Xiaoyao Pills, a Chinese patent medicine, treats mild and moderate depression: A randomized clinical trial combined with DNA methylation analysis.

BACKGROUND: Xiaoyao pills (XYP) is a commercial Chinese patent medicine used in the treatment of depression. However, the mechanisms underlying its therapeutic effects, as well as the patients who can benefit from XYP, have not been evaluated so far. OBJECTIVES: To this end, we conducted a double-blinded, random, and placebo-controlled clinical trial of orally administered XYP in patients with depression. METHODS: The 17-item Hamilton Depression Rating Scale (HAMD-17) scores were recorded at baseline, and every 2 weeks after the start of treatment. To further elucidate the epigenetic mechanism of XYP, we performed mRNA sequencing and genome-wide DNA methylation sequencing using peripheral blood leukocytes of patients and healthy. RESULTS: XYP effectively alleviated the symptoms in patients with mild or moderate depressive disorders, particularly that of psychomotor retardation. XYP restored aberrant gene expression and DNA methylation patterns associated with depression, and the normalization of DNA methylation correlated with downregulation of several genes. In addition, altered DNA methylation levels in the XYP-treated samples were attributed to increased expression of the DNA methyltransferase DNMT1. CONCLUSIONS: Our study provides new insights into the epigenetic mechanism underlying depression and the therapeutic effects of XYP, along with an experimental basis for using XYP in the treatment of depression. TRIAL REGISTRATION INFORMATION: The name of the registry and number: U.S. CLINICAL TRIALS REGISTRY: The link to the registration: ClinicalTrials.gov ISRCTN12746343 (https://www.isrctn.com/ISRCTN12746343). The name of the trial register is "Efficacy and safety of the Xiaoyao pill for improving the clinical symptoms of stagnation of liver qi (chi) and spleen deficiency". The clinical trial registration number is ISRCTN12746343.

Gut microbiota-based metabolites of Xiaoyao Pills (a typical Traditional Chinese medicine) ameliorate depression by inhibiting fatty acid amide hydrolase levels in brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicines (TCMs) are often prepared in oral dosage forms, making TCMs interact with gut microbiota after oral administration, which could affect the therapeutic effect of TCM. Xiaoyao Pills (XYPs) are a commonly used TCM in China to treat depression. The biological underpinnings, however, are still in its infancy due to its complex chemical composition. AIM OF THE STUDY: The study aims to explore XYPs' underlying antidepressant mechanism from both in vivo and in vitro. MATERIALS AND METHODS: XYPs were composed of 8 herbs, including the root of Bupleurum chinense DC., the root of Angelica sinensis (Oliv.) Diels, the root of Paeonia lactiflora Pall., the sclerotia of Poria cocos (Schw.) Wolf, the rhizome of Glycyrrhiza uralensis Fisch., the leaves of Mentha haplocalyx Briq., the rhizome of Atractylis lancea var. chinensis (Bunge) Kitam., and the rhizome of Zingiber officinale Roscoe, in a ratio of 5:5:5:5:4:1:5:5. The chronic unpredictable mild stress (CUMS) rat models were established. After that, the sucrose preference test (SPT) was carried out to evaluate if the rats were depressed. After 28 days of treatment, the forced swimming test and SPT were carried out to evaluate the antidepressant efficacy of XYPs. The feces, brain and plasma were taken out for 16SrRNA gene sequencing analysis, untargeted metabolomics and gut microbiota transformation analysis. RESULTS: The results revealed multiple pathways affected by XYPs. Among them, the hydrolysis of fatty acids amide in brain decreased most significant via XYPs treatment. Moreover, the XYPs' metabolites which mainly derived from gut microbiota (benzoic acid, liquiritigenin, glycyrrhetinic acid and saikogenin D) were found in plasma and brain of CUMS rats and could inhibit the levels of FAAH in brain, which contributed to XYPs' antidepressant effect. CONCLUSIONS: The potential antidepressant mechanism of XYPs by untargeted metabolomics combined with gut microbiota-transformation analysis was revealed, which further support the theory of gut-brain axis and provide valuable evidence of the drug discovery.

Structure identification and content analysis of active components in Xiaoyao pills / 药学学报

Xiaoyao pills are a famous traditional Chinese medicine collected in Welfare Pharmacy, which is a classic prescription for treating liver depression and spleen deficiency. However, its composition is complex. In order to better control the quality of Xiaoyao pills, in this study, HPLC-ion-trap time-of-flight mass spectrometry (LC-IT-TOF/MS) was used to identify the main ingredients of Xiaoyao pills, paeoniflorin, albiflorin, glycyrrhizic acid, saikosaponin A and saikosaponin B2. Then a liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed for simultaneous determination and quantification of the main compounds. Fragmentation pathways of five active components were obtained. The method was validated. Five active ingredients in Xiaoyao pills had a good linear relationship, and the values of RSD (%) of repeatability were all less than 5%, the recovery ranges were between 90% and 115%, and the values of RSD (%) of each substance were less than 10% after the sample solution is placed for 24 hours. Three batches of Xiaoyao pills (concentrated pellets) and two batches of Xiaoyao pills (water pellets) were determined, the contents of paeoniflorin in concentrated pills were more than 4.0 mg·g-1, and those in water pills were more than 2.5 mg·g-1, which was accordance with Chinese Pharmacopoeia. However, other compounds behave differently. This method has high sensitivity and reliable measurement results, which provides basis for quality control of Xiaoyao pills and material basis for pharmacology research.

Xiaoyao Pills Ameliorate Depression-like Behaviors and Oxidative Stress Induced by Olfactory Bulbectomy in Rats via the Activation of the PIK3CA-AKT1-NFE2L2/BDNF Signaling Pathway.

Numerous studies have revealed that oxidative stress is closely associated with the occurrence and development of depression. Xiaoyao Pills (XYW) are included in the Chinese Pharmacopoeia and are frequently used for treating anxiety and depression by smoothing the liver, strengthening the spleen, and nourishing the blood. However, the antidepressant effects of XYW have not yet been thoroughly investigated. The objective of our study was to investigate the antidepressant-like effects of XYW and the underlying molecular mechanism in the olfactory bulbectomized (OB) rat model of depression using the open field test (OFT), sucrose preference test (SPT), splash test (ST), and novelty suppressed feeding test (NSFT). Results showed that XYW (0.93 and 1.86 g·kg-1) significantly alleviated depression-like behaviors in rats, which was indicated by increased sucrose preference in the SPT, prolonged grooming time in the ST, decreased horizontal movement in the OFT, and shorter feeding latency in the NSFT. In addition, XYW treatment dramatically reversed the reduced activity of superoxide dismutase and the decreased level of glutathione, while also lowering levels of malondialdehyde, an inflammatory mediator (nitric oxide), and pro-inflammatory cytokines (interleukin-6 and 1ß) in the serum and cortex of OB rats. Mechanistically, XYW induced marked upregulation of mRNA and protein expression levels of NFE2L2, KEAP1, GPX3, HMOX1, SOD1, NQO1, OGG1, PIK3CA, p-AKT1/AKT1, NTRK2, and BDNF, and downregulation of ROS in the cortex and hippocampus via the activation of the NFE2L2/KEAP1, PIK3CA/AKT1, and NTRK2/BDNF pathways. These findings suggest that XYW exert antidepressant-like effects in OB rats with depression-like symptoms, and these effects are mediated by the alleviation of oxidative stress and the enhancement of neuroprotective effects through the activation of the PIK3CA-AKT1-NFE2L2/BDNF signaling pathways.

Xiaoyao Pills Prevent Lipopolysaccharide-Induced Depression by Inhibiting Inflammation and Protecting Nerves.

Lipopolysaccharides are pro-inflammation mediators that can induce inflammation in the serum, hippocampus, and cortex of animals. And lipopolysaccharide-induced neuroinflammatory state resulted in significant depression-like behaviors, including reduced locomotor activity in the open field test, reduced saccharin preference, added immobility time in tail suspension test and forced swimming test, decreased comb time in the splash test, and increased latency to food in the novelty suppressed feeding test time, and reduced the levels of neurotrophic factors and synaptic proteins, and decreased Nissl bodies. Treatment with Xiaoyao Pills ameliorated the depression-like behavior, decreased the levels of inflammatory indicators, increased those of neurotrophic factors and synaptic proteins, and restored Nissl bodies. Our study suggests that lipopolysaccharides induce inflammation and nerve injury, thereby leading to depression. Xiaoyao Pills could be considered a potential therapeutic candidate for inflammation-induced depression.

Effect Of Chinese Herb Danzhi Xiaoyao Pills On Pharmacokinetics Of Venlafaxine In Beagles.

OBJECTIVE: To investigate the effects of Chinese herb Danzhi Xiaoyao pills on the pharmacokinetics of venlafaxine and its metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in beagles by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: Six beagles (half male, half female) were chosen to test, being fasted before the experiment but having free access to drinking water 1 day before being fed drugs. After oral administration of venlafaxine hydrochloride tablets (10.28 mg/kg), the blood samples were collected in succession at different points in time. After 1-week washout period, Danzhi Xiaoyao pills (0.6g/kg) were given through oral administration to the six beagles every morning until the 7th day, venlafaxine hydrochloride tablets (10.28 mg/kg) were given after feeding Danzhi Xiaoyao pills (0.6g/kg) half an hour and blood samples were collected continuously at different points. All samples were analyzed by UPLC-MS/MS, and the main pharmacokinetic parameters of venlafaxine, ODV and NDV were computed by DAS 2.0. RESULTS: The Cmax of the venlafaxine group (control group) and the combination group (experimental group) were (2267.26±252.89) ng/mL and (1542.64±190.73) ng/mL, respectively. The AUC(0-∞) of the two groups were (13,934.79±3609.23) ng·h/mL and (8001.91±2167.58) ng·h/mL, respectively. The ODV Cmax of the two groups were (2253.80±215.81) ng/mL and (2721.37±118.20) ng/mL, and AUC(0-∞) were (13,974.99±2784.04) ng·h/mL and (17,539.44±1894.29) ng·h/mL, respectively. The NDV Cmax of the two groups were (50.98±5.76) ng/mL and (58.74±12.33) ng/mL, and AUC(0-∞) were (179.26±34.94) ng·h/mL and (220.68±51.41) ng·h/mL, respectively. After administration of Danzhi Xiaoyao pills, the Cmax and AUC(0-∞) of venlafaxine decreased significantly, indicating that the plasma exposure of venlafaxine decreased. The increase of Cmax and AUC(0-∞) of ODV and NDV indicated a rise in plasma exposure. CONCLUSION: Danzhi Xiaoyao pills can accelerate the metabolism of venlafaxine in beagles. In clinical, when venlafaxine was co-administrated with Danzhi Xiaoyao pills, dose adjustment of venlafaxine should be taken into account.

Determination of Six Index Components in Xiaoyao Pills by Quantitative Analysis of Multi-components by Single Marker / 中国药师

Objective:To develop a method of quantitative analysis of multi-components by single marker (QAMS) for six active constituents in Xiaoyao pills. Methods:Using glycyrrhizic acid as the reference, an HPLC method with a Kromasil C18 column (250 mm ×4.6 mm,5 μm) was applied,the mobile phase was acetonitrile(A)-0.1% phosphoric acid solution(B) with gradient elution (0-10 min,5 % A;10-25 min,5 % →20 % A;20-70 min,15 % →60 % A and 40-60 min,60 % A). The detection wavelength was 280nm,the column temperature was 35℃ and the injection volume was 10 μl. The relative correction factors among the six index components were detected by QAMS. The contents of the six index components were determined by an external standard method and QAMS to compare the results obtained from the two different methods and verify the practicability and stability of QAMS. Results:The established QAMS was used to determine the six index components in Xiaoyao pills,and totally 6 batches of Xiaoyao pills were determined.There were no significant differences in the calculated values and the determined ones (P > 0.05). Conclusion:The QAMS method is simple,effective and accurate in determining the contents of the six index components in Xiaoyao pills,which can be used for the quality control of Xiaoyao pills and provide reference for the further study.

Influences of Xiaoyao Pills on peripheral blood and hypothalamic-pituitary- adrenocortical axis in rats with syndrome of stagnation of liver qi and blood deficiency / 中草药

Objective: To study the effect of Xiaoyao Pills (containing Paeoniae Alba Radix) on peripheral blood and hypothalamic- pituitary-adrenocortical (HPA) axis in rats with syndrome of stagnation of liver qi and blood deficiency. Methods: The model of syndrome of stagnation of liver qi and blood deficiency was established by chronic restraint stress combined with radiation, and healthy male rats were randomly divided into four groups such as normal group, model group, Paeoniae Alba Radix group, and Xiaoyao Pills group. The rats in treatment groups and normal group were fed with medicines and pure water from day 1 of the experiment, respectively. The weight, peripheral blood, glucose consumption, and hypothalamic-pituitary-adrenal axis were measured. Results: Compared with the model group, the weight and amount of WBC of model group were decreased significantly (P < 0.001), and the amounts of ACTH, CORT, and CRH were increased significantly (P < 0.01 or 0.001). Glucose consumption test showed that the ability of model group to explore the novel environment was reduced (P < 0.01), Xiaoyao Pills group on the above indicators had a corresponding regulatory role (P < 0.001 or P < 0.01). Conclusion: The effect of Xiaoyao Pill on peripheral blood and HPA axis may be one of the mechanisms of its function of soothing liver to resolve depression and nourishing blood to invigorate the spleen.

Clinical effect of large speckle modulated Q1064nm laser combined with Xiaoyao pills in treatment of chloasma / 中华医学美学美容杂志

Objective To investigate the clinical efficacy of large spot modulated Q 1064 nm laser combined with Xiaoyao pills in the treatment of chloasma.Methods 90 patients with chloasma were treated in the hospital.The patients were randomly divided into 3 groups:treatment group were treated with Q 1064 nm laser combined with large spot Xiao Yao pill;the control group 1 was treated with large spot Q 1064 nm laser only,and control group 2 was treated with Xiaoyao Pill only.Results After 8 weeks of treatment,the effective rate was higher in the treatment group than the control group 2 (P＜0.05);after 16 weeks of treatment,the efficacy was better in the treatment group than the control group 1 and control group 2 (P＜0.05).The combination of the two methods can improve the curative effect and inhibit the recurrence.Conclusions The treatment of chloasma with large speckle modulated Q 1064 nm laser combined with Xiaoyao pills is effective and worthy of clinical promotion.

Content Determination of Paeoniflorin and Liquoritin in Jiawei Xiaoyao Pills by HPLC / 中国药房

OBJECTIVE:To establish the HPLC method for the content determination of paeoniflorin and liquoritin in Jiawei xiaoyao pills. METHODS:It was performed on Hypersil-ODS C18 with the mobile phase of acetonitrile-water(14∶86,V/V)at the flow rate of 1.0 ml/min,the detection wavelength was 230 nm,the column temperature was room temperature and the volume was 10 μl. RESULTS:The linear range was 16.12-80.6 μg/ml for paeoniflorin(r=0.999 6)and 6.08-30.4 μg/ml for liquoritin(r=0.999 8);the RSDs of precision,repeatability and stability tests were all less than 2.0%;the average recovery was respectively 98.51%(RSD=1.94%,n=6)and 98.08%(RSD=1.29%,n=6). CONCLUSIONS:The method is simple,accurate and reliable, and can be used for the content determination of paeoniflorin and liquoritin in Jiawei xiaoyao pills.

Effect of auricular point sticking on menstrual distending pains of breasts / 现代临床护理

Objective To observe the curative effect of auricular point sticking on menstrual distending pains of breasts. Methods Sixty-two patients with hyperplasia of mammary glands were randomized evenly into experiment group and control group:the former were managed with auricular point sticking and the latter with oral administration of Xiaoyao Pills for three treatment courses, each menstrual cycle as a course.The two groups were compared in terms of menstrual distending pains of breasts.Results The experiment group was significantly better than the control group in terms relieves of pains as well as the curative effect after the trial (P<0.01 for both).Conclusions Auricular point sticking is effective in relieving the menstrual distending pains of breasts.It is simple for manipulation and thus worth clinical spreading.