The Tannins from Sanguisorba officinalis L. (Rosaceae): A Systematic Study on the Metabolites of Rats Based on HPLC–LTQ–Orbitrap MS2 Analysis

Sanguisorba tannins are the major active ingredients in Sanguisorba ofJicinalis L. (Rosaceae), one of the most popular herbal medicines in China, is widely prescribed for hemostasis. In this study, three kinds of tannins extract from Sanguisorba officinalis L. (Rosaceae), and the metabolites in vivo and in vitro were detected and identified by high-pressure liquid chromatography, coupled with linear ion trap orbitrap tandem mass spectrometry (HPLC–LTQ–Orbitrap). For in vivo assessment, the rats were administered at a single dose of 150 mg/kg, after which 12 metabolites were found in urine, 6 metabolites were found in feces, and 8 metabolites were found in bile, while metabolites were barely found in plasma and tissues. For in vitro assessment, 100 μM Sanguisorba tannins were incubated with rat liver microsomes, liver cytosol, and feces, after which nine metabolites were found in intestinal microbiota and five metabolites were found in liver microsomes and liver cytosol. Moreover, the metabolic pathways of Sanguisorba tannins were proposed, which shed light on their mechanism.


Introduction
The Sanguisorba officinalis L. (Rosaceae), with a long history of cultivation, is widely distributed in Asia, Western Europe, and North America and is specifically native to the temperate zones of the Northern Hemisphere, a kind of plant that is available for medicine in China, Europe, Japan, and Korea [1]. It is a member of subfamily Rosoideae and family Rosaceae, and all parts of the plant can be used as an effective astringent, but the root is the most active [2]. The dried root of Sanguisorba is recorded in various versions of Chinese Pharmacopoeia, European Pharmacopoeia, Russian Pharmacopoeia [3,4]. Since the Sanguisorba contains phenolic compounds, such as tannic acid, with a function of "cooling blood for hemostasis, detoxification, and astringency", it has been used for metrorrhagia and metrostaxis, hematemesis, hematochezia, bleeding hemorrhoids, pudendum eczema, menopathy, and leukorrheal diseases [5]. The main constituents include triterpenoid saponins, flavonoids, phenols, etc., among which the phenols are highest and are quite active in the treatment for increasing white blood cell, along with having anticancer, antioxidant, and antibacterial benefits [6].
According to the chemical structure of tannins, they can be divided into three categories: (i) hydrolyzable tannins, which generally refer to tannins that can produce gallic acid or various gallic acid polymers (CG, S-HHDP, DHDDP, S, S-gallagyl, lactone valoneoyl) and glucose after hydrolysis; (ii) condensed tannins, which are a class of compounds in which catechins or their derivatives and other flavan-3-ols are polymerized with C-C bonds [7,8]. Since there is no glycoside bond and ester bond in their structure, they cannot be hydrolyzed by acid or base [9]; and (iii) complex tannins, which are composed of the components of condensed tannins, flavan-3-ols, and the hydrolyzed tannins by carboncarbon bonding. These tannins were first isolated from the Fagaceae [10] and have been widely found in plants containing both hydrolyzable tannins and condensed tannins [11].
At present, the limited studies available mainly focus on the metabolites and tissue distribution of Ziyuglycoside I and II [12], the major active ingredients isolated from Sanguisorba officinalis, with few studies about Sanguisorba tannins.
In the present study, high-performance liquid chromatography, coupled with highresolution mass spectrometry (HPLC-HRMS), has been used as an effective and reliable analytical tool to identify metabolites due to its high resolution and high sensitivity [13,14]. Metabolism research can identify metabolites and major metabolic pathways, which can help to understand the pharmacological mechanisms of food and drugs [15]. Recently, the intestinal microbiota has been considered to be a "hidden organ" of the body [16], which can be regarded as a systematic research approach to study the intestinal microbiota metabolism in vitro [17]. In addition, Sanguisorba tannins can be metabolized by liver microsomes, cytosol, and rat primary hepatocytes in vitro [18], which has been regarded as a valuable model to study food and drug metabolism in vitro due to the biotransformation organ of the liver [19].
Therefore, in this study, we conducted a comprehensive in vivo-in vitro investigation on the metabolism of Sanguisorba tannins using HPLC-LTQ-Orbitrap MS 2 . In vitro, Sanguisorba tannins mediated metabolism was studied in the intestinal microbiota and liver microsomal culture system of rats. In vivo, Sanguisorba tannins metabolites were characterized in various biological samples, including urine, feces, plasma, bile, and various tissues, after oral administration of Sanguisorba tannins in adult rats.

Establishment of the Analytical Strategy
In this study, an effective strategy was taken for metabolite identification of Sanguisorba tannins in vivo and in vitro using HPLC-LTQ-Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA) [20], coupled with multiple data processing methods. Firstly, fragmental patterns of the tannins from Sanguisorba L. were analyzed based on the MS 1 and MS 2 information obtained by HPLC-LTQ-Orbitrap MS 2 to acquire the cleavage pathways and diagnostic product ions (DPIs) for metabolite identification. Then, metabolite templates (known and identified metabolites) were summarized and established using the reported metabolic transforms of ingredients in the literature. Thirdly, compared with blank biosamples including urine, bile, plasma, feces, liver microsomes, liver cytosol, and intestinal microbiota, the metabolites from Sanguisorba L. in biosamples were identified using multiple metabolite templates, extracted ion chromatograms (EICs), and DPIs. Furthermore, we also used ChemDraw 14.0 (Thermo Fisher Scientific, Waltham, MA, USA) to calculated the Clog p values and the isomers were selected with different retention times. Often, the compound had a longer retention time when the Clog p value was greater in the reverse-phase chromatographic system.

Identification of Total Tannins Compounds Extracted from Sanguisorba Tannins
After concentration using HDP-400 macroporous resin column, the total tannins content in the final product was 65.5% and the main compounds extracted from Sanguisorba tannins were measured by using HPLC-LTQ-Orbitrap MS 2 . The main compounds detected are shown in Table 1, the total ion chromatograms of Sanguisorba tannins are shown in Figure 1, the summary analytical strategy diagram is shown in Figure 2 and their structures are shown in Figure 3.       Figure 3. The summary contents structure in Sanguisorba tannins.

Identification of Metabolite of Sanguisorba Tannins In Vivo and In Vitro
According to the above analysis method, a large number of metabolites were identified in biological samples. All the detected metabolites of Sanguisorba tannins in vivo and in vitro are listed in Tables 2-4, respectively, the structures of the metabolites in vivo and in vitro are shown in Figure 4, and the metabolic pathways in vivo are shown in Figure 5.

Identification of Metabolite of Sanguisorba Tannins In Vivo and In Vitro
According to the above analysis method, a large number of metabolites were identified in biological samples. All the detected metabolites of Sanguisorba tannins in vivo and in vitro are listed in Tables 2-4, respectively, the structures of the metabolites in vivo and in vitro are shown in Figure 4, and the metabolic pathways in vivo are shown in Figure 5.  − ) were generated by the loss of H 2 O (18 Da) unit and CO 2 (44 Da) unit from the precursor ion, respectively. Based on the mass spectra data and comparison with their reference substances, M3 was absolutely identified as ellagic acid [22].        16 Da. Based on the mass spectra data, it is speculated that M5 and M6 were formed by M4 dehydroxylation. The UV absorption spectrum of M6 shows two absorption peaks in the region of 240-400 nm, with an absorption peak of 349 nm, a second absorption peak of 263 nm, and a third absorption peak of 291 nm, similar to that of M4, and no significant redshift in band II. According to the literature [23], if no hydroxyl group is substituted on the para position (nine-position) of carbonyl group on urolith parent nucleus, there will be a redshift (displacement in the direction of longwave) in band I. Therefore, it is speculated that M6 retains the phenolic hydroxyl group in the para position of the carbonyl group, which is urolith D. The UV absorption spectrum of M5 showed three major absorption peaks in the region of 240-400 nm, respectively, at 251 nm, 275 nm, and 361 nm, with a significant redshift of band 2, compared with M4 (349 nm), suggesting that M5 is a nine-position hydroxyl removal metabolite of M4, which was named urolith M6. M7

The Identification and Characterization of Metabolites In Vitro
Some metabolites in intestinal microbiota, liver microsomes, and liver cytosol can also be detected in vivo metabolism; moreover, new compounds were detected in vitro metabolism, proving that in vitro-in vivo metabolism is different. For example, metabolite N5, metabolite N8, and metabolite N9 were detected in intestinal microbiota.

Identification of Phase II Metabolites
Its mass spectrometry lost 176 Da, which proved to be phase II of combined with glucuronic acid metabolites. Metabolite N5, eluted at 9. − ) was also observed. Therefore, N9 was urolithin C methyl.

Metabolic Pathways of Sanguisorba Tannins
The metabolism of Sanguisorba tannins in rats after oral administration in vivo (plasma, urine, bile, feces, and tissues) and in vitro (liver microsomes, liver cytosol, and intestinal microbiota) through incubation was elaborated in this study. Results are presented in what follows.
HPLC-LTQ-Orbitrap MS 2 identified 19 metabolites in rats, including 4 metabolites from plasma, 9 metabolites from bile, 10 metabolites from urine, 5 metabolites from feces, 1 metabolite from kidney, 5 metabolites from liver microsomes and cytosol, and 9 metabolites from intestinal microbiota. According to the main components in the alcohol extract of Sanguisorba tannins contained and the structures of its metabolites, it can be inferred that after oral administration of tannins, it rapidly decreased in the blood, entered the liver, and expelled from bile, during which nine metabolites were detected. Figure 6 shows the proposed metabolic profiles of Sanguisorba tannins in rats, in liver microsomes, and intestinal microbiota, respectively. Gallic acid and ellagic acid were detected in liver microsomes and cytosol, and their conjugation of methyl was also detected, then absorbed into the blood and excrete from bile for the next metabolic reaction. M4-M10 were dehydroxylation metabolites of phase I, which were mainly detected in feces. Eventually, urolithin A is formed in feces. The phase II metabolites were urolithins of their derivatives and the combination with glucuronic acid, which were mainly detected in urine. Among them, urolithin A was also detected in intestinal microbiota; it is speculated that after oral sanguisorba tannins, the metabolites of phase I appeared first in intestinal microbiota, and a kind of smaller polarity such as urolithins was formed, then absorbed into the body; afterward, conjugation of phase II was formed, which became more polar and excreted from the urine. These results manifested that tannins mainly underwent reduction, hydrolysis, glucuronide conjugation, sulfate conjugation, and methylation changes. It should be noted that reduction reactions were the main metabolic steps. The conversion was further carried out by sulfation, followed by glucuronidation. liver, and expelled from bile, during which nine metabolites were detected. Figure 6 shows the proposed metabolic profiles of Sanguisorba tannins in rats, in liver microsomes, and intestinal microbiota, respectively. Gallic acid and ellagic acid were detected in liver microsomes and cytosol, and their conjugation of methyl was also detected, then absorbed into the blood and excrete from bile for the next metabolic reaction. M4-M10 were dehydroxylation metabolites of phase Ⅰ, which were mainly detected in feces. Eventually, urolithin A is formed in feces. The phase Ⅱ metabolites were urolithins of their derivatives and the combination with glucuronic acid, which were mainly detected in urine. Among them, urolithin A was also detected in intestinal microbiota; it is speculated that after oral sanguisorba tannins, the metabolites of phase Ⅰ appeared first in intestinal microbiota, and a kind of smaller polarity such as urolithins was formed, then absorbed into the body; afterward, conjugation of phase Ⅱ was formed, which became more polar and excreted from the urine. These results manifested that tannins mainly underwent reduction, hydrolysis, glucuronide conjugation, sulfate conjugation, and methylation changes. It should be noted that reduction reactions were the main metabolic steps. The conversion was further carried out by sulfation, followed by glucuronidation.

Comparison of Metabolites In Vitro and In Vivo
Metabolism research plays an important role in understanding the configuration of food and drugs and provides a basis for the study of the safety and toxicity of food and drugs [26]. The approach in vivo is quantitative and very effective in drug metabolism studies [27]. However, because of complex biomatrix, drug metabolites are often difficult to characterize in vivo [28]. Therefore, in vitro metabolic model is necessary to avoid the influence of other biomatrixes on metabolism in vivo. The identification of in vitro metabolites can supplement in vivo metabolites in complex biological samples. In vitro, culture methods are generally applicable to targeted studies and often predict the risk of real harm [29].
Sanguisorba tannins are the main tannins in Sanguisorba, play the main role in Sanguisorba, and have made a great contribution to the biological activity of Sanguisorba. However, research on its metabolic transport modes and pathways in the body is relatively weak, and the biotransformation of Sanguisorba tannins is mostly focused on hydrolyzable tannins. Research on the metabolism of condensed tannins in the body is relatively superficial. What is the relationship between the metabolic mechanism of Sanguisorba tannins in the body and the performance of its pharmacological activities? It needs further investigation.

Plant Materials
The dried Sanguisorba (FY2048) about 1 kg was collected at FEIYUBIO medicine market from Hubei Province, China, in June 2018, identified as Sanguisorba by Professor Zhang Hong of Wuhan University, ground to a fine powder in a grinder, passed through a 60-mesh (pore size in 0.25 mm) sieve and stored at 4 • C until analysis.

Chemicals and Materials
The authentic standards of ellagic acid (EA), pyrogallol, and gallic acid (GA) were purchased from the Chengdu MUST Bio-Technology CO. Ltd.

Instrumentations and Investigation Conditions
Separation was carried out on a Welch Ultimate XB-C 18  The operating conditions of mass spectrometry were as follows: electrospray ionization (ESI) source was employed, and positive and negative ion mode were selected; sheath gas flow, 40 A.U.; auxiliary gas flow, 20 and 10 A.U.; source voltage 3.8 kV; capillary temperature of 300 • C; capillary voltage 25 and −35 V; tube lens, 110 and −110 V. The samples were analyzed using FT full scan with mass in the m/z 100-1500 range. All the raw data were processed using Xcalibur 3.0 software 19 (Thermo Fisher Scientific, Waltham, MA, USA).

Extraction and Purification of the Total Tannins from Sanguisorba
According to the assay reported by [30], ultrasonic (Ningshang Ultrasonic Instrument CO. Ltd. Shanghai, China) assisted ethanol extraction of crude polyphenols was used. Preweighed amounts of Sanguisorba powder were placed into a volumetric flask (100 mL), soak dry ground Sanguisorba powder for 20 g with 70% ethanol solution at 20:1 solvent-tosample ratio (v/w), and the extraction was placed in an ultrasonic cleaning bath at 40 • C for 1 h. Then, filtered through 0.22 µm filter paper to obtain the filtrate, while the residue was extracted again under the same extraction conditions, collected together with the filter, and concentrated by rotary vacuum evaporator (IKA-Werke-GmbH & CO., Staufen, Germany) to obtain the powders. Refer to the purification and separation methods [31] summarized in the previously published results of the research group. The HPD-400 macroporous resin column (Macklin biochemical CO. Ltd., Shanghai, China) was used for purification. Briefly, the crude polyphenolic extract was dissolved in sterile water to obtain the final concentration of 1 mg/Ml. Then, the concentrate was loaded into HPD-400 macroporous adsorption resin column (16 × 300 mm) at a flow rate of 2 mL/min. The final eluent was collected, concentrated by rotary vacuum evaporator, and freeze dried to obtain the tannic powder of Sanguisorba and kept sealed in the dark.

Quantitation and Chemical Analysis of Total Tannins from Sanguisorba
After purified by HDP-400 macroporous resin column, the content of the total tannins in the final product was measured according to the spectrophotometric method from the Chinese Pharmacopoeia, 2010 edition. The total tannins were slightly modified according to the Folin-Ciocalteu method [32]. In brief, 0.2 mL of the extract (2 mg/mL) was mixed with 1 mL of the Folin-Ciocalteu's reagent and 2 mL of 150 mg/mL Na 2 CO 3 . The 10 mL volumetric flasks were shaken and allowed to stand for 2 h at room temperature. The absorbance was measured with a spectrophotometer (Shimadzu CO., Tokyo, Japan) at 765 nm against a reagent blank and gallic acid was used as the standard with the contraction ranging from 2.909 to 6.545 µg/mL. All assays were run in three replicates. The total tannin content value was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (DW) (mg GAE/g DW). The method of determining nonadsorbed phenol content was the same as the determination of total phenol content, except that 0.6 g of casein was added to the sample. As a result, the total tannin content is total phenol content minus nonadsorbed phenol content, which is 65.5%.

Animal Experiments
The male Sprague Dawley (SD) rats, aged 6 weeks (200-220 g), were obtained from the Center of Experimental Animals of Medical College, Wuhan University. The rats were housed in the SPF breeding house of the animal experiment center of the people's hospital of Wuhan university (temperature: 22-25 • C, relative humidity: 55-60%, light/dark cycle for 12 h) for seven days. All rats were fed on a standard pellet diet without tannins and water was freely available.
Sanguisorba tannins were suspended in saline. The 72 rats were randomly divided into 12 groups with 6 rats in each group (group 1, the blank plasma sample group; group 2, the experimental plasma sample group; group 3, the blank bile sample group; group 4, the experimental bile sample group; group 5, the blank urine and feces sample group; group 6, the experimental urine and feces sample group; group 7, the blank liver microsomes and cytosol sample group; group 8, the experimental liver microsomes and cytosol sample group; group 9, the blank intestinal microbiota sample group; group 10, the experimental intestinal microbiota sample group; group 11, the blank tissues sample group; group 12 the experimental tissues group). Before the experiment, the rats in the 12 groups fasted for 12 h with free access to water. The rats in vivo experiments group were orally administered Sanguisorba tannins at a single dose of 150 mg/kg (crude drug weight/rat weight). At the same time, saline was orally administered as blank control group rats. The rats in vitro experiments group, liver microsomes and cytosol, intestinal microbiota were incubated with Sanguisorba tannins (100 mg/mL, suspended in saline).

Sample Collection and Pretreatment In Vivo
Plasma samples collection: After gavage, 200 µL of blood was obtained from the canthus in rats at 0, 1.5, 4, 10, 20, 40, 60, 90, 120, 180, and 240 min. The blood with three volumes of acetonitrile was centrifuged at 4500 rpm for 5 min, then the supernatant was centrifuged at 10,000 rpm/min for 15 min, and the supernatant was taken [33].
The urine and feces collection: a total of 12 rats were divided into two groups at random and housed in individual stainless steel metabolic cages designed for the separation and collection of urine and feces. Urine and feces were collected for 72 h post dosing. After measuring the volume of urine samples and dry weight of feces samples, an aliquot of 3 mL mixed urine was prepared using Grace Pure solid-phase extraction (SPE) C 18 columns (Deerfield, IL, USA). Prior to sample preparation, SPE columns were conditioned using 5 mL methanol and subsequent 5 mL deionized water. Then, urine was loaded on the SPE cartridge, rinsed with 5 mL of deionized water, and eluted with 3 mL of methanol. The methanol eluent was collected and then evaporated to dryness at 40 • C under a gentle stream of nitrogen. Finally, the residues were redissolved using 100 µL 5% acetonitrile and vortex-mixed for 3 min. The 1 g of dried feces were dissolved with deionized water with ultrasonic processing for 60 min, after which the supernatant was collected after centrifuging at 3500 rpm/min for 10 min and concentrated [34].
The bile collection: The rats were orally administered drugs and then anesthetized with 2% pentasorbital sodium (45 mg/kg); the cannulas were surgically inserted into the bile duct to collect bile. An aliquot of 50 µL mixed bile samples were collected before dosing and during 0-4, 4-8, 8-12, 12-24, 24-36, and 36-48 h after dosing. The bile with three volumes of acetonitrile was centrifuged at 10,000 rpm/min for 5 min [35]. The study was carried out in compliance with the ARRIVE guidelines. (https://arriveguidelines.org, accessed on 13 July 2018).
The tissues collection: The rats were sacrificed 4 h after oral administration then anesthetized with 2% pentasorbital sodium (45 mg/kg), collected heart, liver, spleen, lung, kidney, stomach, and small intestine. All tissue samples were washed with saline to remove the blood and content, blotted on filter paper, weighed, and were added three volumes of saline for homogenate. Equal volume methanol was added to the homogenate and vortexmixed for 1 min, then centrifuged at 10,000 rpm/min for 15 min, and the supernatant was collected. The organic phase was dried and extracted with ethyl acetate and centrifuged at 3000 rpm/min for 5 min, the ethyl acetate phase was extracted by the same method, and the organic phase was evaporated to dryness. Finally, the residues were redissolved by using 100 µL 5% acetonitrile and vortex-mixed for 3 min [36]. The study was carried out in compliance with the ARRIVE guidelines. (https://arriveguidelines.org, accessed on 13 July 2018).
All biological samples were stored at −80 • C and filtered through a 0.45 µm membrane filter until analysis.

Sample Preparation and Treatment In Vitro
Preparation of liver microsomes and cytosol: Rat liver microsomes and cytosol were prepared by previous methods [37]. After anesthetized with 2% pentasorbital sodium (45 mg/kg), the liver of the rats was taken, rinsed with ice-cold normal saline, weighed, and homogenized in 0.1 mM phosphate buffer (pH7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 4 • C for 30 min (10,000× g), and the supernatant was centrifuged at 4 • C for 60 min (105,000× g). The supernatant was liver cytosol and the precipitation was liver microsomes. The protein content was determined by the BCA method [37]. The study was carried out in compliance with the ARRIVE guidelines. (https://arriveguidelines.org, accessed on 13 July 2018).
The rat liver microsomes incubation was prepared as follows: 100 µM Sanguisorba tannins, 1.3 mM NADP, 3.3 mM G-6-P, 0.4 U/mL G-6-P-D, 3.3 mM magnesium chloride. The incubation system was achieved with Tris/KCl buffer (pH 7.4) and its volume was 250 µL. After preincubation for 5 min at 37 • C, add NADPH mixture to start the reaction, which including 1.3 mM NADP, 3.3 mM G-6-P, 0.4 U/mL G-6-P-D, and 3.3 mM magnesium chloride. Incubated at 37 • C for two hours and add equal volume acetonitrile to terminated the reaction. The incubation was centrifuged at 10,000× g for 10 min, the supernatant evaporated to dryness at 40 • C under a gentle stream of nitrogen. The residues were dissolved with 80 µL acetonitrile, and 20 µL aliquot was analyzed by HPLC-LTQ-Orbitrap MS 2 .
The optimal incubation conditions (200 µL reaction volume) for rat liver cytosol were as follows: 100 µM Sanguisorba tannins, 5 mM magnesium chloride, 1 mM dithiothreitol, and 300 µM SAM. The incubation system was achieved with Tris/KCl buffer (pH 7.4), and its volume was 200 µL. After preincubation for 5 min at 37 • C, add rat liver cytosol to start the reaction. Incubated at 37 • C for two hours and add equal volume acetonitrile to stop the reaction. The incubation was centrifuged at 10,000× g for 10 min, the supernatant evap-orated to dryness at 40 • C under a gentle stream of nitrogen. The residues were dissolved in an 80 µL acetonitrile, and 30 µL aliquot was analyzed by HPLC-LTQ-Orbitrap MS 2 .
The rat intestinal microbiota incubation was prepared as follows: 100 µM Sanguisorba tannin was added to the intestinal microbiota cultural solution (200 µL). After incubation for 0, 2, 4, 6, 12, 24, 36, 48, and 72 h at 37 • C in an anaerobic environment, equal volume acetonitrile was added to terminate the reaction. The incubation was centrifuged at 10,000× g for 10 min, the supernatant evaporated to dryness at 40 • C under a gentle stream of nitrogen. The residues were dissolved in an 80 µL acetonitrile, and 30 µL aliquot was analyzed by HPLC-LTQ-Orbitrap MS 2 .

Conclusions
In conclusion, using HPLC-LTQ-Orbitrap MS 2 for analysis, high-resolution mass spectrometry solves the deficiencies of the above methods by virtue of its ultra-high resolution and accurate mass function, especially for multicomponent screening. Additionally, a variety of post-acquisition data mining tools were used. This method has good sensitivity and selectivity and is suitable for qualitative analysis for screening purposes; the metabolites in vivo and in vitro of Sanguisorba tannins were successfully identified. A total of 19 metabolites in vivo and 14 metabolites in vitro were recognized in this study; no active drug, ellagic acid, or other metabolites were detected in rat plasma; after the treatment of the tissues, no active drug or metabolites were detected in them, but ellagic acid was detected in the liver and methyl ether glucuronide in the kidney. Moreover, seven metabolites in vitro were also found in vivo; N5, N8, and N9 were detected in vitro but not found in vivo, which indicated metabolic mechanism between in vivo and in vitro was different. The basic metabolic changes that occurred in rats were reduction, which provides a basis for other metabolic reactions such as glucuronide conjugation, sulfate conjugation, and methylation. Finally, the metabolic profile of Sanguisorba tannins was reviewed. High-performance liquid chromatography-mass spectrometry, combined with various data processing techniques, provides valuable information for the identification of metabolites.
Sanguisorba tannins were the main content in Sanguisorba, which have drawn growing attention due to the prominent biological activities of its metabolites. For example, urolithin A, C, and D can reduce the accumulation of triglyceride, reducing the risk of animation atherosclerosis [39]; urolithiasis A and its sulfate conjugation may protect against breast cancer [40]; urolithin A has anti-inflammatory and antioxidant effects of [41]; urolithin A glucuronide and its aglycone urolithin A ameliorate TNF-alpha-induced inflammation [42], which made a great contribution to biological activity. We hope that the strategies developed in this study and the results achieved in this study will be helpful for future studies on the effectiveness and safety of tannins in Sanguisorba. Based on the analysis of its metabolites, the tannin's metabolism and transport pathway can be speculated in vivo, which further elucidated the mechanism of its role.
However, there are many restrictions. The components of traditional Chinese medicine are complex, not a single ingredient, and therefore, it was not possible to trace the metabolic pathways of individual components in the body. Only by combining in vivo and in vitro, by detecting the metabolites in different organs, we can analyze and detect the metabolic pathways in the body. Further study on the activity of its metabolites will be carried out in the following part to provide the basis for the development and utilization of new drugs.