrum of M 7 and −), and 439.3 ([A-H] − ).Accordingly, the structure of the C-3 sugar chain is inferred to be Ara ⟶ Rha ⟶ Ara.Based on the above evidences, M 11 was preliminarily determined as Ara ⟶ Rha ⟶ Ara-A-Glc ⟵ Glc ⟵ Rha (FigureS6).3.3.9.Glucuronidated Metabolites (M7 , M 8 ).7 is GlcA ⟶ Rha ⟶ Ara-A-Glc ← Glc ← Rha (FigureS7).3.4.Metabolic Pathways of Ciwujianoside B.A total of 42 metabolites were tentatively identifed, including 38 metabolites in feces, 17 metabolites in urine, and 11 metabolites