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ObjectiveBased on the antidepressant activity， the optimal ratio of Rehmanniae Radix and Lilii Bulbus in Baihe Dihuangtang was optimized and its possible mechanism was preliminarily explored. MethodA total of 100 male mice were randomly divided into normal group， model group， Rehmanniae Radix single decoction group， Lilii Bulbus single decoction group， and 5 different ratios of Baihe Dihuangtang group （Rehmanniae Radix-Lilii Bulbus 5∶6， 5∶5 ， 5∶4， 5∶3， 5∶2）， fluoxetine hydrochloride group. Except for the normal group， the remaining 9 groups were treated with chronic unpredictable mild stress method （CUMS） for 21 consecutive days to establish the depression mouse model.The Rehmanniae Radix single decoction group， Lilii Bulbus single decoction group and 5 different ratios of Baihe Dihuangtang group were administered with corresponding drugs at a dose of 2 g·kg-1 respectively， and fluoxetine hydrochloride group was administered fluoxetine hydrochloride at a dose of 0.01 g·kg-1， the normal group and the model group were respectively given an equal volume of normal saline by gavage for 21 consecutive days. The antidepressant effect of Baihe Dihuangtang with different ratios was evaluated by behavioral experiments. Western blot was used to detect the expression levels of brain-derived neurotrophic factor （BDNF） and tyrosine kinase receptor B （TrkB） in hippocampus. The level of total superoxide dismutase （SOD） in hippocampus was detected by hydroxylamine method. The level of reduced glutathione （GSH） in hippocampus was detected by spectrophotometric method. The levels of glutathione peroxidase （GPX） and glutathione S-transferase （GST） in hippocampus was detected by colorimetric method. The level of malondialdehyde （MDA） in hippocampus was detected by thiobarbituric acid method， and preliminary exploration of its antidepressant mechanism. ResultCUMS caused a significant decrease in the preference rate of sugar water and a significant prolongation of forced swimming and tail suspension immobility time （P<0.01）， while the different ratios of Baihe Dihuangtang and the single decoction of Rehmanniae Radix and Lilii Bulbus were significantly reversed to varying degrees. Abnormalities of the above indicators were observed （P<0.05， P<0.01）， especially when the ratio of 5∶6 had the most obvious effect on the above indicators， Moreover， CUMS caused the hippocampal BDNF and the expression of TrkB， SOD and glutathione antioxidant-related indexes were significantly decreased （P<0.01）， and the levels of lipid peroxidation product MDA was significantly increased （P<0.01）. However， the superior ratio of Baihe Dihuangtang （Rehmanniae Radix-Lilii Bulbus 5∶6， 5∶5， 5∶4） and the single decoction of Rehmanniae Radix and Lilii Bulbus significantly reversed the abnormality of the above indicators to varying degrees （P<0.05， P<0.01）. ConclusionIn summary， 5 different ratios of Baihe Dihuangtang and the single decoction of Rehmanniae Radix and Lilii Bulbus can produce antidepressant effect， especially when the ratio of 5∶6 had the most obvious effect on the above indicators， and the antidepressant effect gradually weakens with the proportion of Lilii Bulbus in the recipe decreases， and its mechanism may involve enhancing the nutritional level and antioxidant defense ability of hippocampal central nervous system.
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ObjectiveTo establish a traditional Chinese medicine （TCM） syndrome model with yin deficiency and internal heat， discuss the rationality of model evaluation， and analyze differentially expressed genes in multiple dimensions to explore the molecular mechanism-signaling pathways as well as key targets of Baihe Dihuangtang （BHDH） in treating depression with Yin deficiency and internal heat. MethodForty male SD rats were randomly divided into a blank control group，a model group，a fluoxetine group （positive drug），a BHDH group， and a Zhibai Dihuangtang group （positive drug for Yin deficiency and internal heat）. The depression model with Yin deficiency and internal heat was induced by chronic unpredictable mild stress （CUMS）combined with Chinese herbal drugs with warm and heat nature. The model established was comprehensively evaluated by the detection of the basic condition， behavioral performance， and biochemical indicators of rats in each group. The differentially expressed genes were screened out by mRNA sequencing and underwent Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analyses. The protein-protein interaction （PPI） network was plotted and key genes were analyzed to explore the underlying mechanism of BHDH in treating depression with Yin deficiency and internal heat. ResultThe comparison of basic conditions， behavioral assays， energy metabolism， endocrine hormones， cytokines， and neurotransmitters showed that the model was properly induced. BHDH could significantly improve depression with Yin deficiency and internal heat by regulating the pathways related to the nervous system， endocrine system， and inflammatory and immune system. The key genes of the PPI network were Fos， Epha8， Npy2r， Htr2c， and Nr4a1. ConclusionUnder the guidance of TCM theories of treatment based on syndrome differentiation and etiology and pathogenesis，this study established a depression model with yin deficiency and internal heat in animals and evaluation system in accordance with the symptoms and signs of emotional diseases， and further confirmed the scientificity of the modeling method and the underlying mechanism of BHDH in interfering with depression with Yin deficiency and internal heat based on the results of mRNA sequencing.
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ObjectiveTo compare the four preparation methods of Rehmanniae Radix juice described in ancient literature and find the method that is most suitable for the preparation of Rehmanniae Radix juice used in Baihe Dihuangtang. MethodThe ancient medical books record four methods for preparing Rehmanniae Radix juice： crushing fresh Rehmanniae Radix for juice， steaming fresh Rehmanniae Radix for juice， boiling fresh Rehmanniae Radix for juice， and boiling dry Rehmanniae Radix for juice. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS） was employed to detect the compounds in the four juice samples， followed by principal component analysis （PCA）. Result① Totally 27 compounds were identified in the juice samples， including 10 iridoid glycosides， 14 phenylethanoid glycosides， 2 phenolic acids， and 1 irisone. Among them， 15 common compounds were shared by the four juice samples， including 7 iridoid glycosides， 7 phenylethanoid glycosides， and 1 phenolic acid. ② Five common compounds in the four juice samples can be matched with the reference standards， which were catalpol， aucubin， rehmannioside D， ajugol， and purpureaside C. ③ Verbascoside and isoacteoside were not detected in the juice prepared by crushing fresh Rehmanniae Radix， while it was detected in the other three juice samples， which indicated that the two components were produced after heating rather than being the original components in fresh Rehmanniae Radix. ④ The comparison of the ion fragments demonstrated that verbascoside was produced from purpureaside C after the cleavage of the glycosidic bond and removal of a molecule of mannose. ⑤ Isoacteoside could be isomerized from verbascoside， and its relative content increased with the extension of heating time. However， the relative content of verbascoside and purpureaside C did not decrease significantly. Therefore， it was hypothesized that purpureaside C was produced from its upstream component. ConclusionThe juice prepared by crushing fresh Rehmanniae Radix has the chemical composition significantly different from the juice samples prepared with the other 3 methods， while the latter 3 juice samples had similar chemical composition. Although all the four methods can be used， it is more suitable to prepare Rehmanniae Radix juice by steaming fresh Rehmanniae Radix， boiling fresh Rehmanniae Radix， and boiling dry Rehmanniae Radix.
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Objective:To investigate the anti-anxious depression mechanism of Baihe Dihuangtang from the NOD-like receptor thermal protein domain 3 （NLRP3） inflammasome. Method:Fifty SD rats were randomly divided into normal group， model group， venlafaxine group （13.5 mg·kg<sup>-1</sup>）， Baihe Dihuangtang high and low dose group （16，4 g·kg<sup>-1</sup>）， with 10 rats in each group. Chronic restraint stress for 28 days （6 h） combined with subcutaneous injection of corticosterone （30 mg·kg<sup>-1</sup>） was used to establish induce an anxious depression model. From the 8th day of modeling， the rats in the normal group and the model group received distilled water， and those in groups with drug intervention were treated with corresponding drugs by gavage for 21 days. Elevated plus maze and open field test were used to evaluate the behavioral changes of rats. Enzyme- linked immunosorbent assay （ELISA） was used to detect serum and hippocampal interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， interleukin-6 （IL-6） and interleukin-18 （IL-18） levels. Western blot were used to detect the relative expression of hippocampal NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and Caspase-1. The pathological changes of the hippocampus were observed by hematoxylin-eosin（HE） staining， the average fluorescence intensity of NLRP3， ASC， and Caspase-1 was detected by immunofluorescence. The ultrastructure of neurons was observed under electron microscopy. Result:Compared with the normal group， the model group showed reduced total entries （TE）， the ratio of open-arm entries （OE%）， the ratio of open-arm times （OT%）， and the autonomous activity score （<italic>P</italic><0.01）， significant anxiety and depression-like behaviors， increased levels of IL-1<italic>β</italic>， IL-6， and IL-18 in the serum and hippocampus （<italic>P</italic><0.01）， elevated protein expression of NLRP3， ASC， and Caspase-1 （<italic>P</italic><0.01）， activated NLRP3 inflammasomes， and injured hippocampal neurons. Compared with the model group， the high-dose Baihe Dihuangtang group showed improved anxiety and depression-like behaviors （<italic>P</italic><0.01）， and decreased levels of IL-1<italic>β</italic>， IL-6， and IL-18 in the serum and hippocampus （<italic>P</italic><0.05，<italic>P</italic><0.01）， reduced protein expression of NLRP3， ASC， and Caspase-1 （<italic>P</italic><0.01）， and alleviated hippocampal neuron damage. Conclusion:Baihe Dihuangtang can improve neuronal damage in anxious depression by inhibiting the excessive activation of NLRP3 inflammasomes.
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Objective::To study the chemical constituents in Baihe Dihuangtang by ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). Method::The separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm×50 mm, 1.7 μm) by a gradient elution of acetonitrile-0.1%formic acid solution. The flow rate was 0.4 mL·min-1 and the column temperature was 30 ℃, the injection volume was 5 μL. Electrospray ionization was applied and the data were collected via positive and negative ion modes. By using SCIEX OS 1.4 software, the chemical constituents were analyzed based on the retention time, excimer ion peak and fragment ion peak of the compounds, as well as comparison with reference substances and literature data. Result::A total of 49 chemical constituents in Baihe Dihuangtang were identified, including 5 phenolic glycerides, 15 phenylethanoid glycosides, 11 iridoids, 8 lonones, 3 phenylpropanoids, 2 nucleosides, 1 organic acid, and 4 other compounds. Among them, phenolic glycerides belonged to Lilii Bulbus, and other components mainly belonged to Rehmanniae Radix. Four chemical constituents (acteoside, isoacteoside, ferulic acid and caffeic acid) were identified by comparison of reference substances. Conclusion::The established detection method can quickly and accurately analyze the chemical constituents of Baihe Dihuangtang. The information of chemical constituents in Baihe Dihuangtang is comprehensively expounded. The study establishes a foundation for the research of quality control, material foundation of efficacy and the development of compound preparations of Baihe Dihuangtang.
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Objective:To observe the clinical efficacy of addition and subtraction syndrome differentiation method of Huanglian Ejiaotang and Baihe Dihuangtang in the treatment of menopausal syndrome （MPS） and symptoms of deficiency of yin and hyperactivity of fire， and investigate its regulation effects on neuroendocrine network. Method:One hundred and forty-eight patients were divided into control group （74 cases） and observation group （74 cases） according to the random number table method. The patients in control group received estradiol/norethisterone acetate tablets， 3 mg/time， 1 time/day， while the patients in observation group received addition and subtraction of Huanglian Ejiaotang and Baihe Dihuangtang based on the same western medicine treatment as control group， orally 1 dose/day， with a treatment course of 3 months in both groups. Kupperman index （KI） score was evaluated before treatment and at 1 month， 2 months and 3 months after treatment. The Menopausal Quality of Life （MENQOL）， self-rating depression scale （SDS）， Self-rating Anxiety Scale （SAS）， Pittsburgh Sleep Quality Index （PSQI） and score of deficiency of yin and hyperactivity of fire were evaluated before and after treatment. The levels of serum estradiol （E2）， follicle stimulating hormone （FSH）， luteinizing hormone （LH）， norepinephrine （NE）， 5-hydroxytryptamine （5-HT）， nitric oxide （NO）， endothelin-1 （ET-1） and calcitonin gene-related peptide （CGRP） were detected before and after treatment. Result:The clinical efficacy in observation group was better than that in control group （Z=2.115， P<0.05）， and the KI scores in observation group at 1 month， 2 months and 3 months after treatment were lower than those in control group （P<0.01）， and the decrease of KI score in observation group after treatment was larger than that in control group （P<0.01）. The scores of various dimensions of MENQOL scale in observation group were lower than those in control group （P<0.01）. The scores of deficiency of yin and hyperactivity of fire， SAS， SDS and PSQI in observation group were all lower than those in control group （P<0.01）， and the level of E2 in observation group after treatment was higher than that in control group （P<0.01） ，while the levels of FSH and LH were lower than those in control group （P<0.01）.The levels of 5-HT and NE in observation group were higher than those in control group （P<0.01）.The NO level in observation group was higher than that in control group （P<0.01）， while the levels of CGRP and ET-1 were lower than those in control group （P<0.01）. During the study period， there were 5 drop-out cases， 2 excluded cases， and 67 completed cases in the control group. There were 7 drop-out cases， 1 excluded case， 66 completed cases in the observation group. Conclusion:On the basis of hormone replacement therapy， addition and subtraction of Huanglian Ejiaotang and Baihe Dihuangtang for patients with MPS （with symptom of deficiency of yin and hyperactivity of fire） can improve clinical symptoms， relieve depression and anxiety， improve sleep quality and enhance quality of life， and its clinical efficacy is superior to hormone replacement therapy alone. It can also regulate endocrine hormones， vasomotor factors and monoamine neurotransmitters， thereby improving clinical symptoms.