Chemicolome and Metabolome Profiling of Xieriga-4 Decoction, A Traditional Mongolian Medicine, Using the UPLC-QTOF/MS Approach

Background Xieriga-4 decoction (XRG-4) is a classic prescription Mongolian medicine that has potent diuretic and anti-inflammatory activities. However, its functional components remain unknown. Purpose This study aimed to identify the chemical components in XRG-4 and its metabolome in vivo. Methods An ultra-performance liquid chromatography coupled with a quadrupole time-of-flight tandem mass spectrometry based approach was proposed to systematically profile the chemicolome and metabolome of XRG-4. Result A total of 106 constituents were identified in XRG-4. Eighty-nine components were identified in biological samples, including 78 in urine (24 prototypes and 54 metabolites), 26 in feces (19 prototypes and 7 metabolites), and 9 in plasma (5 prototypes and 4 metabolites). In other tissues, only a few compounds, including alkaloids and iridoids, were detected. Conclusion This comprehensive investigation of the chemical and metabolic profiles of XRG-4 provides a scientific foundation for its quality control and administration of clinically-safe medication.


Introduction
Xieriga-4 decoction (XRG-4) is a classical Mongolian medicine prescription, which comprises four plants, including phellodendron chinensis cortex (PCC), tribuli fructus (TF), curcumae longae rhizoma (CLR), and gardenia fructus (GF). It is described in the Mongolian Medicine Volume of the Drug Standard of the Ministry of Public Health of the People's Republic of China (Commission, 1998), becoming a national registered standard preparation. XRG-4 has various pharmacological activities, such as diuresis and detumescence, renal protection, antiinfammation, labor pain, and bacteriostasis [1,2]. It is widely adopted in Mongolian medicine to treat urinary infectious diseases such as nephritis, cystitis, benign prostatic hyperplasia, and urinary tract infection. [3,4]. After oral administration, components of XRG-4 are absorbed by the gastrointestinal tract to be further distributed, metabolized, and excreted. Despite their advantages in multipletarget and synergistic efects of Mongolian medicine, the specifc in vivo targets of active ingredients remain unclear. Terefore, the pharmacodynamic bioactive components and the further mechanism are worth exploring.
Mass spectrometry is a novel, comprehensive method for rapid chemicolome analysis and has unique advantages in metabolism studies of traditional Chinese medicine (TCM) [5,6]. Ultra-performance liquid chromatography coupled with Quadrupole time-of-fight tandem mass spectrometry (UPLC-QTOF/MS) has the characteristics of high sensitivity, mass accuracy, resolution, and wide scanning range. Analysis of the relative molecular mass, elemental composition, and fragmentation information of compounds can lead to the identifcation of various complex components and chemical structures of TCM to provide further technical support for the modern study of traditional Chinese medicine [7,8].
In this research, the UPLC-QTOF/MS technique was used to identify and characterize the compounds in XRG-4. First, the components in XRG-4 were identifed by UPLC-QTOF-MS and by using information from the in-house database. Next, the absorbed prototypes and their phase-I and phase-II metabolites were characterized, and their in vivo distribution was investigated. Finally, a network was built to reveal the relationship between metabolites and prototypes. Our research provides a scientifc basis for further investigating pharmacological bioactive components and quality markers in XRG-4.  . 0.2 g XRG-4 was ultrasound three times for 30 min in a conical bottle with 20 mL of 70% methanol, and the supernatant was centrifuged at 13000 rpm for 10 min. 400 μL supernatant was transferred into a fresh tube and dried under nitrogen gas. Te dried supernatant was redissolved with 400 μL of 50% acetonitrile and centrifuged at 13000 rpm for 10 min. Te supernatant was transferred into a new tube, and a 2.0 μL aliquot was injected for UPLC-QTOF-MS.

Animal Experiments.
Male Wistar rats (weight 150-200 g) were purchased from Jiangsu Ji Cui Yao Kang Biotechnology Co., Ltd. Animals were housed at 23 ± 2°C with 12 h light/dark cycle and had free access to a standard diet and water. A total of 12 rats were randomly categorized into two groups, six each for the control group and the administration group. After 3 days of acclimatization, the rats that were fasted for 16 h before dosing were administered an oral dose of 1.5 g/kg of XRG-4. All procedures were performed as per the guidance of the Provisions and General Recommendation of the Chinese Experimental Animals Administration Legislation.
Feces: Fecal samples were collected at 600 min pre-and post-administration and stored at -80°C before pretreatment. Feces were weighed and nearly 300 mg of the sample was mixed with 1 mL of methanol and magnetic beads. Te mixture was ground at 40 Hz six times with a 5 s interval, with a total running time of 80 s. After grinding, the samples were centrifuged at 13000 r/min (4°C) for 10 min. Nearly 200 μL of supernatant was obtained and dried under nitrogen gas; the residue was redissolved in 200 μL of 50% acetonitrile. After centrifugation at 13,000 rpm for 10 min, a 2.0 μL aliquot of the supernatant was obtained for UPLC-QTOF-MS analysis.
Urine: Urine samples were collected at 600 min pre-and post-administration and stored at -80°C. Te urine was freeze-thawed and centrifuged at 4000 r/min for 10 min, and 1 mL of supernatant was loaded onto an activated C18 SPE column (waters). Te loaded column was washed with 1 mL of ultrapure water, eluted with 1 mL of methanol and collected and centrifuged at 13000 rpm for 10 min at 4°C. Nearly 400 μL of supernatant was transferred and dried under nitrogen gas. Te residue was redissolved in 400 μL of acetonitrile (50%), centrifuged at 13000 rpm for 10 min at 4°C, and a 2.0 μL of aliquot was injected into UPLC-QTOF-MS.
Tissue: Te rats were sacrifced 600 min post-dosing. Ten the heart, liver, lungs, spleen, kidneys, and brain were harvested and homogenized. Te tissue samples were weighed (300 mg) and mixed with 1 mL of methanol and magnetic beads in a 2 mL centrifuge tube. Te mixture was ground six times at 40 Hz with a 5 s interval for a total run time of 80 s. After grinding, the samples were centrifuged at 13000 r/min (4°C) for 10 min. Te supernatant (200 μL) was dried under nitrogen gas, and the residue was redissolved in 200 μL of acetonitrile (50%). Tis was centrifuged at 13,000 rpm for 10 min, and a 2.0 μL of aliquot of the supernatant was used for UPLC/Q-TOF MS analysis.

Analysis Condition for Ultra-performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem
Mass Spectrometry. Te supernatant (2.0 μL) obtained as described in the previous step was used for chromatographic separation on an Exion LC system (AB Sciex, Foster City, CA, USA). Te Waters Acquity HSS T3 column (2.1 × 150 mm; 1.7 μm) was used at 35°C. For the mobile phase, eluent A (water with 0.1% formic acid, v/v) and eluent B (acetonitrile) were used. Te linear elution gradient program was optimized as follows: 0-5 min from 3% to 8% B, 5-11 min from 8% to 30% B, 11-20 min from 30% to 80% B, 20-21 min from 80% to 95% B, 21-29 min at 95% B, then back to the initial ratio of 3% B and maintained for additional 4.5 min for re-equilibration.

Identifcation of Chemical Components in XRG-4.
Te base peak chromatograms in positive and negative modes of XRG-4 in UPLC-QTOF-MS analysis are shown in Figure 1. A total of 106 compounds in XRG-4 were identifed. Among them, 32 compounds exist in PCC, 21 in TF, 16 in GLR, and 47 in GF. As shown in Table S1. Te representative components of each plant are shown in Figure 2. Te main characteristic components in PC were alkaloids, limonin, and phenylpropanoids (organic acid); TF contains more saponins with a molecular weight >900 (Da), some alkaloids, and a few favonoids. Te main characteristic components of GLR were the curcumins, the phenylpropanoid derivatives. A variety of chemical types were found in GF, such as triterpenoids, phenylpropanoid derivatives, favonoids, and iridoids.

Mechanism of Fragmentation of Representative Structures.
Te proposed fragmentation patterns of typical compounds in XRG-4 are shown in Figure 3.

Phellodendron Chinensis
Cortex. PCC has various pharmacological actions, such as anti-infammatory, anti-tumor, and hypoglycemic. Te most characteristic components found in PCC were alkaloids, which generally have a good response in the positive ion mode, considering the fragmentation patterns of Phellodendrine as an example. As shown in Figure 3(a), the fragmentation of heterocyclic rings produced ions at m/z 192, and subsequent demethylation (△m � 15) resulted in product ions at m/z 177. A total of 32 active components were identifed in PCC, including 18 alkaloids such as candicine (P3), phellodendrine oxide (P26), N-methylhigenamine-7-glucopyranoside (P27), and berberine (P77) ( Table S2).

Identifcation of Prototypes and Metabolites of XRG-4 in
Rat Biological Samples. Some compounds (prototypes and some metabolic types), after being administered, are absorbed in the gastrointestinal tract and then sent to the liver through the portal vein for further metabolism. Te circulation system allows for the systemic distribution of the components in various tissues and organs. Finally, the chemicals are excreted in the urine by the kidneys, and other components that are not absorbed by the gastrointestinal tract are excreted in the feces.
In the current study, accurate mass measurement was performed to characterize the XRG-4 compounds, their retention times, and ms/ms fragmentation behaviors; some compounds were identifed by corresponding reference standards. Prototypical components were extracted from plasma, urine, and feces through the rules of phase I and phase II metabolism and showed similar secondary mass spectrometry profles. Te base peak   Figure 5(b) shows the secondary map of P51 (demethyleneberberine), which is also one of the metabolites (produced by loss of CH2 and hydrogenation) of berberine and exhibits the same fragmentation mode as berberine. Figure 5(c) presents the secondary mass spectrometry map of M1 (C25H26NO10, demethyleneberberine, and glucuronidation). Tere is a neutral loss of glucoside (−176 Da), and the parent nucleus is highly similar to demethyleneberberine; hence, M1 was classifed as one of the metabolites of berberine. According to this principle, seven metabolites matched with the prototype berberine, and the structure and biotransformation correlation diagram are presented in Figure 6. A total of 11 representative structures, berberine (P77, alkaloids), curcumin (P100, curcumin), isoquercetin/ hyperoside (P46, favonoids), rutin (P42, favonoids), geniposide (P30, iridoids), genipin 1-gentiobioside (P20, iridoids), jsminoside B/F (P14, 2-ISObutylglutaric acid (P65, organic acids), 3-O-feruloyl quinic acid (P36, 2-Oferuloyl quinic acid, organic acids), 4-sinapoyl-5-caffeoylquinic acid (P70, organic acids), and dioscin (P98, nasal saponins), were selected for metabolite identifcation and prototype-metabolic matching. A total of 56 metabolic components are fnally matched, and the associated network between related prototypes and metabolic compounds is prepared, as shown in Figure 7 and Table S2.  Evidence-Based Complementary and Alternative Medicine Mongolian Medicine. Presently, XRG-4 has diuretic [12,13] and anti-infammatory [14][15][16] efects, but its potential hypoglycemic activities and bioactive components have not been reported [17,18]. Eleven representative structures were selected from the experiment. Tese natural compounds have a potential role in preventing or controlling diabetes mellitus. Te underlying mechanism of the antidiabetic efects of these compounds include improvement in insulin secretion, decrease in insulin resistance, enhanced glycogen synthesis in the liver, and antioxidant and anti-infammatory activities [19,20]. Berberine, an alkaloid, is the main component of PCC. It efectively reduces fasting plasma glucose, postprandial blood glucose, and glycosylated hemoglobin by participating in insulin resistance, antiinfammatory, antioxidation, regulating lipid metabolism disorders and intestinal fora, and other methods. Berberine is mainly used in the treatment of type 2 diabetes, obesity, and metabolic diseases [21][22][23][24][25][26][27]. We detected berberine in plasma, feces, and biological samples, indicating that the prototype and metabolites of berberine are involved in the hypoglycemic mechanism. It is the main bioactive component of XRG-4 in hypoglycemia. Curcumin is a characteristic component of CLR and exhibits anti-infammatory, antioxidation, antitumor, and immunoregulatory properties. Moreover, it improves insulin resistance, obesity, and other FFA-related diseases [11,[28][29][30][31]. In this study, isoquercetin and rutin were also screened as representative compounds. Agarwal [32] reported the actions of isoquercetin and rutin, including antihyperglycemia and their efects on diabetic complications. Geniposide is a new type of iridoid glycoside, which is the main active ingredient of gardenia. Recent studies have found a variety of pharmacological and biological activities of Geniposide, including liver protection, antiosteoporosis, antitumor, antidiabetes, neuroprotection, and so on. In summary, the 11 key compounds detected in this experiment have certain effcacy and potential in addressing diabetes and its complications.

Evidence-Based Complementary and Alternative Medicine
A variety of small molecular compounds were identifed by UPLC-QTOF-MS. Te bioactive components and metabolites of XRG-4 in plasma, urine, feces, and tissue samples in vivo, the metabolic pathways of XRG-4, and the prototype compounds were matched. In addition, the absorption, distribution, metabolism, and excretion pathways of XRG-4 were also determined in vivo. We could also summarize the distribution of prototype and structural representative metabolites in biological samples. Eighty-nine compounds were detected in biological samples and 78 in urine, including 24 prototypes and 54 metabolites. 26 compounds were detected in feces, including 19 prototypes and 7 metabolites, and 9 compounds were detected in plasma, including 5 prototypes and 4 metabolites. Te distribution of bioactive components in vivo is shown in Table S3. In the metabolic process, urine is created by kidney fltration and reabsorption of blood through the glomerulus. Terefore, compounds excreted through the urine have undergone a systematic circulation system.
Only a few compounds, mainly alkaloids and iridoids, were detected in other tissues. Berberine was detected in all tissues and was probably the most widely distributed chemical component. Columbamine, palmatine, and jatrorrhizine were detected in the heart and kidney, and berberrubine was detected in the liver. Among the metabolites with tissue distribution, M1 is the metabolite of berberine, M16 and M17 are the metabolites of geniposide, and M43 is a metabolite of jasminoside B/F. Tese compounds may be worth further study for quality control and pharmacodynamic activities.
We detected only a few compounds in the plasma, possibly due to (1) the rapid excretion process of the chemicals and short residence time resulting in no   Figure 6: Te network association diagram of P77 and its metabolic components. detection in the blood; and (2) compounds combined with plasma protein at a higher rate, and after sample preparation, the low concentration in the plasma limits the detection of the object under test. In summary, the latest experimentation technology was used to discover the bioactive components of XRG-4 in vivo and in vitro, which helped to reveal the potential components of Mongolian medicine in vivo, further clarify its hypoglycemic activities and lay an experimental foundation for a clinical search of efective natural hypoglycemic drugs with few toxic and side efects.

Data Availability
Te raw data required to reproduce these fndings cannot be shared here, as the data also form part of an ongoing study.

Disclosure
Yuanyuan Ma and Ruiting Ma are the co frst authors. Tumenwuliji is the corresponding author.