Identification of Chemical Components of Qi-Fu-Yin and Its Prototype Components and Metabolites in Rat Plasma and Cerebrospinal Fluid via UPLC-Q-TOF-MS

Qi-Fu-Yin, a traditional Chinese medicine formula, has been used to treat Alzheimer's disease (AD, a neurodegenerative disorder) in clinical setting. In this study, the chemical components of Qi-Fu-Yin and its prototype components and metabolites in rat plasma and cerebrospinal fluid, after oral administration, were preliminarily characterized via ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS). A total of 180 compounds, including saponins, flavonoids, organic acids, sucrose esters, oligosaccharide esters, phthalides, phenylethanoid glycosides, alkaloids, xanthones, terpene lactones, ionones, and iridoid glycoside, were tentatively characterized. For the first time, 51 prototypical components and 26 metabolites, including saponins, phthalides, flavonoids, sucrose esters, organic acids, alkaloids, ionones, terpene lactones, iridoid glycoside, and their derivatives, have been tentatively identified in the plasma. Furthermore, 10 prototypical components (including butylidenephthalide, butylphthalide, 20(S)-ginsenoside Rh1, 20(R)-ginsenoside Rh1, and zingibroside R1) and 6 metabolites were preliminarily characterized in cerebrospinal fluid. These results were beneficial to the discovery of the active components of Qi-Fu-Yin anti-AD.


Introduction
Traditional Chinese medicine (TCM) plays a vital role in the treatment of various complex chronic diseases owing to the synergistic effects of the formulations and has, accordingly, garnered increasing attention worldwide [1,2]. Qi-Fu-Yin, a TCM prescription, was first recorded in the book Jingyue Encyclopedia written by Jingyue Zhang during the Ming Dynasty. It is composed of seven herbs-Ginseng Radix et Rhizoma (GRR), Rehmanniae Radix Preparata (RRP), Angelicae Sinensis Radix (ASR), Atractylodis Macrocephala Rhizoma Preparata (ARP), Glycyrrhizae Radix et Rhizoma Preparata cum Melle (GRP), Ziziphi Spinosae Semen (ZSS), and Polygalae Radix Preparata (PRP)-in a ratio of 6 : 9 : 9 : 5 : 3 : 6 : 5 [3]. Qi-Fu-Yin has shown significant effects on Alzheimer's disease (AD) in clinical studies [4,5]. Owing to its remarkable therapeutic effects and pharmacological activities, Qi-Fu-Yin has attracted the attention of various researchers. Previous studies showed that Qi-Fu-Yin improves the learning ability and memory of rats injected with advanced glycation end products [6,7] or β-amyloid protein [8,9]. Furthermore, 154 chemical components were unambiguously identified or tentatively characterized in Qi-Fu-Yin using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) [10]. However, it remains unknown which components are absorbed into the plasma and brain after oral administration of Qi-Fu-Yin, which hinders the elucidation of its potentially bioactive constituents and the underlying action mechanisms.
AD is a neurodegenerative disease characterized by the deposition of Aβ and the formation of neurofibrillary tangles in the brain [11]. e ingredients absorbed into blood and that reach a certain concentration can reportedly exert pharmacodynamic effects [12]. e blood-brain barrier (BBB) allows different components to reach the brain and prevents harmful substances from entering the brain. Drugs passing through the BBB can play important roles in brain diseases [13]. Some biotransformed metabolites possess substantial bioactivities and can act as active components [14]. us, it is essential to detect components absorbed into blood and elucidate their metabolic profile, which could reveal the pharmacologically active substances and provide potential resources for discovering new drugs from TCM. In this study, a three-step approach based on UHPLC-Q-TOF-MS was implemented to analyze the multicomponent metabolic profiles of Qi-Fu-Yin in rat plasma and cerebrospinal fluid. First, the Qi-Fu-Yin in vitro chemical component database was established by consulting literature on Qi-Fu-Yin and its seven constituent herbs. e components in vitro were identified by their corresponding MS/ MS fragment ions in standard solutions and databases. Second, the database of the prototype components was established to characterize the prototypical components in rat plasma and cerebrospinal fluid after oral administration of Qi-Fu-Yin. Under the same LC-MS conditions, the prototype components were identified by comparing the standard solutions, extracts, control, and administered biological samples in parallel. Finally, according to the metabolic pathway and secondary mass spectrometry data of prototype components reported in the literature, the metabolites of Qi-Fu-Yin in plasma and cerebrospinal fluid were tentatively characterized ( Figure 1).

Preparation of Samples of Qi-Fu-Yin and the Seven Herbs.
Qi-Fu-Yin was prepared in the laboratory according to the prescribed protocol [3]. Dried pieces of GRR, RRP, ASR, ARP, GRP, ZSS (crushed), and PRP were accurately weighed and immersed in 9 times amount of water for 30 min; then, the samples were serially decocted with 9 times and 7 times amount of water. After mixing and filtering, the extracts were concentrated to a small volume and lyophilized. An appropriate amount of the lyophilized powder was accurately weighed, dissolved in ultrapure water (equivalent to 50 mg crude drug per mL) in a 25 mL volumetric flask, and mixed evenly via ultrasonication for 30 min. en, the extracts were centrifuged at 13000 rpm and 4°C for 10 min and filtered through a 0.22 µm membrane. e seven herb samples of Qi-Fu-Yin were prepared in the same manner as the prescribed method.

Animals and Drug Administration.
Male SD rats, weighing 200 ± 20 g, were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co., Ltd. (Beijing, China). All animal procedures were approved by the Shandong University of Traditional Chinese Medicine Institutional Animal Experimentation Committee (SDUTCM20210119001). All rats were housed at an ambient temperature of 20 ± 1°C with a 12 h light/dark cycle and fed a standard diet and water ad libitum for 3 days before the experiment. e rats were then divided into a control group (orally administered deionized water) and a Qi-Fu-Yin group (orally administered Qi-Fu-Yin) (n � 12). To detect the prototype components and metabolites of Qi-Fu-Yin in the rat plasma and cerebrospinal fluid, an 8-fold clinical dosage (1.72 g crude drug per mL, 10 mL per kg, twice daily) was selected as the oral dose [6,7]. All groups received intragastric administration twice daily for three consecutive days. Before the experiments, the animals fasted for 12 h, with free access to water.

Biological Sample Collection and Preparation.
After the last intragastric administration, 500 μL aliquots of serial blood samples were collected from the postorbital venous plexus vein of each rat at 0.5, 1.0, 2, and 4 h. en, approximately 100 μL of cerebrospinal fluid from each rat was collected at 4 h via percutaneous puncture of the cerebellar medulla cistern [15]. e biological samples collected in heparinized polythene tubes were centrifuged at 3000 rpm at 4°C for 15 min. Subsequently, the supernatant was transferred into new tubes and immediately stored at −80°C before preliminary treatment.
After unfreezing the biological samples in an ice-water mixture, plasma or cerebrospinal fluid was mixed at four different times to enrich the biological samples of each group. To each tube containing 1 mL of plasma or cerebrospinal fluid, 4 mL of methanol was added. e mixture was then vortexed for 2 min and centrifuged at 13000 rpm and 4°C for 10 min. Subsequently, the supernatant was transferred to another tube and dried using sanitary nitrogen gas at room temperature. en, the residue was redissolved in 100 μL of 30% methanol, vortexed for 2 min, and centrifuged at 13000 rpm and 4°C for 10 min.

RRP.
Iridoid glycosides are considered the main components of RRP. e negative ion mode was selected to characterize the RRP components because the fragmentation pathway of glycosyl was easier to detect in the negative ion mode ( Figure S1). According to the fragmentation rules, 12 phenylethanoid glycosides, 2 iridoid glycosides, 3 ionone glycosides, and 1 organic acid were identified. e loss of acyl residues is a characteristic fragmentation pattern of phenylethanoid glycosides. Compound  Step 1 Step 2 Step Evidence-Based Complementary and Alternative Medicine  [22] 4 Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 5 Evidence-Based Complementary and Alternative Medicine 7  Evidence-Based Complementary and Alternative Medicine 9 10 Evidence-Based Complementary and Alternative Medicine  (Table 1). Other compounds were also preliminarily characterized according to MS 1 /MS 2 data and retention times available in the literature.  (Table 1).

GRP.
Flavonoids and saponins are the primary components of GRP. Flavonoids have a cyclohexene structure, which readily occurred owing to reverse Diels-Alder (RDA) cleavage in the negative ion mode. Except for the aglycones of compounds 77 and 127, all flavonoids were flavonoid glycosides, which were subdivided into O-glycosides and C-glycosides owing to the different bonding types between glycosyl and aglycones (Table 1). e former can only be detected by the loss of different types of glycosyl groups (Glc, Api, and others), whereas the latter can also be detected by the fragments of C n H 2n O n generated from crossring cleavage reactions. Saponins can be easily detected by the characteristic fragments of glucuronic acid residues (GlcA) at m/z 351.05 and 193.03 in the negative ion mode. Seventeen flavonoids, 18 saponins, and 1 organic acid were identified according to the fragmentation rules.  3.1.6. ZSS. Flavonoids and saponins are the main components of ZSS. A total of 10 flavonoids, 2 saponins, 9 alkaloids, and 2 organic acids were identified.
Most of the identified flavonoids contained a structure nucleus of spinosin, and a few of them were the common C-glycosyl flavonoids. Fragmentation ions at m/z 327.08 represented the flavonoid base peak of spinosin in the positive ion mode, and m/z 445.11, 427.10, 325.07, and 307.06 were detected in the negative ion mode (Table 1) (Table 1). According to the MS 1 / MS 2 data, eight isoquinoline alkaloids and one cyclopeptide alkaloid were identified.

PRP.
e main components of PRP are xanthones, sucrose esters, oligosaccharide esters, and saponins. Both sucrose esters and xanthones have low molecular weights, whereas oligosaccharide esters and saponins are larger. Based on the fragmentation characteristics of the different types of components, 16 sucrose esters, 14 oligosaccharide esters, 11 saponins, 6 xanthones, and 2 organic acids were identified. e main characteristic of sugar esters in the negative mode is the neutral loss of acyl (acetyl, feruloyl, p-coumaroyl, sinapoyl, and p-hydroxy benzoyl) residues. e presence of Z 2 − , Z 1 − and Z 0 − ions indicated that 3,4,5trimethoxycinnamic acid and sinapoyl moieties were situated on the glucose and fructose residues, respectively. erefore, compound 90 was deduced to be tenuifoliside C, as shown in Figure S3. e fragmentation rule of oligosaccharide esters was similar to that of sucrose esters.      (Table 1). e remaining 15 sucrose esters and 13 oligosaccharide esters were characterized on the basis of fragmentation rules and the literature. e basic structure of saponins in PRP mainly comprised an aglycone substituted at C-3 with a mono-glucosyl saccharide (A-chain) and at C-28 with a second complex oligosaccharide (B-chain). Saponins produced characteristic fragments at m/z 455 and 425 in the negative ion mode because of the easy elimination of CH 2 OH (30 Da) on C-14. erefore, compound 107 was deduced to be polygalasaponin XXVIII (Table 1). According to the fragmentation rules, the remaining 10 saponins were preliminarily characterized.

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Characteristic fragments of C n H 2n O n were found for xanthones due to cross-ring cleavage. erefore, compound 10 was identified as polygalaxanthone III, as shown in Figure 2.

Characterizing the Prototype Components in Plasma after Oral Administration of Qi-Fu-Yin.
e identification process for the prototype components was similar to that used in vitro. Using the same UPLC-Q-TOF-MS conditions, 51 prototype components were preliminarily identified by comparing the components of Qi-Fu-Yin in vitro, including 24 triterpene saponins, 10 phthalides, 8 flavonoids, 4 sucrose esters, 1 organic acid, 1 alkaloid, 1 xanthone, 1 terpene lactone, and 1 ionone. Among them, 10 components were compared with the reference standards, and others were identified by comparing the retention times, fragmentation pathways, and MS/MS spectra (Table 2, Figure 3). Some saponins with low molecular weights can be directly absorbed into blood.  glucuronic acid residues, which were readily detected at m/z 351.056 and 193.0351 in the negative ion mode (Figure 4(b)).  Figure S4).

Characterization of Metabolites in Plasma after Oral
Administration of Qi-Fu-Yin. Twenty-six metabolites were preliminarily identified by comparing with data from the metabolite database, mainly including oxidation, reduction, glucuronidation, and sulfation (Table 2, Figure 5). e pathways of some metabolites are shown in Figure 6. e product ions at m/z 257.08 were also approximately 2 Da more than those at 255.07. Combining these data with the retention time, M5, M7, M8, and M11 were deduced to be hydrogenation and sulfate conjugates of (iso)liquiritigenin ( Figure 6).
M9 and M13 produced the same fragment ions at m/z 267.07, which were believed to be metabolites of formononetin; according to the adduct ions of m/z 443.0984 and 347.0230, they were identified as formononetin-7-Oglucuronide and formononetin-7-O-sulfate, respectively ( Figure 6)  (C 17 H 23 NO 6 S), M12 was identified as an acetylcysteine conjugate of ligustilide I or H (  (44 Da), and were identified as hydroxylate conjugates of glycyrrhetinic acid (Table 2).

Characterization of Prototypical Components and Metabolites in the Cerebrospinal Fluid after Oral
Administration of Qi-Fu-Yin. Using the same UPLC-Q-TOF-MS conditions, 10 prototype components (P8-P10, 23,24,30,32,34,48, and 51) and 6 metabolites (M3, 4, 10, 23, 25, and 27) were preliminarily identified by comparing the components of the drugged rat plasma, among which two components were compared with the reference standards, and others were identified by comparing the retention times, fragmentation pathways, and MS/MS spectra (Table 2 and Figure 7).

Discussion
In recent years, LC-MS technology has been widely used in the analysis of components of TCM, combining the high separation ability of liquid chromatography with the high sensitivity of mass spectrometry [47,48]. Up to now, the only research on the identification of components in Qi-Fu-Yin was based on UPLC-Q-TOF-MS in vitro [10]. In this present study, the same 110 components were detected consistent with previous studies [10], and 70 components were preliminarily identified for the first time in vitro ( Table 1, Table S1). Among them, forty-four reported  components [10] were undetected, and 18 of them were lost due to different scanning ranges (Table S1).
Qi-Fu-Yin consists of seven herbs, but there is no research on the similarities and differences of components between them after decocting. For the first time, upon comparing Qi-Fu-Yin with the seven herbs, the categories of chemical components were found to be unanimous, and the number of flavonoids and organic acids in Qi-Fu-Yin was more than the sum of seven herbs; however, the opposite was true for phenylethanoid glycosides ( Figure S5). Most of the chemical components could be detected in both, but 9 and 13 chemical components were only detected in the seven herbs and Qi-Fu-Yin, respectively, and the configuration of some components changed ( Figure S5, Table 1). is showed that the chemical composition of Qi-Fu-Yin is not a simple addition of compounds in its single herbs.
As far as we know, the prototype components and metabolites of the seven herbs, not Qi-Fu-Yin, in the plasma after oral administration have been reported. For example, saponins in GRR [49], GRP [46], ZSS [50], flavonoids in GRP [51], ZSS [50], phthalides in ASR [36,52], sugar esters in PRP [53], phenylethanoid glycosides, and iridoid glycoside in RRP [54] are the main components in plasma after oral administration of herbs. In this research, 51 prototypical components and 26 metabolites of Qi-Fu-Yin, including saponins, phthalides, flavonoids, sucrose esters, organic acids, alkaloids, ionones, terpene lactones, iridoid glycoside, and their derivatives have been tentatively identified in the plasma for the first time.
Similarly, the prototype components and metabolites in the cerebrospinal fluid after oral administration of Qi-Fu-Yin have not been reported. Several research showed that some saponins in GRR [55,56], GRP [57], and phthalides in ASR [58,59] can be absorbed into the cerebrospinal fluid. In addition, saponins in GRR [60] and GRP [61], flavonoids in ZSS [62], and source esters in PRP [53] have been determined in the brain tissue homogenate. In this research, 10    Evidence-Based Complementary and Alternative Medicine prototypical components and 6 metabolites were preliminarily characterized in the rat cerebrospinal fluid after oral administration of Qi-Fu-Yin. Among them, butylidenephthalide, butylphthalide, 20(S)-ginsenoside Rh 1 , 20(R)-ginsenoside Rh 1 , zingibroside R 1 , and six other metabolites were detected in the cerebrospinal fluid for the first time. Some prototype components, as saponins, phthalides, and sucrose esters, could be directly absorbed into plasma and cerebrospinal fluid, and phthalides had a higher absorption rate ( Figure 8). Some flavonoids, organic acids, alkaloids, xanthones, terpene lactones, and iridoid glycosides could be absorbed into the plasma, whereas other categories of chemical components were not detected in the plasma and cerebrospinal fluid. Studies have shown that glycyrrhetinic acid [57], 3,6′disinapoyl sucrose [63], tenuifolin [64], and senkyunolide I and H [65] can be absorbed into cerebrospinal fluid. Some components have been determined in the brain tissue homogenate [66][67][68], but whether these components can penetrate the BBB is unknown, and they may only exist in the astrocytes and/or vascular endothelial cells constituting the BBB. In this study, 3,6′-disinapoyl sucrose, ginsenoside Rh 1 , butylphthalide, glycyrrhetinic acid, tenuifolin, and senkyunolide I and H were detected in cerebrospinal fluid. Many studies showed that they had promising effects on neuroprotection, antiapoptosis, anti-inflammation, or antioxidative stress (Table 3).
is suggested that these compounds might be potentially active components of Qi-Fu-Yin for treating AD.

Conclusions
In this study, the chemical components of Qi-Fu-Yin in the plasma and cerebrospinal fluid after oral administration of Qi-Fu-Yin were preliminarily characterized using UPLC-Q-TOF-MS. To our knowledge, this is the first systematic investigation of the metabolic profiles of the constituents of Qi-Fu-Yin. In total, 51 prototypical components and 26 metabolites were tentatively identified in plasma. e major phase I metabolic pathway of Qi-Fu-Yin involved hydrogenation and oxidation, whereas that of phase II reactions included sulfate and glucuronic acid conjugation. Furthermore, 10 prototypical components and 6 metabolites, which might be responsible for the potential activity of Qi-Fu-Yin, were preliminarily characterized in the cerebrospinal fluid. is study provides a chemical basis for elucidating the active components of Qi-Fu-Yin that play roles in the treatment of AD and should further motivate research on the mechanisms underlying the anti-AD activity of Qi-Fu-Yin. Protein expression of Bax and caspase-3↓ Antiapoptosis [76] Tenuifolin Aβ 1-42 -induced BV2 cells Amounts of TNF-α, IL-6, and IL-1β↓ Antiinflammation [77] mRNA expression of iNOS and COX-2↓ Amount of NO↓ Antioxidative stress [77] Senkyunolide I Glutamate-induced Neuro2a cells Amount of caspase-3↓ Antiapoptosis [78] Glycyrrhetinic acid BACE1 FRET assay Activity of BACE1↓ Neuroprotection [79] ↓, decrease; ↑, increase; Aβ, amyloid-β; CREB, cyclic AMP response element binding protein; BDNF, brain-derived neurotrophic factor; Bax, Bcl-2 associated X protein; Bcl-2, B cell lymphoma/leukemia-2; NO, nitric oxide; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; IL-6, interleukin 6; IL-1β, interleukin 1β; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MAPK, mitogen-activated protein kinase; BACE1: β-site APP cleaving enzyme 1.

Data Availability
e data used to support the findings of this study are included within the article and are available from the corresponding author upon request.

Ethical Approval
All animal procedures were approved by the Shandong University of Traditional Chinese Medicine Institutional Animal Experimentation Committee (SDUTCM20210119001).

Disclosure
Hengyu Li and Hongwei Zhao are co-first authors. Xiaorui Cheng and Jiafeng Wang are conjointly designated as corresponding authors.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Xiaorui Cheng, Jiafeng Wang initiated and designed the study. Hengyu Li, Hongwei Zhao, and Xiaorui Cheng developed the method and drafted the manuscript. Dongmei Qi and Yong Yang provided experimental platform and equipment. All authors read and approved the final manuscript.