LU101867B1

LU101867B1 - Method for preparing antidepressive standard baihe dihuang decoction and use thereof

Info

Publication number: LU101867B1
Application number: LU101867A
Authority: LU
Inventors: Ke Ma; Shijun Wang; Yuan Wang; Haijun Zhao; Hongxiu Zhang; Shan Ma
Original assignee: Univ Shandong Traditional Chinese Medicine
Priority date: 2019-07-25
Filing date: 2019-12-17
Publication date: 2021-01-20
Also published as: WO2021012589A1; CN110448636A

Abstract

The present invention relates to the field of traditional Chinese medicine (TCM), and in particular to a method for preparing an antidepressive standard Baihe Dihuang Decoction. First, records of the dosage, places of origin of genuine medicinal materials, classical formula preparation methods of the Baihe Dihuang Decoction are searched from ancient literature to prepare a "standard Baihe Dihuang Decoction". After intervention, the Baihe Dihuang Decoction increases the content of monoamine neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NE), and dopamine (DA), lowers the content of inhibitory neurotransmitters, and raises the content of excitatory neurotransmitters. Finally, active pharmaceutical ingredients (APIs) are analyzed by high performance liquid chromatography (HPLC), providing a modern scientific basis for the mechanism underlying the efficacy of the Baihe Dihuang Decoction in depression and formulas. It is of high theoretical significance and clinical value.

Description

i 1 METHOD FOR PREPARING ANTIDEPRESSIVE STANDARD BATHE DIHUANG 101867

DECOCTION AND USE THEREOF

[0001] TECHNICAL FIELD

[0002] The present invention relates to the field of traditional Chinese medicine (TCM), and in particular to a method for preparing an antidepressive standard Baihe Dihuang Decoction and use thereof.

[0003] BACKGROUND

[0004] Depression is mental disease clinically manifested as emotional depression, thought retardation, decreased or slow speech and activity, including postpartum depression, menopause depression, dysthymia, etc., which seriously influence the quality of life of patients. A co-study of the World Health Organization (WHO) indicated that depression had been the second serious disease burden in China. Depression has complex etiological factors; there are a plurality of hypotheses of pathogenesis thereof; various kinds of antidepressants developed accordingly merely benefit some patients, and there are defects, such as slow improvement of clinical symptoms, easy recurrence, and numerous side effects. These suggest that the existing pathogenesis of depression is not enough to explain the multicausal heterogeneous disease. However, Chinese herbal compound formulas are administered under the guidance of syndrome differentiation and treatment. Characterized by numerous components, multiple targets, and overall regulation, these formulas are consistent with the essence that diseases are caused by the complexity of the body, and show unique advantages and good prospects in the prophylaxis and treatment of depression.

[0005] There is no so-called depression in TCM, but considerable similar descriptions of symptoms of the disease are found in ancient literature, for example, lily disease recorded in the Golden Chamber Synopsis. Eating disorder, insanity, dyssomnias, and abnormal behavior manifested by the lily disease are very similar to cardinal clinical manifestations of depression. The disease is mostly caused by unremitted residual evil gi of the heat illness to compromise the heart and lungs, by heart-lung yin deficiency with endogenous heat caused by continuous anxiety and slow loss of yin and blood, or by development of various diseases caused by deficiency-heat “ disturbing mind. Treatment principles are clearing away the heart fire to moisten the lung, and ’ nourishing yin for tranquillization. Baihe Dihuang Decoction should be prescribed. Clinical use and - fundamental research have found that Baihe Dihuang Decoction has an excellent therapeutic effect on depression. ; [0006] SUMMARY . [0007] To normalize the preparation of famous classical formulas of TCM, it is particularly ° -. important to clarify the mechanisms and active pharmaceutical ingredients (APIs) of classical formulas and strengthen the research on quality standards of TCM. Therefore, the present invention provides a method for preparing an antidepressive standard Baihe Dihuang Decoction. ITH¥ 867 antidepressant effect and pharmacological mechanism of the “standard Bathe Dihuang Decoction” are investigated by determining related ancient measurement units and selecting a modified model of lipopolysaccharide (LPS)-induced depression of endogenous heat.

[0008] A method for preparing an antidepressive standard Baihe Dihuang Decoction includes the following steps: (1) the dosage and administration of Baihe Dihuang Decoction: taking seven pieces or a segment of Lilii Bulbus, and measuring one sheng of Rehmanniae Radix juice; washing the Lilii Bulbus with water, and soaking in water overnight; removing the water until foams appear; changing the water with two sheng of spring water and decocting to one sheng, and removing dregs; pouring the Rehmanniae Radix juice into the resulting decoction, and decocting to 1.5 sheng; taking the warm decoction in divided doses; (2) standardizing: one sheng is 200 ml; the Rehmanniae Radix is selected from Jiaozuo, Henan Province; the Lilii Bulbus from Shennongjia, Hubei Province is used as a measured medicinal material; and (3) using the amount obtained in step (1) to obtain a standard decoction according to the standards in step (2).

[0009] The preparation method includes the following steps: (1) juicing fresh Rehmanniae Radix from Jiaozuo, Henan Province to obtain fresh Rehmanniae Radix juice; (2) washing fresh Lilii Bulbus from Shennongjia, Hubei Province with water, soaking in water overnight, and decocting with spring water to obtain a Lilii Bulbus water decoction; and (3) adding the decoction obtained in step (2) to the juice obtained in step (1), and decocting to obtain a standard Baihe Dihuang Decoction.

[0010] A principal component analysis of the standard Baihe Dihuang Decoction includes the following steps: (a) chromatographic conditions of the Lilii Bulbus and preparation of test solution; mobile phase: acetonitrile (A)-0.05% phosphoric acid solution (B); elution gradient: 0-6 min, 5% A; 6-15 min, 5%-14% A; 15-25 min, 14% A; 25-30 min, 14%-21% A; 30-35 min, 21% A; 35-45 min, 21%-36% A; 45-51 min, 36% A; 51-63 min, 36%-66.3% A; 63-70 min, 66.3% A; 70-83 min,

66.3%-100% A; and 83-90 min, 90% A; flow rate: 1.0 mL/min; column temperature: 30°C; detection wavelength of ferulic acid fraction: 330 nm; and detection wavelength of dioscin fraction: 205 nm;

[0011] preparation of reference solutions: accurately weighing ferulic acid chemical reference substance (CRS) (g.s.) and dioscin CRS

| (q.s.) in 80% methanol to prepare 0.0200 mg/mL reference solutions, respectively; shaking We 1867 sonicating to dissolve either reference solution fully, and filtering through a microporous membrane to collect subsequent filtrates, namely, the reference solutions; preparation of test solutions: preparation of test solutions of ferulic acid and dioscin: breaking 400 g of fresh Lilii Bulbus into pieces, washing clean, wiping dry, weighing accurately, and decocting in a casserole for 1.5 h; condensing the decoction to 200 ml; placing the concentrated decoction in an evaporating dish and evaporating to dryness in a water bath; grinding residues in a mortar into a powder; accurately weighing 2 g of powder in a conical flask with stopper, adding 50 ml of methanol accurately, stoppering tightly, weighing, sonicating, cooling, weighing again, making up to weight with methanol, and shaking well; filtering through a funnel and a microporous membrane successively to collect subsequent filtrates, namely, the reference solutions;

[0012] (b) chromatographic conditions of the Rehmanniae Radix and preparation of test solution; mobile phase: acetonitrile-0.1% phosphoric acid solution (1:99); detection wavelength: 210 nm; flow rate: 1.0 mL/min; injection volume: 10 uL; and column temperature: 25°C; mobile phase of verbascoside: methanol (A)-0.1% phosphoric acid solution (B); gradient elution program: 0-5 min, 40%-43% A; 5-10 min, 43%-56% A; 10-20 min, 56%-60% A; and 20-25 min, 60%-40% A; detection wavelength: 334 nm; flow rate: 1.0 mL/min; injection volume: 20 uL; and column temperature: 25°C; preparation of reference solutions: accurately weighing catalpol CRS (q.s.), adding the mobile phase to prepare a 0.5 mg/ml solution, shaking well, sonicating for 5 min to dissolve the catalpol CRS fully, filtering through a microporous membrane to collect a subsequent filtrate; accurately weighing verbascoside CRS (q.s.), adding 50% methanol to prepare a 0.06 pg/ml solution, dissolving, and filtering;

[0013] preparation of test solutions: preparation of test solutions of catalpol and verbascoside: weighing 400 g of fresh Rehmanniae Radix, washing clean, wiping dry, removing fibrous roots, cutting into pieces, weighing accurately, and juicing in a juicer to obtain 200 ml of juice; placing the juice in an evaporating dish, and evaporating to dryness in a water bath; grinding residues in a mortar into a powder; accurately weighing 2 g of powder in a conical flask with stopper, adding 50 ml of methanol accurately, stoppering tightly, weighing, sonicating, cooling, weighing again, making up to weight with methanol, and shaking well; filtering through a funnel and a microporous membrane successively to collect subsequent filtrates, namely, the reference solutions;

[0014] Use of the principal component analysis in determining the quality of the Baihe Dihuang Decoction is disclosed.

[0015] Use of the antidepressive standard Baihe Dihuang Decoction as an antidepressant {31867 disclosed.

[0016] First, records of the dosage, places of origin of genuine medicinal materials, classical formula preparation methods of the Baihe Dihuang Decoction are searched from ancient literature to prepare a “standard Baihe Dihuang Decoction”. Next, a model of LPS-induced depression is selected to investigate the antidepressant effect and pharmacological mechanism of the “standard Baihe Dihuang Decoction”. Finally, APIs thereof are analyzed by high performance liquid chromatography (HPLC), providing a modern scientific basis for the mechanism underlying the efficacy of the Baihe Dihuang Decoction in depression and formulas. It is of high theoretical significance and clinical value.

[0017] Compared with LPS model group, after intervention, the Baihe Dihuang Decoction increases the content of monoamine neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NE), and dopamine (DA), lowers the content of inhibitory neurotransmitters, and raises the content of excitatory neurotransmitters; also, the Baihe Dihuang Decoction restores the balance between gamma-aminobutyric acid (GABA) and glutamic acid and lowers levels of pro-inflammatory cytokines interleukin-1B (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) through double regulation. By high-throughput sequencing plus bioinformatics, preliminary analysis shows that the Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) up-regulates the expression of GABAergic neuron information coding output genes GAD-67, VGAT, and GAT-3 mRNAs in prefrontal cortex (FIGS. 4 and 5) after intervention.

[0018] Four antidepressive APIs in fresh Lilii Bulbus and Rehmanniae Radix juice are analyzed by HPLC; after qualitative determination of content thereof, each of the four APIs is assayed in the same TCM decoction; results show that delivery of dioscin and ferulic acid in the compound decoction shows a downward trend compared with that in fresh products, whereas verbascoside and catalpol show an upward trend (Tables 1 and 2).

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 illustrates a preparation process of a standard Baihe Dihuang Decoction.

[0021] FIG. 2 illustrates significant improvement of LPS-induced depression-like behaviors after intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula). Treatment with both Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) and fluoxetine significantly increases the sucrose intake (A), shortens the immobility time in the forced swim test (B) and tail suspension test (C), and improves social behaviors in the Y-maze (D). * represents a comparison with the control group, *P<0.01, **P<0.01, and ***P<0.001. * represents a comparison with the LPS + Saline group, *P<0.01, #P<0.01, and #*P<0.001 (9-12 rats in each group). LPS+Saline: LPS + Saline group; LPS+Flu: LPS + Fluoxetine group; LPS+LBRD: LPS + Baihe Dihuang Decoction

(Zhang Zhongjing’s classical formula) group; and LPS+XDF: LPS + modern Baihe Dihuat(è! 867 Decoction group.

[0022] FIG. 3 illustrates modeling and an intervention schedule.

[0023] FIG. 4 illustrates changes in neurotransmitters and cytokines in sera of mice with LPS- induced depression-like behaviors after intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula). Compared with the LPS + Saline group, intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) significantly increases the content of 5- HT (A), NE (B), DA (C), and GABA (D) in model mice, and lowers levels of glutamic acid (E), corticosterone (F), Il-1B (G), IL-6 (H), and TNF-o (I). * represents a comparison with the control group, *P<0.01, **P<0.01, and ***P<0.001. * represents a comparison with the LPS + Saline group, #p<0.01, #P<0.01, and *##P<0.001 (9-12 rats in each group). LPS+Saline: LPS + Saline group; LPS+Flu: LPS + Fluoxetine group; and LPS+LBRD: LPS + Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) group.

[0024] FIG. 5 illustrates the expression of mRNAs in the prefrontal lobes of rats in each group. (A) Mouse prefrontal lobe mRNA expression profiles after different interventions are detected by high- throughput sequencing. Genes with a >1.5-fold difference between groups are screened for cluster analysis. (B) Compared with the LPS + Saline group, here is a heat map of differentially expressed genes in the prefrontal cortex after intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) (four rats in each group).

[0025] DETAILED DESCRIPTION

[0026] Example 1

[0027] A method for preparing a standard decoction included the following steps: (1) Fresh Rehmanniae Radix was selected from Jiaozuo, Henan Province to juice repeatedly. The juice yield of the fresh Rehmanniae Radix was approximately 50%, i.e., 400 g of fresh Rehmanniae Radix was juiced to a volume of approximately 200 ml. Fresh Lilii Bulbus from Shennongjia, Hubei Province was used as a measured medicinal materials. After measurement, seven pieces of Lilii Bulbus (washed with water and soaked overnight) had a fresh weight of approximately 400 g and a dry weight of approximately 175 g.

[0028] (2) Approximately 400 g of seven fresh Lilii Bulbus from Shennongjia, Hubei Province (washed with water and soaked overnight) were decocted with 400 ml of spring water to obtain 200 ml of Lilii Bulbus water decoction.

[0029] (3) Two hundred milliliters of fresh Rehmanniae Radix juice (obtained from approximately 400 g of fresh Rehmanniae Radix) was mixed for further decoction, to obtain 300 ml of standard Baihe Dihuang Decoction.

[0030] Example 2

[0031] 1. Chromatographic conditions of the Lilii Bulbus and preparation of test solution lu101867

[0032] Mobile phase was acetonitrile (A)-0.05% phosphoric acid solution (B); elution gradient was performed (0-6 min, 5% A; 6-15 min, 5%-14% A; 15-25 min, 14% A; 25-30 min, 14%-21% A; 30-35 min, 21% A; 35-45 min, 21%-36% A; 45-51 min, 36% A; 51-63 min, 36%-66.3% A; 63-70 min, 66.3% A; 70-83 min, 66.3%-100% A; and 83-90 min, 90% A); flow rate was 1.0 mL/min; column temperature: was 30°C; detection wavelength of ferulic acid fraction was 330 nm; detection wavelength of dioscin fraction was 205 nm; injection volume was 20 pL.

[0033] Preparation of reference solutions:

[0034] Ferulic acid CRS (g.s.) and dioscin CRS (g.s.) were accurately weighed in 80% methanol to prepare 0.0200 mg/mL reference solutions, respectively; after shaking well, the reference solutions were sonicated for 5 min to dissolve fully, and filtered through a microporous membrane (0.45 um oil film) to collect subsequent filtrates, namely, the reference solutions.

[0035] Preparation of test solutions:

[0036] Preparation of test solutions of ferulic acid and dioscin: 400 g of fresh Lilii Bulbus (broken into pieces, washed clean, and wiped dry) was weighed accurately, and decocted in a casserole for

1.5 h; the resulting decoction was condensed to 200 ml; the concentrated decoction was placed in an evaporating dish and evaporated to dryness in a water bath; residues were ground in a mortar into a powder; 2 g of powder was accurately weighed in a conical flask with stopper, and 50 ml of methanol was added accurately; the conical flask was stoppered tightly, weighed, sonicated for 15 min, cooled, weighed again, made up to weight with methanol, and shaken well; the solution was filtered through a funnel and a microporous membrane (0.45 um oil film) successively to collect subsequent filtrates, namely, the test solutions.

[0037] 2. Chromatographic conditions of the Rehmanniae Radix and preparation of test solution

[0038] Mobile phase was acetonitrile-0.1% phosphoric acid solution (1:99); detection wavelength was 210 nm; flow rate was 1.0 mL/min; injection volume was 10 pL; and column temperature was 25°C; mobile phase of verbascoside was methanol (A)-0.1% phosphoric acid solution (B); gradient elution program was 0-5 min, 40%-43% A; 5-10 min, 43%-56% A; 10-20 min, 56%-60% A; and 20-25 min, 60%-40% A. Detection wavelength was 334 nm; flow rate was 1.0 mL/min; injection volume was 20 pL; and column temperature was 25°C.

[0039] Preparation of reference solutions:

[0040] Catalpol CRS (g.s.) was accurately weighed, mixed with the mobile phase to prepare a 0.5 mg/ml solution, shaken well, sonicated for 5 min to dissolve the catalpol CRS fully; the resulting solution was filtered through a microporous membrane (0.45 pm water film) to collect a subsequent filtrate; verbascoside CRS (q.s.) was accurately weighed, mixed with 50% methanol to prepare a

0.06 pg/ml solution, dissolved, and filtered (did the same operations as the catapol).

[0041] Preparation of test solutions: lu101867

[0042] Preparation of test solutions of catalpol and verbascoside: 400 g of fresh Rehmanniae Radix (washed clean, wiped dry, removed fibrous roots, cut into pieces) was weighed accurately, and juiced in a juicer to obtain 200 ml of juice; the juice was placed in an evaporating dish and evaporated to dryness in a water bath; residues were ground in a mortar into a powder; 2 g of powder was accurately weighed in a conical flask with stopper, and 50 ml of methanol was added accurately; the conical flask was stoppered tightly, weighed, sonicated for 15 min, cooled, weighed again, made up to weight with methanol, and shaken well; the resulting solution was filtered through a funnel and a microporous membrane (0.45 pm oil film) successively to collect subsequent filtrates, namely, the test solutions.

[0043] Results

[0044] Three aliquots of the same sample were placed in respective sample vials and detected under the above chromatographic conditions; the retention time of the samples coincided approximately with that of standard, and effective separation from other substances was done under this condition. Content of dioscin and ferulic acid was calculated by the external standard method based on peak areas. Results are shown in Table 1.

[0045] Table 1 Content of dioscin and ferulic acid in test solutions time solution mg/ml solution mg/ml

[0046] Three aliquots of the same sample were placed in respective sample vials and detected under the above chromatographic conditions; the retention time of the samples detected coincided approximately with that of standard, and effective separation from other substances was done under this condition. Content of catalpol and verbascoside was calculated by the external standard method based on peak areas. Results are shown in Table 2.

[0047] Table 2 Content of catalpol and verbascoside in test solutions time

[0048] Comparative Example 1

[0049] Preparation process of modern Baihe Dihuang Decoction: 45 g of Lilii Bulbus (dried product) and 45 g of Rehmanniae Radix (dried product) were decocted with spring water twice, each for 30 min; after combining, 300 ml of extract was obtained for use.

[0050] Effect Example 1

[0051] An equivalent dose (150 g-kg!) of a rat was converted by the body surface area method. Each rat was administered at a volume of 10 ml-kg' by gavage daily. Models of depression of endogenous heat were constructed by the modified LPS method. Each rat was administered for two consecutive weeks while modeling, with fluoxetine as a positive control. Experimental results are shown in FIG. 2. “Standard Baihe Dihuang Decoction” can improve LPS-induced depression-like behaviors. By searching ancient medical books, textual research is conducted on the composition, dosage, places of origin of medical materials of the Baihe Dihuang Decoction; then a “standard decoction” is prepared to verify the antidepressant effect, providing a reference for simplified registration of classical Baihe Dihuang Decoction.

[0052] From Days 1 to 14, rats in the LPS model group, fluoxetine positive control group, and Baihe Dihuang Decoction treatment group were given 0.3 mg/kg LPS intraperitoneally, 0.5 ml each time; blank controls were operated as above, but not given the corresponding drug.

[0053] Blank control group, i.e., Control group, were conventionally fed under the above laboratory conditions and given normal saline equivalent to the dose of Baihe Dihuang Decoction by gavage from Days 4 to 17 of the experiment.

[0054] LPS model group, i.e., LPS + Saline group, were given normal saline equivalent to the dose of Baihe Dihuang Decoction by gavage from Days 4 to 17 of the experiment.

[0055] Baihe Dihuang Decoction treatment group, i.e., LPS + LBRD group, were given 95 g/kg Baihe Dihuang Decoction by gavage from Days 4 to 17 of the experiment, and the dosage was calculated by the body surface area method.

[0056] Modern Baihe Dihuang Decoction treatment group, i.e., LPS + XDF group, were given 95 g/kg Baihe Dihuang Decoction by gavage from Days 4 to 17 of the experiment, and the dosage was calculated by the body surface area method. lu101867

[0057] Fluoxetine positive control group, i.e., LPS + Flu group, were given 10 mg/kg fluoxetine by gavage from Days 4 to 17 of the experiment, and the dosage was calculated by the body surface area method.

[0058] Modeling and a treatment schedule are shown in FIG. 3.

[0059] Sucrose preference test (SPT)

[0060] A bottle of tap water and a bottle of freshly prepared 1% sucrose solution were prepared in each cage. The bottle wall was wiped dry. Bottles were weighed by an electronic balance and then placed in RVC cages. After rats were given free access to water for 4 h, the positions of both bottles were switched gently; after 4 h normal activities of rats, the bottle wall was wiped dry, and bottles were weighed again; intake of either drink by rats was measured within 8 h of the testing, and sucrose preference was calculated.

[0061] Forced swim test (FST)

[0062] Rats were placed in an aquarium, and struggles thereof were recorded within 6 min by using a camera. Immobility time thereof was recorded in the latter 4 min by a program.

[0063] Tail suspension test (TST)

[0064] The test was performed at night to ensure normal activities of rats. Rats, fixing at approximately 5 cm from the end of the tail, were suspended by a beam around 30 cm from the ground. The fixed rats would struggle to overcome the uncomfortable position, and would appear immobile discontinuously and desperate after a while. The tail was suspended for 6 min for each rat, but each rat was first acclimatized for 2 min. Foe each rat, therefore, only the sum of immobility time within the last 4 min was recorded.

[0065] Y-maze

[0066] Y-maze test was conducted in two steps: step 1, isolating the arm to explore first, placing an experimental rat in the center where the rat was free to move in other two arms, and removing the rat after acclimatization for 5 min; step 2, placing the rat on one side of the arm to explore, and using a program to record how the rat moves freely. After each rat completed the task, the instrument was wiped with alcohol to avoid the interference of the previous rat. After the completion of the task, the ratio of the time a rat stays in the arm to explore to the sum of the time the rat stays in three arms was analyzed using software.

[0067] Results

[0068] Compared with the blank control group, Sprague-Dawley (SD) rats in the LPS model group showed a significant decrease in sucrose preference (in SPT) and a decrease in intake (p<0.01); next, compared with the blank control group, the SD rats in the LPS model group showed a rise in rectal temperature (p<0.01), demonstrating that LPS showed an acute febrile inflammatory response; thus, further practice demonstrated that rat models of LPS-induced depression exhibitdd®d 867 syndrome of endogenous heat. Results are illustrated in FIGS. 4A and 4E.

[0069] Compared with the LPS model group, intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) significantly increased the sucrose intake (A), shortened the immobility time in the FST (B) and TST (C), and improved social behavior time in the Y-maze (D). Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) had an excellent antidepressant effect, and had better efficacy than modern Baihe Dihuang Decoction. Detailed results are illustrated in FIGS. 4A, 4B, 4C, 4D, and 4E.

[0070] Effect Sample 2

[0071] 1. Cytokine assay

[0072] Dilution of standard: The kit provided a vial of original standard, which was diluted in a small test tube.

[0073] Sample spiking: A blank well (neither sample nor conjugate reagent was added to the blank control well, the remaining steps had the same operations), a standard well, and an analyte well were set up, respectively. On a coated ELISA plate, 50 pl of standard was added accurately; 40 ul of sample diluent and 10 pul of analyte (final dilution was 5-fold) were added successively. During sample spiking, the sample was added to the bottom of the well of the ELISA plate and did not touch the wall of the well as far as possible, followed by shaking well gently.

[0074] Incubation: The ELISA plate was blocked with a microplate sealer and incubated for 30 min at 37°C.

[0075] Preparation of diluent: 30x Washing Concentrate was diluted 30-fold with distilled water for use.

[0076] Washing: The microplate sealer was peeled off carefully, and the fluid was discarded; the ELISA plate was spin-dried; each well was filled with wash buffer and allowed to stand for 30 s; then the wash buffer was discarded. The procedure was repeated in quintuplicate. The ELISA plate was pat-dried.

[0077] Enzyme addition: Except the blank well, 50 pl of conjugate reagent was added to each well.

[0078] Incubation: The operations were the same as that of the previous incubation.

[0079] Washing: The operations were the same as that of the previous washing.

[0080] Color development: 50 ul of chromogenic agent A, followed by 50 ul of chromogenic agent B, was added to each well, shaken well gently, and placed in the dark at 37°C for color development for 10 min.

[0081] Stopping: 50 ul of stop solution was added to stop the reaction (during which the color changed from blue turn to yellow immediately).

[0082] Measurement: Setting the blank well to zero, optical density (OD value) of each well was measured at 450 nm successively. The measurement should be conducted within 15 min 481867 addition of the stop solution.

[0083] 2. High-throughput sequencing and bioinformatics analysis

[0084] After rats were anesthetized on ice, prefrontal lobe tissues were extracted and placed in a refrigerator at -80°C. Tissue RNAs were extracted with TRIzol TM LS Reagent and subjected to high-throughput sequencing.

[0085] RNAs (mass >10 pg, concentration >200 ng/ul, RIN>8, 28/18s>1) were used to construct a transcriptome and small RNA libraries. Transcriptome sequencing procedure was as follows: after total RNA with desirable quality was extracted and DNA was digested with DNase I, eukaryotic mRNA was enriched with magnetic beads with Oligo (dT); in a thermomixer at moderate temperature, the mRNA was randomly cleaved into short fragments (approximately 200 bp) using a cleaving agent; the cleaved mRNAs, as template, were synthesized into a single-stranded cDNA; then, a double-stranded synthetic reaction system was prepared to synthesize a double-stranded cDNA, and a kit was used for purification, recovery, repair of cohesive end, addition of base “A” to the 3’-terminal of cDNA, ligation of linker, fragment size selection, and finally PCR amplification (15 cycles); after the quality control of the constructed libraries was qualified by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System, paired-end sequencing was conducted by using Illumina HiSeqTM 2500 System; the sequencing length was 100 bp; 4 G Clean data were acquired for each libraries.

[0086] The original image data acquired by sequencing were converted into the sequence data by base calling, called raw data or raw reads. Subsequently, raw reads were subjected to quality control (QC) to determine whether the sequencing data were suitable for subsequent analysis. DynamicTrim Perl scripts implemented in SolexaQA package were executed to control the quality of the raw sequencing data based on the following criteria. Data filtering had the following steps: 1) removing adapter-containing reads; 2) removing reads with a percentage of N (represents uncertain base information) of >10%; and 3) removing poor-quality reads (the number of bases with a quality value (Q) of <10 accounting for at least 50% of the whole read). After filtering, the remaining data were called “clean reads”. Subsequently, clean reads were mapped to a reference genome (UCSC mm10) by alignment software BWA; clean reads were mapped to reference genes by Bowtie. Erroneous base pairing alignment was set as 3. Expression of genes and transcripts was quantified by RNASeq by Expectation Maximization (RSEM) tool. In RSEM, a maximum likelihood abundance estimation model was established by using Paired-end relationship, reads length, fragment length distribution, and quality value based on the expectation-maximization algorithm, in order to distinguish which transcripts were different subtypes of the same gene. FPKM is used as a unit for the result of quantification of expression, and a specific calculation formula is as follows:

. 106€ lu101867 FPKM= WLf10P (0087] Differential expression analysis aims to find out different expression genes (DEGs) among different samples and further functionally excavate DEGs. Noiseq software package was used to analyze DEGs for inter-group screening, and intra-group samples were required to be biologically repetitive. Criteria for screening DEGs included >1.5-fold changes and diverge probability of >0.8. Subsequently, enrichment analyses were made on the pathway of the correlation between DEGs and physiological or biochemical process. In these enrichment analyses, hypergeometric test was implemented in the tool WebGestalt (Version 2), and the database was from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Through this analysis, the enriched metabolic pathways or signal transduction pathways in DEGs compared with the whole genome background were identified. P values were adjusted using the Benjamini-Hochberg method. The corrected p <0.05 was regarded as significantly enriched.

[0088] Results are shown in FIGS. 4 and 5. Intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) changes neurotransmitters and cytokines in sera of rats with LPS- induced depression-like behaviors. Compared with the LPS + Saline group, intervention with Baihe Dihuang Decoction (Zhang Zhongjing’s classical formula) significantly increases the content of 5- HT, NE, DA, and GABA and lowers levels of glutamic acid, corticosterone, IL-1B, IL-6, and TNF-a in model rats. After treatment, Baihe Dihuang Decoction up-regulates the expression of GABAergic neuron information coding output genes GAD-67, VGAT, and GAT-3 mRNAs in the prefrontal cortex. Thus, the antidepressant effect of Baihe Dihuang Decoction closely relates to the restoration of balance between inhibitory and excitatory neurotransmitters and the reduction of levels of pro- inflammatory cytokines. This finding provides an experimental basis for the modern development of the Baihe Dihuang Decoction.

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Claims

WHAT IS CLAIMED IS: lu101867

1. A method for preparing an antidepressive standard Baihe Dihuang Decoction, comprising the following steps: (1) the dosage and administration of Baihe Dihuang Decoction: taking seven pieces or a segment of Lilii Bulbus, and measuring one sheng of Rehmanniae Radix juice; washing the Lilii Bulbus with water, and soaking in water overnight; removing the water until foams appear; changing the water with two sheng of spring water and decocting to one sheng, and removing dregs; pouring the Rehmanniae Radix juice into the resulting decoction, and decocting to 1.5 sheng; taking the warm decoction in divided doses; (2) standardizing: one sheng is 200 ml; the Rehmanniae Radix is selected from Jiaozuo, Henan Province; the Lilii Bulbus from Shennongjia, Hubei Province is used as a measured medicinal material; and (3) using the amount obtained in step (1) to obtain a standard decoction according to the standards in step (2).

2. The preparation method according to claim 1, comprising the following steps: (1) juicing fresh Rehmanniae Radix from Jiaozuo, Henan Province to obtain fresh Rehmanniae Radix juice; (2) washing fresh Lilii Bulbus from Shennongjia, Hubei Province with water, soaking in water overnight, and decocting with spring water to obtain a Lilii Bulbus water decoction; and (3) adding the decoction obtained in step (2) to the juice obtained in step (1), and decocting to obtain a standard Baihe Dihuang Decoction.

3. A principal component analysis of the standard Baihe Dihuang Decoction according to claim 1 or 2, comprising the following steps: (a) chromatographic conditions of the Lilii Bulbus and preparation of test solution; mobile phase: acetonitrile (A)-0.05% phosphoric acid solution (B); elution gradient: 0-6 min, 5% A; 6-15 min, 5%-14% A; 15-25 min, 14% A; 25-30 min, 14%-21% A; 30-35 min, 21% A; 35-45 min, 21%-36% A; 45-51 min, 36% A; 51-63 min, 36%-66.3% A; 63-70 min, 66.3% A; 70-83 min,

66.3%-100% A; and 83-90 min, 90% A; flow rate: 1.0 mL/min; column temperature: 30°C; detection wavelength of ferulic acid fraction: 330 nm; and detection wavelength of dioscin fraction: 205 nm; preparation of reference solutions: accurately weighing ferulic acid chemical reference substance (CRS) (¢.s.) and dioscin CRS (g.s.) in 80% methanol to prepare 0.0200 mg/mL reference solutions, respectively; shaking well, sonicating to dissolve either reference solution fully, and filtering through a microporous membrane to collect subsequent filtrates, namely, the reference solutions; PT iu preparation of test solutions: lu101867 preparation of test solutions of ferulic acid and dioscin: breaking 400 g of fresh Lilii Bulbus into pieces, washing clean, wiping dry, weighing accurately, and decocting in a casserole for 1.5 h; condensing the decoction to 200 ml; placing the concentrated decoction in an evaporating dish and evaporating to dryness in a water bath; grinding residues in a mortar into a powder; accurately weighing 2 g of powder in a conical flask with stopper, adding 50 ml of methanol accurately, stoppering tightly, weighing, sonicating, cooling, weighing again, making up to weight with methanol, and shaking well; filtering through a funnel and a microporous membrane successively to collect subsequent filtrates, namely, the test solutions; (b) chromatographic conditions of the Rehmanniae Radix and preparation of test solution; mobile phase: acetonitrile-0.1% phosphoric acid solution (1:99); detection wavelength: 210 nm; flow rate: 1.0 mL/min; injection volume: 10 pL; and column temperature: 25°C; mobile phase of verbascoside: methanol (A)-0.1% phosphoric acid solution (B); gradient elution program: 0-5 min, 40%-43% A; 5-10 min, 43%-56% A; 10-20 min, 56%-60% A; and 20-25 min, 60%-40% A; detection wavelength: 334 nm; flow rate: 1.0 mL/min; injection volume: 20 pL; and column temperature: 25°C; preparation of reference solutions: accurately weighing catalpol CRS (g.s.), adding the mobile phase to prepare a 0.5 mg/ml solution, shaking well, sonicating for 5 min to dissolve the catalpol CRS fully, filtering through a microporous membrane to collect a subsequent filtrate; accurately weighing verbascoside CRS (q.s.), adding 50% methanol to prepare a 0.06 ug/ml solution, dissolving, and filtering; preparation of test solutions: preparation of test solutions of catalpol and verbascoside: weighing 400 g of fresh Rehmanniae Radix, washing clean, wiping dry, removing fibrous roots, cutting into pieces, weighing accurately, and juicing in a juicer to obtain 200 ml of juice; placing the juice in an evaporating dish, and evaporating to dryness in a water bath; grinding residues in a mortar into a powder; accurately weighing 2 g of powder in a conical flask with stopper, adding 50 ml of methanol accurately, stoppering tightly, weighing, sonicating, cooling, weighing again, making up to weight with methanol, and shaking well; filtering through a funnel and a microporous membrane successively to collect subsequent filtrates, namely, the test solutions;

4. Use of the principal component analysis according to claim 3 in determining the quality of the Baihe Dihuang Decoction according to claim 1

5. Use of the antidepressive standard Baihe Dihuang Decoction according to claim 1 in the preparation of an antidepressant.