Investigation on Spectrum-Effect Correlation between Constituents Absorbed into Blood and Bioactivities of Baizhu Shaoyao San before and after Processing on Ulcerative Colitis Rats by UHPLC/Q-TOF-MS/MS Coupled with Gray Correlation Analysis

Baizhu Shaoyao San (BSS) is a crucial traditional Chinese medicinal formula widely applied for the treatment of painful diarrhea, diarrhea-predominant irritable bowel syndrome, ulcerative colitis, and some other gastrointestinal diseases. Corresponding to the clinical medication, the three medicinal herbs (Atractylodis Macrocephalae Rhizoma, Paeoniae Radix Alba, and Citri Reticulatae Pericarpium) included in BSS should be processed using some specific methods of stir-frying. To find the underlying correlations between serum chemical profiles and curative effects of crude and processed BSS on ulcerative colitis rats, and further explore for the effective material basis of processing, an UHPLC/Q-TOF-MS/MS technique coupled with gray correlation analysis (GCA) was developed. A total of 134 compounds were identified in rat sera after oral administration of BSS, among which 24 compounds were prototypes and 110 compounds were metabolites. Meanwhile, an ulcerative colitis model was established in rats by enema with 2,4,6-trinitrobenzene sulfonic acid, and the pharmacodynamic indicators for drug efficacies were evaluated as well. According to the results, processed BSS showed better efficacy than crude BSS. The top 10 potential effective components with high degree of correlation were identified based on GCA results, which were thought to be the crucial compounds that contributed to the enhancement of therapeutic effects in BSS after processing.


Introduction
As a famous and classic herbal formula in traditional Chinese medicine (TCM), Baizhu Shaoyao San (BSS) is composed of Atractylodis Macrocephalae Rhizoma (Asteraceae), Paeoniae Radix Alba

UHPLC/Q-TOF-MS/MS Analysis of Constituents Absorbed into Blood
The typical total ion chromatograms (TICs) of compounds in rat sera after 3 days of administration of crude and processed BSS in negative and positive ion modes are shown in Supplementary Figure S1. The MetabolitePilot TM and PeakView TM softwares were used to extract and identify the constituents absorbed into blood. By comparing with the chromatogram of control serum sample, 134 peaks were identified in drug-containing serums, which consisted of 24 prototype components in BSS and 110 metabolites. The detailed information of the identification results and MS data are shown in Table 1. It could be concluded from the results that the metabolic pathways of lactones from Atractylodis Macrocephalae Rhizoma in rats mainly include glucuronidation, hydroxylation, hydrolysis, and hydrogenation, and reciprocal transformation is possible; the metabolic pathways of glycosides from Paeoniae Radix Alba mainly include methylation, deglycosylation, benzoyloxy group loss, glycine conjugation, and glutamine conjugation; the metabolic pathways of flavonoids from Citri Reticulatae Pericarpium mainly include demethylation, demethoxylation, hydroxylation, methylation, glucuronidation, and deglycosylation; the metabolic pathways of chromones from Saposhnikoviae Radix mainly include deglycosylation, demethoxylation, hydroxylation, and deacetylation; the metabolic pathways of Coumarins from Saposhnikoviae Radix mainly include methylation, hydroxylation, and isomerization.
As a typical example, the proposed metabolic pathways of atractylenolide I in rat sera are illustrated in Figure 1.   The results of IL-6, IL-10, TGF-β1, and TNF-α levels in rat sera after consecutive administration of crude and processed BSS on day 3, 6, 9, 13, 17, and 21 are shown in Figure 2.

Effects of BSS on Serum Cytokines before and after Processing
The results of IL-6, IL-10, TGF-β1, and TNF-α levels in rat sera after consecutive administration of crude and processed BSS on day 3, 6, 9, 13, 17, and 21 are shown in Figure 2.  The results showed that the levels of TNF-α, IL-6, IL-10, and TGF-β1 in rat sera of the 2,4,6-trinitrobenzenesulfonic acid (TNBS) control group were significantly different from those of the sham control group, indicating that the model of ulcerative colitis was successfully established. Specifically, compared with the sham control group on day 3, 6, 9, 13, 17, and 21, the levels of IL-6 and TNF-α in rat serums were significantly elevated (p < 0.001) and the levels of IL-10 and TGF-β1 in rat serums were significantly decreased (p < 0.01), indicating that the rats developed extensive inflammatory responses. However, compared with the TNBS control group, all serum cytokine levels in crude and processed BSS groups were improved to some extents. Especially after drug administration on day 13, the BSS groups exhibited significant difference on cytokine levels from the TNBS control group, among which the processed BSS group led to a better efficacy than the crude BSS group. The results proved that BSS could inhibit the development of ulcerative colitis by reducing the expression of pro-inflammatory cytokines and increasing the expression of anti-inflammatory cytokines in serums, which verified the theory that processing can increase curative effects.

Effects of BSS on Disease Activity Index (DAI) before and after Processing
After one day of modeling, the TNBS-treated rats showed positive reaction in hemoccult test and displayed varying degrees of loose stool. Mental slackness, lazy eating, weight loss, vertical hair, and arched back gradually appeared in the TNBS control group throughout the experiment process. After about seven days of treatment, the conditions of activity, diarrhea, blood stool, and weight loss in the crude BSS, processed BSS, and salazosulfapyridine (SASP) groups were all alleviated to some extent. Table 2 shows the DAI score results after 20 days of drug administration. Compared with the sham control group, DAI was significantly higher in the TNBS control group (p < 0.001). Treatments with SASP, crude, and processed BSS significantly prevented TNBS colitis-induced weight loss and DAI increasement (p < 0.001). Besides, DAI was markedly lower in the processed BSS group than the crude processed BSS group (p < 0.001).

Effects of BSS on Macroscopic and Histopathological Alterations before and after Processing
Compared with the sham control group, the macroscopic colon damage scores of the TNBS control group were markedly increased (p < 0.001). However, the damage scores showed a significant decrease in the crude BSS, processed BSS, and SASP groups (p < 0.001), among which the processed BSS group exhibited better efficacy than the crude BSS group (p < 0.01). The results are shown in Table 2.
The hematoxylin-eosin (HE) staining of pathological sections of rat colon tissues is shown in Figure 3, where the obvious histopathological differences of each group can be observed. The colon tissue structures from the sham control group were complete and continuous, the glands arranged regularly with clear structures, and there were no inflammatory cell infiltration or ulceration. The colon tissues from the TNBS control group exhibited hyperemia, vascular hyperplasia, edema, ulcer formation, massive inflammatory cell infiltration, and partial mucosal necrosis. Intestinal glands and crypts were also disappeared. Compared with the TNBS control group, colonic tissues of rats administered with crude BSS, processed BSS, and SASP were all recovered to some extent. The colon structures of the SASP group were generally clear and intact, the glands were well arranged again, with a small amount of inflammatory cell infiltration and mucosal congestion. An improved colon structure was observed in the crude BSS group, the inflammatory cell infiltration and vascular hyperplasia were mild, but still accompanied with moderate congestion of mucosa. Compared with crude BSS, the processed BSS group was demonstrated better performance in repairing colon injury, the tissue structures were relatively complete and continuous with significant reduction of inflammatory cell infiltration and mucosal congestion.
crypts were also disappeared. Compared with the TNBS control group, colonic tissues of rats administered with crude BSS, processed BSS, and SASP were all recovered to some extent. The colon structures of the SASP group were generally clear and intact, the glands were well arranged again, with a small amount of inflammatory cell infiltration and mucosal congestion. An improved colon structure was observed in the crude BSS group, the inflammatory cell infiltration and vascular hyperplasia were mild, but still accompanied with moderate congestion of mucosa. Compared with crude BSS, the processed BSS group was demonstrated better performance in repairing colon injury, the tissue structures were relatively complete and continuous with significant reduction of inflammatory cell infiltration and mucosal congestion.

Effects of BSS on Protein Expression in TLR4 Signaling Pathway before and after Processing
The influence of BSS on the activation of TLR4 signaling pathway was examined. We assessed a series of signaling molecules, including TLR4, IL-1R1, MyD88, and NF-κB p65 by western Blot, and the results are shown in Figure 4.

Effects of BSS on Protein Expression in TLR4 Signaling Pathway before and after Processing
The influence of BSS on the activation of TLR4 signaling pathway was examined. We assessed a series of signaling molecules, including TLR4, IL-1R1, MyD88, and NF-κB p65 by western Blot, and the results are shown in Figure 4. crypts were also disappeared. Compared with the TNBS control group, colonic tissues of rats administered with crude BSS, processed BSS, and SASP were all recovered to some extent. The colon structures of the SASP group were generally clear and intact, the glands were well arranged again, with a small amount of inflammatory cell infiltration and mucosal congestion. An improved colon structure was observed in the crude BSS group, the inflammatory cell infiltration and vascular hyperplasia were mild, but still accompanied with moderate congestion of mucosa. Compared with crude BSS, the processed BSS group was demonstrated better performance in repairing colon injury, the tissue structures were relatively complete and continuous with significant reduction of inflammatory cell infiltration and mucosal congestion.

Effects of BSS on Protein Expression in TLR4 Signaling Pathway before and after Processing
The influence of BSS on the activation of TLR4 signaling pathway was examined. We assessed a series of signaling molecules, including TLR4, IL-1R1, MyD88, and NF-κB p65 by western Blot, and the results are shown in Figure 4.   As can be seen from the figure, the TLR4, IL-1R1, MyD88, and NF-κB p65 protein expression in the inflamed colon was significantly increased in the TNBS control group compared with the sham control group (p < 0.001). Treatment with Crude BSS, processed BSS, and SASP markedly inhibited TNBS-induced increase of these TLR4-relevant protein expressions (p < 0.05). Compared with crude BSS, processed BSS exhibited a stronger ability in regulating the high expression of TLR4, MyD88, and NF-κB p65 (p < 0.05). Processed BSS also decreased the IL-1R1 level more in the inflamed colon compared with crude BSS, but the difference was not significant.

Results of Gray Correlation Analysis
The calculated results of the inhibition and promotion rates of the cytokine levels after administration of crude and processed BSS on day 3, 6, 9, 13, 17, and 21 are shown in Table 3. GCA was used for investigating the spectrum-effect relationships between the area values of 134 compound peaks detected by UHPLC fingerprints and the anti-ulcerative colitis efficiency characterized by the inhibition and promotion rates of inflammatory cytokines at different time points. Then, the gray correlation degree between these two sets of parameters were calculated, in which the top 10 potential effective components associated with each cytokine sorted according to the degree of correlation are shown in Table 4. As can be seen from the figure, the TLR4, IL-1R1, MyD88, and NF-κB p65 protein expression in the inflamed colon was significantly increased in the TNBS control group compared with the sham control group (p < 0.001). Treatment with Crude BSS, processed BSS, and SASP markedly inhibited TNBS-induced increase of these TLR4-relevant protein expressions (p < 0.05). Compared with crude BSS, processed BSS exhibited a stronger ability in regulating the high expression of TLR4, MyD88, and NF-κB p65 (p < 0.05). Processed BSS also decreased the IL-1R1 level more in the inflamed colon compared with crude BSS, but the difference was not significant.

Results of Gray Correlation Analysis
The calculated results of the inhibition and promotion rates of the cytokine levels after administration of crude and processed BSS on day 3, 6, 9, 13, 17, and 21 are shown in Table 3. GCA was used for investigating the spectrum-effect relationships between the area values of 134 compound peaks detected by UHPLC fingerprints and the anti-ulcerative colitis efficiency characterized by the inhibition and promotion rates of inflammatory cytokines at different time points. Then, the gray correlation degree between these two sets of parameters were calculated, in which the top 10 potential effective components associated with each cytokine sorted according to the degree of correlation are shown in Table 4. Table 3. Inhibition/promotion rates of TNF-α, IL-6, IL-10, and TGF-β1 levels after consecutive administration of crude and processed BSS on day 3, 6, 9, 13, 17, and 21. According to the gray correlation grades listed in Table 4, for each different pharmacodynamic index, the constituents absorbed into blood with relatively high correlation were also different. Among these correlative constituents, it was found that atractylenolide I-M5, a hydroxylated metabolite of atractylode I, was highly correlated with all inflammatory cytokines including TNF-α, IL-6, IL-10, and TGF-β1. In addition, atractylenolide II, atractylenolide III, hydrogenated metabolite of atractylenolide III (atractylenolide III-M5), two hydroxylated metabolites of atractylenolide III (atractylenolide III-M3 and atractylenolide III-M4), hydroxylated and hydrolyzed metabolite of atractylenolide I (atractylenolide I-M2), and di-hydroxylated metabolite of atractylenolide II (atractylenolide II-M2) also demonstrated strong correlation with anti-colitis effect. These atractylenolide-related compounds were all belong to the main ingredients of Atractylodis Macrocephalae Rhizoma (the monarch drug of BSS), and exhibited extensive pharmacological activities including protecting intestinal mucosa, inhibiting inflammation, regulating gastro-intestinal peristalsis, and boosting immunity, according to numerous relative literature [21][22][23][24]. At the same time, based on our previous study [14], the contents of atractylenolide I, atractylenolide II, and atractylenolide III in BSS were significantly increased after processing, and thus the increasement of these components eventually led to the enhancement of in vivo effects, which validated the ancient theory of TCM that 'processing enhancing efficacy'. Meanwhile, it could be interestingly found that most of the metabolites possessed higher grades of correlation in treating colitis compared with the prototype compounds. Through some recent literature [25,26], the lack of hydrophilic groups in the chemical structures of atractylenolide I and atractylenolide II resulted in their low bioavailability after oral administration, while atractylenolide III contained a hydrophilicity structure of hydroxyl, which greatly increased its absorption into blood. Similarly, these atractylenolide compounds increased water solubility through in vivo metabolisms, and thus increased the ratio of blood absorption and enhanced the bioavailability, which could be partially explained that why the metabolites of atractylenolides owned relative higher relevance with the treatment of ulcerative colitis and suggested that the drug metabolism of BSS played a vital role during the process of treatment.

Cytokine
In Meanwhile, it was reported that albiflorin, hesperidin, nobiletin, tangeretin, and prim-O-glucosylcimifugin have the effects of protecting intestinal barrier and resisting experimental colitis [27][28][29][30][31], which were definitely in accordance with the results in our study. According to our previous study [14], among the compounds above, only the content of albiflorin in BSS significantly increased after processing, while other compounds did not change significantly before and after processing, nevertheless, all these components were positively correlated with the enhancement of efficacy after processing. There may be two reasons for this interesting phenomenon. The first reason could be inferred that the intestinal microbial compositions and hepatic drug metabolic enzymes in vivo are too much complicated, and thus some structurally similar constituents with increased contents after processing could be transformed into the above-mentioned metabolites through a series of complex metabolic procedures in the body. For example, catechin, luteolin-7-O-rutinoside, 6-methoxy-naringenin, and nobiletin isomer are flavonoids with increased contents in BSS after processing based on a previous study [14], they share the same parent structure with the flavonoid metabolites that correlated with efficacy enhancement, and probably could be transformed into the target metabolites through some unknown pathways. The second reason might be related to the drug interaction effects in vivo. Although some components in BSS might not exert therapeutic effects by themselves, the variation of contents after processing might influence the absorption and metabolism of other components by affecting the binding sites of membrane transport proteins and the biological activities of drug-metabolizing enzymes, which was closely related to the compatibility theory of TCM according to some published literature [32][33][34][35].
Processing and compatibility together constitute the characteristics of clinical medication in TCM, and that is what TCM can be distinguished from absolute natural medicine [36,37]. As a splendid treasure of science and culture in China, TCM embodies profound and sapiential wisdom and holistic and systematic health concepts. The results of our study provided supporting evidence for the theory concerning the synergistic effects of processing and compatibility in TCM systems. Additionally, the GCA results demonstrated the material basis of BSS, which could be helpful for deeper exploration on its mechanism of action. Also, it would be considered as a primary evaluation standard of BSS for quality control during its clinical application.

Materials and Reagents
Medicinal herbs of Crude Atractylodis Macrocephalae Rhizoma, Paeoniae Radix Alba, Citri Reticulatae Pericarpium, and Saposhnikoviae Radix were all obtained from Jingwan TCM Pieces Factory (Bozhou, China), and were authenticated by Professor Hao Cai, who is the expert in the TCM field. Processed samples of Atractylodis Macrocephalae Rhizoma, Paeoniae Radix Alba, and Citri Reticulatae Pericarpium were prepared corresponding to the processing specifications in the Chinese Pharmacopoeia. Voucher specimens of all medicinal herbs were deposited in School of Pharmacy, Nanjing University of Chinese Medicine (Nanjing, China).

Preparation of Sample Solutions
The solution of crude BSS was prepared according to a previously optimized method [38]. Firstly, crude medicinal herbs of Atractylodis Macrocephalae Rhizoma, Paeoniae Radix Alba, Citri Reticulatae Pericarpium, and Saposhnikoviae Radix were crushed into rough powders, and then mixed together as a ratio of 6:4:3:4. The mixed medicinal herbs were macerated for 1 h in 70% ethanol aqueous solution, and then decocted twice with 70% ethanol aqueous solution (1:12, w/v) using reflux extraction, 2 h for each time. Subsequently, the solution was filtered through a filter paper. The filtrates were combined together and concentrated under vacuum at 55 • C to a density of 1.6 g/mL for oral administration of rats. Similarly, the solution of processed BSS was prepared in the accordant procedure using processed Atractylodis Macrocephalae Rhizoma, processed Paeoniae Radix Alba, processed Citri Reticulatae Pericarpium, and crude Saposhnikoviae Radix.

Animals
Male Sprague-Dawley rats weighing 240-260 g were supplied by Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China, Specific Pathogen-Free grade, certificate No.: SCXK-2016-0011). The humidity and temperature of the experimental environment were kept constant, and light/dark conditions were altered for each 12 h every day. Standard food and water were provided ad libitum. Prior to the experiments, all the rats were acclimatized to the laboratory environment for one week. Before model establishment, rats were fasted for 24 h with water taken freely. The animal experiment was conducted according to the Guide for the Care and Use of Laboratory Animals. The animal facilities and protocols were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine.

Induction of Ulcerative Colitis and Experimental Design
In this study, experimental ulcerative colitis was induced with TNBS enema by a published method with some modification [39]. Briefly, 24 h fasted rats were anesthetized with intraperitoneal injection of 1.5% pentobarbital sodium aqueous solution (3 mL/kg) and residual feces at the end of the rectum were gently squeezed out. Then a disposable silicone catheter with an external diameter of 2.0 mm was inserted rectally into the colon at 8 cm proximal to the anus verge by vaseline lubrication. TNBS dissolved in 30% (v/v) ethanol solution was slowly instilled into the colon via the catheter at a dose of 100 mg/kg to induce ulcerative colitis. In the sham control experiments, rats received saline alone using the same method. Rats were kept in a head-down position for 1 min after the instillation, in order to prevent leakage and distribute the agents within the entire colon.
After colon instillation, rats were randomly divided into five groups with 10 rats of each. Group 1 (sham control) and group 2 (TNBS control) were administrated with saline, group 3 (crude BSS) and group 4 (processed BSS) were given crude and processed BSS respectively at a dose of 16 g/kg, and group 5 (SASP) was given SASP at a dose of 300 mg/kg. All the treatment drugs, including saline, SASP, and crude and processed BSS, were given to rats by gavage once a day for 21 consecutive days. Blood samples (about 0.8 mL) of rats in groups 1-4 were collected from the orbital vein in centrifuge tubes at 90 min of day 3,6,9,13,17, and 21 of administration, respectively. The collected blood samples were then kept for 30 min at room temperature and centrifuged at 4500 r/min for 5 min, and the supernatant serums were stored at -80 • C for further analysis. 24 h after the last treatment, all rats were sacrificed under anesthesia. The distal 10 cm portion of colon was excised, dissected along the longitudinal mesentery, freed of adherent adipose tissue, rinsed with ice-cold saline, and stored at -80 • C for efficacy evaluation.

Instruments
For analysis of the multiple constituents absorbed into blood, a Shimadzu LC-30A UHPLC system (Shimadzu, Kyoto, Japan) coupled to a hybrid quadrupole time-of-flight tandem mass spectrometer (LC/MS-Triple TOF TM 5600 + , AB Sciex, Concord, ON, Canada) equipped with an electrospray ionization (ESI) interface was employed. The Analyst TF 1.6 software was installed for high efficiency data collection and processing.

Mass Spectrometry
The mass spectrometry was operated in both positive and negative ion modes. The MS method consisted of a TOF MS scan and IDA-fragmentation TOF MS/MS scan with mass ranges of m/z 100-1500 and 50-1000, respectively. The parameter settings were optimized as follows: ion spray voltage floating, +5500/-4500 V; turbo spray temperature, 550 • C; declustering potential, 60 V; nebulizer gas (gas 1), 55 psi; heater gas (gas 2), 55 psi; curtain gas, 35 psi. Nitrogen was used as the nebulizer and auxiliary gas.

Preparation of Rat Serum Samples
Liquid-liquid extraction was applied to prepare the rat serum samples. Rat serum (100 µL) was spiked into a centrifuge tube and then mixed with 1000 µL of ethyl acetate by vortex mixing for 5 min. The mixture was separated by centrifugation at 12,000 r/min for 5 min, and the upper layer was then transferred to another tube and evaporated to dryness under high purity nitrogen at room temperature. The residue was reconstituted with 100 µL of methanol, vortexed for 3 min, and centrifuged at 12,000 r/min for 5 min. The final supernatant was used for UHPLC/Q-TOF-MS/MS analysis.

Identification of Constituents Absorbed into Blood
After data acquisition, MetabolitePilot TM (version 2.0, AB Sciex, Concord, ON, Canada) and PeakView TM software (version 1.2, AB Sciex) were employed to search for the constituents absorbed into blood. Firstly, by searching from such databases as SciFinder, PubMed, and CNKI, the chemical compounds reported in the literature [40][41][42][43][44][45][46] belonging to Atractylodis Macrocephalae Rhizoma, Paeoniae Radix Alba, Citri Reticulatae Pericarpium, and Saposhnikoviae Radix were all summarized to establish an in-house compounds library, which includes names, molecular formulas, and chemical structures. Meanwhile, a metabolic pathway template including phase I and phase II metabolism were concluded based on the properties of chemical structures of constituents in BSS. Then, these databases of compounds and metabolite pathways were imported into MetabolitePilot TM software to analyze the prototypes and metabolites in serum of each sample using the functions of generic peak finding, predicted metabolites, isotope pattern, and mass defect. Identification of peaks was based on retention time (RT) and m/z data pairs. When ions in different samples demonstrated both the same RT (tolerance of 0.1 min) and the same m/z value (tolerance of 10 ppm), they were then defined as the same ions. These procedures were completed by comparing a control sample to eliminate those chromatographic peaks belonging to endogenous components. Afterwards, PeakView TM software was used to confirm the validity of the identified compounds according to mass accuracy and matching rate of MS/MS fragmentation profiles.

Evaluation of Anti-Ulcerative Colitis Effects
3.6.1. Determination of TNF-α, IL-6, IL-10, and TGF-β1 Levels in Rat Serums Blood samples in group 1-4 collected at different time were used to assess the cytokine levels after administration of crude and processed BSS. The levels of TNF-α, IL-6, IL-10, and TGF-β1 in rat serums were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instruction (Jin Yibai Biological Technology Co., Ltd.). Absorbance was measured with a microplate reader at 450 nm.

Assessment of Disease Activity Index
Disease activity index (DAI) was assessed based on weight loss, stool consistency, and blood in stool. Severity of the DAI was scored on a scale of 0-4 according to the previously scoring system [47] described by Table 5. All groups of rats were monitored after 20 days of administration to assess the disease process and treatment effect. The results of DAI were expressed as the average score of weight loss, stool consistency, and blood in stool. Macroscopic colon damage was scored on a scale of 0-5 by the pathologists who were blinded to the group, using a previously validated scoring system with slight modifications [48]. Briefly, scoring of macroscopic colon damage was as follows: 0, no ulcer and no inflammation; 1, local hyperemia, no ulcer, and smooth colon surface; 2, hyperemia edema, slight ulcer, and ankylenteron; 3, moderate hyperemia edema, local colonic wall thickening, and mucosal necrosis and ulcer extending less than 1 cm 2 ; 4, severe hyperemia edema, colonic wall thickening or necrosis (2-4 cm 2 ), and mucosal necrosis and ulcer extending more than 1 cm 2 ; 5, severe hyperemia edema, extensive colonic wall thickening or necrosis (>4 cm 2 ), and mucosal necrosis and ulcer extending more than 1 cm 2 . After macroscopic observation, about 0.5 cm of the representative colon tissue in each rat was taken from the region of the inflamed site and was fixed in 4% paraformaldehyde for histopathological section. The fixed tissues were dehydrated, embedded in paraffin, and finally 4 µm thick sections were obtained. The samples were then deparaffinized, hydrated, cleared with xylene, and stained with hematoxylin and eosin (HE) for histological assessment.

Western Blot Analysis
The protein concentration was determined using the BCA procedure (Thermo, Waltham, MA, USA). Protein in the colon tissue from each sample was resolved by SDS-PAGE electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, and then blocked with 5% non-fat dry milk at room temperature for 120 min. After washing with PBS containing 0.05% Tween-20 (PBST) for three times, membranes were incubated overnight at 4 • C with TLR4, IL-1R1, MyD88, NF-κB p65, and β-tubulin antibodies. The membranes were then washed three times in PBST and incubated at room temperature for 120 min with the corresponding secondary antibody conjugated to horseradish peroxidase. The densities of protein bands were quantified by the average ratios of integral optic density following normalization to the β-tubulin expression.

Statistical Analysis
All the results were expressed as mean ± SD. Statistical analysis was performed with SPSS 22.0 software. One way analysis of variance and Bonferroni t test were used to analyze the statistical significance of any difference among groups. p-values less than 0.05 were considered significantly different.

Gray Correlation Analysis
The basic idea of GCA is to investigate on the correlation degree between two sets of variations. In this work, GCA was applied to find out the spectrum-effect relationships between peak areas of diverse compounds absorbed into blood in UHPLC fingerprints after oral administration of crude and processed BSS and their corresponding biological activities in ulcerative colitis rats at different time-points during the treatment process.
Then, the correlation coefficient is defined as follows: ρ is the distinctive coefficient lying between 0-1, and it is generally set as 0.5. The gray correlation degree is the arithmetic mean of the correlation coefficient at different time-points, and is formulated as follows: ζ(X 0 , X i ) = 1 n n ∑ k=1 ζ(X 0 (k), X i (k)) (2) In this work, serum cytokine levels were selected as the pharmacodynamic indexes for GCA study. Prior to the GCA fitting, data pre-processing procedure should be made to the pharmacodynamic results due to the difference of positive and negative correlation between TNF-α, IL-6, IL-10, and TGF-β1 levels and the drug efficacy. Accordingly, the inhibition rate of TNF-α and IL-6 and the promotion rate of IL-10 and TGF-β1 were used to perform the GCA calculations instead of the absolute values of these inflammatory cytokines [49]. Specifically, the algorithm formula was as follows: Inhibition/promotion rate = cytokine levels in TNBS group−cytokine levels in treatment group cytokine levels in TNBS group−cytokine levels in sham control group (3) Then, the inhibition and promotion rates of serum cytokine levels in crude and processed BSS groups at different time-points were set as the sequence of system behavior characteristics, while the UHPLC peak areas of diverse compounds absorbed into blood were set as sequence of system associated factors. Due to the different dimensions of two sets of data, dimensionless processing should be carried out on the raw data to eliminate the adverse effects brought by inconsistent units. Specially for dimensionless processing, each value of data was divided by the average value of the corresponding sequence, and the resultant data would be employed to calculate the correlation coefficients.

Conclusions
In this study, a GCA statistical method combining UHPLC/Q-TOF-MS/MS chemical profile and biological effect evaluation was first developed and successfully applied to investigate the underlying correlation between constituents absorbed into blood and bioactivities of crude and processed BSS. The results showed that processed BSS had better efficacy than crude BSS for the treatment of ulcerative colitis. The material basis of processing that contributed to the difference of therapeutic effects in crude and processed BSS was also found. In a word, our study can provide a scientific foundation for safety and efficacy of BSS in the clinic. Moreover, the strategy of this research could provide a valuable reference for revealing the therapeutic material basis of other TCMs or complex drug systems.
Supplementary Materials: The following are available online, Figure S1: Typical total ion chromatograms (TICs) of constituents absorbed into blood of crude and processed BSS in both positive and negative ion modes. (A): crude BSS; (B): processed BSS.