In Vitro And in Vivo Metabolism Of Cistanche Tubulosa Extract in Normal And Chronic Unpredictable Stress-induced Depressive Rats

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Yang Li, et al

ABSTRACT

Cistanche tubulosa, one species of Cistanches Herba, was recently confirmed to have antidepressant efficacy in chronic unpredictable stress (CUS) rats by restoring homeostasis of intestinal microbiota. In this paper, we aim to explore the metabolic profile of C. tubulosa in normal and CUS-induced depressive model rats in vitro and in vivo. Using UPLC-Q-TOF-MS, the in vitro gastrointestinal metabolism of Cistanche tubulosa extract (CTE) was evaluated in both normal and CUS rats. At the same time, in vivo metabolism of CTE (Cistanche tubulosa extract) in normal and depressed rats was also investigated in rat urine and feces. A total of 20 and 26 metabolites were characterized from in vitro and in vivo metabolism in normal and CUS rats, respectively. CTE (Cistanche tubulosa extract) was metabolized to aglycones and degradation products of phenylethanoid glycosides (PhGs) and iridoid glycosides whether by normal or depressed rat intestinal microbiota in vitro. Phase II metabolites of aglycones and degradation products of PhGs and iridoid glycosides were the main metabolites in rat urine and feces. Additionally, the metabolic capability to generate secondary glycosides and aglycones in depressive rat intestinal microbiota was much weaker than that in normal rat intestinal microbiota, which was attributed to the disordered glycoside hydrolases produced by intestinal microbiota in CUS depressed rats. The results of this study laid the foundation for understanding the metabolic process and therapeutic mechanism of CTE's antidepressant property.

Keywords: Cistanche tubulosa, Depression Metabolism, In vitro, In vivo, Intestinal microbiota

1. Introduction

Cistanches Herba is officially recorded as the dried succulent stems of Cistanche deserticola (Y. C. Ma) and C. tubulosa (Schrenk), which is used to treat kidney deficiency, impotence, female infertility, morbid leucorrhea, profuse metrorrhagia, and senile constipation [1]. Modern pharmacological studies have shown that Cistanches Herba possesses various biological activities such as anti-neurodegeneration, immunoregulation, and anti-inflammation [2,3]. Our previous investigations have verified that Cistanche tubulosa extract (CTE), which are consisted of 48.6% phenylethanoid glycosides (PhGs), 6.9% iridoid glycosides, and 20.0% total saccharides, could markedly alleviate depressive symptoms of chronic unpredictable stress (CUS)-induced depressive rats by restoring homeostasis of gut microbiota [4]. Recent studies indicate that changes in the intestinal microbiota composition were associated with the development and progression of depression [5,6]. The relative abundances of the microbial genera were markedly disturbed in CUS depressive model rats compared with normal controls [7]. In depressed patients, the diversity and richness of intestinal microbiota were also significantly altered [8]. Moreover, various compounds including phenylethanoid glycosides (PhGs) and iridoid glycosides were considered as the main constituents of Cistanches Herba [2,3], which were easily metabolized into their secondary glycosides and aglycones including hydroxytyrosol (HT), 3,4-dihydroxyphenethyl glycoside, deglycosylated geniposidic acid, etc. by human intestinal microbiota. These metabolites are more easily absorbed through the intestine and exert biological activity consistent with the prototype component [9–11]. Thus, we believe that during the occurrence and development of depression, the disturbance of intestinal microflora structure will inevitably affect the metabolism of oral traditional Chinese medicines (TCMs) in the gastrointestinal tract, in addition to affecting the physiological state of the host. Most of the existing metabolic data of Cistanches Herba come from metabolic studies on healthy animals [12–15]. Therefore, it would be of more clinical significance to investigate the metabolic profile of CTE (Cistanche tubulosa extract) in the pathological state in elucidating its bioactive components and understanding the mechanism of action for its anti-depressive efficacy.

In the current study, we aim to characterize the metabolic profiles of CTE (Cistanche tubulosa extract) in both healthy and CUS-induced depressive model rats by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Gastric juice, intestinal fluid, and microbiota of normal and depressive pathological rats have been used to simulate the metabolic process of CTE (Cistanche tubulosa extract) in the gastrointestinal tract in vitro, independently and sequentially. In vivo metabolites are also elucidated after oral administration of CTE (Cistanche tubulosa extract) in normal and CUS rats. This study provides new insights into the metabolism and active metabolites of CTE (Cistanche tubulosa extract) for depression.

2. Material and methods

2.1. Material

Dried stems of C. tubulosa were collected from Hetian County (Xinjiang, China). The voucher specimen samples were authenticated by Prof. Xiaobo Li and deposited at the herbarium of the School of Pharmacy, Shanghai Jiao Tong University (Shanghai, China). The extraction method was used as specified in our previous publication [4]. TheCistanche tubulosa extract (CTE) samples were stored at 4 °C and re-dissolved with sterile water before use. The sterile water solutions of the CTE (Cistanche tubulosa extract) sample were then filtered through a 0.22 μm membrane, and the filtrates were collected in sterile tubes.

Echinacoside was provided by Dr. Pengfei Tu's laboratory, Peking University (Beijing, China). Acteoside, isoacteoside, 2′-acetylacteoside, and cistanoside A were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Hydroxytyrosol, caffeic acid, 3, 4-dihydroxybenzenepropionic acid, 3-hydroxyphenylpropionic acid, and 3-phenylpropionic acid were purchased from Aladdin Industrial Inc. (Shanghai, China). The purity of each component was determined to be > 95% by HPLC-UV. HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Deionized water was prepared from distilled water using a Milli-Q water purification system (Millipore, Bedford, MA, USA). All other reagents and chemicals used were of analytical grade.

2.2. Animal experiments

Male Sprague-Dawley rats (200 ± 20 g) were procured from Beijing Vital River Laboratory Animal Technology Company (Beijing, China), and housed in the Laboratory Animal Center of Shanghai Jiao Tong University (Shanghai, China). The animals were group-housed under controlled room temperature (25 ± 2 °C, 55 ± 10% relative humidity) with a 12:12 h light-dark cycle. The rats were allowed free access to regular laboratory rats chow and water for 1 week. The animal facilities and protocols were approved by the Animal Ethics Committee of Shanghai Jiao Tong University (Shanghai, China).

After one-week acclimation, twelve naive rats were randomly divided into two groups (n = 6), the control group and the chronic unpredictable stress (CUS) group. CUS rats were developed as in our previous report [4], which were subjected to a variety of stressors: white noise (100 dB) for 1 h, overnight low-intensity stroboscopic illumination (120 flashes/min), water deprivation for 24 h, empty water bottles for 1 h (after water deprivation), food deprivation for 24 h, physical restraint (1−2 h), forced swimming (5 min), soiled cage for 24 h (200 mL water in 100 g sawdust bedding), tail pinch (1 min), shock for 30 min, 45° cage tilt for 24 h, and overnight illumination (12 h). Stressors were applied continuously and randomly for 4 weeks, detailed arrangement is described in Table S1. After 4 weeks of stress, the sucrose preference test, open-field test, and novelty-suppressed feeding test were performed as described previously [4]. The outline of the CUS and the behavioral test is shown in Fig. S1. After behavioral tests, at least 4 fecal pellets were obtained from each rat and placed in sterile conical tubes for in vitro analysis of CTE (Cistanche tubulosa extract) metabolism.

2.3. Gastrointestinal metabolism of CTE (Cistanche tubulosa extract) by normal and CUS rat in vitro

2.3.1. Metabolism of CTE (Cistanche tubulosa extract) in simulated gastric and intestinal juices

Fifty milligrams of CTE (Cistanche tubulosa extract) were added to 10 mL of simulated gastric juice and intestinal juice, respectively. Then CTE (Cistanche tubulosa extract) was incubated at 37 °C for 4 h in gastric juice and 6 h in intestinal juice. The cultured mixture (1 mL) was quenched with 3 mL of water-saturated n-butanol immediately at 0 and 4 h in gastric juice, and at 0 and 6 h in intestinal juice. The processing method of the sample used was as previously described [9].

2.3.2. Metabolism of CTE (Cistanche tubulosa extract) by normal and CUS rat intestinal microbiota

Fresh normal and CUS rat fecal samples were firstly mixed and homogenized with 25 times the volume of GAM broth. Sediments were removed by filtration through three pieces of gauze. The suspension was then incubated at 37 °C in an anaerobic incubator in which the air was replaced by a gas mixture (H2 5%, CO2 10%, N2 85%). Fifty milligrams of CTE were added to 5 mL normal and CUS rat fecal suspension separately, and the suspension was incubated at 37 °C for 48 h. The cultured mixture was removed and extracted with water-saturated nbutanol at 0, 12, 24, and 48 h. The sample processing method was as previously described [9].

2.3.3. Sequential metabolism of CTE (Cistanche tubulosa extract) by the gastric juice, intestinal juice, normal and CUS rat intestinal microbiota

Firstly, 100 mg of CTE (Cistanche tubulosa extract) was added to 10 mL simulated gastric juice and incubated at 37 °C for 4 h. The whole reaction was quenched by 3 times the volume of water-saturated n-butanol, and centrifuged at 3000 rpm for 15 min, followed by evaporation of the supernatant under a stream of nitrogen gas at 37 °C. Secondly, the residue was re-dissolved in 0.4 mL of sterile water, added to 8 mL of simulated intestinal juice, and incubated at 37 °C for 6 h. The sample with gastric juice was pre-pared in the same manner. Finally, the residue was re-dissolved in 0.3 mL of sterile water, added to 6 mL normal and CUS rat fecal suspensions respectively, and incubated at 37 °C for 48 h in an anaerobic incubator. One milliliter of the reaction was quenched by 3 mL of water-saturated n-butanol immediately at 0 and 4 h in gastric juice, at 0 and 6 h in intestinal juice, and at 0, 12, 24, and 48 h in rat intestinal microbiota. The sample was processed identically CTE (Cistanche tubulosa extract) in simulated gastric juice.

2.4. Metabolism of CTE (Cistanche tubulosa extract) by normal and CUS rat in vivo

Each rat in the two groups was then housed in an individual metabolic cage. Following overnight fasting that only allowed free access to water, all rats were orally administered with 2 mL of water via a gastric tube. Blank urine and fecal samples were collected from all rats from 0 h to 12 h. Further, CTE (Cistanche tubulosa extract) (400 mg/kg) was administered by gavage. Urine and fecal samples were collected from 0 h to 24 h. All urine and fecal samples were stored at −80 °C immediately.

Urine and fecal samples from normal and CUS rat were pretreated as previously described [12]. All resulting samples were analyzed by UPLC-Q-TOF-MS.

2.5. Analytical method

UPLC was performed on a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm, Waters Corp., USA) by gradient elution using 0.1% formic acid acetonitrile (A) and 0.1% formic acid in water (B) at a flow rate of 0.4 mL/min. The gradient profile was: 0–5 min (A: 5–20%), 5–7.5 min (A: 20–30%), 7.5–10 min (A: 30–70%), 10–11 min (A: 70–100%), and was held for 1.5 min. The gradient was recycled back to 5% in 0.5 min and was held for 2.5 min for the next run. The injection volume was 3 μL. The temperature of the column oven was set to 35 °C.

Mass spectrometry was carried out using a Waters Vion IMS mass spectrometer (Waters Corp., Milford, MA, USA). Ionization was performed in the negative electrospray (ESI−) mode. The MS parameters were as follows: capillary voltage, −2.0 kV; cone voltage, 20 V; source temperature, 120 °C; desolvation temperature, 500 °C; gas flows of cone and desolvation, 50 and 1000 L/h, respectively. For accurate mass measurement, leucine-enkephalin was used as the lock mass to generate an [M–H]− ion (m/z 554.2615). An MSE (Mass SpectrometryElevated Energy) experiment in two scan functions was carried out as follows: function 1 (low energy): m/z 50–1000, 0.25 s scan time, 0.02 s interscan delay, 6 eV collision energy; function 2 (high energy): m/z 50–1000, 0.25 s scan time, 0.02 s inter-scan delay, collision energy ramp of 20–45 eV.

2.6. Data processing

The data were processed using UNIFI 1.8.1 software (Waters Corp., Milford, MA, USA) for metabolites identification within the accurate mass full-scan raw data collected through MSE. Compounds were identified based on accurate mass, fragments in high-energy mass spectrometry. The intensity threshold was set as 100.0 counts. The target identification, fragment match tolerance, and other parameters were automatically set.

3. Results

3.1. Behavioral changes in the CUS induced depression rat

The rats with CUS-induced depressive symptoms were assessed by behavioral tests including sucrose preference test, open-field test, and novelty-suppressed feeding test. Student's t-test revealed that sucrose preference in sucrose preference test (p < .001), total distance covered in the open-field test (p < .001), and latency to eat in novelty-suppressed feeding test (p < .01) were significantly different compared with the control group after 4-week CUS treatment (Fig. 1). These findings indicated that the chronic unpredictable stress model was successfully developed.

3.2. Characterization of chemical constituents of CTE (Cistanche tubulosa extract)

A comprehensive analysis of prototype constituents of CTE (Cistanche tubulosa extract) was carried out by UPLC-Q-TOF-MS. In total, 27 constituents from CTE (Cistanche tubulosa extract) were detected and tentatively characterized, including 20 PhGs, 5 iridoids and iridoid glycosides, and 2 oligosaccharides. Detailed information including retention time, accurate MS, and MS/MS fragment ions are listed in Supporting information (Table S2) to provide insight into the structure of these chemical constituents. UPLC-Q-TOF-MS total ion chromatogram (TIC) of CTE was shown in Fig. S2.

3.3. Gastrointestinal metabolism of CTE (Cistanche tubulosa extract) by normal and CUS rat in vitro

In this study, the potential metabolites of CTE (Cistanche tubulosa extract) by normal and CUS rats in vitro were detected from the TICs and identified with a combination of elemental compositions and MS/MS fragment mass spectra after comparing them to the control samples. All of the metabolites from CTE (Cistanche tubulosa extract) in simulated gastric and intestinal juices, normal and CUS rat intestinal microbiota are listed in Table 1.

3.3.1. Metabolism of CTE (Cistanche tubulosa extract) in simulated gastric and intestinal juices

Seven metabolites of CTE (Cistanche tubulosa extract) in simulated gastric juice were tentatively identified by accurate mass and MSE fragment information: M1 (m/z 315.1074, C14H20O8, 1.66 min), M4 (m/z 459.1501, C20H28O12, 2.36 min), M5 (m/z 445.1715, C20H30O11, 2.60 min), M7 (m/z 179.0338, C9H8O4, 2.85 min), M12 (m/z 785.2481, C35H46O20, 4.77 min), M16 (m/z 827.2580, C37H48O21, 5.73 min), and M18 (m/z 623.1968, C29H36O15, 5.81 min). Deglycosylation, dehydroxylation, dehydrogenation, and isomerization were considered as the main metabolic pathways for CTE in gastric juice. M4 and M5 were found to have a molecular weight of 2 Da and 16 Da lower than their prototype component, decaffeoylacteoside, and thus identified as its dehydrogenated and dehydroxylated products, respectively. M12 was identified as an isomer of echinacoside, producing the same ions as echinacoside at m/z 623.2178, 477.1601, 315.1055, 161.0237.

Same metabolites were detected after CTE (Cistanche tubulosa extract) incubation in intestinal juice. It is noteworthy that the caffeoyl group at the C-6′ position in PhGs was readily metabolized by digestive enzymes in intestinal juice to produce its decaffeyl metabolites and caffeic acid.

3.3.2. Metabolism of CTE (Cistanche tubulosa extract) by normal and CUS rat intestinal microbiota

A total of 20 metabolites bio-transformed from CTE (Cistanche tubulosa extract) in normal rat intestinal microbiota were detected and identified (Fig. 2). From the results, it was observed that PhGs were degraded to aglycone hydroxytyrosol (HT) M2 (m/z 153.0550, C8H10O3, 1.78 min), and caffeic acid (CA) M7 (m/z 179.0338, C9H8O4, 2.85 min), then they were further metabolized to M3 (m/z 163.0390, C9H8O3, 2.02 min), M6 (m/z 181.0501, C9H10O4, 2.76 min), M10 (m/z 195.0655, C10H12O4, 4.35 min), and M11 (m/z 165.0552, C9H10O3, 4.36 min) through dehydroxylation, reduction, and methylation. In addition, the central metabolic pathways that produced direct metabolites of PhG prototype compounds from CTE in normal rat intestinal microbiota are reduction, methoxylation, deglycosylation, decaffeoyl, dehydrogenation, and isomerization.

After incubation in CUS-induced depression rat intestinal microbiota, CTE (Cistanche tubulosa extract) was converted to 20 metabolites through the same metabolic pathways as normal rats.

3.3.3. Sequential metabolism of CTE by the gastric juice, intestinal juice, normal and CUS rat intestinal microbiota

After sequential incubation in gastric juice, intestinal juice, normal, and CUS rat intestinal microbiota, CTE (Cistanche tubulosa extract) was metabolized to 14 metabolites (including 8 with gastric juice, 7 with intestinal juice, 11 with normal, and 10 with CUS rat intestinal microbiota). Among these, M2 (HT) and M11 (3-hydroxyphenylpropionic acid, 3-HPP) were the final metabolites of PhGs after sequential incubation of CTE (Cistanche tubulosa extract) in gastric juice, intestinal juice, and intestinal microbiota. There was no significant difference in metabolites between normal and CUS rat.

M8, M9, M14, M17, M19, and M20 were only detected in the in-dependent metabolism of CTE by normal and CUS rat intestinal microbiota. These metabolites were mainly metabolic intermediates that had been completely metabolized to final metabolites in the study of the sequential metabolism of CTE and are therefore difficult to be detected.

3.3.4. Differences between the metabolic rate of CTE by normal and CUS rat intestinal microbiota

To elucidate the differences between the metabolic rate of CTE (Cistanche tubulosa extract) by normal and CUS rat intestinal microbiota, the relative contents of 27 prototype compounds and 20 metabolites after incubation with gastric juice, intestinal juice, normal and CUS rat intestinal microbiota were determined separately and sequentially (Tables S3 and S4). The results indicated that although there were no significant differences between CTE (Cistanche tubulosa extract) metabolites of normal and depressive rats, a significant difference was observed in their metabolic rates. For example, C2 and C5 were identified as 8-epiloganic acid or its isomer. They were completely metabolized in normal samples within 12 h incubation. In pathologically depressed rat intestinal microbiota, however, they were thoroughly metabolized after 48 h incubation. It was evident that the metabolic rate in the normal rat was faster than that in the CUS rat. Similar results were discovered from C18 (isoacteoside). Moreover, it is noteworthy that the peak area of M12 (isomerization of echinacoside) and M16 (isomerization of tubuloside A) in normal samples were much larger than that in CUS samples, indicating that the isomerization reaction of CTE (Cistanche tubulosa extract) was more prevalent in normal rat intestinal microbiota than in depression rat intestinal microbiota.

3.4. Metabolism of CTE by normal and CUS rat in vivo

By comparing biological samples of the CTE-treated group with blank biological samples, a total of 26 metabolites (compound 1–26) of CTE (Cistanche tubulosa extract) in normal and CUS rats were detected (Table 2). Typical UPLC chromatograms of normal and CUS rat urine samples are presented in Fig. 3.

3.4.1. Characterization of the metabolites of CTE in normal and CUS rat urine

A total of 18 in vivo metabolites of CTE (Cistanche tubulosa extract) in normal rat urine samples were tentatively identified. Degradation metabolites of PhGs including HT and CA, and their further sulfation (compound 1, 2, 3, 5, 8, and 16), methylation (6, 21, 22, and 24), and methoxylation (13 and 14) metabolites were the main metabolites in normal rat urine. Iridoid glycosides were readily metabolized to aglycones (23, 25, and 26) through deglycosylation. It is noteworthy that no prototype component was detected in the normal rat urine sample.

In the depressive rat urine sample, 22 metabolites of CTE (Cistanche tubulosa extract) were detected and characterized. One prototype compound, 8-epiloganic acid, was detected in pathological rat urine. Other metabolites were in accordance with those found in normal rat urine, including sulfated metabolites (1, 2, 3, 8, 10, and 16), methylated metabolites (6, 11, 19, and 22), methoxylated metabolites (13 and 14) of HT and CA, and the aglycones of iridoid glycosides (25 and 26).

3.4.2. Characterization of the metabolites of CTE (Cistanche tubulosa extract) in normal and CUS rat feces

In this study, only one metabolite (compound 20, 3-HPP) of CTE(Cistanche tubulosa extract) was identified in normal rat feces. Most PhGs were first degraded to CA and consequently underwent further metabolism to its major microbial metabolite, 3-HPP. In the fecal sample from the CUS rat, 3 metabolites were tentatively characterized, including sulfated 3-HPP (compound 16), and sulfated HT (compounds 2 and 3).

3.4.3. Differences between in vivo metabolites of CTE in normal and CUS rats

After oral administration of CTE (Cistanche tubulosa extract), the in vivo metabolites showed obvious differences in healthy and depressive model rats. 21 metabolites (compound 1–3, 5, 6, 8–14, 16, 17, and 19–26) were detected in both healthy and CUS rat samples. Compound 23 (deglycosylated geniposidic acid) was identified only in healthy rat samples, while compounds 4 (HT), 7 (8-epiloganic acid), 15 (3, 4-dihydroxybenzenepropionic acid), and 18 (3-HPP glucuronide conjugation) were only detected in CUS model rat samples. In summary, prototype constituents were only detected in CUS rats, whereas, more phase II metabolites were discovered in normal rats.

4. Discussion

In this study, three in vitro incubation models including gastric juice, intestinal juice, normal and CUS rat intestinal microbiota were employed independently and sequentially to investigate the gastrointestinal metabolic profile of CTE (Cistanche tubulosa extract) in vitro. It was found that PhGs and iridoid glycosides in CTE (Cistanche tubulosa extract) were readily metabolized to their secondary glycosides and aglycones by CUS-induced depressive rat intestinal microbiota. After that, in vivo metabolism of CTE (Cistanche tubulosa extract) in normal and CUS rats was also verified. The proposed metabolic pathways for CTE(Cistanche tubulosa extract) in healthy and CUS-induced depressive rats are shown in Fig. 4. PhGs, such as echinacoside and acteoside, were metabolized to HT and CA, and CA underwent further metabolism to its major microbial metabolite, 3-HPP. HT, CA, and 3-HPP were then metabolized to their sulfated, methylated, and methoxylated metabolites. Iridoid glycosides including geniposidic acid, kankanoside A, and kankanoside N were metabolized to their aglycones through deglycosylation. These further demonstrated that PhGs and iridoid glycosides in CTE (Cistanche tubulosa extract) were readily metabolized to secondary glycosides and aglycones in CUS rats. These metabolites normally exhibit better intestinal absorption and bioavailability to be further absorbed into the blood to exert biological activity [16–18]. It's worth noting that isomerization was prevalent for PhGs in the gastrointestinal tract, relevant metabolites were identified after being compared with the UPLC retention time of their prototype compounds based on an optimized ideal UPLC gradient profile.

Caffeic acid was the primary degradation product of CTE (Cistanche tubulosa extract) by depressive pathological rat intestinal microbiota. Previous publications reported that caffeic acid produces antidepressive-like effects in the forced swimming test in mice. Both brain-derived neurotrophic factor (BDNF) mRNA level in the frontal cortex and TrkB mRNA level in the amygdala was significantly decreased after the forced swimming test, and the former reduction was significantly inhibited by caffeic acid [19]. Hydroxytyrosol was the aglycone of PhGs, which protects neurogenesis and cognitive function by preventing stress-induced downregulation of neural protein BDNF [20]. Thus, it is necessary to pay more attention to some bioactive metabolites (i.e., HT and CA) transformed by intestinal microbiota after oral administration.

In addition, the present findings provide evidence that in depressive rat intestinal microbiota, the metabolic capability to generate secondary glycosides and aglycones was markedly weaker than that in normal rat intestinal microbiota. The reason is likely to be attributed to depression-induced structural changes of the intestinal microbiota, which leads to decreased activity of metabolic enzymes produced by intestinal microbiota [21]. Interestingly, a previous study showed that phylum Bacteroidetes encoded the most abundant glycoside hydrolase and polysaccharide lyase genes for the glycoside hydrolysis and the cleavage of complex carbohydrates with an elimination mechanism [22]. Specifically, Bacteroides spp. including B. caccae, B. dorei, B. finegoldii, B. fragilis, B. intestinalis, B. ovatus, B. thetaiotaomicron, B. uniformis, and B. xylanisolvens showed a dominant total number of genes encoding GHs and PLs. Parabacteroides distasonis also possesses the same characteristics [22]. Our previous studies confirmed that 28-day chronic unpredictable stress stimulation decreased relative abundance of genera Bacteroides, Parabacteroides, Butyricimonas, and Weissella, whereas, increased Ruminococcus and Deinococcus in rats [4]. It is noteworthy that Bacteroides and Parabacteroides were the two most abundant microbial taxa that accounted for approximately 20% relative abundance in normal rats. After CUS treatment, relative abundances of Bacteroides and Parabacteroides were sharply dropped to approximately 5% in depressive model rats. Therefore, this will inevitably lead to a reduction in the total number of GH and PL enzymes in CUS rats, and further disturbs the deglycosylated reaction by CUS depressive intestinal microbiota after oral administration of CTE (Cistanche tubulosa extract) in model rats.

5. Conclusion

In the present study, the UPLC-Q-TOF-MS technique was established and applied to screen and identify metabolites of Cistanche tubulosa extract in normal and CUS depressive rats in vitro and in vivo. The results showed that CTE (Cistanche tubulosa extract) was metabolized to aglycones and degradation products of PhGs and iridoid glycosides by both healthy and depressed rat intestinal microbiota. After oral administration of CTE (Cistanche tubulosa extract), phase II metabolites of aglycones and degradation products of PhGs and iridoid glycosides were predominantly found in rat urine. The metabolic capability to generate secondary glycosides and aglycones in depressive rat intestinal microbiota was much weaker than that in the normal rat's intestinal microbiota, which was attributed to the disordered glycoside hydrolases produced by intestinal microbiota in CUS depressed rats. This study provides a new perspective for the later development of CTE (Cistanche tubulosa extract) as a potential antidepressant.

Acknowledgments

This work was supported by grants from the National Key Research and Development Program of China (2017YFC1702400).

Appendix A. Supplementary data

Supplementary data to this article can be found online at HTTPS:// doi.org/10.1016/j.jchromb.2019.121728

From: ' In vitro and in vivo metabolism of Cistanche tubulosa extract in normal and chronic unpredictable stress-induced depressive rats' by Yang Li, et al

--Journal of Chromatography B 1125 (2019) 121728

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